Genotoxicity of environmental tobacco smoke: a review

Environmental tobacco smoke (ETS), or second-hand smoke, is a widespread contaminant of indoor air in environments where smoking is not prohibited. It is a significant source of exposure to a large number of substances known to be hazardous to human health. Numerous expert panels have concluded that there is sufficient evidence to classify involuntary smoking (or passive smoking) as carcinogenic to humans. According to the recent evaluation by the International Agency for Research on Cancer, involuntary smoking causes lung cancer in never-smokers with an excess risk in the order of 20% for women and 30% for men. The present paper reviews studies on genotoxicity and related endpoints carried out on ETS since the mid-1980s. The evidence from in vitro studies demonstrates induction of DNA strand breaks, formation of DNA adducts, mutagenicity in bacterial assays and cytogenetic effects. In vivo experiments in rodents have shown that exposure to tobacco smoke, whole-body exposure to mainstream smoke (MS), sidestream smoke (SS), or their mixture, causes DNA single strand breaks, aromatic adducts and oxidative damage to DNA, chromosome aberrations and micronuclei. Genotoxicity of transplacental exposure to ETS has also been reported. Review of human biomarker studies conducted among non-smokers with involuntary exposure to tobacco smoke indicates presence of DNA adducts, urinary metabolites of carcinogens, urinary mutagenicity, SCEs and hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene mutations (in newborns exposed through involuntary smoking of the mother). Studies on human lung cancer from smokers and never-smokers involuntarily exposed to tobacco smoke suggest occurrence of similar kinds of genetic alterations in both groups. In conclusion, these overwhelming data are compatible with the current knowledge on the mechanisms of carcinogenesis of tobacco-related cancers, occurring not only in smokers but with a high biological plausibility also in involuntary smokers.     

Carcinogen: DNA adducts in tobacco smoke-associated cancer of the upper respiratory tract.

Mortality connected with tobacco smoke-associated laryngeal cancer in Poland markedly exceeds the relevant epidemiological data from other European countries. The main groups of genotoxic agents considered as potential carcinogens present in tobacco smoke are polycyclic aromatic hydrocarbons, aromatic amines, N-nitrosoamines and reactive oxygen species. Aromatic DNA adducts, N7-alkylated guanosines and oxidative DNA damage derived from tobacco smoke exposure were detected in laryngeal and oral (tumour and non-tumour) biopsies, and white blood cells of cancer subjects. Further, DNA lesions were analysed to estimate the significance of such confounders as intensity of smoking, subject's sex, age, topography of larynx, cancer staging and genetic factor. The number of cigarettes smoked per day was found to be the main determinant of an individual's DNA adduct level. The occurrence of DNA lesions was established as a reliable marker of former exposure to tobacco smoke genotoxicants. On the other hand, a comparison of DNA lesion levels in various regions of larynx indicates limited usefulness of DNA adduct analysis as an estimate of cancer risk. For a better risk estimation one has to take into account DNA lesions in proto-oncogenes and tumour suppressor genes and the efficacy of DNA repair. Altogether, DNA adducts formation and removal has to be considered as a single stage in the multistep carcinogenesis.     

Genotoxicity of tobacco smoke and tobacco smoke condensate: a review

This report reviews the literature on the genotoxicity of mainstream tobacco smoke and cigarette smoke condensate (CSC) published since 1985. CSC is genotoxic in nearly all systems in which it has been tested, with the base/neutral fractions being the most mutagenic. In rodents, cigarette smoke induces sister chromatid exchanges (SCEs) and micronuclei in bone marrow and lung cells. In humans, newborns of smoking mothers have elevated frequencies of HPRT mutants, translocations, and DNA strand breaks. Sperm of smokers have elevated frequencies of aneuploidy, DNA adducts, strand breaks, and oxidative damage. Smoking also produces mutagenic cervical mucus, micronuclei in cervical epithelial cells, and genotoxic amniotic fluid. These data suggest that tobacco smoke may be a human germ-cell mutagen. Tobacco smoke produces mutagenic urine, and it is a human somatic-cell mutagen, producing HPRT mutations, SCEs, microsatellite instability, and DNA damage in a variety of tissues. Of the 11 organ sites at which smoking causes cancer in humans, smoking-associated genotoxic effects have been found in all eight that have been examined thus far: oral/nasal, esophagus, pharynx/larynx, lung, pancreas, myeoloid organs, bladder/ureter, uterine cervix. Lung tumors of smokers contain a high frequency and unique spectrum of TP53 and KRAS mutations, reflective of the PAH (and possibly other) compounds in the smoke. Further studies are needed to clarify the modulation of the genotoxicity of tobacco smoke by various genetic polymorphisms. These data support a model of tobacco smoke carcinogenesis in which the components of tobacco smoke induce mutations that accumulate in a field of tissue that, through selection, drive the carcinogenic process. Most of the data reviewed here are from studies of human smokers. Thus, their relevance to humans cannot be denied, and their explanatory powers not easily dismissed. Tobacco smoke is now the most extreme example of a systemic human mutagen.     

Damage to DNA in cervical epithelium related to smoking tobacco.

OBJECTIVE--To determine whether tobacco smoking causes increased DNA modification (adducts) in human cervical epithelium. DESIGN--Comparison of DNA adducts measured by the technique of postlabelling with phosphorus-32 in normal ectocervical epithelium of smokers and non-smokers. A questionnaire on smoking habit and a urinary cotinine assay were used to identify smokers and non-smokers. SETTING--Cytology unit in large teaching hospital. SUBJECTS--39 women (11 current smokers, seven former smokers, and 21 who had never smoked) undergoing gynaecological treatment (colposcopy or hysterectomy). Nineteen members of staff who did not smoke as controls. INTERVENTIONS--Biopsy of normal ectocervical epithelium. Urine sample. MAIN OUTCOME MEASURES--Measurement of DNA adducts in cervical epithelial tissue of smokers and non-smokers. Smoking habit derived from results of questionnaire and urinary cotinine:creatinine ratio. Proportion of adducts in women with abnormal and normal results of cervical smear test. RESULTS--DNA samples from smokers (identified from questionnaire) had significantly higher median proportions of DNA adducts that non-smokers (4.62 (95% confidence interval 4.04 to 7.74) v 3.47 (2.84 to 4.78) adducts/10(8) nucleotides; p = 0.048). Exclusion of women whose urinary cotinine:creatinine ratio did not confirm their self reported smoking habit (smoker or non-smoker) increased this difference (4.7 (3.85 to 8.08) v 3.52 (2.32 to 4.95) adducts/10(8) nucleotides; p = 0.03). Women who had abnormal results of cervical smear tests had significantly higher proportions of adducts than those with normal results (4.7 (3.90 to 8.13) v 3.47 (3.06 to 5.36) adducts/10(8) nucleotides; p = 0.03). CONCLUSIONS--Tobacco smoking by women leads to increased modification of DNA in cervical epithelium, suggesting biochemical evidence consistent with smoking as a cause of cervical cancer.     

Enhancement of pre-existing DNA adducts in rodents exposed to cigarette smoke

Exposure to tobacco smoke has been implicated in the increased incidence of cancer and cardiovascular diseases. This report describes various experimental studies in animals that were carried out to determine the ability of cigarette smoke to form DNA adducts and to define chromatographic nature of the major adducts. Tissues from rodents exposed to mainstream or sidestream cigarette smoke in nose-only and whole-body exposure systems, respectively, for different durations were analyzed for DNA adducts by 32P-postlabeling assay. The results showed essentially similar qualitative patterns in various respiratory (lung, trachea, larynx) and non-respiratory (heart, bladder) tissues of smoke-exposed rats. However, adduct pattern in the nasal mucosa was different. The mean total DNA adducts in various tissues expressed as per 1010 nucleotides exhibited the following order: heart (700)>lung (420)>trachea (170)>larynx (150)>bladder (50). Some qualitatively identical adducts were routinely detected in tissues from sham-treated rats but at greatly reduced levels (5- to 25-fold). The levels of lung DNA adducts increased with the duration of exposure up to 23 weeks and returned to control levels 19 weeks after the cessation of exposure. Species-related differences in adduct magnitude and patterns were observed among rats, mice and guinea pigs; mouse being the most sensitive to DNA damage and guinea pig the least sensitive. Whole-body exposure of rats to sidestream cigarette smoke also enhanced the pre-existing DNA adducts by several fold in different tissues. Selective chromatography, and extractability in butanol suggested lipophilic nature of smoke-associated DNA adducts, which were, however, recovered significantly better in nuclease P1 than butanol enrichment procedure. The major smoke-associated adducts were chromatographically different from any of the reference adducts of polycyclic aromatic hydrocarbons (PAHs) co-chromatographed with the smoke DNA samples. Because PAH-DNA adducts are recovered with equal efficiency by the two enrichment procedures, the above observations suggested that smoke-associated adducts are not related to typical PAHs, like benzo[a]pyrene. It is concluded that cigarette smoke increased the levels of pre-existing endogenous DNA adducts (the so-called I-compounds) in animal models and that these adducts are unrelated to those formed by typical PAHs.     

A protocol for detecting and scavenging gas-phase free radicals in mainstream cigarette smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking (5,6,7,12). Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens(9,10). Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung(3). Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke(4). A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke(14). However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine(8). In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy(1,2,14). We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively(11,13).     

Smoking-related DNA adducts in anal epithelium

Several studies have identified tobacco smoking as a risk factor for anal cancer in both women and men. Samples of anal epithelium from haemorrhoidectomy specimens from current smokers (n = 20) and age-matched life-long non-smokers (n = 16) were analysed for DNA adducts by the nuclease P(1) digestion enhancement procedure of 32P-postlabelling analysis. The study included 14 men and 22 women. Both qualitative and quantitative differences in the adduct profiles were observed between the smokers and non-smokers. The mean adduct level was significantly higher in the smokers than in the non-smokers (1.88 +/- 0.71) (S.D.) versus 1.36 +/- 0.60 adducts per 10(8) nucleotides, P = 0.02, two-tailed unpaired t-test with Welch's correction); furthermore, the adduct pattern seen in two-dimensional chromatograms revealed the smoking-related diagonal radioactive zone in 17/20 smokers, but not in any of the non-smokers (P < 0.00001, Fisher's exact test). These results indicate that components of tobacco smoke inflict genotoxic damage in the anal epithelium of smokers and provide a plausible mechanism for a causal association between smoking and anal cancer.     

Bronchial malondialdehyde DNA adducts, tobacco smoking, and lung cancer

Tobacco smoking is a major risk factor for lung cancer causing, among other effects, oxidative stress and lipid peroxidation. Malondialdehyde (MDA)-DNA adducts can be induced by direct DNA oxidation and by lipid peroxidation. We measured the relationship between bronchial MDA-DNA adducts and tobacco smoking, cancer status, and selected polymorphisms in 43 subjects undergoing a bronchoscopic examination for diagnostic purposes. MDA-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 increased 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)). Lung cancer cases with levels of MDA-DNA adducts over the median showed a worse, but not statistically significant, survival, after adjusting for age, gender, and packyears (hazard ratio = 2.48, 95% CI 0.65-9.44). Our findings reinforce the role of smoking in lung carcinogenesis through oxidative stress. Subjects who carry at least one variant allele of the CCND1 gene could accumulate DNA damage for altered cell-cycle control and reduced DNA repair proficiency.     

Research on adducts of benzo(a)pyrene and DNA in different cells using synchronous fluorescence spectrophotometry]

The levels of benzo(a)pyrene diolepoxide(BPDE)-DNA adducts were measured in rat and human cells by synchronous fluorescence spectrophotometry. BPDE-DNA adducts detected in human pulmonary alveolar macrophages were related to current smoking habits, in contrast to the adducts found in peripheral blood leukocytes. BP administration to rats produced BPDE-DNA adducts in both liver and lungs. Although small yet repeatable signals were also detected in lung DNA from rats treated for 3 days with tobacco smoke. None of the samples obtained from untreated animals was positive. The detection of BPDE-DNA adducts may be used in biomonitoring and experimental studies for determining of exposure to BP even when applied as a constituent of complex mixtures.     

Population burden of lung cancer due to environmental tobacco smoke

The population burden of lung cancer due to environmental tobacco smoke is significant because a large fraction of the population is exposed. The risks are, of course, lower than those to smokers themselves; but smoking is self-inflicted, passive smoking is involuntary. Making various assumptions, the proportion of lung cancer cases among non-smokers that could reasonably be attributed to environmental tobacco smoke can be calculated to be about 20–30% in western countries. Thus, non-smokers in the society could benefit considerably from diminishing exposures to other people's smoke.     

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

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

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

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

Influence of glutathione S-transferase polymorphisms on genotoxic effects induced by tobacco smoke

Genotoxicity of tobacco smoke has long been investigated and tobacco smoke is considered to be one of the principal human carcinogens. Although its role in DNA-damage induction and cancer development has been documented, the mechanisms by which this happens are not well understood. Many chemical constituents of tobacco smoke are enzymatically metabolized by phase-I and phase-II enzymes, but modifications in coding and regulating sequences of these genes could influence their ability to detoxify these compounds. In this work, we studied several enzymes involved in the metabolism of xenobiotics, viz. the glutathione S-transferases (GST) M1, T1, P1 and A1, with respect to their influence on the genotoxic effects induced by cigarette smoking. We assessed the genotoxic effects of tobacco smoke on peripheral blood lymphocytes of 72 healthy caucasians by use of the chromosomal aberration (CA) assay and the micronucleus (MN) test. Genotypes of GST M1, T1, P1 and A1 were determined by means of the polymerase chain reaction and methods based on restriction fragment length polymorphism (RFLP). We found that smoke and gender are the two variables that most influence the DNA damage. In particular, we observed that female smokers seem to be more sensitive than male smokers, having a significantly higher frequency of CAs. Moreover, a significant increase in frequency of micronuclei in bi-nucleated cells (BNMN) was found in smokers, but not in non-smokers. This increase seems to be influenced not only by age and gender, but also by genetic constitution. Subjects carrying GSTM1-null genotype seemed to have an higher susceptibility to DNA damage induced by tobacco smoke than GSTM1-positive ones. When considering a combination of GST genotypes, we found a lower BNMN frequency in subjects with GSTP1 variant allele plus GSTM1-positive genotypes, while the most damaged cells are found in subjects bearing GSTM1-null plus GSTP1-wild type. Our results suggest that investigation of the association between several gene polymorphisms and important endpoints of DNA damage could contribute to better understanding the role of gene-gene interaction.     

Formation of 8-nitroguanine in tobacco cigarette smokers and in tobacco smoke-exposed Wistar rats

Our previous studies have shown that 8-nitroguanine (8-NO(2)-G) could serve as a specific biomarker of DNA damage induced by gaseous nitrogen oxides (NO(x)) exposure. To evaluate the effect of tobacco cigarette smoking on the DNA damage in peripheral lymphocytes of cigarette smoke ones, we randomly collected and determined the level of 8-NO(2)-G in DNA extracted from peripheral lymphocyte of 15 each of light-smoking healthy volunteer (L-S, less than one pack per day), moderate-smoking healthy volunteers (M-S, one to two pack per day for 5-10 years), heavy-smoking healthy volunteers (H-S, over two packs per day for 10 years), lung cancer patients with heavy smoking (cancer H-S) and non-smoking healthy controls. Both of the mean level of the 8-NO(2)-G levels in peripheral lymphocyte (0.90+/-1.0, 1.23+/-1.14, 1.43+/-0.79, 3.62+/-1.38 ng per microg DNA) and serum nitrite (38.99+/-9.58, 46.70+/-9.38, 55.46+/-10.45, 70.1+/-18.54 microM) of L-S, M-S, H-S and cancer H-S groups were higher than that of non-smoking healthy controls (0.02+/-0.04 and 18.96+/-4.31 for 8-NO(2)-G level and serum nitrite, respectively). Furthermore, in animal experiment, a dose-dependent increase in 8-NO(2)-G was observed in rat lung and peripheral lymphocyte DNA of Wistar rats after tobacco cigarette smoke exposure twice a day, for 1 month. The level of 8-NO(2)-G is 0.17+/-0.41, 1.65+/-3.15, 23.50+/-20.75 and 37.58+/-17.55 ng per microg lung DNA for rat exposed with tobacco cigarette smoke from 0, 5, 10, 15 cigarettes per day, respectively. It was also found that count of peripheral lymphocytes and nitrite concentration in serum of rat increased after the tobacco smoke exposure. It is postulated that tobacco cigarette smoking could induce DNA damage (8-NO(2)-G formation) by exo- and endogenous NO(x).     

O(6)-methylguanine-DNA methyltransferase (MGMT): impact on cancer risk in response to tobacco smoke

Tobacco, smoked, snuffed and chewed, contains powerful mutagens and carcinogens. At least three of them, N-dimethylnitrosamine, N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, attack DNA at the O(6)-position of guanine. The resulting O(6)-alkylguanine adducts are repaired by the suicide enzyme O(6)-methylguanine-DNA methyltransferase (MGMT), which is known to protect against the mutagenic, genotoxic and carcinogenic effects of monofunctional alkylating agents. While in rat liver MGMT was shown to be subject to regulation by genotoxic stress leading to adaptive changes in its activity, in humans evidence of adaptive modulation of MGMT levels is still lacking. Several polymorphisms are known, which are suspected to impact on the risk of developing cancer. In this review we focus on three questions: (a) Has tobacco consumption by smoking or chewing an impact on MGMT expression and MGMT promoter methylation in normal and tumor tissue? (b) Is there an association between MGMT polymorphisms and cancer risk and is this risk related to smoking? (c) Does MGMT protect against tobacco-associated cancer? There are several lines of evidence for an increase of MGMT activity in the normal tissue of smokers compared to non-smokers. Furthermore, in tumors developed in smokers a tendency towards an increase of MGMT expression was found. The data points to the possibility that agents in tobacco smoke are able to trigger upregulation of MGMT in normal and tumor tissue. For MGMT promoter methylation data is conflicting. There is some evidence for an association between MGMT polymorphisms and smoking-induced cancer risk. The key question whether or not MGMT protects against tobacco smoke-induced cancer is difficult to answer since prospective studies on smokers versus non-smokers are lacking and appropriate animal studies with MGMT transgenic mice exposed to the complex mixture of tobacco smoke have not been performed, which indicates the need for further explorations.     

Children's exposure to environmental pollutants and biomarkers of genetic damage. II. Results of a comprehensive literature search and meta-analysis

The present review is based on findings from 178 publications retrieved through an extensive search of the MedLine/PubMed database for a 25 years time period (1980-2004) and 10 manually identified papers. Among the cytogenetic biomarkers that are frequently used in field studies, chromosome aberrations (CA) and micronuclei (MN) but not sister chromatid exchanges (SCE) were found consistently increased in children exposed to environmental pollutants. Meta-analysis of the studies reporting SCE in cord blood showed similar levels of SCE in exposed and in non-exposed newborns. Exposure to airborne pollutants, soil and drinking water contaminants, mostly increased CA and, to a lesser extent, MN levels in children. The effect of exposure to airborne urban pollutants was consistently reported by field studies measuring DNA, albumin and hemoglobin adducts. Prenatal (in utero) and postnatal exposure (environmental tobacco smoke, ETS) to tobacco smoke compounds were associated with increased frequencies of DNA and hemoglobin adducts and CA. The limited number of field studies measuring DNA fragmentation (Comet assay), hypoxanthine-guanine phosphoribosyltransferase (HPRT) and the glycophorinA (GPA) mutation frequency in environmentally exposed children precluded a meaningful evaluation of the usefulness of these assays. Meta-analyses performed in children exposed to ETS and in newborns exposed in utero to their mothers' smoke showed 1.3 and 7 times higher levels of hemoglobin adducts compared to referent subjects, respectively. These increases are consistent with the epidemiological evidence of higher lung cancer risks reported in adults who had never smoked and were exposed to ETS during childhood and with 7-15 times higher lung cancer risks reported in smokers than in non-smokers. Higher levels of PAH-DNA adducts were found in fetal than in maternal tissue, suggesting a specific susceptibility of the fetus to this class of ubiquitous environmental pollutants. According to these findings, future research and biomonitoring programs on children would greatly benefit from the inclusion of selected biomarkers that could provide biologically based evidence for the identification of intervention priorities in environmental health.     

Immunoperoxidase detection of 4-aminobiphenyl- and polycyclic aromatic hydrocarbons-DNA adducts in induced sputum of smokers and non-smokers

Tobacco smoke constituents, 4-aminobiphenyl (4-ABP) and polycyclic aromatic hydrocarbons (PAH) possess carcinogenic properties as their reactive metabolites form DNA adducts. We studied the formation of 4-ABP- and PAH-DNA adducts in induced sputum, a non-invasively obtainable matrix from the lower respiratory tract, of smokers (n=20) and non-smokers (n=24) utilizing a semi-quantitative immunohistochemical peroxidase assay. Smokers had significantly higher levels of 4-ABP-DNA adducts as compared to non-smokers (0. 08+/-0.02 versus 0.04+/-0.01, P=0.001, density of immunohistochemical staining), and the levels of adducts were related to current smoking indices (cigarettes/day: r=0.3, P=0.04 and tar/day: r=0.4, P=0.02). Likewise, smokers had elevated levels of PAH-DNA adducts as compared to non-smokers, however, the differences was not statistically significant (0.13+/-0.02 versus 0. 08+/-0.02, P=0.07). The levels of PAH-DNA adducts were only significantly related to the amount of tar consumed per day (r=0.4, P=0.04) but not to the number of cigarettes smoked per day. Neither the levels of 4-ABP-DNA adducts nor those of PAH-DNA adducts were related to smoking history index (pack years). Further, the levels of 4-ABP-DNA adducts were correlated with those of PAH-DNA adducts (r=0.4, P=0.02). We conclude that immunohistochemistry of 4-ABP-DNA adducts in induced sputum is a specific approach to assess current exposure to tobacco smoke in the lower respiratory tract, however, in the case of PAH-DNA adducts, such analysis is less specific as it does not explicitly reflect the magnitude of the exposure.     

Polycyclic aromatic hydrocarbon-DNA adducts in cervix of women infected with carcinogenic human papillomavirus types: an immunohistochemistry study

Among women infected with carcinogenic human papillomavirus (HPV), there is a two- to five-fold increased risk of cervical precancer and cancer in women who smoke compared to those who do not smoke. Because tobacco smoke contains carcinogenic polycyclic aromatic hydrocarbons (PAHs), it was of interest to examine human cervical tissue for PAH-DNA adduct formation. Here, we measured PAH-DNA adduct formation in cervical biopsies collected in follow-up among women who tested positive for carcinogenic HPV at baseline. A semi-quantitative immunohistochemistry (IHC) method using antiserum elicited against DNA modified with r7,t8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) was used to measure nuclear PAH-DNA adduct formation. Cultured human cervical keratinocytes exposed to 0, 0.153, or 0.331microM BPDE showed dose-dependent increases in r7,t8,t9-trihydroxy-c-10-(N(2)deoxyguanosyl)-7,8,9,10-tetrahydro-benzo[a]pyrene (BPdG) adducts. For BPdG adduct analysis, paraffin-embedded keratinocytes were stained by IHC with analysis of nuclear color intensity by Automated Cellular Imaging System (ACIS) and, in parallel cultures, extracted DNA was assayed by quantitative BPDE-DNA chemiluminescence immunoassay (CIA). For paraffin-embedded samples from carcinogenic HPV-infected women, normal-appearing cervical squamous epithelium suitable for scoring was found in samples from 75 of the 114 individuals, including 29 cases of cervical precancer or cancer and 46 controls. With a lower limit of detection of 20 adducts/10(8) nucleotides, detectable PAH-DNA adduct values ranged from 25 to 191/10(8) nucleotides, with a median of 75/10(8) nucleotides. PAH-DNA adduct values above 150/10(8) nucleotides were found in eight samples, and in three samples adducts were non-detectable. There was no correlation between PAH-DNA adduct formation and either smoking or case status. Therefore, PAH-DNA adduct formation as measured by this methodology did not appear related to the increased risk of cervical precancer and cancer among carcinogenic HPV-infected smokers.     

Smoking-related DNA adducts in anal epithelium

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

Oxidative DNA base modifications and polycyclic aromatic hydrocarbon DNA adducts in squamous cell carcinoma of larynx

Tobacco smoke, recognized as a major etiological factor for cancers of the upper aerodigestive tract, represents an abundant source of reactive oxygen species (ROS), which are believed to play a significant role in mutagenesis and carcinogenesis. An additional source of ROS in tissues exposed to tobacco smoke may be metabolic oxidation of polycyclic aromatic hydrocarbons (PAH). To investigate the relationships between oxidative DNA lesions and aromatic DNA adducts, six modified DNA bases 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine and 4,6-diamino-5-formamidopyrimidine and the total level of PAH-related DNA adducts were measured in cancerous and the surrounding normal larynx tissues (68 subjects), using gas chromatography/isotope-dilution mass spectroscopy with selected ion monitoring and the 32 P-postlabeling-HPLC assay, respectively. The levels of oxidative DNA lesions in cancerous and adjacent tissue were comparable; the differences between the two types of tissue were significant only for 5-hydroxypyrimidines (slightly higher levels were observed in the adjacent tissue). Comparable levels of DNA lesions in cancerous and the surrounding normal tissues observed in the larynx tumors support a field cancerization theory. The surrounding tissues may still be recognized as normal by histological criteria. However, molecular alterations resulting from the chronic tobacco smoke exposure, which equally affects larynx epithelia, may lead to multiple premalignant lesions. Thus, a demonstration of similar levels of DNA damage in cancerous and the adjacent tissue could explain a frequent formation of secondary tumors in the larynx and the frequent recurrence in this type of cancer. A weak, but distinct effect of tumor grading and metastatic status was observed in both kinds of tissue in the case of 5-hydroxyuracil, 5-hydroxycytosine, 7,8-dihydro-8-oxoguanine, 7,8-dihydro-8-oxoadenine. This effect was displayed as a gradual shift in the data distribution toward high values from G1 through G2-G3 and from non-metastatic to metastatic tumors. Since the levels of oxidative DNA base modifications tended to increase with the tumor aggressiveness, we postulate that the oxidative DNA lesions increase genetic instability and thus contribute to tumor progression in laryngeal cancer. No associations between aromatic adduct levels and oxidative DNA lesions were present, suggesting that the metabolism of PAH does not contribute significantly to the oxidative stress in larynx tissues, remaining the tobacco smoke ROS as a major source of oxidative DNA damage in the exposed tissue.