Reliability of bulky DNA adducts measurement by the nuclease P1 32P-post-labelling technique

The aim was to assess the reliability of bulky DNA adducts measurement by means of the ³²P-post-labelling assay. The research design consisted of an intramethod reliability study. Buffy coats from 41 subjects were used to obtain two aliquots of 1–5 μg DNA for each subject; bulky DNA adducts were measured using the nuclease P1 ³²P-post-labelling technique. The reliability of the measurement was assessed by means of the intraclass correlation coefficient (ICC), the distribution of the differences between the two measurements and the limits of agreement. The estimated ICC was 0.977, with a 95% confidence interval between 0.921 and 0.977. The limits of agreement were ±0.44 (DNA adducts per 10⁸ nucleotides). Only three subjects had differences lying out of such limits. Bulky DNA adduct levels measured by the ³²P-post-labelling technique showed good reliability. Only one measurement is needed to use DNA adducts as a biomarker of exposure and, possibly, cancer risk. Besides, as a validation analysis, ³²P-post-labelling measurements can be repeated in only 20–30% of samples.     

Analysis of tamoxifen-induced DNA adducts by 32P-postlabelling assay using different chromatographic techniques

DNA isolated from livers of rats receiving tamoxifen was analysed by the ³²P-postlabelling method. The postlabelled DNA hydrolysis mixture was analysed both by reversed-phase HPLC with ³²P on-line detection and by TLC on polyethyleneimine plates followed by autoradiography. Using the HPLC method, five well separated adduct peaks could be detected, while by the TLC method, two groups of adduct spots were observed. The detection limit of the TLC assay was lower (0.5 adducts/10¹⁰ nucleotides) than that of the HPLC assay (3 adducts/10¹⁰ nucleotides). Thus, the TLC assay is more sensitive but also more laborious. The advantages of the HPLC assay were, in addition to better resolution, the ease of quantification and operation.     

Capillary zone electrophoresis with on-line blotting for separation and detection of 32P-postlabelled DNA adducts

A new technique for the detection of ³²P-postlabelled DNA adducts separated by capillary electrophoresis was developed. By direct transfer from the capillary outlet to a positively charged moving filter paper, eluted radioactive peaks can be quantified using a phosphor imaging detector. With this method it is possible to separate DNA adducts from different carcinogens after ³²P-postlabelling of the modified and unmodified nucleotides with high sensitivity approaching 1 adduct per 10⁹ nucleotides.     

32P-postlabelling analysis of 1,3-butadiene-induced DNA adducts in vivo and in vitro

Butadiene monoepoxide (BMO), epoxybutanediol (EBD) and diepoxybutane (DEB) are reactive metabolites of 1,3-butadiene (BD), an important industrial chemical classified as a probable human carcinogen. The covalent interactions of these metabolites with DNA lead to the formation of DNA adducts which may induce mutations or other types of DNA damage, resulting in tumour formation. In the present study, two pairs of diastereomeric N-1-BMO-adenine adducts were identified in the reaction of BMO with 2´-deoxyadenosine-5´-monophosphate (5´-dAMP). The major products formed by reacting EBD with 2´-deoxyguanosine-5´-monophosphate (5´-dGMP) were characterized as diastereomeric N-7-(2´,3´,4´-trihydroxybut-1´-yl)-5´-dGMP by UV and electrospray mass spectrometry. The formation of N-7-BMO-guanine adducts (1´-carbon, 60; 2´carbon, 54/10⁴ nucleotides) in BMO-treated DNA was about four times higher than that of N-1-BMO-adenine adducts (1´-carbon, 20; 2´-carbon, 8.7/10⁴ nucleotides). However, the recovery of N-1-BMO-adenine adducts in DNA (45 ± 5%) was two times higher than that of N-7-guanine adducts (20 ± 4%) by 32P-postlabelling analysis. Using the 32P-postlabelling/ HPLC assay, N-1-BMO-adenine, N-7-BMO-guanine and N-7-EBDguanine adducts were detected in BMO- or DEB-treated DNA and in liver DNA of rats exposed to BD by inhalation. The amount of N-7-EBD-guanine adducts (11/10⁸ nucleotides) in rat liver was about three-fold higher than N-7-BMO-guanine adducts (4.0/10⁸ nucleotides). The novel finding of N-1-BMO-adenine adducts formed in vivo may contribute to the understanding of the mechanisms of BD carcinogenic action.     

Comparison of DNA binding between the carcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene

We used ³²P-postlabelling to compare DNA binding between the potent hepatocarcinogen 2,6-dinitrotoluene and its noncarcinogenic analog 2,6-diaminotoluene. The two compounds were compared to determine whether differences in DNA binding could partly explain the differences in their carcinogenicity. Fischer-344 rats were administered 1.2 mmol/kg of a compound by single i.p. injection and examined for DNA adduct formation in the liver. Four adducts were detected following administration of 2,6-dinitrotoluene, with a total adduct yield of 13.5 adducted nucleotides per 10⁷ nucleotides. Qualitatively identical adducts were also detected after treatment with the derivative 2-amino-6-nitrotoluene. Adduct yields from 2,6-dinitrotoluene were 30 times greater than from 2-amino-6-nitrotoluene. No adducts were observed following treatment with 2,6-diaminotoluene. 2,6-Dinitrotoluene and 2,6-diaminotoluene were also compared for qualitative differences in hepatotoxicity. 2,6-Dinitrotoluene produced extensive hemorrhagic necrosis in the liver, whereas no evidence of hepatocellular necrosis was detected following administration of the latter. The differences between the two compounds in both DNA binding and cytotoxicity were consistent with the differences in their carcinogenicity.     

Carcinogen DNA adducts and the risk of colon cancer: case–control study

Colorectal cancer represents 8.5% of all tumours at the King Faisal Specialist Hospital & Research Centre. Environmental and dietary carcinogens such as polycyclic aromatic hydrocarbons (PAHs) and heterocyclic amines (HCAs) have long been suspected to play a prominent role in colon cancer aetiology. We designed a case–control study to test the hypothesis of whether or not the presence of DNA adducts can play a role in the aetiology of colon cancer. DNA adducts were measured in 24 cancerous and 20 non-cancerous tissue samples of newly diagnosed colon cancer patients by ³²P-post-labelling technique. Normal tissue from 19 hospital patients served as controls. The mean levels of adducts per 10¹⁰ nucleotides in cancerous and non-cancerous tissue were 151.75±217.27 and 114.81±186.10, respectively; however, only adducts in cancerous tissue were significantly higher than controls (32.78±57.51 per 10¹⁰ nucleotides) with p-values of 0.017. No BPDE-DNA adducts were found. No relationship was found between urinary cotinine as a marker of tobacco smoke and 1-hydroxypyrene as an indicator of an individual's internal dose of PAHs and DNA adducts. In a logistic regression model, only adducts in cancerous tissue were associated with the subsequent risk of colon cancer, with an odds ratio of 3.587 (95% confidence interval 0.833–15.448) after adjustment for age and the duration of living in the current region, but of a borderline significance (p=0.086). Although it is difficult to arrive at a definite conclusion from a small dataset, our preliminary results suggest the potential role of DNA adducts in the colon carcinogenesis process. Additional studies with larger sample sizes are needed to confirm our preliminary finding. It is also important to identify the structural characterization of these unknown DNA adducts in order to have a better understanding of whether or not environmental carcinogens play a role in the aetiology of colon cancer.     

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.     

High-resolution anion-exchange and partition thin-layer chromatography for complex mixtures of P-postlabeled DNA adducts

³²P-Postlabeling has emerged as a major tool for detecting DNA adducts resulting from exposure to complex carcinogen mixtures. An integral component of this assay is multi-directional PEI-cellulose TLC in which lipophilic ³²P-adducts are resolved in high-salt, high-urea solvents following removal of the bulk of non-adduct radioactivity. This TLC system is very effective for adducts formed following exposure to individual carcinogens; however, adducts resulting from exposure to complex mixtures (e.g. cigarette smoke) generally appear in the form of the so-called diagonal radioactive zones. By using mixtures of polycyclic aromatic hydrocarbon- and aromatic amine-DNA adducts as well as adducts in mouse skin treated with cigarette smoke condensate, we have demonstrated that a combination of 0.3–0.4 M NH₄ OH and isopropanol-4 M NH₄OH (1-1.4:1) solvents can provide more sharply defined adduct spots than the commonly used urea solvents. The non-urea solvents also result in excellent resolution of many adducts which otherwise may remain buried in diagonal radioactive zones when using the urea solvents. In addition, the signal-to-noise ratio is increased 2- to 5-fold over the urea solvents enabling detection of discrete adducts at ≤3 adducts per 10¹⁰ nucleotides. These partition TLC solvents also involve fewer manipulations (e.g. no water washes to remove salt and urea), and are likely to be more informative with regards to the type of individual adducts detected in the biomonitoring of humans than has hitherto been possible.     

High-resolution anion-exchange and partition thin-layer chromatography for complex mixtures of 32P-postlabeled DNA adducts

³²P-Postlabeling has emerged as a major tool for detecting DNA adducts resulting from exposure to complex carcinogen mixtures. An integral component of this assay is multi-directional PEI-cellulose TLC in which lipophilic ³²P-adducts are resolved in high-salt, high-urea solvents following removal of the bulk of non-adduct radioactivity. This TLC system is very effective for adducts formed following exposure to individual carcinogens; however, adducts resulting from exposure to complex mixtures (e.g. cigarette smoke) generally appear in the form of the so-called diagonal radioactive zones. By using mixtures of polycyclic aromatic hydrocarbon- and aromatic amine-DNA adducts as well as adducts in mouse skin treated with cigarette smoke condensate, we have demonstrated that a combination of 0.3–0.4 M NH₄ OH and isopropanol-4 M NH₄OH (1-1.4:1) solvents can provide more sharply defined adduct spots than the commonly used urea solvents. The non-urea solvents also result in excellent resolution of many adducts which otherwise may remain buried in diagonal radioactive zones when using the urea solvents. In addition, the signal-to-noise ratio is increased 2- to 5-fold over the urea solvents enabling detection of discrete adducts at ≤3 adducts per 10¹⁰ nucleotides. These partition TLC solvents also involve fewer manipulations (e.g. no water washes to remove salt and urea), and are likely to be more informative with regards to the type of individual adducts detected in the biomonitoring of humans than has hitherto been possible.     

DNA adducts,genetic polymorphisms,and K-ras mutation in human pancreatic cancer

To test the hypothesis that carcinogen exposure and oxidative stress are involved in pancreatic carcinogenesis in susceptible individuals, aromatic DNA adducts and 8-hydroxyguanosine (8-OH-dG) were measured by ³²P-postlabeling and HPLC–EC, respectively, in 31 pancreatic tumors and 13 normal tissues adjacent to the tumor from patients with pancreatic cancer. Normal pancreatic tissues from 24 organ donors, from six patients with non-pancreatic cancers, and from five patients with chronic pancreatitis served as controls. It was found that tissue samples from patients with pancreatic cancer had significantly higher levels of both aromatic DNA adducts and 8-OH-dG compared with control samples. The mean (±S.D.) levels of aromatic DNA adducts were 101.8±74.6, 26.9±26.6, and 11.2±6.6 per 10⁹ nucleotides in adjacent tissues, tumors, and controls, respectively. The mean (±S.D.) levels of 8-OH-dG were 11.9±9.6, 10.8±10.6, and 6.7±4.6 per 10⁵ nucleotides in adjacent tissues, tumors, and controls, respectively. Polymorphisms of the CYP1A1, CYP2E1, NAT1, NAT2, GSTM1, MnSOD, and hOGG1 genes were determined in these patients. The level of aromatic DNA adducts was significantly associated with polymorphism of the CYP1A1 gene. No significant correlation was found between the level of 8-OH-dG and the MnSOD, GSTM1, and hOGG1 polymorphisms. However, one novel polymorphism/mutation of the hOGG1 gene was found in a pancreatic tumor. Mutation at codon 12 of the K-ras gene was found in 25 (81%) of 31 pancreatic tumors, including three G-to-A transitions and 22 G-to-T transversions. Patients with the G-to-T mutation had a significantly higher level of aromatic DNA adducts than those with G-to-A or wild-type codon (P=0.02). On the other hand, the K-ras mutation profile was not related to the level of 8-OH-dG. Given the limitation of sample size, these preliminary data lend further support the hypothesis that carcinogen exposure and oxidative stress are involved in pancreatic carcinogenesis.     

Comparative study of DNA adducts levels in white sucker fish (Catostomus commersoni) from the basin of the St. Lawrence River (Canada)

The levels of DNA adducts in the hepatic tissue of the white sucker fish speciesCatostomus commersoni were determined by³²P-postlabelling. The fish were caught at four sites: two sites near the city of Windsor (Québec, Canada) on the St. François River, a downstream tributary of the St. Lawrence River, and two sites in the St. Lawrence River itself, near the city of Montréal (Québec, Canada). The latter sites are known to be contaminated by many pollutants including polycyclic aromatic hydrocarbons. Total adduct levels in all fish ranged from 25.1–178.0 adducts per 10⁹ nucleotides. White sucker from the selected sites of the St. Lawrence River had a significantly higher mean level of DNA adducts than those of the St. François River (129.4 vs 56.8, respectively). These results suggest that the effluents of many heavy industries (e.g. from a Soderberg aluminium plant) flowing in the St. Lawrence River are more likely to produce genotoxic damage to fish than those released in one of its tributary, and mainly associated to the activities of a small town and a nearby pulp and paper mill.     

DNA adduct levels in fish from pristine areas are not detectable or low when analysed using the nuclease P1 version of the 32P-postlabelling technique

In order to understand and apply DNA adduct formation in fish liver as a biomarker for aquatic pollution, information concerning the natural background levels in non-contaminated organisms, caused by endogenous compounds, is of fundamental importance. In this study, DNA adducts were analysed in liver of 11 fish species from arctic and sub-arctic areas in the northern Atlantic using the nuclease P1 version of the ³²P-postlabelling technique. The collected fish were assumed not to have been influenced by anthropogenic pollution apart from possible long-range transported pollutants. As polycyclic aromatic hydrocarbons (PAHs) are thought to be fundamental in forming the type of DNA adducts detected by the method used, biliary PAH metabolite levels were measured in a selection of the investigated species. In all investigated individuals, the levels of PAH metabolites were undetectable. Controlled on-site exposure experiments with benzo[a]pyrene (polar cod) and laboratory experiments with crude oil (polar cod and Atlantic cod) were conducted. DNA adducts were formed in both these species. The field-sampled fish showed undetectable levels of DNA adducts or levels just above the detection limit. The present study supports the assumption that when DNA adducts are detected by the nuclease P1 version of the ³²P-postlabelling method in fish liver, it can be interpreted as DNA damage caused by pollutants.     

Enzymes involved in the repair of damaged DNA

The multitude of enzymes responsible for removing damaged nucleotides from DNA in an error-free manner is reviewed. The most direct mechanisms include enzymatically catalyzed photoreversal of cyclobutane dimers and the removal of the O⁶-methylguanine adduct from alkylated DNA by an enzyme whose presence is dependent on adaptation. The direct removal of either damaged purines or pyrimidines or partial removal of photochemically induced diadducts is catalyzed by DNA glycosylases in the absence of phosphodiester bond hydrolysis. Incision of DNA containing apurinic or apyrimidinic sites arising either spontaneously or by the action of DNA glycosylases is catalyzed by specific endonucleases. The incision of DNA containing bulky adducts is attributed to a multigenically controlled uvr system in Escherichia coli. The mechanisms of damaged nucleotide excision and reinsertion of nucleotides are controlled by unique exonuclease functions in either direct or indirect association with DNA polymerases.     

Effects of dietary fish oil on the depletion of carcinogenic PAH-DNA adduct levels in the liver of B6C3F1 mouse

Many carcinogenic polycyclic aromatic hydrocarbons (PAHs) and their metabolites can bind covalently to DNA. Carcinogen-DNA adducts may lead to mutations in critical genes, eventually leading to cancer. In this study we report that fish oil (FO) blocks the formation of DNA adducts by detoxification of PAHs. B6C3F1 male mice were fed a FO or corn oil (CO) diet for 30 days. The animals were then treated with seven carcinogenic PAHs including benzo(a)pyrene (BaP) with one of two doses via a single intraperitoneal injection. Animals were terminated at 1, 3, or 7 d after treatment. The levels of DNA adducts were analyzed by the ³²P-postlabeling assay. Our results showed that the levels of total hepatic DNA adducts were significantly decreased in FO groups compared to CO groups with an exception of low PAH dose at 3 d (P = 0.067). Total adduct levels in the high dose PAH groups were 41.36±6.48 (Mean±SEM) and 78.72±8.03 in 10⁹ nucleotides (P = 0.011), respectively, for the FO and CO groups at 7 d. Animals treated with the low dose (2.5 fold lower) PAHs displayed similar trends. Total adduct levels were 12.21±2.33 in the FO group and 24.07±1.99 in the CO group, P = 0.008. BPDE-dG adduct values at 7 d after treatment of high dose PAHs were 32.34±1.94 (CO group) and 21.82±3.37 (FO group) in 10⁹ nucleotides with P value being 0.035. Low dose groups showed similar trends for BPDE-dG adduct in the two diet groups. FO significantly enhanced gene expression of Cyp1a1 in both the high and low dose PAH groups. Gstt1 at low dose of PAHs showed high levels in FO compared to CO groups with P values being 0.014. Histological observations indicated that FO played a hepatoprotective role during the early stages. Our results suggest that FO has a potential to be developed as a cancer chemopreventive agent.     

Nucleotide incorporation by human DNA polymerase gamma opposite benzo[a]pyrene and benzo[c]phenanthrene diol epoxide adducts of deoxyguanosine and deoxyadenosine

Mitochondria are major cellular targets of benzo[a]pyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N²-deoxyguanosine (dG) and N⁶-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzo[c]phenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol γ) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DE–dG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DE–dA adducts derived from both BaP and BcPh. In the presence of p55 the polymerase incorporated all four nucleotides and catalyzed limited translesion synthesis past BaP DE–dG adducts but not past BaP or BcPh DE–dA adducts. Thus, all these adducts cause erroneous purine incorporation and significant blockage of further primer elongation. Purine misincorporation by pol γ opposite the BaP DE–dG adducts resembles that observed with the Y family pol η. Blockage of translesion synthesis by these DE adducts is consistent with known BaP inhibition of mitochondrial (mt)DNA synthesis and suggests that continued exposure to BaP reduces mtDNA copy number, increasing the opportunity for repopulation with pre-existing mutant mtDNA and a resultant risk of mitochondrial genetic diseases.     

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.     

The relationship between aryl hydrocarbon hydroxylase activity and DNA adducts measured by 32P-postlabelling assay in lymphocytes of lung cancer patients

We have investigated the correlation between DNA adduct levels and aryl hydrocarbon hydroxylase (AHH) activity in peripheral lymphocyte samples obtained from 42 lung cancer patients. DNA adducts and AHH activity were determined by the ³²P-postlabelling technique and the fluorometric method, respectively. The mean +/- SD of DNA adduct level was 0.88 +/- 0.37 (ranged from 0.22 to 1.90) per 10⁸ nucleotides. The geometric means of non-induced and 3-methylcholanthrene (MC)-induced AHH activity, as well as AHH inducibility (MC-induced AHH activity/non-induced AHH activity) were 0.029, 0.228 pmol min^-1 10^-6 cells, and 7.776, respectively. There was no statistically significant correlation between DNA adduct levels and non-induced or MC-induced AHH activity. A tendency of positive correlation was found between DNA adduct levels and AHH inducibility for the all subjects (n = 42, r = 0.25, p = 0.11). Such a positive correlation reached statistical significance in the subjects with squamous cell carcinoma (n = 13, r = 0.70, p < 0.01). In addition, similar correlation of DNA adducts with AHH inducibility was also observed in the GSTM1 present genotype (n = 17, r = 0.44, p = 0.07) and GSTP1-AA genotype (n = 29, r = 0.37, p = 0.05) individuals. These findings suggest that DNA adduct levels are mediated by CYP1A1 enzyme, and AHH inducibility may be a more relevant indicator than specific AHH activity for explaining the variation of DNA adduct levels in lymphocytes.     

32P-base analysis of DNA

A ³²P-labeling method for the base composition analysis of nonradioactive DNA was developed consisting of the digestion of DNA to deoxynucleoside 3′-monophosphates by incubation with a mixture of micrococcal nuclease and spleen phosphodiesterase, transfer of ³²P-label from [γ-³²P]ATP to the 5′-hydroxyl groups of the mononucleotides by T4 polynucleotide kinase, two-dimensional anion-exchange thin-layer chromatography on PEI-cellulose of the resultant [5′-³²P]deoxynucleoside 3′,5′-bisphosphates, autoradiography, and scintillation counting. The method was standardized to afford quantitative digestion of DNA to mononucleotides as well as to give quantitative incorporation of ³²P-label into the nucleotides in the DNA hydrolysate so as to make the method an accurate means for determining the base composition of eucaryotic DNA containing adenine, guanine, thymine, cytosine, and 5-methylcytosine.     

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.     

Discrimination against major groove adducts by Y-family polymerases of the DinB subfamily

Y-family DNA polymerases bypass DNA adducts in a process known as translesion synthesis (TLS). Y-family polymerases make contacts with the minor groove side of the DNA substrate at the nascent base pair. The Y-family polymerases also contact the DNA major groove via the unique little finger domain, but they generally lack contacts with the major groove at the nascent base pair. Escherichia coli DinB efficiently and accurately copies certain minor groove guanosine adducts. In contrast, we previously showed that the presence in the DNA template of the major groove-modified base 1,3-diaza-2-oxophenothiazine (tC) inhibits the activity of E. coli DinB. Even when the DNA primer is extended up to three nucleotides beyond the site of the tC analog, DinB activity is strongly inhibited. These findings prompted us to investigate discrimination against other major groove modifications by DinB and its orthologs. We chose a set of pyrimidines and purines with modifications in the major groove and determined the activity of DinB and several orthologs with these substrates. DinB, human pol kappa, and Sulfolobus solfataricus Dpo4 show differing specificities for the major groove adducts pyrrolo-dC, dP, N⁶-furfuryl-dA, and etheno-dA. In general, DinB was least efficient for bypass of all of these major groove adducts, whereas Dpo4 was most efficient. DinB activity was essentially completely inhibited by the presence of etheno-dA, while pol kappa activity was strongly inhibited. All three of these DNA polymerases were able to bypass N⁶-furfuryl-dA with modest efficiency, with DinB being the least efficient. We also determined that the R35A variant of DinB enhances bypass of N⁶-furfuryl-dA but not etheno-dA. In sum, we find that whereas DinB is specific for bypass of minor groove adducts, it is specifically inhibited by major groove DNA modifications.