• DNA adducts;
  • protein adducts;
  • tobacco;
  • smoking;
  • human biomonitoring


  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

Tobacco smoke contains a variety of genotoxic carcinogens that form adducts with DNA and protein in the tissues of smokers. Not only are these biochemical events relevant to the carcinogenic process, but the detection of adducts provides a means of monitoring exposure to tobacco smoke. Characterization of smoking-related adducts has shed light on the mechanisms of smoking-related diseases and many different types of smoking-derived DNA and protein adducts have been identified. Such approaches also reveal the potential harm of environmental tobacco smoke (ETS) to nonsmokers, infants and children. Because the majority of tobacco-smoke carcinogens are not exclusive to this source of exposure, studies comparing smokers and nonsmokers may be confounded by other environmental sources. Nevertheless, certain DNA and protein adducts have been validated as biomarkers of exposure to tobacco smoke, with continuing applications in the study of ETS exposures, cancer prevention and tobacco product legislation. Our article is a review of the literature on smoking-related adducts in human tissues published since 2002.

Tobacco smoking is the major preventable cause of cancer deaths in many countries. It contributes to one in five deaths in developed countries and an estimated 50% of lifelong smokers die prematurely of a smoking-related disease, with an average reduction in life expectancy of 10 years.1 Smoking is causally linked to cancer of the lung, bladder, renal pelvis, oral cavity (including the lip), oropharynx, hypopharynx, larynx, esophagus, pancreas, ureter, liver, stomach, uterine cervix, tongue, nasal cavity and paranasal sinuses, nasopharynx, bone marrow (myeloid leukemia), colorectum and ovary, according to the most recent evaluation by the International Agency for Research on Cancer.2 There is also limited evidence that tobacco smoking causes breast cancer. Furthermore, in the last 10 years, it has become established that environmental tobacco smoke (ETS) causes lung cancer in nonsmokers, and also there is limited evidence for an association of ETS exposure with cancer of the larynx and pharynx.2 Other adverse health effects of ETS include acute myocardial infarction, while for children exposure is associated with sudden infant death syndrome (SIDS), asthma, middle ear infection and lower respiratory tract illness.3 There are also concerns about the effects of prenatal exposure to tobacco smoke on both the physical and mental development of children.3

Tobacco smoke is a complex mixture of several thousand chemicals, with at least 60 of them classified as carcinogens.4 The continuing accumulation of evidence for the harm to human health of tobacco smoke necessitates the development and application of robust biomarkers to assess exposure and, in appropriately designed studies, to contribute to risk assessment. A biomarker is a substance or effect that is measurable in a human bodily fluid or tissue. For exposures related to smoking, this includes components of tobacco smoke and their metabolites, their interactions with cellular macromolecules and the biological effects of such interactions, such as mutation. Much work has been done on this in the last few decades and various aspects of the subject have been reviewed, both with reference to occupational and environmental exposures to carcinogens in general5–9 and in terms of tobacco smoke exposure in particular.3, 10–18 Some of these biomarkers will be useful in the newer area of tobacco product regulation, as the tobacco industry develops and markets new products for delivering nicotine to those addicted to this drug.19, 20

The formation of DNA adducts by reaction of electrophilic species generated by metabolism of exogenous chemicals is the first step in the genotoxic mechanism through which many carcinogens exert their biological effects. DNA adducts may induce mutations that initiate carcinogenesis.21 Reactive electrophiles may also interact with nucleophilic sites in other cellular macromolecules, including protein; as a more abundant cellular constituent, protein can thus serve as a useful surrogate material for detecting human exposure to carcinogens. In practice, DNA and protein adducts can both serve as useful biomarkers for detecting human exposure to carcinogens.

In a review published in 2002 (Ref. 11), the relationship between smoking-related DNA and protein adducts was considered on a tissue-by-tissue basis, and it was concluded that identification and quantification of adducts in human tissues provided valuable insights into the mechanisms of tobacco-related diseases and could increase understanding of how inhalation of tobacco smoke induces several chronic and often mortal diseases at multiple sites. Our article reviews work on tobacco-related adducts published since 2002 and takes the chemical nature and origin of adducts as a starting point. It examines how DNA repair, genetic polymorphisms and gene mutation can be influenced by, and can operate on, tobacco-related adducts. To compile the review, we performed a systematic search of Medline from January 2002 to March 2012, using the search item “smoking OR tobacco AND adducts”, filtered for human studies. This identified 488 publications. Only those that reported the analysis of DNA or protein adducts in human tissues with information on the smoking status of the subjects, or their exposure to secondhand tobacco smoke, were considered for inclusion in this review. However, it is important to note that smoking is frequently a confounding factor in studies of, for example, human exposure to air pollution, occupational exposure to carcinogens and the effect of diet on DNA adduct formation in human tissues. It is thus important to understand the nature and origins of smoking-induced DNA damage, and to take account of its effects, whenever conducting human biomonitoring studies.

Detection Methods for Smoking-Induced DNA and Protein Damage

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

The armory for measuring DNA and protein adducts in human tissues and blood is now diverse. The main types of smoking-related adducts, their chemical parents and detection methods are summarized in Table 1. Most of these chemicals have other environmental sources besides tobacco smoke, the exceptions being the so-called tobacco-specific nitrosamines, of which 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N′-nitrosonornicotine (NNN) are prime examples.14

Table 1. Smoking-related adducts and methods for their detection
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For DNA adducts, a commonly used detection method is 32P-postlabelling, which remains the most sensitive method, although the sensitivity of other methods is improving steadily. However, the method does not characterize the adducts detected, which means there remain uncertainties about what is meant by “smoking-related” adducts. It was earlier assumed that these were formed by bulky, aromatic carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), because of the chromatographic similarity of these adducts to those formed by complex mixtures of PAHs in experimental systems. Hence, they are frequently referred to as “bulky adducts” or “aromatic adducts”. Nevertheless, the nature of the adducts detected, and the contents of the so-called “diagonal radioactive zone” resolved on thin-layer chromatography when DNA from smokers is analyzed by this method remain in doubt.54

Antibodies, both polyclonal and monoclonal, have been raised against a number of carcinogen–DNA adducts, and these have been used in a number of sensitive and semiautomated assays analyzing DNA from human tissues. Uniquely among DNA adduct detection methods, the antibody approach can localize adducts within tissues, when incorporated into immunohistochemical protocols.55

Antibodies raised against benzo[a]pyrene diol-epoxide (BPDE)-DNA adducts will also recognize a number of other PAH-DNA adducts. Thus the use of these antibodies in human biomonitoring studies provides a semiquantitative estimate of adducts formed by PAHs as a class, rather than by benzo[a]pyrene specifically.

Fluorescence detection of DNA adducts, or of the hydrolysis products thereof, has also been widely applied, particularly for benzo[a]pyrene.23, 56 Mass spectrometry, however, offers the best opportunity for unequiviocal identification of adducts, and methods have been developed for a growing number of carcinogen–DNA adducts related to tobacco smoke carcinogens.57

For protein adducts, both hemoglobin (from red blood cells) and albumin (from blood plasma) have been analyzed. A variety of adducts formed at the N-terminal valine of hemoglobin have been identified (Table 1), generally after hydrolytic release of the modified amino acid or of the carcinogen itself, followed by detection by mass spectrometry. For albumin, mass spectrometry analysis of albumin peptides, and also immunoassay using antibodies raised against some carcinogen–protein adducts, have been successfully used.11 A novel mass spectrometry method for detecting adducts formed specifically at the Cys34 residue of albumin has recently been described.58

Smoking-Related DNA Adducts in Human Tissues

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

Bulky/aromatic adducts

PAHs are products of the incomplete combustion of organic material, which are widespread environmental pollutants found in air, soil and water. Inhalation of tobacco smoke is a major source of exposure for smokers and to a lesser extent, for passive smokers. The most studied PAH is benzo[a]pyrene, which is metabolically activated to BPDE, which forms a major adduct with DNA at the N2 position of guanine.59

When 32P-postlabelling, fluorescence or immunological methods (with antibodies raised against BPDE-modified DNA) have been used to compare DNA from smokers with that from nonsmokers, the adduct levels detected by this technique are frequently higher in the former, as reviewed earlier.11 In more recent studies this trend has continued, with the phenomenon observed with 32P-postlabelling studies of larynx,33 bladder,60 anal epithelium,61 and with immunohistochemical analysis of liver62 and oral tissue.63 However, in an immunohistochemical analysis of formalin-fixed cervical tissue, there was no correlation between PAH-DNA adducts and smoking status,64 in contrast to earlier reports of smoking-related DNA adducts in human cervix.11 A similar analysis of human placentas also found no difference in extent of DNA damage between smokers and nonsmokers.65

Adduct levels are also consistently higher in lung tissue of smokers compared with nonsmokers.32, 54, 66 Varkonyi et al.67 reported a correlation between adduct levels in blood mononuclear cells and lung tissue from lung cancer patients, and they also reported the detection of adducts chromatographically similar to those derived from hydroquinone and benzenetriol, metabolites of benzene. Gyorffy et al.68 compared DNA adducts in normal lung and tumor tissue by both 32P-postlabelling and immunoassay (with antibodies to BPDE-DNA) and found a statistically significant correlation between levels in the two samples for both smokers and nonsmokers by both methods. Associations between normal lung and blood DNA adducts correlated only for nonsmokers, and the levels in normal lung did not correlate between the two methods, even though levels were significantly elevated in smokers by both methods of analysis. These discrepancies are not so surprising, given the very different ways of detecting DNA adducts inherent in the 32P-postlabelling and immunoassay methods, and continuing uncertainty about the nature of the smoking-related adducts detected by 32P-postlabelling.54

Several studies investigating environmental exposures to carcinogens in general populations have demonstrated the influence of smoking, as well as other lifestyle factors, including dietary habits and air pollution, on adduct levels in peripheral blood cells.69–75 In other studies, a significant correlation with smoking was not apparent.76–78 With several possible sources of adduct-forming compounds evident from these studies, it is perhaps not surprising that the influence of smoking on adduct levels is not observed in some populations, but is in others.

Even though the association between smoking and levels of bulky adducts in peripheral blood is inconsistent, high-adduct levels appear to be predictive of lung and bladder cancer risk.79, 80 A pooled analysis of studies of adducts in blood cells found the sources of interindividual variation largely unexplained.81

Some studies have suggested that higher levels of PAH-DNA adducts in peripheral blood cells is associated with increased risk of breast cancer,82 but the evidence that this is related to smoking is also not consistent.83

Several studies have found that increased levels of DNA damage and DNA adducts are associated with sperm motility and impaired male fertility, but there are inconsistencies in the evidence for a link with smoking. For example, in one study, levels of bulky DNA adducts detected by 32P-postlabelling showed a significant inverse correlation with sperm concentration and motility, but bulky adducts were only 1.2-fold higher in smokers, which was not statistically significant.84 In another study, occupational exposure to PAHs, but not smoking, was significantly associated with higher levels of PAH-DNA adducts detected by immunofluorescence.85 In contrast, another study has reported significantly higher levels of BPDE-DNA adducts, detected by fluorescence imaging, in the sperm of smokers than in nonsmokers.86

Aromatic and heterocyclic aromatic amines

4-Aminobiphenyl (4-ABP) is an aromatic amine with a variety of environmental and occupational sources of exposure, including tobacco smoke, and it is a known bladder carcinogen in both experimental animals and humans. Its major DNA adduct is formed at the C-8 position of guanine. Sensitive and specific mass spectrometry and immunologic methods for detecting 4-ABP-DNA adducts have been applied to a number of human tissues (see Table 1). In breast tissue, adduct levels in tumor-adjacent tissue, but not in tumor tissue itself, correlated with smoking status.87 In bladder biopsies from cancer patients, adduct levels were associated with current smoking and with tumor grade.88 However, in a small study of human pancreas (n = 12), there was no correlation of adducts with smoking, age or gender.28 A more recent study has called into question the conclusions regarding the presence of 4-ABP-DNA adducts in breast tissue, based on immunohistochemistry. Using a specific LC-MS method, Gu et al.89 failed to detect the presence of 4-ABP-DNA adducts in any of 70 breast biopsies of tumor-adjacent tissue. Although there was no systematic enquiry into the smoking status of these patients, 37 were described as never smokers, 13 as former smokers and 4 as current smokers.

Heterocyclic amines, formed in cooked meat and also present, in some cases, in tobacco smoke, also form adducts predominantly with the C-8 position of guanine in DNA. Bessette et al.90 investigated the formation of a number of adducts formed by these compounds in the saliva of 37 human volunteers. The C-8-dGuo adduct of 2-amino-1-methyl-6-phenylimidazo[4,4-b]pyridine (PhIP) was detected in 13/29 ever smokers (former and current smokers) and 2/8 never smokers. In contrast, adducts formed by 2-amino-9H-pyrido[2,3-b]indole (AαC) and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) were detected only in the saliva of three current smokers, and 4-ABP-DNA adducts were also detected in two current smokers. However, in another study by these investigators,91 in which the number of subjects was not specified, adducts formed by these carcinogens were not detectable in buccal cell DNA from smokers.


Acrolein (2-propenal), a highly reactive α,β-unsaturated aldehyde, occurs widely in cooked foods and in the environment. Its ubiquity is attributed to incomplete combustion of petrol, wood, and plastic, to smoking tobacco, frying of foods in oils, endogenous lipid peroxidation, and to endogenous polyamine metabolism. Cigarette smoke contains about 180 μg of acrolein per cigarette4 and is considered to account for a large proportion of total human exposure to acrolein.92 Acrolein is mutagenic in bacteria and in cultured human cells. There is inadequate evidence for its human carcinogenicity.93 Chen94 has reviewed the use of 32P-postlabelling- and MS-based methods for the analyses of acrolein-derived DNA adducts in human tissues.

Acrolein reacts with deoxyguanosine in DNA to form two pairs of stereoisomers of cyclic 1,N2-propanodeoxyguanosine adducts (Acr-dGuo). Of the α-OH-Acr-dGuo and γ-OH-Acr-dGuo isomers, the α-isomer is particularly mutagenic in human cells and induces predominantly G to T transversions.39

It is noteworthy that in an earlier study using 32P-postlabelling /HPLC, Nath et al.38 found that the mean Acr-dGuo levels in gingival tissue DNA from 11 smokers (4 male and 7 female) was significantly higher than that in 12 nonsmokers (8 male and 4 female) (1.36 ± 0.90 μmol/mol guanine in smokers versus 0.46 ± 0.26 μmol Acr-dGuo/mol guanine in nonsmokers; p = 0.003).

Because Acr-dGuo adducts, like PAH-DNA adducts, act to induce predominantly G to T transversions in human cells, Feng et al.95 hypothesized that Acr-dGuo adducts could be responsible for TP53 mutations in cigarette-related lung cancer. They mapped the distribution of Acr-dGuo adducts at the sequence level in the TP53 gene of lung cells and found that the Acr-dGuo binding pattern is similar to the TP53 mutational pattern in human lung cancer. They also found that acrolein greatly reduces the DNA repair capacity for damage induced by BPDE. They suggested that acrolein is a major etiological agent for lung cancer caused by cigarette smoke and that it contributes to lung carcinogenesis through two detrimental effects: DNA damage and inhibition of DNA repair.

To determine whether Acr-dGuo adducts could be detected in human lung, Zhang et al.39 developed a specific liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) method for the quantitative analysis of these DNA adducts. Thirty DNA samples from normal lung tissue obtained at surgery were analyzed, and Acr-dGuo adducts were detected in all samples. Both α-OH- and γ-OH-Acr-dGuo were observed in most of the samples; total adduct concentrations ranged from 16 to 209 adducts/109 nucleotides. However there was no difference in adduct levels between confirmed current smokers (N = 5) and nonsmokers (N = 9), nor was there any relationship of adduct levels to self-reported time since cessation of smoking, gender, age, urinary nicotine and cotinine.

Both these adducts were reported to be detectable by mass spectrometry in buccal cell DNA of smokers.91 In a later study96 Acr-dGuo adducts were analyzed in peripheral blood leukocytes from 25 smokers and 25 nonsmokers whose smoking history was known and whose smoking status was confirmed by exhaled carbon monoxide. The predominant isomer in all samples was γ-OH-Acr-dGuo, while α-OH-Acr-dGuo was detected in only three subjects. Again, there was no significant difference between the total Acr-dGuo levels in smokers (7.4 ± 3.4 adducts/109 nucleotides) and nonsmokers (9.8 ± 5.5 adducts/109 nucleotides). However, the mean level of γ-OH-Acr-dGuo was significantly higher in nonsmokers (9.7 ± 5.5) than in smokers (7.0 ± 2.5). Based on this and other studies of acrolein-derived mercapturic acids in the urine of smokers and nonsmokers, the authors concluded that glutathione conjugation effectively removes acrolein from external exposures such as cigarette smoking, protecting leukocyte DNA from damage.


Acetaldehyde is one of the most prevalent carcinogens in tobacco smoke, and also found widely in the environment, for example in food and fuel combustion products. It is also formed endogenously during the catabolism of threonine and metabolism of ethanol. It reacts with DNA to form primarily N2-ethylidene-dGuo, which when DNA is subjected to enzyme hydrolysis in the presence of NaBH3CN is converted to N2-ethyl-dGuo, detectable by LC-ESI-MS/MS-SRM.37 N2-ethyl-dGuo was detected thus in the blood leukocyte DNA of 25 smokers at levels of 1,310 ± 720 (range: 124–7,700) and 1,120 ± 1,140 (range: 138–5,760) fmol/μmol dGuo at two baseline points.94

Theoretically, subsequent reaction of N2-ethylidene-dGuo in DNA would give rise to 1,N2-propano-dGuo (1,N2-εdGuo, i.e., Acr-dGuo). This adduct has been detected in human cells exposed in vitro to micromolar concentrations of acetaldehyde. This raises the possibility that such a “double adduct” could play a role in the genotoxicity of acetaldehyde, and that this carcinogen could, in part, account for the presence of 1,N2-εdGuo (Acr-dGuo) in human DNA.97

Alkyl adducts

There is evidence for a direct-acting ethylating agent in tobacco smoke, whose structure is as yet unknown.98 In a study of nontumorous lung tissue from lung cancer patients, O4-ethylthymidine was detectable in 10 of 13 smokers, but only 3 of the 11 nonsmokers.32 In another small study, the adduct was detected in cells obtained by sputum induction from two of four smokers, but from none of three nonsmokers.40 Both these studies used an immunoenriched 32P-postlabelling method. In a third study using this technique, Anna et al.99 found the adduct to be 1.7-fold higher in normal lung tissue, from lung cancer patients at surgery, of smokers than of long-term ex-smokers. There was no correlation with levels of bulky adducts determined by 32P-postlabelling, in contrast to the study by Godschalk et al.,32 where a correlation between the two adduct types was observed (R = 0.65, p < 0.05). A mass spectrometry method has been developed to measure 7-ethylguanine in DNA; when leukocyte DNA from 30 smokers and 30 nonsmokers was compared, the difference in adduct levels was not significant (49.6 ± 43.3 vs. 41.3 ± 43.3).41

Tobacco-specific nitrosamines

The representative tobacco nitrosamine NNK is metabolically activated to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL). Both NNK and NNAL convert to methanediazohydroxide, which reacts with DNA to form the methyl adducts O6-methyl-dGuo, 7-methyl-dGuo and O4-methyl-dThd. Such adducts can also be formed by many methylating agents, but a more specific pathway leading to adducts from both NNK and NNN is one that yields 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) on neutral thermal or acid hydrolysis of DNA. Such adducts, including O6-, 7-, N2 dGuo, O2-dThD and O2-dCyd, are formed at multiple sites in DNA.18

HPB-releasing DNA adducts were at significantly higher levels in lung DNA from 21 self-reported smokers (404 ± 258 fmol/mg DNA) than in 11 self-reported nonsmokers (59 ± 56 fmol/mg).100 However adduct levels in esophagus did not differ significantly between smokers and nonsmokers, leading to speculation that the alkaloid myosine, which occurs in many food items as well as in tobacco, might also be a source of HPB-releasing adducts.101, 102 in another study, HPB-releasing adducts were detectable in only 6 of 58 samples of pancreatic DNA (four of them from smokers).103


Formaldehyde is an industrial chemical with a wide array of uses and is produced in high volumes. It occurs in humans endogenously, is rapidly metabolized and is also formed through the metabolism of many xenobiotic agents. Formaldehyde is ubiquitous in the environment and has been detected in indoor and outdoor air; in treated drinking water, bottled drinking water, surface water, and groundwater; on land and in the soil; and in numerous types of food.104 It is also a constituent of tobacco smoke; based on an analysis of 48 cigarette brands under ISO conditions, it has been established that mainstream cigarette smoke contains 14–28 μg/cigarette of formaldehyde.105 Formaldehyde is classified as a human carcinogen and is genotoxic and forms DNA adducts and crosslinks.106

In a study of leukocyte DNA samples from 32 smokers and 30 nonsmokers, Wang et al.36 used LC-ESI-MS/MS to quantify the formaldehyde-DNA adduct N6-hydroxymethyldeoxyadenosine (N6-HOMe-dAdo). N6-HOMe-dAdo was detected in 29 of 32 smoker samples (mean ± SD, 179 ± −205 fmol/μmol dAdo). In contrast, it was detected in only 7 of 30 nonsmoker samples (15.5 ± 33.8 fmol/μmol dAdo; p < 0.001). The authors speculated on the source of the elevated levels of formaldehyde-DNA adducts in smokers and suggested that inhalation of formaldehyde in cigarette smoke as the simplest explanation.

Oxidative damage to DNA

Exposure to tobacco smoke can provoke the formation of reactive oxygen species (ROS) that can oxidize DNA, causing single-strand and double-strand breaks, and generating genotoxic electrophiles by lipid peroxidation of polyunsaturated fatty acids. These highly reactive species include 2,3-epoxy-4-hydroxynonanol, and can form a variety of mutagenic DNA adducts, including the exocyclic adducts 1,N6-ethenoadenine (εAde), 3,N4-ethenocytosine (εCyt) and N2,3-ethenoguanine (N2,3-εGua).107, 108 Malondialdehyde (MDA) is another reactive electrophile generated by lipid peroxidation and forms a cyclic pyrimidopurinone N-1,N2-malondialdehyde-2′-deoxyguanosine DNA adduct (M1dG). Another consequence of the attack of DNA by ROS is the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) which is formed by hydroxylation of deoxyguanosine in DNA.

In a study of several types of DNA adduct in tumor-adjacent but uninvolved lung tissues of 13 smoking and 11 nonsmoking obtained at surgery from lung cancer patients, levels of εAde and εCyt, determined by immunoenriched 32P-postlabelling, did not differ between smokers and nonsmokers, but large interindividual variations were observed (80- and 250-fold differences for εAde and εCyt, respectively).32

Whether levels of 8-oxodG are higher in smokers than in nonsmokers remains uncertain. For example, Singh et al.,31 using liquid chromatography–tandem mass spectrometry selected reaction monitoring (LC–MS/MS SRM), found no significant differences in 8-oxodG adduct levels in lymphocyte DNA from individuals, from three European countries, that could be attributed to smoking status. Earlier studies (reviewed by Singh et al.) have produced conflicting results, but several studies support the view that there is no consistent and reliable evidence that 8-oxodG levels are higher in smokers than nonsmokers.

There is also a lack of consistency in the case of M1dG adducts in smokers compared to nonsmokers. However, this may be related to the diversity of tissues and organs that have been examined.

Using an immunohistochemical method, employing a monoclonal antibody specific for MDA-DNA adducts, Zhang et al.35 observed a mean 1.3-fold (p = 0.02) excess of adduct-related staining in oral mucosa cells from 25 smokers compared to 25 age-, race- and sex-matched nonsmokers.

In a study of laryngeal biopsies, levels of malondialdehyde-DNA adducts with chromatographic properties consistent with exocyclic MDA-deoxyadenosine (MDA-dA) and MDA-deoxyguanosine (MDA-dG) were detected in laryngeal biopsies from 30 patients, 13 with larynx cancer and 17 controls.33 Of the 30, 9 were nonsmokers and 21 were smokers. Mean total MDA-DNA adducts, MDA-dA and MDA-dG adducts per 108 nucleotides in smokers were higher than in nonsmokers (6.7 ± 1.0 vs. 4.0 ± 1.4, p < 0.05; 3.3 ± 0.7 vs. 2.1 ± 1.0, p < 0.05; 3.4 ± 0.4 vs. 1.9 ± 0.5, p = 0.078, respectively) after controlling for age, sex, larynx cancer, residence and alcohol consumption. There was evidence of a positive dose-response relationship between cigarette consumption and adduct levels but this did not achieve statistical significance.

Munnia et al.34 measured the relationship between bronchial M1dG adducts (using 32P-postlabelling) and tobacco smoking, cancer status, and selected polymorphisms in 43 subjects undergoing diagnostic bronchoscopic examination. M1dG-DNA adducts were higher in current smokers than in never smokers [frequency ratio (FR) = 1.51, 95% confidence interval (CI): 1.01–2.26]. MDA-DNA adducts were also higher in lung cancer cases with respect to controls, but only in smokers (FR = 1.70, 95% CI: 1.16–2.51). Subjects with GA and AA cyclin D1 (CCND1) genotypes showed higher levels of MDA-DNA adducts than those with the wild-type genotype [FR = 1.51 (1.04–2.20) and 1.45 (1.02–2.07)].

Singh et al.31 found no significant difference between smokers and nonsmokers in the levels of lymphocyte M1dG adducts measured by an immunoslot blot assay employing a murine M1dG monoclonal primary antibody. The adduct level (per 108 nucleotides) in smokers was 32.8 ± 30.5 and in nonsmokers, 30.4 ± 23.8.

In a cross-sectional study comparing the prevalence of M1dG adducts, detected by 32P-postlabelling, in the peripheral leukocytes of groups of subjects experiencing various degrees of air pollution in Thailand, Peluso et al.109 concluded that “formation of DNA damage [M1dG adducts] tended to be associated with tobacco smoking, but without reaching statistical significance.” Adduct levels per 108 nucleotides were 3.7 ± 0.4 in nonsmokers, 4.2 ± 0.7 in ex-smokers and 4.8 ± 0.4 in current smokers.

In a study of M1dG adduct levels, analyzed by 32P-postlabelling, in leukocytes of pathologists occupationally exposed to formaldehyde and nonexposed control subjects, Bono et al.110 found no significant differences in adduct levels per 108 nucleotides between nonsmokers (n = 27, 3.8 ± 0.9) and smokers (n = 13, 4.5 ± 1.3, p = 0.494).

Using 32P-postlabelling of breast fine-needle aspirate samples, Peluso et al.111 measured M1dG adduct levels in 22 patients with breast cancer, at different clinical stages, and 13 controls. Multivariate analysis revealed that increasing severity of breast cancer was significantly and positively associated with M1dG adduct levels but that there were no significant effects of age and smoking habit on adduct levels (MR (mean ratio) = 1.58, 95% CI: 0.92–2.72 and MR = 1.68, 95% CI: 0.88–3.20, respectively).

Smoking-Related Protein Adducts

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

Hemoglobin adducts formed by 4-ABP are significantly higher in the red blood cells of smokers than of nonsmokers, and there is generally no overlap in levels between the two groups.11 More recent studies confirm this,112–114 making 4-ABP-Hb adducts a biomarker distinguishing between smokers and nonsmokers. The levels of 4-ABP-Hb in smokers' blood were found by Dallinga et al.115 to be inversely related to the antioxidant status of the subjects, as measured by the trolox equivalent antioxidant capacity (TEAC).

4-Hydroxy-1-(3-pyridyl)-1-butanone (HPB)-releasing hemoglobin adducts, derived from NNK, were reported to be only marginally higher in 12 self-reported smokers than in 7 self-reported nonsmokers, and there was no correlation with the levels of HPB-releasing DNA adducts.100

Acrylamide is found in tobacco smoke and food, and there are also documented examples of occupational exposure. It forms adducts with hemoglobin, as does its metabolite glycidamide, which is mutagenic and clastogenic. Studies measuring hemoglobin adducts of acrylamide (AAHb) and glycidamide (GAHb) have found that smoking is the main contributor to their formation.116–126 Thus in these studies, most of which are concerned primarily with assessing dietary exposure to acrylamide, it is clear that smoking is a confounding factor that needs to be taken into consideration. Similarly, in an occupational setting, where exposure to acrylamide and also acetonitrile was monitored, levels of the hemoglobin adducts formed by these agents were mainly accounted for by smoking and diet.127

The validity of smoking machine-derived tar yields in determining the levels of human exposure has long been discredited; nevertheless, studies on the relationship between biomarkers of exposure and tar yields have continued. One such study reported a statistically significant effect of machine-measured tar yield on several biomarkers in US smokers, including 4-ABP-Hb adducts, although in this case the percentage difference in levels between highest and lowest tar was only ∼10%.128 Another study found no difference in 4-ABP-Hb adduct levels between smokers of “light” and “regular” cigarettes.129 In a German study, the levels of several hemoglobin adducts (methyl-, hydroxyethyl-, cyanoethyl- and carbamoylethylvaline) were no more than weakly related to the machine-derived tar yields of the cigarettes smoked.43

Repair of DNA Adducts

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

There are a number of biological consequences of DNA damage. First, the surveillance and corrective DNA repair mechanisms in mammalian cells may detect the damage and restore the genome sequence to its original state. Second, the DNA damage may trigger mechanisms leading to programmed cell death (apoptosis). Third, erroneous replication of the damaged DNA template may lead to mutagenesis. If mutations that occur alter the function of genes critical for cellular homeostasis (e.g., tumor suppressor genes, proto-oncogenes and mismatch repair genes) then the consequence for the cell of mutation may be the acquisition of the characteristics of malignancy.

There is evidence from a number of studies that not only does tobacco smoke induce premutagenic DNA damage (the main theme of this article) but also that the capacity of cells to repair such damage is reduced in patients with tumors. Thus, reduced repair capacity of lymphocytes from patients with squamous cell carcinoma of head and neck, measured using a host-cell reactivation assay for BPDE-modified DNA, has been reported.130 A similar assay on bladder cancer cases and controls, this time using DNA modified by 4-ABP, showed significantly reduced repair capacity for the cases (p = 0.006).131 In patients with adenocarcinoma of the lung, but not with squamous cell carcinoma, there was reduced capacity of normal lung tissue and blood leukocytes to repair εAde and εCyt.132 However, in smokers with non-small cell lung carcinoma, activity of hOGG1, a DNA glycosylase was increased in tumor biopsies relative to normal lung tissue, while the activity of hNTH1, an AP endonuclease, was decreased.133

Using another approach, DNA damage and repair capacity in lymphocytes from non-small cell lung cancer cases and controls was assessed by the comet assay.134 In this study, it was found that constitutive DNA damage, BPDE-induced damage and repair following BPDE-induced damage were all significantly higher in cases than in controls.

Some components in tobacco smoke, or agents produced endogenously through oxidative and lipid peroxidation, that are capable of forming DNA adducts have also been shown to inhibit nucleotide excision repair (e.g., of BPDE-DNA adducts). They include acrolein,95 trans-4-hydroxy-2-nonenal (4-HNE)135 and malondialdehyde.136 They could therefore contribute to lung carcinogenesis in smokers through such mechanisms. Other components in tobacco smoke, such as heavy metals that interfere with the fidelity of DNA polymerases or inhibit DNA repair, may also enhance the mutagenic potential of the DNA- damaging compounds in tobacco smoke.137

O6-alkylguanine lesions in DNA, which can be formed, for example, by tobacco-specific nitrosamines, are repaired by O6-alkylguanine-DNA alkyltransferase (MGMT). A study of peripheral blood mononuclear cells and lung bronchial epithelial cells of smokers and nonsmokers revealed that MGMT activity was significantly lower in the lung cells, but not the blood cells, of the current smokers.138 As activity was higher in the blood cells than in the lung cells the latter may be more sensitive to alkylation damage, although in this study, there was no correlation between activity in the two cell types.

Adducts and Mutations

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

In lung cancer the spectrum of TP53 mutations differs significantly between smokers and nonsmokers, with the prevalence of G to T transversions at endogenously methylated CpG dinucleotides in the gene higher in smokers. A number of tobacco carcinogens, including PAHs, can induce such a mutation. BPDE preferentially damages TP53 codons that are hotspots for mutation in smokers' lung cancer.139 It also preferentially modifies codons 12 and 14 of the KRAS gene, which are also hotspots for mutation in human tumors.140 G to T transversions are also induced by other tobacco carcinogens whose DNA adducts have been detected in smokers' lung tissue, including NNK, 4-aminobiphenyl and ROS.139 Indeed, in vitro studies with NNK have shown that it preferentially forms adducts at the second position of codon 12 of KRAS, which is a major hotspot for G to A and G to T mutations in smoking-induced lung cancer.141 Acrolein is yet another tobacco carcinogen that binds preferentially to TP53 mutation hotspots in smoking-related lung cancer, and it too was reported to induce predominantly G to T transversion mutations in human cells.95 These mutations were detected in the supF gene borne on a shuttle vector that was transformed into E. coli. When, in another study,142 acrolein-induced supF mutagenesis was investigated in human fibroblasts, or in the cII gene in Big Blue mouse embryonic fibroblasts, mutagenic activity was not detected. However, in the hands of the authors of the original study,95 mutagenicity of acrolein in the supF system in mammalian cells was confirmed, with the predominant sites of adduct formation mapping to the mutational hotspots, where G to T and G to A substitutions were the most common.143 Acetaldehyde, another constituent of tobacco smoke, has also been investigated for its binding distribution to KRAS and TP53 sequences, but in contrast to these other agents, it showed little sequence selectivity.144

In a study of Hungarian lung cancer patients, TP53 mutations were detected in 45% (47/104) of the tumors, with the smoking-associated G to T transversion being exclusive to smokers (8/43, 19%), and most of these cases had high levels of bulky DNA adducts in nontumorous lung tissue.145

In contrast, the relationship between breast cancer, TP53 mutation and PAH-DNA adducts in peripheral mononuclear cells is more complex. TP53 mutations were less common in women assessed to have been exposed to PAHs (according to smoking status, consumption of grilled/smoked meat and adduct levels), although some specific mutations, including G to T transversions at CpG dinucleotides, were more common in the exposed women.146 Nevertheless, the overall frequency of TP53 mutation in the study was low (15%).

Using massively parallel sequencing technology, Pleasance et al.147 have sequenced a small-cell lung cancer cell line, NCI-H209, to explore the mutational burden associated with tobacco smoking. Overall, 22,910 somatic mutations were found, of which 1,134 were in coding exons. Multiple mutation signatures attest to the complex mixture of carcinogens present in tobacco smoke and point the way to future mechanistic understanding of the nature and origins of mutations in human tumors, whether smoking-related or not.

A molecular link between DNA damage, inhibition of DNA repair by cadmium and mutagenesis during fetal development was suggested by a study of smoking and nonsmoking mothers.137 Levels of zinc, lead, cadmium and DNA adducts in cord blood and in the peripheral blood of mothers, and HPRT mutations in cord blood only, were measured. Adduct levels were significantly higher in the smokers' than in the nonsmokers' peripheral blood, but not cord blood. Nevertheless, mutation frequency was 3-fold higher in cord blood from smoking mothers than from nonsmokers (p = 0.03). When expressed as mutations per adduct, there was a good correlation with cadmium levels. This implies that heavy metals, known to interfere with DNA repair processes, may potentiate the mutagenic potential of PAH-like DNA damage in neonates.

Influence of Genetic Polymorphisms on Smoking-Related Adducts

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

Despite recent emphasis on the genetic origins of cancer, it remains the case that most cancers arise from a combination of environmental factors and inherited susceptibilities, with the environment playing the predominant role.148 This is very apparent in the case of tobacco smoking, yet not all smokers develop cancer and those that do can succumb to a wide variety of different cancers, as mentioned in the Introduction. It is conceivable that genetic differences between smokers may play a role in determining who does and does not get cancer, and in what organ of the body.

In recent years, the influence of host factors and human genetic variability in tobacco-related cancers has thus been extensively studied.149 These studies divide into three main types: the analysis of genes that encode for enzymes that metabolize the carcinogenic components of tobacco smoke; the analysis of genetic variations in genes that encode DNA repair enzymes; and genes involved in cell cycle regulation, DNA replication and/or apoptosis. Here we consider those studies published since 2002 that have investigated the influence of such polymorphisms on smoking-related DNA and protein adducts in human tissues. These studies vary greatly in the numbers of individuals studied (see Tables 2 and 3), which has a bearing on the importance that can be attached to each finding. Furthermore, there are instances in which the phenotypic consequences of a genetic polymorphism are unclear.150

Polymorphisms in genes for xenobiotic metabolism enzymes

Many studies have investigated whether genetic polymorphisms in phase I metabolizing enzymes cytochrome P450, microsomal epoxide hydrolase, myeloperoxidase (CYP1A1, CYP1A2, mEH and MPO) and others, or whether genes for phase II detoxifying enzymes, glutathione S-transferase (GSTM1, GSTP1 and GSTT1) and N-acetyltransferase (NAT1 and NAT2) influence levels of DNA adducts in smokers. The studies covered by the period of this review are summarized in Table 2.

Table 2. Influence of genetic polymorphisms in xenobiotic metabolizing genes on smoking-related adducts
inline image

Studies published before 2002 have been reviewed by Alexandrov et al.168 and by Beseratinia et al.169 The influence of these low-penetrance genes is variable and, in general, small in magnitude. Defining individual genetic risk from exposure to tobacco carcinogens is likely to be governed by a combination of genetic polymorphisms in different xenobiotic metabolizing genes and, indeed, this is evident both in the earlier studies and in some of the more recent ones summarized in Table 2. Thus, for example, Firozi et al.151 reported an interaction between CYP1A1 and GSTM1, Perera et al.162 found an interaction between GSTM1 and GSTP1, Lewis et al.165 between GSTM1, GSTP1 and GSTT1, and Ketelsleger et al.156 between GSTM1, mEH and GPX1 in determining DNA adduct levels in smokers.

It is apparent from this body of literature that polymorphisms in xenobiotic metabolism genes can influence DNA adduct formation from smoking and other environmental sources, and it is assumed that this may have a bearing on individual susceptibility. However, the heterogeneity of the study populations and their total environmental exposure in the different studies makes comparisons and conclusions difficult. In addition, the variety of adduct endpoints measured and the plethora of techniques used to detect them add further complexity and heterogeneity to these studies. As the evidence stands at the moment, there is little to suggest that some individuals are protected to any significant extent from the genotoxic effects of smoking due to genetic polymorphism in genes responsible for the activation or detoxication of carcinogenic constituents of tobacco smoke.

Polymorphisms in genes for DNA repair enzymes

Because DNA adducts are subject to DNA repair processes, it is logical to hypothesize that genetic polymorphisms in DNA repair genes with the potential to alter activity of the enzymes might influence the levels of adducts in individuals. Several genes have been investigated. Studies covered by the period of this review are summarized in Table 3.

Table 3. Influence of genetic polymorphisms in DNA repair genes on smoking-related adducts
inline image

In most cases there is no observable effect of genetic polymorphism on DNA adducts, and even where observed the numerical differences between individuals with different genotypes are relatively small. As with xenobiotic metabolizing genes, it is perhaps more likely that a combination of genes, rather than a single one, may be more influential, but this has not been extensively studied, and study power is a limiting factor to studying large numbers of genes simultaneously. It can be seen from Table 3 that in some cases a significant difference is observed when a combination of a DNA repair gene and xenobiotic metabolism gene is considered. Furthermore, it should be noted that most of these studies have measured DNA adducts in peripheral blood lymphocytes and, as already mentioned, in many studies smoking is at best a weak correlate with adduct levels; other environmental sources may also contribute.

Polymorphisms in other genes

Genome-wide association studies have provided evidence that variation at 5p15.33 (TERT-CLPTM1L), 6p21.33 and 15q25.1 (CHRNA5-CHRNA3) influences lung cancer risk. In lung tissue of smokers with lung cancer (n = 204) the risk allele of rs402710 (TERT-CLPTM1L locus) was associated with significantly higher levels of bulky DNA adducts (p = 0.02).174 This demonstrates a potential association between the TERT-CLPTM1L variant and bulky DNA adducts and hence a basis for susceptibility to lung cancer.

Polymorphism in the interleukin-1 β gene (IL1B), influencing chronic inflammation responses, has also been associated with lung cancer risk. Individuals homozygous for the associated IL1RN*1 allele (interleukin 1 receptor antagonist) and also carrying the IL1B-31T allele were found to have an increased risk of lung cancer and also nearly 2-fold higher levels of bulky/hydrophobic DNA adducts, detected by 32P-postlabelling, in their lung tissue (n = 209 lung cancer cases).175

A series of studies of breast cancer in the Long Island Breast Cancer Study Project have investigated the presence of PAH-DNA adducts, detected by competitive ELISA, in blood cell DNA of cases and controls. Subjects were classified as having detectable or nondetectable levels of adducts, and association with smoking was investigated and also the modifying effect of a number of genetic polymorphisms. In this cohort (although not in some others), an association between smoking and adduct levels has been observed, but neither cancer risk nor the presence of adducts were found to be significantly influenced by polymorphism in IGHMBP2 (immunoglobulin-binding protein 2, involved in DNA repair, replication and recombination) (n = 866 cases, 938 controls).176 Second, there was no association found between polymorphisms in Fas and Fasl, genes involved in apoptotic signaling, and breast cancer risk, although there was a 36% increase in risk among those with detectable adducts and a variant allele of FAS1377 (n = 873 cases, 941 controls).177 Third, although some polymorphisms in TP53 modulated breast cancer risk in the cohort, there was no correlation with the presence or absence of detectable DNA adducts in 578 cases and 390 controls.178

Exposure to ETS

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

There is now a wealth of evidence showing the adverse health effects, including cancer, of exposure of nonsmokers to tobacco smoke (referred to as passive smoking, secondhand smoke and ETS).4 There is thus an increasing need to control and monitor such exposures. A number of biomarkers have been employed, with varying success.3, 179 Most studies do not show that DNA adducts levels in peripheral blood cells are sufficiently different between unexposed and ETS-exposed nonsmokers for them to be reliable biomarkers of passive smoking.11 This is not surprising given the numerical difference (generally 2-fold or less) in adduct levels in blood cells between smokers and nonsmokers. While target tissues might show greater differences, these are not generally obtainable from healthy subjects.

There are many adverse health effects in newborn babies that are a consequence of maternal smoking during pregnancy, and even due to exposure of nonsmoking mothers to ETS during pregnancy.180–182 BPDE-DNA adducts in neonatal buccal cells, determined by immunoassay, have been reported to be a reliable biomarker of in utero exposure to smoking.183 In a series of studies of mother-newborn pairs and the influence of prenatal environmental exposures, including ETS, on birth outcomes and child development, children born to nonsmoking mothers in New York had cord blood DNA adduct levels (BPDE-DNA adducts, measured by HPLC-fluorescence) that correlated with a number of adverse effects. Although combined exposure to high ETS and high-adduct levels affected birth outcomes, there was no correlation between adducts and ETS exposure.184, 185 The same result was obtained in a separate study of mothers and newborns living within one mile of the World Trade Center disaster on September 11, 2001.186, 187 Another study found no difference in the levels of 8-oxo-dG in newborns (in placenta) of smoking and nonsmoking mothers,188 but bulky adducts (determined by 32P-postlabelling and by ELISA using BPDE-DNA antibodies) were elevated.189 In a different study, a similar proportion of placental samples from ETS-exposed and nonexposed mothers had detectable levels of PAH-DNA adducts (by ELISA), although paradoxically none of the samples from smoking mothers (n = 6) was positive.190

Thus in those studies where nonsmoking mothers, with and without exposure to ETS, are compared, differences in adduct levels in the newborns are generally not found, while there are examples where infants born to smoking mothers have higher levels of adducts than those born to nonsmokers.

A study of children exposed to ETS did not find a relationship between DNA adduct levels in white blood cells, measured by 32P-postlabelling, and other markers of exposure, namely levels of cotinine in hair or serum.191

One study in which a qualitative change in DNA adducts was observed involved biomonitoring adult nonsmokers before and after visits to a public house.192 The profile of DNA adducts in induced sputum, but not peripheral blood lymphocytes, showed evidence of an extra adduct spot (by 32P-postlabelling) after exposure.

A similar picture emerges from studies of protein adducts, although there are some exceptions. Under controlled conditions (i.e., in an environmental chamber) exposure of 40 nonsmokers to sidestream tobacco smoke for 4 hr resulted in a small (16%) but significant (p < 0.0001) increase in 4-ABP-Hb adducts.193 In a study of bladder cancer among never smokers, women exposed to ETS had a higher (but not significantly) mean level of 4-ABP-Hb adducts than unexposed women (23.6 vs. 16.4 pg/g), and those currently exposed were nine times more likely to have a higher than median level of adducts (p for trend = 0.046).194

For children exposed to ETS, N-(2-hydroxyethyl)valine in hemoglobin (OHEtVal-Hb, formed by ethylene oxide) has been found to be at elevated levels in those exposed,195 and earlier studies of fetal exposure to maternal smoking, reviewed by Neri et al.,196 showed increased hemoglobin adduct levels (4-ABP-Hb and OHEtVal-Hb) in those so exposed.

In a study examining biomarkers of bladder cancer risk, nonsmokers with bladder cancer exhibited higher levels of 4- and 3-ABP-Hb adducts than disease-free nonsmokers, although the possible contribution of ETS is not specified;197 as discussed elsewhere,5 other environmental sources of 4-ABP may contribute to adduct levels in nonsmokers.

In a study of 104 nonsmokers, higher levels of CyEtVal (formed by acrylonitrile) were found in 12 ETS-exposed subjects (p = 0.07) than in the 92 self-reported unexposed subjects, while the levels of OHEtVal (from ethylene oxide), hydroxpropylvaline (from propylene oxide), AAVal (from acrylamide) and GAVal (from glycidamide) did not differ between exposed and unexposed groups.44

Comparisons of paired maternal blood and cord blood levels of hemoglobin adducts derived from acrylamide, glycidamide and ethylene oxide indicate that the placenta gives little protection to the fetus from exposure to these compounds in utero.45 Nonsmoking mothers exposed to ETS during pregnancy had detectable levels of HPB-releasing hemoglobin adducts, as did the fetal cord blood samples analyzed.198

Sex Differences

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

Various epidemiologic studies have indicated a higher susceptibility to lung cancer for women, compared to men, after adjustment for number of cigarettes smoked, although the issue remains controversial and the mechanism(s) are unclear.199, 200 The expression of CYP1A1 is reported to be higher in the lungs of female smokers, correlating with levels of bulky DNA adducts.201 However, results of in-vitro studies to investigate a possible role of the estrogen receptor (ERα) on CYP1A1 expression did not support the hypothesis of a role for estrogen or ERα in regulating PAH activation in lung.202

The urinary levels of etheno-DNA adducts, measured by GC-MS, among smokers also showed sex differences.203 Female smokers excreted significantly higher levels of 1,N6-ethenoadenine (εAde) and 3,N4-ethenocytosine (εCyt), products of lipid peroxidation, than male smokers (p = 0.002 and 0.005, respectively. Adduct levels were also significantly higher than in nonsmokers.

Sex differences have also been reported with respect to DNA damage in nonsmokers exposed to ETS.204 Blood cells were analyzed by the comet assay with and without hOGG1 digestion (to detect oxidative lesions) and 8-oxo-dG levels in blood serum were also measured. The levels of general DNA damage (by the comet assay without hOGG1) were significantly higher with ETS exposure in males but not in females. In contrast the oxidative-specific damage did not increase in either sex with increasing exposure, nor did 8-oxo-dG in serum correlate with ETS exposure or differ between men and women. In contrast, 4-ABP-Hb adduct levels were found to be higher in nonsmoking women than nonsmoking men, suggesting that women may be more susceptible to the carcinogenic effects of ETS exposure than men,205 although the overall contribution of ETS to 4-ABP-Hb adducts in nonsmokers, relative to other potential sources, is not clear.5 Nevertheless, in a US study of bladder cancer risk, levels of 4-ABP-Hb varied with ETS exposure status among female controls; as mentioned above, those exposed to ETS were nine times more likely to have a higher than median level of adducts than the never-exposed women, but there was no relationship between adducts levels and ETS exposure in men.194 Finally, an intervention study involving vitamin supplementation resulted in a reduction in blood DNA adducts in women smokers, but not in male smokers206 (see below).

Relationship of Adducts to Chronic Diseases Other than Cancer

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

Atherosclerosis is causally linked to smoking, and there is much experimental evidence to suggest a role for PAHs in formation of atherosclerotic plaques.207 Prior to 2002, the presence of smoking-related DNA adducts in cardiovascular tissue was well established.208, 209 More recently, although autopsy tissue from thoracic aortas of subjects with atherosclerotic damage had higher levels of bulky DNA adducts than subjects without damage, there was no difference between smokers and nonsmokers in either group.210 In contrast, the levels of εAde and εCyt in abdominal aorta smooth muscle cells of a small group of atherosclerotic patients were marginally significantly higher in smokers than in nonsmokers, and the levels correlated with levels of bulky DNA adducts detected by 32P-postlabelling.211

With low body mass index (BMI) reportedly a risk factor for smoking-induced lung cancer, adduct levels, determined by 32P-postlabelling, in peripheral blood lymphocytes of a group of 24 smokers were found to be inversely correlated with BMI (p = 0.02).212

Chemoprevention and Smoking Cessation

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

Many agents have been suggested to have the potential to inhibit the metabolic activation of carcinogenic components of tobacco smoke and thereby to protect against DNA damage from smoking, but only a few have been tested so far in human trials for such effects. The discussion here is limited to those that have been tested.

Oltipraz is a drug that induces phase II detoxifying enzymes and that has been demonstrated to decrease DNA adduct formation in rats exposed to benzo[a]pyrene, aflatoxin and tobacco smoke. When chronic smokers took 400 or 200 mg/week oltipraz or placebo for 12 weeks, however, no differences were found between groups in levels of PAH-DNA or BPDE-DNA adducts in lung epithelial cells, determined by immunohistochemistry or by MS, respectively, despite some subjects experiencing oltipraz-related gastrointestinal toxicity. Levels of BPDE-hemoglobin adducts in peripheral blood, measured by MS, were also unaffected by oltipraz.213

In a double-blind Phase II trial healthy smokers were supplemented daily with N-acetyl-L-cysteine (NAC) or placebo for 6 months, and DNA adducts and hemoglobin adducts were measured.214 There was an inhibition by NAC of the induction of lipophilic-DNA adducts (detected by 32P-postlabelling) and 8-oxo-dG in bronchoalveloar lavage (BAL) cells, but no effect on lipophilic-DNA adducts in peripheral blood lymphocytes or PAH-DNA adducts (detected by immunohistochemistry) in mouth floor/buccal mucosal cells or 4-ABP-hemoglobin adducts.

In a randomized trial investigating dietary influences on smoking-related DNA adducts, detected by 32P-postlabelling, in exfoliated bladder cells and white blood cells, groups of smokers were assigned to a diet rich in flavanoids (e.g., from cruciferous vegetables) or a diet supplemented by flavanoid-rich green tea and soy products, or a control isocalorific diet.215 A slight decrease (nonsignificant) in bladder cell adducts was observed after 1 year in the supplementation group and after 1 month in white blood cells. Multiple regression analysis did reveal, however, a significant decrease in adducts associated with increased flavanoid intake after 1 year after adjusting for smoking (p = 0.02).

Another study that used 32P-postlabelling analysis investigated the effect of consumption of Tahitian Noni Juice on DNA adducts levels in peripheral blood lymphocytes of smokers.216 A reduction of up to 45% was seen after drinking 1 or 4 oz per day for 1 month. The juice extract, from Morinda citrifolia (Noni) is documented to have strong antioxidant and anti-inflammatory activity.

Consumption for 4 weeks of green tea by smokers in a pilot study (n = 3) had the effect of reducing both PAH-DNA adducts and 8-oxo-dG (both detected by immunohistochemistry) in oral cells.217 In another study, the effect of decaffeinated green tea consumption on levels of urinary 8-oxo-dG was also monitored in heavy smokers who were genotyped for GSTM1 and hOGG1.218 Four cups of tea per day for 4 months significantly reduced excretion levels (by 31%) in GSTM1-positive smokers regardless of hOGG1 status. Consumption of black tea did not reduce levels of urinary 8-oxo-dG.

A pooled analysis of studies on DNA adducts and dietary vitamins was conducted by Ragin et al.219 A total of 2,758 subjects from seven studies were included, from which it was found that vitamin E was inversely associated with DNA adducts in smokers' white blood cells or lymphocytes, measured by 32P-postlabelling, in both ever and never smokers, while an inverse association between adducts and vitamin A was apparent only for studies measuring adducts in lymphocytes, and then only for ever-smokers.

However, in another study, vitamin supplementation did not, overall, reduce BPDE-DNA adduct levels (determined by HPLC-fluorescence) in white blood cells of smokers, although there were some sex differences.206 The study involved a double-blinded placebo-controlled trial of vitamin A (500 mg daily) and vitamin E (400 IU daily) supplementation for 15 months, with blood samples collected every 3 months. Overall and among men, there was no effect of treatment on adduct levels, but among women only, adduct levels decreased by 31% compared to the controls (p = 0.03). For protein adducts, smokers had a significant (p = 0.05) decrease in 4-ABP-Hb adduct levels with increasing intake of carotenoids.220

Thus there is fragmentary evidence that dietary intervention can reduce to some extent the levels of protein and DNA damage caused by smoking. Whether such interventions can seriously counteract the adverse health effects of smoking has yet to be determined, but it seems unlikely. Perhaps most effort should be expended in stopping people smoking.

Smoking cessation leads to a fall in DNA adduct levels. For bulky DNA adducts detected by 32P-postlabelling the half-life in peripheral blood lymphocytes was estimated to be 11 weeks.212 In a study of the effect of smoking cessation on 25 volunteers, the major DNA adduct formed by acetaldehyde, N2-ethylidene-dGuo, was detected by MS as N2-ethyl-dGuo detected in leukocyte DNA after it had been subjected to enzyme hydrolysis in the presence of NaBH3CN. It was measured during a 2-week smoking period and again after 4 weeks of abstinence from smoking.37 The median levels of the adduct decreased by 28% as a result of smoking cessation, which was statistically significant (p = 0.02). Another study found that smoking cessation for 1 month reduced the concentration of 7-methylguaine in urine to 54% (mean value for 18 male subjects) of the smoking levels.221 Switching from conventional cigarettes to electrically heated smoking devices has also been reported to result in reduced levels of a number of biomarkers, including 4-ABP hemoglobin (on average by 43%).222

Concluding Remarks

  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References

The delivery of an addictive drug, nicotine, to large sections of the human population over many decades by means of the inhalation of tobacco smoke and by application of unburnt tobacco to mucous membranes has resulted in an unintentional experiment in chemical carcinogenesis on an epic scale and the experiment continues.20 Fundamental studies of chemical carcinogenesis have revealed the central role of DNA adducts in the genesis of cancer and our review has revealed that some tobacco-related DNA adducts appear to be causally related to some tobacco-related cancers. However, the data are sometimes conflicting and confusing. This is not surprising, given the myriad of toxicants and carcinogens in tobacco smoke, its ability to provoke irritation, oxidative damage and to inhibit DNA repair, and the variety of different tissues and organs in which it induces cancer. The plethora of different methods of assay, target tissues and organs, the wide variation in data between individuals, and the lack of uniformity in the methods used for measuring exposure to tobacco smoke add to the difficulties of making well-founded and useful generalizations about the current status of DNA and protein adducts as biomarkers for the prevention or alleviation of tobacco-related disease. Moreover, many of the adducts that have been associated with tobacco smoke are also engendered by other exposures and by endogenous processes and it is often difficult to distinguish between the effects of tobacco and these other confounding variables. Nevertheless, based on the data of the type under review, and combined with results of urinary excretion of tobacco-related metabolites and adducts, suggestions have been made for a battery of biomarkers—including DNA and protein adducts—considered to be of the required sensitivity and specificity to qualify as tools for the regulation of tobacco products and the identification and protection of people at risk from tobacco-related diseases.20


  1. Top of page
  2. Abstract
  3. Detection Methods for Smoking-Induced DNA and Protein Damage
  4. Smoking-Related DNA Adducts in Human Tissues
  5. Smoking-Related Protein Adducts
  6. Repair of DNA Adducts
  7. Adducts and Mutations
  8. Influence of Genetic Polymorphisms on Smoking-Related Adducts
  9. Exposure to ETS
  10. Sex Differences
  11. Relationship of Adducts to Chronic Diseases Other than Cancer
  12. Chemoprevention and Smoking Cessation
  13. Concluding Remarks
  14. Acknowledgements
  15. References
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