Carcinogenesis, Vol. 23, No. 12, 1979-2004,
December 2002
© 2002 Oxford University Press
REVIEW |
Smoking-related DNA and protein adducts in human tissues
Institute of Cancer Research, Haddow Laboratories, Cotswold Road, Sutton SM2 5NG, UK Email: davidp{at}icr.ac.uk
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Abstract |
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 Top Abstract IntroductionSmoking-related DNA adducts in...Respiratory tractOral and nasal cavitiesUrogenital tissuesOther tissuesBlood cellsSmoking-related DNA adducts in...Smoking-related protein adducts...Smoking-related protein adducts...ConclusionsReferences |
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Tobacco smoking causes not only lung cancer but also cancer of the oral and nasal cavities, oesophagus, larynx, pharynx, pancreas, liver, kidney, stomach, urinary tract and cervix. Tobacco smoke contains many carcinogens that exert their biological effects through interaction of reactive intermediates with DNA to form DNA adducts. The same electrophilic species also react with cellular proteins. The effects of smoking are evident by the detection of elevated levels of carcinogen–DNA adducts in many human tissues and of carcinogen–protein adducts in blood. Components of tobacco smoke also induce oxidative DNA damage. Systemic exposure to tobacco-derived carcinogens is demonstrated by the observation of elevated levels of DNA adducts in tissues not directly exposed to tobacco smoke. For many of these tissues there is epidemiological evidence, varying from comprehensive to preliminary, that smoking is a causative factor in cancer of that site. The effects of passive smoking, which also causes lung cancer in non-smokers, is also evident in elevated levels of protein adducts in exposed non-smokers so exposed, relative to non-exposed non-smokers. This paper reviews the literature on smoking-related DNA and protein adducts in human tissues and shows how such studies have provided mechanistic insight into the epidemiological associations between smoking and cancer.
Abbreviations: 3-ABP, 3-aminobiphenyl; 4-ABP, 4-aminobiphenyl; BAL, bronchoalveolar lavage; BPDE, benzo[a]pyrene diol-epoxide; CEVal, N-(2-cyanoethyl)valine; DRZ, diagonal radioactive zone; ECD, electrochemical detection; ELISA, enzyme-linked immunosorbent assay; ETS, environmental tobacco smoke; GC-MS, gas chromatography-mass spectrometry; HPB, 4-hydroxy-1-(3-pyridyl)-1-butanone; HOEtVal, N-(2-hydroxyethyl)valine; 7-MedG, 7-methyldeoxyguanosine; 8-oxo-dG, 8-oxo-deoxyguanosine; PAH, polycyclic aromatic hydrocarbon; WBC, white blood cell
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Introduction |
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TopAbstractIntroductionSmoking-related DNA adducts in...Respiratory tractOral and nasal cavitiesUrogenital tissuesOther tissuesBlood cellsSmoking-related DNA adducts in...Smoking-related protein adducts...Smoking-related protein adducts...ConclusionsReferences |
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Tobacco is a uniquely dangerous consumer product that is addictive and harmful to human health when used as intended. Tobacco smoking is the greatest single cause of preventable illness and premature death, killing half of all people who continue to smoke for most of their lives (1). Half of these will die before the age of 69. Thirty per cent of all cancer deaths, including nearly 90% of deaths from lung cancer are caused by smoking. The cancers for which epidemiological studies have established a causal association with tobacco smoking are lung, urinary tract, kidney, oral cavity, sinonasal, nasopharynx, hypopharynx and oropharynx, oesophagus, larynx, pancreas, stomach, liver, colorectal, cervix and myeloid leukaemia (2).
Tobacco smoke contains many thousands of chemicals including a plethora of carcinogens (3). Many carcinogens undergo metabolic activation in mammalian tissues to reactive intermediates that interact with and modify informational macromolecules, such as DNA (4,5), with potentially mutagenic consequences. The exposure of human tissues and organs to these carcinogens, and their metabolic activation therein, is the mechanism by which smoking-related cancer is initiated. Over the past 20 or so years the systemic nature of the exposure of the human body to carcinogens inhaled in tobacco smoke has become apparent. This is evident from the widespread formation of DNA and protein adducts in human tissues, and the detection of tobacco-related carcinogens and their metabolites in various bodily fluids. In a recent article Hecht (6) reviewed the detection of human urinary carcinogen metabolites as biomarkers for investigating the relationship between tobacco smoking and cancer. The current article reviews the literature on smoking-related DNA and protein adducts and their uses in monitoring human exposure to, and assessing cancer risk from, tobacco smoking.
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Smoking-related DNA adducts in human tissues |
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TopAbstractIntroductionSmoking-related DNA adducts in...Respiratory tractOral and nasal cavitiesUrogenital tissuesOther tissuesBlood cellsSmoking-related DNA adducts in...Smoking-related protein adducts...Smoking-related protein adducts...ConclusionsReferences |
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Because DNA damage, frequently in the form of chemically stable adduct formation, is an early and essential step in the process by which genotoxic carcinogens initiate the carcinogenic process, the detection of DNA adducts in human tissues is a useful and appropriate means to assess human exposure to such agents. A number of different procedures have been used for this. 32P-Postlabelling analysis, in which DNA is digested and the resultant carcinogen-modified nucleotides are radiolabelled enzymatically with [32P]orthophosphate, constitutes a sensitive method for which prior knowledge of structures of the adducts formed is not required. A common feature of postlabelled DNA from cells or tissues exposed to complex mixtures of carcinogens, such as tobacco smoke, is the appearance of a diagonal radioactive zone (DRZ) when the material is resolved by 2-dimensional thin-layer chromatography. Although this material has not been fully characterized, its properties are compatible with those of a complex mixture of aromatic and/or hydrophobic DNA adducts. Another method used to detect DNA adducts in human tissues employs antibodies raised against various carcinogen-modified DNA or nucleotides. Immunohistochemistry allows localization of adducts within a tissue specimen and measurement of fluorescent staining intensity permits a semi-quantitative estimate of adduct levels. Thirdly, fluorescence detection of adducts in DNA, or of products released from DNA by hydrolysis, has been used for such carcinogens that have strongly fluorescent properties, for example polycyclic aromatic hydrocarbons (PAHs). Fourthly, mass spectrometry has been used for the chemical-specific detection of certain adducts in DNA samples and finally, electrochemical detection (ECD) has been used for some smaller DNA lesions, for example 8-oxo-deoxyguanosine (8-oxo-dG), formed in DNA by oxidative processes. The uses, strengths and limitations of these techniques have been widely described (7–13). Earlier studies of tobacco-related DNA adducts in human tissues have been reviewed (14).
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Respiratory tract |
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TopAbstractIntroductionSmoking-related DNA adducts in...Respiratory tractOral and nasal cavitiesUrogenital tissuesOther tissuesBlood cellsSmoking-related DNA adducts in...Smoking-related protein adducts...Smoking-related protein adducts...ConclusionsReferences |
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Lung and bronchus
A large number of studies have been published in which the levels and characteristics of DNA adducts in the lung and bronchus of smokers and non-smokers have been compared. These are summarized in Table I
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In most of these studies, significantly elevated levels of DNA adducts were detected in the peripheral lung, bronchial epithelium or bronchioalveolar lavage (BAL) cells of the smokers. This is the case for total bulky DNA adducts (as detected by the 32P-postlabelling method) and for more chemical-specific methods including HPLC/fluorescence and gas chromatography-mass spectrometry (GC-MS). However, 4-aminobiphenyl(4-ABP)–DNA adduct levels in lung did not correlate with smoking status (15). In some cases where smoking status was not known but inferred from plasma cotinine levels (indicating smoking habit at the time of death of the subjects) there was generally a poor correlation with the detection of one specific type of adduct, such as 7-methyldeoxyguanosine (7-MedG) (16) or those formed by benzo[a]pyrene (17).
Some studies have found a linear correlation between adduct levels and daily or lifetime consumption of cigarettes (18–21), but in other studies this relationship was not found (22,23). Among a group of male smokers all with lung cancer, there was a linear relationship between adduct levels and the inverse of years of smoking (24); this result implies that individuals with higher levels of adducts may get cancer from smoking in a shorter time than smokers with lower levels. Mean levels of adducts in ex-smokers (usually with at least a 1 year interval since smoking cessation) are found generally to be intermediate between the levels of smokers and life-long non-smokers. From these comparisons estimations of the half-life of adducts in lung tissue are put at ~1–2 years. This is somewhat longer than would be estimated from a consideration of the biochemical processes of DNA repair and the rate of cell turnover, and may be a consequence of the slow clearance of carcinogen-containing tar and particulate deposits in the lung, resulting in continued metabolic activation of tobacco carcinogens after smoking cessation.
In a Norwegian cohort, adduct levels were higher in the lungs of women smokers than in men's, a difference that was greater after adjustment of the lower smoking exposure of the women (24,25). However, in a Hungarian cohort, adduct levels in male and female smokers with lung cancer were the same (22).
Larynx
32P-Postlabelling analysis of DNA from larynx mucosa revealed the presence of hydrophobic adducts (the DRZ) in smokers (n = 21) but not non-smokers (n = 4). The ability of both the nuclease P1 digestion and the butanol extraction procedures to detect these adducts suggested that they were formed primarily by PAHs rather than aromatic amines. Adduct levels correlated with levels of CYP1A, 2C and 3A4, but not with levels of expression of CYP2E1 and 2A6 (26).
In another study, adduct levels in laryngeal tumours were compared with those in non-tumour tissue from 43 patients, most of them smokers. Adduct levels, detected by 32P-postlabelling, were higher in tumour tissue than in normal, and the few non- or ex-smokers (n = 5) had lower levels than the smokers (not significant) (27). Subsequently, 32P-postlabelling analysis was used to measure levels of N7-alkylguanine in DNA from laryngeal biopsies. Levels in heavy smokers (>40 cigarettes/day) were 2.5-fold higher than in moderate smokers (~20 cigarettes/day) and 6-fold higher than in non- and ex-smokers, and there was a significant correlation between N7-alkylguanine levels and aromatic/hydrophobic adduct levels in these tissue samples (28).
An immunohistochemical study of laryngeal biopsies from 38 patients, using antibodies raised against 4-ABP–DNA adducts, included analysis of tumours, polyps and surrounding tissue. The study showed significant differences in staining intensity of polyps and surrounding tissue from smokers compared with that from non-smokers. Differences between smokers and non-smokers in tumour tissue were not significant (limited number of samples). The study shows that 4-ABP–DNA adducts, related to smoking exposure, are formed in the larynx (29).
Analysis of laryngeal biopsies from 33 Polish patients, all of whom had tumours, was carried out by 32P-postlabelling. DNA from tumour, non-tumour and the interarytenoid area was analysed separately. Large inter-individual differences in adduct levels were seen, with highest levels in the interarytenoid area. Adduct levels were reported to be correlated with age, sex (28 of the patients were men, five were women; adduct levels were higher in the males), cigarette smoking (all patients were smokers, 19 smoked 20 cigarettes/day, 14 smoked 30–40) and stage of tumour progression (P values not reported) (30).
Sputum
DNA isolated from sputum, induced by inhalation of nebulized saline solution, was compared in a group of smokers (n = 20) and non-smokers (n = 24). Using the nuclease P1 digestion method of 32P-postlabelling analysis, all smokers and only one non-smoker showed a DRZ in their adduct patterns, and adduct levels were significantly higher in smokers than in non-smokers (3.1 ± 1.4 versus 0.6 ± 0.8/108 nt; P = 0.0007) (31). In another report, the same authors found that the adduct levels in the induced sputum of smokers (n = 9, monitored on three occasions), were significantly higher than the levels in non-smokers (n = 9, monitored once) (32). When immunohistochemical analysis was carried out on induced sputum, the cells of smokers (n = 20) had significantly greater staining using 4-ABP–DNA antibodies than cells of non-smokers (n = 24) (P = 0.001), but not using benzo[a]pyrene diolepoxide(BPDE)–DNA antibodies (P = 0.07) (33).
DNA damage in human lung cells in vitro
In cultured human lung cells, the effect of bubbling cigarette smoke through the medium (phosphate-buffered saline) is to induce DNA single strand breaks and formation of 8-oxo-dG in DNA, and evidence was presented that this was mediated, in part, through the formation of reactive oxygen species (34). It was shown that cigarette tar promotes neutrophil-induced DNA damage in human lung cells and this activity is further enhanced by iron and inhibited by catalase (35).
Treatment of human fetal lung cells with the tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(N-methyl-N-nitrosamino)-1-butanone (NNK) caused strand breaks in DNA. Inhibition by oxygen radical scavengers suggested that the hydroxyl radical was an important intermediate in the process (36).
DNA damage, as measured by the single-cell gel electrophoresis (comet) assay, has also been shown to result in human embryo lung cells and in human lymphoid cells treated with water-soluble compounds from cigarette smoke (37,38).
In another study, treatment of tracheobronchial epithelial cells with gas-phase cigarette smoke caused DNA strand breakage accompanied by increases in the levels of a number of DNA lesions, including 8-oxo-dG, xanthine and hypoxanthine. These latter lesions can arise from the deamination of guanine and adenine, by a mechanism involving reactive nitrogen species. Thus, DNA damage induced by cigarette smoking may be mediated by both reactive oxygen species and reactive nitrogen species (39).
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Oral and nasal cavities |
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TopAbstractIntroductionSmoking-related DNA adducts in...Respiratory tractOral and nasal cavitiesUrogenital tissuesOther tissuesBlood cellsSmoking-related DNA adducts in...Smoking-related protein adducts...Smoking-related protein adducts...ConclusionsReferences |
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In two early 32P-postlabelling studies of DNA from oral mucosal cells, a variety of aromatic and/or hydrophobic adducts were seen at varying levels in smokers and non-smokers, but none of them were specific to tobacco smoke exposure (40,41).
DNA from clinically normal oral tissue (mucosa) from patients undergoing surgery for intraoral squamous cell carcinoma was analysed by 32P-postlabelling using both nuclease P1 digestion and butanol extraction enhancement methods. Both the range and levels of adducts were greater with the latter method, and there was a statistically significantly higher mean level of adducts in the smokers (n = 20) than in the ex-smokers (n = 4) and non-smokers (n = 9) combined (42). A comparison between DNA from oral biopsies and from buccal mucosa from the same individuals showed that there was a good correlation between adduct levels in the two sources, with a significantly higher level in DNA from smokers (n = 20) than from non-smokers (n = 10) (43).
In a small study of nasal mucosal DNA from smokers (n = 9), ex-smokers (n = 2) and non-smokers (n = 10), 32P-postlabelling analysis detected the DRZ in eight smokers, one ex-smoker and one non-smoker. The mean adduct level in smokers was significantly higher than that in the non-smokers (P < 0.01; 4.8 versus 1.4/108 nucleotides) and a linear relationship between adduct levels and daily cigarette consumption was observed among the smokers (44). In another study, the levels of DNA adducts detected in smokers' nasal cells (n = 6) by 32P-postlabelling analysis was also significantly higher than the levels in non-smokers' cells (n = 14) (mean value 17.0 versus 6.8/108 nucleotides) (45).
Using a 32P-postlabelling method developed to detect cyclic adducts such as 1,N2-propanodeoxyguanosine, possible products of the reaction of tobacco smoke constituents such as acrolein and crotonaldehyde with DNA, significantly higher levels of each of three such adducts were detected in gingival tissue DNA of smokers (n = 11) than of non-smokers (n = 12); the total levels of the adducts were 4.4-fold higher in the smokers than in the non-smokers (46).
Using the comet assay to detect DNA strand breaks in exfoliated buccal cells, significantly increased DNA migration was found with the cells of smokers (n = 11) than with those of non-smokers (n = 9) (47).
A study of exfoliated oral cells from healthy volunteers used an immunohistochemical approach to stain cells with antibodies raised against BPDE–DNA adducts. Staining intensity was significantly greater with cells from smokers (n = 33) than from non-smokers (n = 64), and increasing staining was observed with increasing numbers of cigarettes smoked per day among the former group (48). Similar results were obtained in another study using this technique, in which both buccal cavity and mouth floor cells were investigated; staining intensity in both cell types was significantly higher in smokers (n = 26) than in non-smokers (n = 22) (49). When antibodies to 4-ABP–DNA adducts were used, staining intensity was also significantly greater in exfoliated oral cells of smokers (n = 12) compared with non-smokers (n = 12) (50).
Immunohistochemical staining of human oral mucosal cells, using antibodies to BPDE–DNA adducts, revealed significantly higher levels of staining in smokers (n = 16) than in matched non-smokers (n = 16) (51). A second study, using both these antibodies and ones derived from 4-ABP-modified DNA, showed significantly higher levels of DNA damage in smokers (n = 20) than in matched non-smokers (n = 20) in both circumstances (52). Subsequently, an analogous analysis using antibodies to malondialdehyde–DNA adducts has demonstrated elevated levels of this adduct, derived from lipid peroxidation, in the oral mucosal cells of smokers (n = 25) compared with matched non-smokers (n = 25) (53).
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Urogenital tissues |
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TopAbstractIntroductionSmoking-related DNA adducts in...Respiratory tractOral and nasal cavitiesUrogenital tissuesOther tissuesBlood cellsSmoking-related DNA adducts in...Smoking-related protein adducts...Smoking-related protein adducts...ConclusionsReferences |
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Bladder
Investigations of the presence of smoking-related DNA adducts in bladder cells have made use of autopsy and biopsy specimens and also exfoliated epithelial cells excreted in urine.
When DNA adduct levels were determined in bladder biopsy samples using 32P-postlabelling with both butanol extraction and nuclease P1 enhancement procedures, the mean levels of several specific adducts were elevated in smokers (n = 13) compared with ex-smokers (n = 20) and non-smokers (n = 9); some adducts were present at similar levels in all groups and thus were not smoking-related. One of the smoking-related adducts was chromatographically identical to the major adduct formed in DNA by 4-ABP (54).
Exfoliated urothelial cells recovered from urine of 73 individuals yielded sufficient DNA for 32P-postlabelling in 40 of the samples. The DNA was found to contain multiple DNA adducts, of which four were present in smokers' DNA at two to 20 times higher levels than in non-smokers' DNA. One of these corresponded chromatographically to N-(deoxyguanosin-8-yl)–4-ABP (55). In a further study, two adducts detected in exfoliated uroepithelial cells from healthy male subjects were specifically associated with smoking and with the levels of 4-ABP–haemoglobin adducts (50 smokers, 50 non-smokers) (56).
In a study of bladder biopsies of 20 bladder cancer patients and exfoliated bladder cells of 36 healthy individuals, a dose–response relationship between smoking and adduct levels was found for both groups (statistics not given; note many groups are small). Adduct levels were not higher in the cases than in the controls (57).
Analysis by 32P-postlabelling of DNA of bladder tissue samples taken at autopsy from 56 individuals revealed a positive but weak association (P = 0.09) between adduct levels and tobacco smoking, and a correlation between adduct levels in lung and bladder (P < 0.01, Spearman rank correlation test) (58).
Comparison of the adducts detected by 32P-postlabelling in bladder biopsies from 30 smokers with those from 24 non-smokers found that, overall, adduct levels were not significantly different in the two groups; 3–5-fold higher levels of adducts were detected by the butanol extraction procedure than by the nuclease P1 digestion procedure, implying a preponderance of aromatic amine-like adducts over those formed by PAHs. However, one minor adduct was found to be present at a 2-fold higher level in the smokers' samples analysed (n = 17) than in non-smokers (n = 8) (P < 0.005, one-tailed) (59).
Human bladder biopsy samples were analysed by immunohistochemical analysis with antibodies to 4-ABP–DNA adducts and the levels of staining compared with smoking history. Adduct levels were significantly higher in current smokers (n = 24) than in non-smokers (n = 22) (P < 0.0001). There was also a linear relationship between mean levels of relative adduct staining and numbers of cigarettes smoked per day (60). In another study using the same technique, higher levels of staining were also observed with bladder specimens from smokers (n = 30) than from ex-smokers (n = 41) and non-smokers (n = 24), but significance was borderline (P = 0.07) (61).
Immunohistochemical staining of human exfoliated urothelial cells, using antibodies to BPDE–DNA adducts or to 4-ABP–DNA adducts, revealed significantly higher levels of staining in smokers (n = 20) than in matched non-smokers (n = 20) in both cases (52).
In a more recent study, DNA adducts of 4-ABP were measured in bladder biopsies from cancer patients by GC-MS. Detectable levels were found in 37/75 samples, which included smokers (24/46), ex-smokers (9/17) and non-smokers (5/8). However, among the patients with higher grade (i.e. grade 3) tumours, there was a significantly greater probability of the smokers having adducts than the ex- or non-smokers (10/11 versus 5/11) (62).
The presence of 3-alkyladenines in human urine was determined in two smokers who underwent a period of voluntary abstinence from smoking. In one of them, the excretion of 3-ethyladenine was significantly lower on non-smoking days (P < 0.01). Smoking-dependent differences in levels of 3-methyladenine were only apparent when volunteers consumed a diet low in 3-alkyladenines during the study period. Thus, both adducts are produced by alkylating agents in tobacco smoke, but natural dietary levels of 3-methyladenine make this an insensitive biomarker of smoking (63).
In a study of depurinated adducts of benzo[a]pyrene in urine, detectable levels were found in the urine of 3/7 women smokers (0.1–0.6 fmol/mg creatinine), but not in any of 13 non-smokers (64).
Analysis of bladder cells for DNA damage, using the single-cell gel electrophoresis (comet) assay, revealed significantly increased comet tail moments for smokers and ex-smokers (n = 18) compared with non-smokers (n = 12) (P < 0.03) (65).
In a study of oxidative damage measured by means of 8-oxo-dG excretion in urine, smokers (n = 30) were reported to have 50% higher levels of the nucleoside (corrected for body weight) than non-smokers (n = 53) (66).
Taken together, the studies analysing DNA adducts in bladder tissue or exfoliated urothelial cells demonstrate that smoking-related DNA adduct formation is detectable in them, but that some of the adducts present derive from other sources of environmental carcinogens.
Cervix
A pilot study of DNA isolated from cervical scrapes of 22 women and analysed by the butanol extraction enrichment procedure of 32P-postlabelling showed the presence of the diagonal radioactive zone and elevated levels of adducts in 3/6 smokers compared with the levels in 13 non-smokers (67). In a subsequent study, sufficient DNA was isolated from 33/38 cervical smear samples and subjected to 32P-postlabelling analysis. Adduct levels were significantly higher in DNA from smokers (n = 18) than from non-smokers (n = 15) (68).
Using DNA isolated from cervical biopsies, 32P-postlabelling analysis with butanol extraction was carried out on samples from 39 women (11 smokers, seven ex-smokers and 21 never smokers). Adduct levels were significantly higher in the smokers and ex-smokers than in the non-smokers (P = 0.048). Exclusion of those women whose urinary cotinine levels did not confirm their self-reported non-smoking status (n = 7) increased the differences between smokers and non-smokers (P = 0.03) (69).
A study of cervical biopsy samples from 35 women (19 smokers, five ex-smokers and 11 non-smokers) found that DNA adduct levels, determined by 32P-postlabelling analysis with the butanol extraction procedure, were significantly higher in the smokers than in the non-smokers, with an intermediate mean value obtained for the ex-smokers (70). This study population was subsequently increased to 22 smokers, four ex-smokers and 14 non-smokers and analysed by both butanol extraction and nuclease P1 digestion procedures of 32P-postlabelling. Adduct levels were significantly higher in smokers when the former procedure was used, but not with the latter (71). The lack of a difference between smokers and non-smokers using nuclease P1 digestion was also reported in another small study (16 smokers, eight non-smokers) (72). However, in another larger study, 32P-postlabelling analysis using nuclease P1 digestion enrichment showed significantly higher levels in DNA from histologically normal cervical biopsies from current smokers (n = 48) than from non-smokers (n = 48) (73).
Immunohistochemical analysis of human cervical cells, using antibodies to BPDE–DNA adducts, demonstrated significantly increased staining intensity in smears from smokers (n = 16) compared with smears from non-smokers (n = 16) (74).
GC-MS analysis of BPDE–DNA adducts in cervical epithelial cells showed an ~2-fold higher level in smokers (n = 7) than in non-smokers (n = 7) and this difference was statistically significant (P = 0.02) (75).
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Other tissues |
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TopAbstractIntroductionSmoking-related DNA adducts in...Respiratory tractOral and nasal cavitiesUrogenital tissuesOther tissuesBlood cellsSmoking-related DNA adducts in...Smoking-related protein adducts...Smoking-related protein adducts...ConclusionsReferences |
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Breast
In a pilot study, a total of 31 human breast tissue samples were analysed by 32P-postlabelling using nuclease P1 digestion. These included tumour and tumour-adjacent samples from 15 women and four samples from reduction mammoplasties. The characteristic DRZ was detected in 5/15 cancer patients, all of whom were tobacco smokers. None of the eight ex-smokers and non-smokers displayed this pattern (P < 0.01) (76).
In another 32P-postlabelling study of DNA from breast tissue from breast cancer patients and non-cancer patients undergoing reduction mammoplasty, the characteristic DRZ was detected in 17/17 smokers, 4/8 ex-smokers and 4/52 non-smokers. In addition, a particular adduct spot was observed in 36 normal tissue samples (27 of them from non-smokers) but was not significantly associated with smoking (77).
Putative malondialdehyde-derived DNA adducts, detected by 32P-postlabelling, were detected in tumour-adjacent tissue from breast cancer patients (n = 51) at higher levels (P = 0.0001) than in tissue from controls (reduction mammoplasty, n = 28). The levels of these adducts were also associated with the previously detected major DNA adduct (see above), but significantly lower in smokers than in ex-smokers and non-smokers (78).
Immunohistochemical staining of breast tumour tissue using antibodies raised against BPDE–DNA adducts was not significantly different between smokers (n = 35), ex-smokers (n = 72) and non-smokers (n = 75). However, among smokers and ex-smokers, there was a trend towards higher levels of staining with greater levels of exposure or earlier age of starting (79). Positive staining was also obtained with breast tumour tissue (n = 48) and benign disease tissue (n = 30) from inhabitants of Upper Silesia, a region of Poland that has received high levels of environmental pollution. However, in neither group of patients were staining levels elevated in smokers, relative to non-smokers (80).
In a study of PAH–DNA adducts in breast tissue of cancer patients (n = 119) and of those with benign breast disease (n = 108), immunohistochemical analysis indicated a significant association between adduct levels and breast cancer, but there was no relationship between smoking status and adduct levels or disease status (81).
Pancreas
32P-Postlabelling analysis of 20 pancreatic tumours and 13 normal tissue samples, using nuclease P1 digestion, led to the detection of significantly higher levels of adducts in these normal tissues than in either the tumour material or in normal pancreatic tissue from 19 previously healthy organ donors. A number of qualitative and quantitative differences between the adduct profiles of smokers and non-smokers was observed, including some that were statistically significant (82).
In another study, fluorescence spectral analysis for the presence of BPDE–DNA adducts in pancreatic DNA did not reveal detectable levels in any of 11 samples (six smokers, five non-smokers) (83).
Colon
Fluorescence spectral analysis of colon DNA for the presence of BPDE–DNA adducts resulted in the detection of adducts in three of four samples from smokers and one of three samples from non-smokers. Adduct levels were estimated to be in the range 0.2–1.0 adducts/108 nucleotides (83).
Stomach
Tumour DNA from 26 patients with gastric cancer was analysed for DNA adducts using 32P-postlabelling with nuclease P1 digestion. For males only, DNA adducts were significantly higher in the material from smokers (n =14) than in that of non-smokers (n = 4) (84), although it should be noted that the number of non-smokers analysed was small.
Anal epithelium
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 32P-postlabelling. Qualitative and quantitative differences were observed in the adduct profiles of the two groups. The mean adduct level was significantly higher in the smokers than in the non-smokers (1.88 ± 0.71 versus 1.36 ± 0.60 adducts/108 nucleotides, P = 0.02); furthermore, the adduct pattern revealed the smoking-related DRZ in 17/20 smokers, but not in any of the non-smokers (P < 0.00001) (85).
Placenta and fetal tissue
In the first study of placental DNA for the presence of DNA adducts, 32P-postlabelling analysis and a competitive enzyme-linked immunosorbent assay (ELISA) with antibodies to BPDE–DNA adducts were used. The immunological method showed a small but non-significant increase in adducts in material from smokers compared with non-smokers. Using 32P-postlabelling analysis, one particular adduct was found to be present in the DNA from 16/17 smokers, but in only 3/14 non-smokers (86). In a subsequent study, up to seven different adducts were detected by 32P-postlabelling in 53 specimens of human placental tissue, three of which were found `almost exclusively' in smokers (87).
The combined use of immunological, fluorescence spectral and mass spectrometric methods has led to the detection and characterization of BPDE–DNA adducts in human placental DNA and also provided evidence for the presence of adducts formed by other PAHs (88,89). Synchronous fluorescence spectroscopy for the detection of BPDE-modified DNA gave positive results with 10/28 human placental samples, but there was no correlation with smoking status (90). In a subsequent study, 5/7 placentas from smokers and three/nine from non-smokers were positive for these adducts (91).
In another small study, placental DNA from smokers (n = 5) was found to contain adducts, detected as a DRZ by 32P-postlabelling, at a 1.8-fold higher mean level than that in placental DNA from non-smokers (n = 5) (92) (4.3 ± 1.7 versus 2.3 ± 0.4 adducts/108 nucleotides, statistical significance not stated).
Using 32P-postlabelling with nuclease P1 digestion, DNA adducts were detected in placental and umbilical cord DNA regardless of whether the mothers were smokers or not. Adduct levels were significantly higher in maternal than in fetal tissue and, combining data for all tissues, total DNA adduct levels were significantly higher in smokers (n = 8) than in non-smokers (n = 11). Although individual tissues showed a trend towards increased adduct levels in smokers, the differences were not statistically significant (93,94). DNA adducts derived from PAH were detected by ELISA in 6/14 placentas and in 5/12 matched fetal lung samples from spontaneous abortions. None of the samples were from women who reported smoking during pregnancy, suggesting that smoking was not a likely source of the adducts (95). However, in another study using ELISA analysis using antibodies to BPDE–DNA adducts, there was a linear correlation between adduct levels and urinary cotinine levels for both placental DNA and umbilical cord DNA, the former tissue having the higher adduct levels. Overall, adducts were detected in 13/15 placental samples from smokers and three/10 from non-smokers (96).
When placental DNA was analysed both for bulky DNA adducts by 32P-postlabelling and for 8-oxo-dG by ECD, neither method showed a difference between 11 smokers, 10 non-smokers and nine non-smokers exposed to passive smoking (97).
Thus, while some studies indicate the presence of smoking-related DNA adducts in human placenta, overall the association between smoking status and adduct levels, determined by a variety of different methods, is weak. The results would seem to indicate that there are significant sources of environmental carcinogens other than tobacco smoke that result in DNA adducts being formed in this tissue.
Sperm
In a study in which sperm DNA from 12 heavy smokers (>20 cigarettes/day), 12 light smokers (1–19 cigarettes/day) and 12 non-smokers was subjected to 32P-postlabelling analysis, no discernible differences were observed between the patterns or levels of DNA adducts between the three groups of subjects (98).
However, in a study in which immunohistochemical staining of sperm, using antibodies to BPDE–DNA adducts, was carried out, staining intensity was significantly higher in the sperm of smokers (n = 11) than in the sperm of non-smokers (n = 12) (99). Furthermore, in vitro fertilization experiments using smokers' sperm indicated the transmission of BPDE-modified DNA to the embryos of non-smoking partners (100). In related studies, granulosa-lutein cells of women undergoing in vitro fertilization were analysed. Immunostaining for BPDE–DNA adducts was confined to the nucleus and significantly greater in cells from smokers (n = 14) than in those of passive smokers (n = 7) and non-smokers (n = 11) (101). (See also section on passive smoking.)
It was found that the sperm DNA of smokers (n = 28) contained about 1.5 times the level of 8-oxo-dG found in the sperm DNA of age-matched non-smokers (n = 32), a difference that was statistically significant (6.19 ± 1.71 versus 3.93 ± 1.33 8-oxo-dG/105 dG, P < 0.001) (102). The same authors have reported a correlation between sperm defects and levels of 8-oxo-dG in sperm DNA, indicating that this lesion is useful for assessing sperm quality and male fertility (103). In a study of sperm from heavy smokers (>20 cigarettes/day, n = 10), light smokers (<20 cigarettes/day, n = 11) and non-smokers (n =10), the frequency of disomy was increased among the smokers for some chromosomes (e.g. 13) but not others (21, X, Y) (104).
In another study, the sperm DNA of smokers (n = 35) was found to be more sensitive to acid-induced denaturation (P < 0.02) and to possess higher levels of DNA strand breaks (P < 0.05) than the DNA of non-smokers (n = 35) (105).
Cardiovascular tissues
In a pilot study investigating the presence of DNA adducts by 32P-postlabelling in a number of human tissues at autopsy, the diagonal radioactive zone characteristic of smoking-related DNA damage was observed in heart tissue of two smokers, but not in that of a non-smoker. Moreover, the levels of adducts were highest in heart of all the tissues examined, which included lung and bronchus (106). A study comparing 32P-postlabelling, HPLC-fluorescence detection of BPDE–DNA adduct hydrolysis products, and synchronous fluorescence spectroscopy detection of the same products demonstrated that all three methods were capable of detecting DNA adducts in smooth muscle of human atherosclerotic lesions. However, due to the limited number of samples investigated (four from smokers, three from ex-smokers) no conclusion could be drawn regarding the origins of the adducts detected (107). Another study measured, using 32P-postlabelling, DNA adducts in the thoracic aorta of 133 victims of sudden or accidental death. Those with significant atherosclerotic changes and where atherosclerosis was classified as the main cause of death were designated as cases (n = 76), those without as controls (n = 57). Smoking status was determined by measuring cotinine levels in plasma. DNA adduct levels were significantly higher in the cases than in the controls, but when smokers and non-smokers were compared, significant differences were observed among the controls, but not among the cases (108).
Immunohistochemical staining of endothelial and smooth muscle cells of blood vessels using antibodies to BPDE–DNA adducts resulted in higher staining of the endothelial cells, although the correlation with smoking habits among the 33 subjects studied (9/11 smokers gave positive staining, compared with 12/21 non-smokers) did not reach statistical significance (109).
In a study of DNA from the right atrial appendage of 41 patients undergoing open heart surgery, the levels of adducts detected by 32P-postlabelling were significantly higher in the smokers (n = 15) than in the ex-smokers (n = 15) and in the non-smokers (n = 11) (P < 0.01 and 0.001, respectively). Among the smokers, a significant linear relationship between adduct levels and daily cigarette smoking was observed (110).
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Blood cells |
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TopAbstractIntroductionSmoking-related DNA adducts in...Respiratory tractOral and nasal cavitiesUrogenital tissuesOther tissuesBlood cellsSmoking-related DNA adducts in...Smoking-related protein adducts...Smoking-related protein adducts...ConclusionsReferences |
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Although nucleated peripheral blood cells are not generally considered target cells for tobacco-induced tumourigenesis, the greater ease with which they can be obtained from human subjects, relative to many target organs, has led to a large number of studies exploring the differences between cells from smokers and non-smokers. Studies in which smoking-related DNA adducts have been determined are summarized in Table II
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The picture that emerges from these many studies is an inconsistent one. Measurements made in the longer-lived cells (lymphocytes and monocytes) are more likely to reveal significant differences between smokers and non-smokers than measurements in whole white blood cell (WBC) preparations (see Table II
). However, a study of adduct levels in smokers following cessation of the habit did show a decline over time (111). It is clear from studies of populations occupationally exposed to environmental agents such as PAHs that there are multiple sources of exposure resulting in DNA adducts in blood cells and whether or not the influence of tobacco smoking is discernible probably depends on the contribution of other occupational or environmental sources in particular study populations. There are also reports of the influence of nutritional factors on DNA adduct levels in blood cells in some studies (112–114) but not others (115). The modulating effects of genetic polymorphisms in xenobiotic metabolizing genes and DNA repair genes have been implicated in several studies. In general, these observations have been made on the basis of rather small numbers of subjects, the magnitude of the effects are not large, and confirmation of the findings is required in larger studies. There have been a limited number of reports to suggest that adduct levels in the blood of smokers correlate with adduct levels in the lung (116–118). However, there are also conflicting findings in which no association was found between blood DNA adduct levels and adducts in lung (119) or BAL cells (120).
In the case of oxidative damage in lymphocytes, a number of studies report an increase in oxidative DNA damage in smokers (121–123). The magnitude of this increase, although significant, is generally small (<2-fold) and its biological significance is unclear. In contrast, there are two reports in which smokers had lower levels of this lesion in their WBC or lymphocyte DNA (124,125).
Several studies that have compared smokers with cancer with smokers who are free of the disease report an ~2-fold higher adduct level in the cases. This has been observed in two lung cancer case-control studies (126,127) and one prospective study (128), but not in another case-control study (129). The effect was also observed in a bladder cancer case-control study (130). This suggests that DNA adducts can be considered as biomarkers of risk in some populations, although the magnitude is small when seen against a background of wide inter-individual variation, making assessment of risk on an individual basis problematic.
Another approach to monitoring human exposure to carcinogens has been to detect the antigenic response to BPDE–DNA adducts in blood cells. Serum antibodies to these adducts have been detected in a number of groups of occupationally exposed workers, but in a study of smokers only a small percentage (8%) of heavy smokers (n = 50) initially tested positive for the antibodies. Paradoxically, the frequency of positivity increased during a cessation programme in which 28 subjects quit smoking and 22 reduced consumption by 75% (131). Nevertheless, in a larger population study of 1345 individuals for the presence of detectable BPDE–DNA antibodies in serum, there was a positive association with smoking (17.8% of non-smokers, 21.5% of ex-smokers and 25.7% of smokers tested positive) and there was a synergism between smoking and family history of lung cancer in determining the prevalence of antibodies (P = 0.02) (132).
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Smoking-related DNA adducts in non-smokers |
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In comparison to the number of studies that have investigated the levels of DNA adducts, or other measures of DNA damage, in the tissues of smokers, ex-smokers and non-smokers (see preceding section), relatively few studies have addressed the issue of using these biomarkers to monitor exposure of non-smokers to environmental tobacco smoke (ETS). Considering that the magnitude of the difference between smokers and non-smokers is often quite small (for example, <2-fold in the case of DNA adducts in WBC), it is probable that in many circumstances this biomarker may lack the sensitivity to distinguish the effects of exposure to ETS from exposure to other sources of environmental carcinogens. Studies in which a distinction has been made between non-smokers exposed to ETS and those unexposed are described below.
In a study in which declining DNA adduct levels in the WBC of smokers enrolled in a smoking cessation programme were measured by ELISA using antibodies to BPDE–DNA adducts, levels of adducts both at baseline and 10 weeks after cessation were significantly associated with hours of exposure to ETS at home and were also higher if the subject lived with another smoker (P = 0.009, 0.02 and 0.02, respectively). However, there was no observable influence of exposure to ETS in the workplace (111).
Using the prevalence of serum antibodies to BPDE–DNA adducts in an Italian population as a biomarker of exposure to environmental PAHs, investigators reported an association between per cent positivity and smoking status (25.7% of smokers, 21.5% of ex-smokers and 17.8% of non-smokers) and living environment (26.0% urban dwellers and 17.9% suburban dwellers), but no association with passive smoking was found (132).
In a study in which significant differences were observed between the levels of adducts, determined by a flow cytometric method using BPDE–DNA antibodies, in peripheral lymphocytes of smokers (n = 40) and non-smokers (n = 35), the mean value for non-smokers with no or low exposure to ETS (n = 17) was lower than that of non-smokers who were exposed (n = 18), but the difference was not statistically significant (133).
A group of five non-smokers were exposed to ETS under controlled conditions (exposure to gas-phase only for 8 h, followed by exposure to whole ETS for 8 h, 40 h later). When their monocyte DNA was analysed by 32P-postlabelling, no changes in the adduct patterns were seen after either exposure period, compared with the samples obtained before exposure (134). Similarly, the frequency of DNA single strand breaks in mononuclear cells of non-smokers was not affected by controlled exposure to ETS for 8 h (135).
In a study of biomarkers of air pollution exposure in two Greek populations, one from an urban location, the other rural, 32P-postlabelling analysis of lymphocyte DNA revealed the presence of DNA adducts that were more abundant in the rural subjects than in the urban ones. The variation in adduct levels was found to significantly (P < 0.001) parallel exposure to ETS, as judged by self-reporting, plasma cotinine concentrations and profiles of personal exposure to PAHs characteristic of ETS rather than other environmental sources (136).
When a group of 15 non-smokers (11 female, five male), who provided blood and sputum samples before, and 1–2 h after, spending 3 h in a smoky environment were studied, their plasma cotinine and nicotine levels were both significantly increased by the exposure to ETS, but exposure did not result in the induction of DNA adducts in peripheral blood lymphocytes. However, changes in the adduct profiles of sputum DNA of some individuals were observed, including the formation of an adduct characteristic of the major BPDE–DNA adduct in three individuals (137).
When placental DNA was analysed both for bulky DNA adducts by 32P-postlabelling and for 8-oxo-dG by ECD, neither method showed a difference between 11 smokers, 10 non-smokers and nine non-smokers exposed to passive smoking (97).
Immunohistochemical staining, using antibodies to BPDE–DNA adducts, of ovarian granulosa-lutein cells of women undergoing IVF was intermediate in intensity for non-smoking women exposed to passive smoking (n = 7) to the values for smokers (n = 14) and unexposed non-smokers (n = 11) and statistically significantly different from both these groups (P < 0.0001) (101).
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Smoking-related protein adducts in human tissues |
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Even though proteins are not the target molecules for mutagenic events, their use as biomarkers can be advantageous because of their greater abundance than DNA and the fact that they are not subject to enzymatic repair. The rate of turnover of the proteins and/or of the cells that harbour them means that carcinogen–protein adducts can provide evidence of exposure on a time-scale of up to several months. In theory, haemoglobin, serum albumin, histones and collagen are suitable proteins for study (138), but in practice only haemoglobin and albumin have been extensively studied as biomarkers of human exposure to environmental carcinogens.
Protein adducts are generally detected in one of three ways: mass spectrometric detection of the carcinogen moiety after its release from protein by mild acid or base hydrolysis, or release and detection of the modified N-terminal valine of haemoglobin; immunochemical analysis using antibodies raised against protein adducts; and HPLC with fluorescence detection of the released carcinogen (139). The use of protein adducts as biomarkers of human exposure to carcinogens has been extensively reviewed elsewhere (9,10,12,138).
Adducts formed by aromatic amines
In an early study of smokers and non-smokers, 4-ABP–haemoglobin adducts were measured by GC-MS. The smokers (n = 22) had adduct levels significantly higher than the non-smokers (n = 24) and there was a significant correlation with average packs/day, pack-years and cumulative lifetime tar exposure. It is noteworthy that the range of values for smokers (75–256 pg/g haemoglobin) did not overlap with that for non-smokers (7–51 pg/g) (140).
Non-overlap in values was also observed in another study involving 19 smokers (range 70–260 pg/g; mean 154) and 26 non-smokers (range 10–50 pg/g; mean 28) and the difference between the two groups was highly significant (P < 0.001). The finding of detectable levels of adducts in all the non-smokers is consistent with other environmental sources of 4-ABP besides tobacco smoking (141).
In a group of male volunteers arising from a case-control study of bladder cancer in Turin, Italy, haemoglobin adducts of 15 different aromatic amines were determined in non-smokers and smokers of blond- or black-tobacco cigarettes. The smokers of the blond tobacco (n = 40) and the black tobacco (n = 18; a further three subjects smoked both types) had significantly higher levels of ABP adducts than non-smokers (n = 25). Furthermore, adduct levels were significantly higher (40–50%) for black tobacco smoking than for blond tobacco. Adduct levels correlated with amount smoked per day for all smokers (P = 0.0015) and this was also significant for blond tobacco smoking only (P = 0.0074). For other aromatic amines, the levels of 3-ABP adducts were also significantly elevated in smokers (P < 0.0001) and associated with numbers of cigarettes smoked by the blond tobacco users (P = 0.02). Adducts derived from a further five aromatic amines, 2-naphthylamine, o-toluidine, p-toluidine, 2-ethylaniline and 2,4-dimethylaniline were significantly higher in the smokers, while those of m-toluidine, 2,5-, 2,6-, 2,3-, 3,5- and 3,4-dimethylaniline and 3- and 4-ethylaniline were not (142). In a subsequent analysis, the authors sought explanations for the inter-individual variability observed. There appeared to be a distinct difference between the values obtained with the binuclear compounds and those obtained with the mononuclear compounds. Correlations between the levels of the three binuclear amines (2-naphthylamine, 4-ABP and 3-ABP) were significant (P < 0.05) and 49/54 of the correlations between different mononuclear amines were significant, but only 2/33 correlations between a binuclear and a mononuclear amine were significant. These results suggest the existence of two distinct pathways of metabolic activation of aromatic amines and explain a part, but not all, of the inter-individual variation in adduct levels observed (143).
As part of a validation exercise in a study of 4- and 3-ABP–haemoglobin adducts in non-smokers exposed to ETS (see following section), seven quitting smokers were monitored on the day they stopped smoking and again at least 2 months later. The mean levels of 4-ABP adducts fell by 75% and the mean level of 3-ABP fell by at least 80% (in four cases of the latter, to levels below the limit of sensitivity of the assay) (144). A further study of smokers enrolled at a cessation clinic found that the 34 subjects at the start had a mean level of 4-ABP–haemoglobin of 120 ± 7 (SE) pg/g which declined to 82 ± 6 pg/g after 3 weeks. In the 15 smokers who remained abstainers after 2 months the level was 34 ± 5 pg/g; the rate of decline was slightly more rapid than would be expected from an assumption that erythrocytes have a life-span of 120 days. At the start of the study there was no correlation with plasma cotinine levels or with the tar content of the cigarette brands that had been smoked (145). Similar findings were reported in another study of smoking cessation involving 40 smokers. Mean levels for 16 subjects were <50 and 25% of those at the start, at 10 weeks and 8 months, respectively, both reductions highly significant. Depending on the model used, the half-life of the adducts was estimated at between 7 and 12 weeks (111).
In a study of 50 non-smokers, 31 smokers of blond tobacco, 16 smokers of black tobacco and three pipe smokers, the relationship between consumption of blond and black tobacco and 4-ABP–haemoglobin adduct levels was confirmed (P = 0.0001 in each case). A linear relationship between adduct levels and the numbers of cigarettes smoked in the preceding 24 h was also observed (P = 0.0001). Furthermore, when the subjects were divided into slow and rapid acetylators (by measuring urinary excretion of caffeine after administration of a test dose), it was found that the ratio of adduct levels slow:rapid was 1.6 for non-smokers, 1.3 for blond tobacco and 1.5 for black tobacco. This approximates to the estimated relative risk of the slow acetylator phenotype for bladder cancer of 1.32 (95% CI 1.02–1.71) (146). A further study of this cohort, while confirming these findings, also found that urinary mutagenicity was associated with the numbers of cigarettes smoked but not with the acetylator phenotype (147). Subsequently, 4-ABP–haemoglobin adduct levels in 39 individuals (21 non-smokers, 11 blond-tobacco smokers and seven black-tobacco smokers) were found to correlate with total DNA adduct levels in exfoliated urothelial cells (P = 0.03) (55).
In a case-control study of lung cancer, there were 53 cases and 56 controls (33 with non-cancer pulmonary disease and 23 with non-pulmonary cancer). Adduct levels were higher in smokers than non-smokers and reflected recent smoking exposure. However, there was no association between adduct levels and lung cancer diagnosis (148). However, in a case-control study of bladder cancer, the mean adduct level in the cases (n = 13, all smokers) was significantly higher than in the controls (n = 13, all smokers) [103 ± 47 (SD) pg/g haemoglobin versus 65 ± 44 pg/g, P = 0.04] (149).
4-ABP–haemoglobin adduct levels were also significantly higher in pregnant women who smoked (n = 15) than in those who did not (n = 40) (183 ± 108 pg/g haemoglobin versus 22 ± 8 pg/g, P<0.001), in a study in which levels of the adduct in fetal blood were also measured (see following section) (150). Similarly, two studies of non-smokers and an equal number of smokers measured paired maternal–fetal blood samples. Levels of 4-ABP–haemoglobin adducts were significantly higher in the smokers, and similar ratios of adduct levels in maternal and cord blood were found to those reported in the previous study (151,152). There was also a correlation between adduct levels in maternal blood and cigarettes smoked per day (152).
Analysis of smokers and non-smokers for haemoglobin adducts of several aromatic amines revealed significantly higher levels in smokers of those formed by 4-ABP (P < 0.001), 3-ABP (P < 0.001) and 2,4-dimethylaniline (P < 0.05), but not by aniline, o-toluidine, m-toluidine, p-toluidine, 2-ethylaniline and o-anisidine. For many of these comparisons the numbers of subjects was small (four to 22 smokers, four to 16 non-smokers) (153).
Comparisons of 3- and 4-ABP–haemoglobin adducts in three different racial groups (white, black and Asian) led to the expected detection of higher levels of both adducts in smokers (n = 61) than in non-smokers (n = 72), and of a correlation with the numbers of cigarettes smoked per day (P < 0.0005). Those with the slow acetylator phenotype had higher adduct levels in all three ethnic groups (2.5-fold higher for 3-ABP, P < 0.0005; 1.2 fold higher for 4-ABP, P = 0.19) (154).
In a cohort of 55 smokers and four non-smokers, 4-ABP–haemoglobin adduct levels were related to numbers of cigarettes smoked, although saturation of adduct formation was apparent at >30 cigarettes/day. A relationship between aromatic DNA adducts in lymphocytes and daily cigarette consumption was not found. In the same study, GSTM1 and NAT2 polymorphisms did not affect protein adduct levels (155). The same investigators went on to investigate additional genetic polymorphisms in 67 smokers and found no overall effects on 4-ABP–haemoglobin adduct levels of polymorphisms in NAT1, NAT2, GSTM1 or GSTT1, except that in smokers of <25 cigarettes/day, NAT2 slow acetylators had significantly higher adduct levels than fast acetylators (P = 0.03) (156).
In another study NAT2 slow acetylators also had significantly higher levels of 4-ABP–haemoglobin adducts than rapid acetylators, but there was no association with possession of the NAT1*10 allele. As before, smokers had significantly higher levels of 3- and 4-ABP–haemoglobin adducts and the levels increased with increasing cigarettes smoked per day (P < 0.0001, n = 68 smokers, 288 non-smokers) (157).
Adducts formed by polycyclic aromatic hydrocarbons
In a study involving 87 mothers and their 87 children in which PAH–albumin adducts were investigated as a biomarker of the children's exposure to ETS from their smoking mothers (see following section) it was also noted that adduct levels were significantly higher in the smoking mothers than in the non-smokers (158).
A GC-MS method was used to measure BPDE–globin adduct in 10 smokers and 10 non-smokers. Subjects were also monitored for formation of globin adducts of another PAH, namely chrysene. In both cases the procedure involved measurement of the respective PAH tetrols released from globin by acid hydrolysis. Levels of BPDE adducts in the smokers were 2.7-fold higher than in the non-smokers (P < 0.01), and although the chrysene diol-epoxide adducts were 25% higher in smokers, this was not statistically significant (P = 0.06) (159).
BPDE adducts with both haemoglobin and with serum albumin were determined by GC-MS of tetrols released by acid hydrolysis in a study of 44 male lung cancer patients. Individuals who were positive for haemoglobin adducts (n = 6) all had detectable albumin adducts, but not vice versa (24 subjects were positive for albumin adducts). Those who were carriers of a CYP1A1 variant allele were more frequently positive for albumin adducts (P = 0.03) and those with two `slow' mEH alleles had a lower frequency of these adducts (160).
In another study, tobacco smokers (n = 27) had significantly higher levels of BPDE–albumin adducts and non-significantly higher levels of BPDE–haemoglobin than non-smokers (n = 42) (161) (see also following section).
Adducts formed by tobacco-specific nitrosamines
Treatment of haemoglobin adducted with NNK or NNN releases 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), which is amenable to detection by GC-MS after purification and derivatization. In the first determination of HPB levels in smokers, a group of 40 smokers had a mean value of 79.6 ± 189 fmol/g haemoglobin (undetectable in 11 individuals; limit of detection was 5 fmol/g) while among 21 non-smokers the mean value was 29.3 ± 25.9 fmol/g (undetectable in four individuals). (Statistical significance not stated, although among 22 snuff dippers HPB levels were 517 ± 538 fmol/g, significantly higher than in smokers, P < 0.0001.) No relationship was apparent between HPB levels in smokers and levels of plasma cotinine or numbers of cigarettes smoked (162).
In a comparison of 20 smokers and 15 non-smokers, HPB levels in haemoglobin were significantly higher in the smokers (69.2 ± 43.9 versus 34.4 ± 16.0 fmol/g) (P < 0.005) (153).
In another study of 18 smokers and ex-smokers and 52 non-smokers, levels of HPB in haemoglobin were significantly higher in the smokers [26 ± 13 (SD) fmol/g haemoglobin] than in the non-smokers (20 ± 8 fmol/g) (P = 0.02). No differences were found between the sexes (14 males, 56 females) or between the smokers (n = 10) and ex-smokers (n = 8) (duration of smoking cessation not stated) (163).
Adducts formed by other compounds
Ethylene is a major gaseous constituent of tobacco smoke that converts, via ethylene oxide, the N-terminal valine of haemoglobin to N-(2-hydroxyethyl)valine (HOEtVal). Eleven smokers of >20 cigarettes/day had HOEtVal in haemoglobin in the range 217–690 pmol/g (mean ± SD, 389 ± 138) while for 14 non-smokers the range was 27–106 pmol/g (58 ± 25). It is noteworthy that there was no overlap in values between the two groups (164). In a controlled experiment, two volunteers who were regular smokers (29 and 18 cigarettes/day, respectively) abstained from smoking for 7 days and then resumed. From the levels of HOEtVal before stopping, 7 days after stopping and 7 days after resuming, it was calculated that in the 29-a-day smoker each cigarette smoked increased the level of HOEtVal by 0.12 pmol/g haemoglobin. For the other smoker the increment was 0.08 pmol/g (165).
In a study of smokers (n = 26) and non-smokers (n = 24), background levels of HOEtVal in the non-smokers averaged 49.9 pmol/g haemoglobin (range 22–106). In the smokers the levels were significantly higher by an estimated 71 pmol/10 cigarettes/day, and the correlation with cigarettes smoked per day was significant (P < 0.01) (166).
As part of a maternal–fetal comparison of HOEtVal in haemoglobin (see following section), samples from 10 non-smoking pregnant women and 13 pregnant women smoking 15 or more cigarettes per day were analysed. The non-smokers had 63 ± 20 (mean ± SD) pmol/g haemoglobin, while the smokers had 361 ± 107 pmol/g (P < 0.01) (167).
In a study of 146 adults (82 males and 64 females), smokers (n = 44) had significantly higher levels of HOEtVal in haemoglobin than non-smokers (n = 74) and self-reported passive smokers (n = 28) (mean ± SD: 59 ± 52, 17 ± 21 and 17 ± 23, respectively). Linear regression analysis of adduct levels and cigarettes smoked per day showed a highly significant correlation coefficient (r = 0.63, P < 0.0001). Adduct levels were also significantly higher in males than in females (mean value 2.6-fold higher, P = 0.00001) (168).
Ethylene oxide is a substrate for GSTT1. To determine whether polymorphisms in GSTT1 modulate HOEtVal formation, blood samples from 10 women and 17 men were analysed. The median level of the adduct in smokers was 5-fold higher in the smokers (five males, one female) than the non-smokers (12 males, nine females) but no correlation with daily cigarette consumption was found. GSTM1 and GSTT1 genotype did not influence adduct levels in the smokers, although there was an effect of GSTT1 genotype on levels in non-smokers (169).
The effect of GSTT1 was also investigated in another study involving 14 non-smokers, 16 one-pack-a-day smokers and 13 two-pack-a-day smokers. HOEtVal levels increased with increasing smoking, and differences between the three groups were statistically significant. In addition, levels of N-(2-cyanoethyl)valine (CEVal), which is formed from acrylonitrile, were also significantly correlated with smoking in these subjects. HOEtVal and CEVal levels were found to be significantly correlated in smokers (P = 0.003). There was no effect of GSTM1 or GSTT1 genotypes on CEVal levels in any of the groups or of GSTM1 on HOEtVal levels; however, GSTT1 null individuals had higher levels of HOEtVal (slope of regression line 50% higher) (170).
A study of acrylamide and acrylonitrile exposure in laboratory workers, smokers and non-smokers included also measurement of ethylene oxide haemoglobin adducts. The mean levels of adducts from acrylamide, acrylonitrile and ethylene oxide in 10 smokers were 116, 106 and 126 pmol/g, respectively, significantly higher than in eight non-smokers (31, <2 and 17 pmol/g, respectively). In smokers, both acrylamide and acrylonitrile adducts correlated with the number of cigarettes smoked per day (171).
Two other studies have reported acrylonitrile–haemoglobin adducts in smokers. In the first, four smokers had 75–106 pmol CEVal/g haemoglobin (mean 88) while in four non-smokers the levels were all below the limit of detection of 20 pmol/g (172). In the second study, 13 smoking mothers had adduct levels in the range 92.5–373 pmol/g (mean ± SD: 217 ± 85.1) but adduct levels were below the limit of detection (1 pmol/g) in 10 non-smoking mothers. Among the smokers, there was a linear correlation with cigarettes smoked per day (P = 0.02) and also with the levels of the adducts in their newborn babies (see following section) (173).
In a study of 43 Chinese workers exposed to benzene and 44 unexposed controls, in which haemoglobin and albumin adducts of two metabolites, benzene oxide and 1,4-benzoquinone were measured, tobacco smoking was found to have an additive effect on 1,4-benzoquinone–albumin formation (P = 0.034) but not on benzene oxide–albumin formation (P = 0.23) (174).
Human haemoglobin contains relatively high background levels of N-methylvaline, which could limit its sensitivity as a biomarker of exposure to environmental carcinogens. However, in a study of 11 pairs of monozygotic twins discordant for tobacco smoking, both HOEtVal and N-methylvaline were found to be higher in the smokers than the non-smokers. For the smokers the levels of HOEtVal and N-methylvaline were 143 ± 26 (mean ± SE) and 268 ± 13 pmol/g, respectively and for the non-smokers 15.6 ± 1.9 and 225 ± 11 pmol/g, respectively. Thus, N-methylvaline adduct levels were significantly different between smokers and non-smokers (P = 0.006) and there was a highly significant correlation with number of cigarettes per day (P < 0.001) (175).
Despite the relatively high background levels of N-methylvaline in human haemoglobin, levels of the adduct were found to be elevated in a group of 32 smokers compared with 37 non-smokers (mean ± SD 1546 ± 432 versus 1175 ± 432 pmol/g, P <0.001); for the smokers there was also a linear correlation between adduct levels and cigarettes smoked per day (P < 0.001). A significant difference between smokers and non-smokers for HOEtVal was also observed in this study (176).
Since cigarette smoke contains reactive species that can cause oxidative and nitrative damage in cellular macromolecules, plasma protein from 52 lung cancer patients (24 smokers, 28 non-smokers) and 43 control subjects (18 smokers, 25 non-smokers) was analysed for nitrotyrosine and carbonyl groups as markers of nitration and oxidation, respectively. Nitrated proteins were significantly higher in the patients than in the controls (P = 0.003) but not related to smoking status. In contrast, oxidized proteins were higher in smokers (P < 0.001) but not related to disease status (177).