Whole DNA methylome profiling in mice exposed to secondhand smoke

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease.     

Whole DNA methylome profiling in mice exposed to secondhand smoke

Aberration of DNA methylation is a prime epigenetic mechanism of carcinogenesis. Aberrant DNA methylation occurs frequently in lung cancer, with exposure to secondhand smoke (SHS) being an established risk factor. The causal role of SHS in the genesis of lung cancer, however, remains elusive. To investigate whether SHS can cause aberrant DNA methylation in vivo, we have constructed the whole DNA methylome in mice exposed to SHS for a duration of 4 mo, both after the termination of exposure and at ensuing intervals post-exposure (up to 10 mo). Our genome-wide and gene-specific profiling of DNA methylation in the lung of SHS-exposed mice revealed that all groups of SHS-exposed mice and controls share a similar pattern of DNA methylation. Furthermore, the methylation status of major repetitive DNA elements, including long-interspersed nuclear elements (LINE L1), intracisternal A particle long-terminal repeat retrotransposons (IAP-LTR), and short-interspersed nuclear elements (SINE B1), in the lung of all groups of SHS-exposed mice and controls remains comparable. The absence of locus-specific gain of DNA methylation and global loss of DNA methylation in the lung of SHS-exposed mice within a timeframe that precedes neoplastic-lesion formation underscore the challenges of lung cancer biomarker development. Identifying the initiating events that cause aberrant DNA methylation in lung carcinogenesis may help improve future strategies for prevention, early detection and treatment of this highly lethal disease.     

Antenatal exposure of maternal secondhand smoke (SHS) increases fetal lung expression of RAGE and induces RAGE-mediated pulmonary inflammation

Background

Receptors for advanced glycation end-products (RAGE) are immunoglobulin-like pattern recognition receptors abundantly localized to lung epithelium. Our research demonstrated that primary tobacco smoke exposure increases RAGE expression and that RAGE partly mediates pro-inflammatory signaling during exposure. However, the degree to which RAGE influences developing lungs when gestating mice are exposed to secondhand smoke (SHS) has not been determined to date.

Methods

Timed pregnant RAGE null and wild type control mice were exposed to 4 consecutive days of SHS from embryonic day (E) 14.5 through E18.5 using a state of the art nose-only smoke exposure system (Scireq, Montreal, Canada). RAGE expression was assessed using immunofluorescence, immunoblotting, and quantitative RT-PCR. TUNEL immunostaining and blotting for caspase-3 were performed to evaluate effects on cell turnover. Matrix abnormalities were discerned by quantifying collagen IV and MMP-9, a matrix metalloprotease capable of degrading basement membranes. Lastly, TNF-α and IL-1β levels were assessed in order to determine inflammatory status in the developing lung.

Results

Pulmonary RAGE expression was elevated in both dams exposed to SHS and in fetuses gestating within mothers exposed to SHS. Fetal weight, a measure of organismal health, was decreased in SHS-exposed pups, but unchanged in SHS-exposed RAGE null mice. TUNEL assessments suggested a shift toward pulmonary cell apoptosis and matrix in SHS-exposed pups was diminished as revealed by decreased collagen IV and increased MMP-9 expression. Furthermore, SHS-exposed RAGE null mice expressed less TNF-α and IL-1β when compared to SHS-exposed controls.

Conclusions

RAGE augmentation in developing pups exposed to maternal SHS weakens matrix deposition and influences lung inflammation.     

Dose responses for DNA adduct formation in tissues of rats and mice exposed by inhalation to low concentrations of 1,3-[2,3-[(14)C]-butadiene

Male Sprague-Dawley rats and B6C3F1 mice were exposed to either a single 6h or a multiple (5) daily (6h) nose-only dose of 1,3-[2,3-(14)C]-butadiene at exposure concentrations of nominally 1, 5 or 20 ppm. The aim was to compare the results with those from a similar previous study at 200 ppm. DNA isolated from liver, lung and testis of exposed rats and mice was analysed for the presence of butadiene related adducts, especially the N7-guanine adducts. Total radioactivity present in the DNA from liver, lung and testis was quantified and indicated more covalent binding of radioactivity for mouse tissue DNA than rat tissue DNA. Following release of the depurinating DNA adducts by neutral thermal hydrolysis, the liberated depurinated DNA adducts were measured by reverse phase HPLC coupled with liquid scintillation counting. The guanine adduct G4, assigned as N7-(2,3,4-trihydroxybutyl)- guanine, was the major adduct measured in liver, lung and testis DNA in both rats and mice. Higher levels of G4 were detected in all mouse tissues compared with rat tissue. The dose-response relationship for the formation of adduct G4 was approximately linear for all tissues studied for both rats and mice exposed in the 1-20 ppm range. The formation of G4 in liver tissue was about three times more effective for mouse than rat in this exposure range. Average levels of adduct G4 measured in liver DNA of rats and mice exposed to 5 x 6 h 1, 5 and 20 ppm 1,3-[2,3-(14)C]-butadiene were, respectively, for rats: 0.79 +/- 0.30, 2.90 +/- 1.19, 16.35 +/- 4.8 adducts/10(8) nucleotides and for mice: 2.23 +/- 0.71, 12.24 +/- 2.15, 48.63 +/- 12.61 adducts/10(8) nucleotides. For lung DNA the corresponding values were for rats: 1.02 +/- 0.44, 3.12 +/- 1.06, 17.02 +/- 4.07 adducts/10(8) nucleotides, and for mice: 3.28 +/- 0.32, 14.04 +/- 1.55, 42.47 +/- 13.12 adducts/10(8) nucleotides. Limited comparative data showed that the levels of adduct G4 formed in liver and lung DNA of mice exposed to a single exposure to butadiene in the present 20 ppm study and earlier 200 ppm study were approximately directly proportional across dose, but this was not observed in the case of rats. From the available evidence it is most likely that adduct G4 was formed from a specific isomer of the diol-epoxide metabolite, 3,4-epoxy-1,2-butanediol rather than the diepoxide, 1,2,3,4-diepoxybutane. Another adduct G3, possibly a diastereomer of N7-(2,3,4-trihydroxybutyl)-guanine or most likely the regioisomer N7-(1-hydroxymethyl-2,3-dihydroxypropyl)-guanine, was also detected in DNA of mouse tissues but was essentially absent in DNA from rat tissue. Qualitatively similar profiles of adducts were observed following exposures to butadiene in the present 20 ppm study and the previous 200 ppm study. Overall the DNA adduct levels measured in tissues of both rats and mice were very low. The differences in the profiles and quantity of adducts seen between mice and rats were considered insufficient to explain the large difference in carcinogenic potency of butadiene to mice compared with rats.     

Modulatory effects of different doses of alpha-tocopherol on benzo(a)pyrene-DNA adduct formation in the pulmonary tissue of cigarette smoke inhaling mice

Cigarette smoke (CS) has been established as one of the major risk factors for many pathologies including lung cancer in humans and experimental animals. In view of the discrepancy about the role of alpha-tocopherol (AT) in carcinogenesis, the present study was designed to investigate the effects of different doses of AT on benzo(a)pyrene-DNA [B(a)P-DNA] adduct formation in lungs of CS inhaling mice. Extent of carcinogen-DNA adduct formation has been considered as an index for carcinogenesis. Feeding of 35 IU AT/kg body weight increased B(a)P-DNA adducts formation significantly whereas feeding of 5 IU AT/kg body weight did not altered much the B(a)P-DNA adduct levels when both were compared to the control counterparts. With CS inhalation, the B(a)P-DNA adducts formation increased in all the groups when compared to their respective sham counterparts. Interestingly, in CS exposed groups, there was least increase in B(a)P-DNA adducts formation in 5 IU AT/kg fed animals followed by the control and 35 IU AT/kg body weight fed groups respectively. The results suggest that higher doses of AT accentuate DNA adduct formation in CS inhaling mice.     

32P-postlabelling detection of aromatic DNA adducts in peripheral blood lymphocytes from aluminium production plant workers

Aluminium production plant workers are exposed to a great number of airborne polycyclic aromatic hydrocarbons and epidemiological studies suggest that these workers are at increased risk of lung and bladder cancer. Blood samples from 46 workers at 2 primary aluminium plants and from 29 occupationally unexposed control individuals were analysed. DNA was isolated from the peripheral blood lymphocytes and aromatic DNA adducts were detected by 32P-postlabelling assay using the nuclease P1 digestion procedure for the enrichment of the adducts. The total levels of DNA adducts of exposed individuals varied from the detection limit of about 0.5 adducts/10(8) nucleotides up to 7.1 adducts/10(8) nucleotides and control adduct levels were up to 2.42 adducts/10(8) nucleotides. There was no significant difference between the mean adduct levels of the control group and of the individuals of one plant. However, the mean DNA adduct level obtained from workers of the second plant was significantly higher than that of the controls (p less than 0.001) and of the first plant (p less than 0.01), respectively. This difference can be attributed to differences in the design of technology and different levels of exposure at the 2 plants. The results of this study encourage further investigations of the use of peripheral white blood cells as marker cells and of 32P-postlabelling analysis for monitoring occupational exposure to mixtures of environmental carcinogenic pollutants.     

DNA adducts of styrene-7,8-oxide in target and non-target organs for tumor induction in rat and mouse after repeated inhalation exposure to styrene

Styrene by inhalation had been shown to increase the lung tumor incidence in mice at 20 ppm and higher, but was not carcinogenic in rats at up to 1000 ppm. Styrene-7,8-oxide, the major metabolic intermediate, has weak electrophilic reactivity. Therefore, DNA adduct formation was expected at a low level and a 32P-postlabeling method for a determination of the two regioisomeric 2'-deoxyguanosyl-O6-adducts at the alpha(7)- and beta(8)-positions had been established. The first question was whether DNA adducts could be measured in the rat at the end of the 2 years exposure of a bioassay for carcinogenicity, even though tumor incidence was not increased. Liver samples of male and female CD rats were available for DNA adduct analysis. Adducts were above the limit of detection only in the highest dose group (1000 ppm), with median levels of 9 and 8 adducts per 10(7) nucleotides in males and females, respectively (sum of alpha- and beta-adducts). The result indicates that the rat liver tolerated a relatively high steady-state level of styrene-induced DNA adducts without detectable increase in tumor formation. The second question was whether different DNA adduct levels in the lung of rats and mice could account for the species difference in tumor incidence. Groups of female CD-1 mice were exposed for 2 weeks to 0, 40, and 160 ppm styrene (6h per day; 5 days per week), female CD rats were exposed to 0 and 500 ppm. In none of the lung DNA samples were adducts above a limit of detection of 1 adduct per 10(7) DNA nucleotides. The data indicate that species- and organ-specific tumor induction by styrene is not reflected by DNA adduct levels determined in tissue homogenate. The particular susceptibility of the mouse lung might have to be based on other reactive metabolites and DNA adducts, indirect DNA damage and/or cell-type specific toxicity and tumor promotion.     

DNA adducts in rats and mice following exposure to [4-14C]-1,2-epoxy-3-butene and to [2,3-14C]-1,3-butadiene

1,3-Butadiene (BD) is a major industrial chemical and a rodent carcinogen, with mice being much more susceptible than rats. Oxidative metabolism of BD, leading to the DNA-reactive epoxides 1,2-epoxy-3-butene (BMO), 1,2-epoxy-3,4-butanediol (EBD) and 1,2:3,4-diepoxybutane (DEB), is greater in mice than rats. In the present study the DNA adduct profiles in liver and lungs of rats and mice were determined following exposure to BMO and to BD since these profiles may provide qualitative and quantitative information on the DNA-reactive metabolites in target tissues. Adducts detected in vivo were identified by comparison with the products formed from the reaction of the individual epoxides with 2'-deoxyguanosine (dG). In rats and mice exposed to [4-14C]-BMO (1-50 mg/kg, i.p.), DNA adduct profiles were similar in liver and lung with N7-(2-hydroxy-3-butenyl)guanine (G1) and N7-(1-(hydroxymethyl)-2-propenyl)guanine (G2) as major adducts and N7-2,3,4-trihydroxybutylguanine (G4) as minor adduct. In rats and mice exposed to 200 ppm [2,3-14C]-BD by nose-only inhalation for 6 h, G4 was the major adduct in liver, lung and testes while G1 and G2 were only minor adducts. Another N7-trihydroxybutylguanine adduct (G3), which could not unambiguously be identified but is either another isomer of N7-2,3,4-trihydroxybutylguanine or, more likely, N7-(1-hydroxymethyl-2,3-dihydroxypropyl)guanine, was present at low concentrations in liver and lung DNA of mice, but absent in rats. The evidence indicates that the major DNA adduct formed in liver, lung and testes following in vivo exposure to BD is G4, which is formed from EBD, and not from DEB.     

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.     

Levelling-off of the risk of lung and bladder cancer in heavy smokers: an analysis based on multicentric case-control studies and a metabolic interpretation

The shape of the dose-response relationship between carcinogenic exposure and cancer risk is a key issue, both from a theoretical (models of carcinogenesis) and practical (risk assessment) point of view. Human populations exposed to Polycyclic Aromatic Hydrocarbons (PAH) via air pollution showed a non-linear relationship between levels of exposure and WBC-DNA adducts. Among highly exposed subjects, the DNA adduct level per unit of exposure was significantly lower than measured at environmental exposures. The same exposure-dose non-linearity was observed in lung DNA from rats exposed to PAH. We have analyzed 11 case-control studies on bladder cancer (4584 incident cases and 9360 hospital controls) and eight case-control studies on lung cancer (5092 incident cases and 6083 population controls), conducted in Europe in recent years. All the studies collected detailed information on smoking histories with a similar methodology. We have estimated the relationship between the number of cigarettes smoked and the risk of cancer, with and without adjustment by duration of smoking. We have observed a levelling-off of the relationship between the number of cigarettes smoked and the relative risks for lung and bladder cancer, both in men and women. The levelling-off occurred at an odds ratio of about 5 for bladder cancer, while it occurs at about 20 for lung cancer (in men). A potential explanation for such levelling-off involves metabolic pathways and individual susceptibility. It has been suggested that some metabolic polymorphisms exert an effect that is more important at low levels of exposure.     

DNA adducts in relation to lung tumour outcome are not markers of susceptibility following a single dose treatment of SWR, BALB/c and C57BL/6J mice with N-nitrosodiethylamine

The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a valuable marker for assessing exposure to carcinogens but as yet the role of DNA adducts as a biomarker of carcinogenic susceptibility still needs to be clearly ascertained. To address this question an animal study was instigated using mice (SWR (high), BALB/c (intermediate) and C57BL/6J (low)) varying in their susceptibility to lung carcinogenesis. Groups of animals from each strain were dosed with a single intraperitoneal injection of saline or N -nitrosodiethylamine (NDEA) at 15 or 90 mg kg^-1 body weight. Lung and liver tissues were removed at different time points following dosing. Further groups of mice dosed with the same regime had urine samples collected 24 h post dosing and were then left up to 18 months to allow for the development of tumours. Immunoslot-blot analysis was used for the determination of N-7 ethylguanine (N-7EtG) and O⁶ ethylguanine (O⁶EtG) adduct levels in the DNA from the tissues and gas chromatography-mass spectrometry (GC-MS) was used to determine N-3 ethyladenine (N-3EtA) adduct levels in the urine samples. Levels of alkyltransferase (ATase) were also determined in the tissues. The results showed that the DNA adduct levels and persistence were similar across the three strains of mice following dosing with 15 and 90 mg kg^-1 NDEA. High levels of adducts were observed in the urine of the BALB/c strain, implying an increased metabolic or repair capacity in this strain. However there were no differences in the levels of ATase in the lung and liver of the three strains of mice following dosing with 15 mg kg^-1 NDEA. The incidence of tumours in C57BL/6J mice was lower compared with the other two strains and showed a dose dependent increase. The results from this study show that the differences in susceptibility to lung carcinogenesis between the three strains of mice do not appear to be linked to the formation of the two adducts detected. These results imply that dosing with NDEA resulted in toxicity which may have led to cell death and induction of tumours by compensatory cell proliferation. Although these results do not allow decisive conclusions to be drawn concerning the relationship between total levels of DNA adducts and differences in carcinogenic susceptibility for the three strains of mice it is clear that the increased presence of a DNA adduct in the target tissue increases the likelihood of tumour development.     

DNA adducts in relation to lung tumour outcome are not markers of susceptibility following a single dose treatment of SWR,BALB/c and C57BL/6J mice with N-nitrosodiethylamine

The formation of DNA adducts by the covalent binding of genotoxic chemicals to DNA represents a valuable marker for assessing exposure to carcinogens but as yet the role of DNA adducts as a biomarker of carcinogenic susceptibility still needs to be clearly ascertained. To address this question an animal study was instigated using mice (SWR (high), BALB/c (intermediate) and C57BL/6J (low)) varying in their susceptibility to lung carcinogenesis. Groups of animals from each strain were dosed with a single intraperitoneal injection of saline or N -nitrosodiethylamine (NDEA) at 15 or 90 mg kg^-1 body weight. Lung and liver tissues were removed at different time points following dosing. Further groups of mice dosed with the same regime had urine samples collected 24 h post dosing and were then left up to 18 months to allow for the development of tumours. Immunoslot-blot analysis was used for the determination of N-7 ethylguanine (N-7EtG) and O⁶ ethylguanine (O⁶EtG) adduct levels in the DNA from the tissues and gas chromatography-mass spectrometry (GC-MS) was used to determine N-3 ethyladenine (N-3EtA) adduct levels in the urine samples. Levels of alkyltransferase (ATase) were also determined in the tissues. The results showed that the DNA adduct levels and persistence were similar across the three strains of mice following dosing with 15 and 90 mg kg^-1 NDEA. High levels of adducts were observed in the urine of the BALB/c strain, implying an increased metabolic or repair capacity in this strain. However there were no differences in the levels of ATase in the lung and liver of the three strains of mice following dosing with 15 mg kg^-1 NDEA. The incidence of tumours in C57BL/6J mice was lower compared with the other two strains and showed a dose dependent increase. The results from this study show that the differences in susceptibility to lung carcinogenesis between the three strains of mice do not appear to be linked to the formation of the two adducts detected. These results imply that dosing with NDEA resulted in toxicity which may have led to cell death and induction of tumours by compensatory cell proliferation. Although these results do not allow decisive conclusions to be drawn concerning the relationship between total levels of DNA adducts and differences in carcinogenic susceptibility for the three strains of mice it is clear that the increased presence of a DNA adduct in the target tissue increases the likelihood of tumour development.     

Correlation of DNA adduct levels with tumor incidence: carcinogenic potency of DNA adducts

The quantitative relationship between DNA adducts and tumor incidence is evaluated in this review. All available data on DNA adduct levels determined after repeated administration of a carcinogen to rats or mice have been compiled. The list comprised 27 chemicals, of all major structural classes of carcinogens. For the correlation with tumor incidence, the DNA adduct levels measured at the given dose were normalized to the dose which resulted in a 50% tumor incidence under the conditions of a 2-year bioassay (TD50 dose). In rat liver, the calculated adduct concentration 'responsible' for a 50% hepatocellular tumor incidence spanned from 53 to 2083 adducts per 108 nucleotides, for aflatoxin B1, tamoxifen, IQ, MeIQx, 2,4-diaminotoluene, and dimethylnitrosamine (in this order). In mouse liver, the respective figures were 812 to 5543 adducts per 108 nucleotides, for ethylene oxide, dimethylnitrosamine, 4-aminobiphenyl, and 2-acetylaminofluorene. The observed span (40-fold in rats, 7-fold in mice) reflects differences between the various DNA adducts to lead to critical mutations. If additional carcinogens fit in with this astonishingly narrow range, the measurement of DNA adduct levels in target tissue has the potential to be not only an exposure marker but an individual cancer risk marker. For toremifen and styrene, low levels of DNA adducts were detected in rat liver at the end of a negative long-term bioassay. This shows that the limit of detection of DNA adducts can be well below the limit of detection of an increased tumor incidence. For a cancer risk assessment at low levels of DNA damage, treatment-related adducts must be discussed in relation to the background DNA damage and its inter- and intraindividual variability.     

A critical evaluation of DNA adducts as biological markers for human exposure to polycyclic aromatic compounds

The causative role of polycyclic aromatic hydrocarbons (PAH) in human carcinogenesis is undisputed. Measurements of PAH-DNA adduct levels in easily accessible white blood cells therefore represent useful early endpoints in exposure intervention or chemoprevention studies. The successful applicability of DNA adducts as early endpoints depends on several criteria: i. adduct levels in easily accessible surrogate tissues should reflect adduct levels in target-tissues, ii. toxicokinetics and the temporal relevance should be properly defined. iii. sources of interand intra-individual variability must be known and controllable, and finally iv. adduct analyses must have advantages as compared to other markers of PAHexposure. In general, higher DNA adduct levels or a higher proportion of subjects with detectable DNA adduct levels were found in exposed individuals as compared with nonexposed subjects, but saturation may occur at high exposures. Furthermore, DNA adduct levels varied according to changes in exposure, for example smoking cessation resulted in lower DNA adduct levels and adduct levels paralleled seasonal variations of air-pollution. Intraindividual variation during continuous exposure was low over a short period of time (weeks), but varied significantly when longer time periods (months) were investigated. Inter-individual variation is currently only partly explained by genetic polymorphisms in genes involved in PAH-metabolism and deserves further investigation. DNA adduct measurements may have three advantages over traditional exposure assessment: i. they can smooth the extreme variability in exposure which is typical for environmental toxicants and may integrate exposure over a longer period of time. Therefore, DNA adduct assessment may reduce the monitoring effort. ii. biological monitoring of DNA adducts accounts for all exposure routes. iii. DNA adducts may account for inter-individual differences in uptake, elimination, distribution, metabolism and repair amongst exposed individuals. In conclusion, there is now a sufficiently large scientific basis to justify the application of DNA adduct measurements as biomarkers in exposure assessment and intervention studies. Their use in risk-assessment, however, requires further investigation.     

High susceptibility of neonatal mice to molecular, biochemical and cytogenetic alterations induced by environmental cigarette smoke and light

Our recent studies have shown that both cigarette smoke and UV-containing light, which are the most widespread and ubiquitous mutagens and carcinogens in the world, cause systemic genotoxic damage in hairless mice. Further studies were designed with the aim of evaluating the induction of genotoxic and carcinogenic effects in Swiss albino mice exposed to smoke and/or light since birth. We observed that a 4-month whole-body exposure of mice to mainstream cigarette smoke, starting at birth, caused an early and potent carcinogenic response in the lung and other organs. Our further experiments showed that exposure of mice to environmental cigarette smoke, during the first 5 weeks of life, resulted in a variety of significant alterations of intermediate biomarkers, including cytogenetic damage in bone marrow and peripheral blood, formation of lipid peroxidation products, increase of bulky DNA adduct levels, induction of oxidative DNA damage, and overexpression of OGG1 gene in lung, stimulation of apoptosis, hyperproliferation and loss of Fhit protein in pulmonary alveolar macrophages and/or bronchial epithelial cells, and early histopathological alterations in the respiratory tract. Moreover, exposure of mice to UV-containing light, mimicking solar irradiation, significantly enhanced oxidative DNA damage and bulky DNA adduct levels in lung, and synergized with smoke in inducing molecular alterations in the respiratory tract. The baseline OGG1 expression in lung was particularly high at birth and decreased in post-weanling mice. Oxidative DNA damage and other investigated end-points exhibited differential patterns in post-weanling mice and adult mice. The findings of these studies provide a mechanistic clue to the general concept that the neonatal period and early stages of life are critical in affecting susceptibility to carcinogens.     

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.     

Biomonitoring of human genotoxicity induced by complex occupational exposures

Sensitivity, specificity and correlations among several biomarkers for monitoring occupational exposure to complex mixtures of genotoxic agents were assessed in occupational environments in Hungarian study populations. The studies have been focused on DNA adduct formation, urinary metabolites, mutations and micronuclei induced by exposures to complex organic mixtures. In two Hungarian aluminium plants, increased DNA adduct and 1-hydroxypyrene (1-OH-PY) levels were observed in workers as compared to controls. However, no association between the biomarker levels was evident on an individual basis. In Hungarian garage mechanics, DNA adduct determinations did not show increased genotoxic exposure as compared to the controls. However, ambient air measurements, significantly enhanced 1-OH-PY levels, and slightly enhanced frequency of micronuclei indicated increased polycyclic aromatic hydrocarbon (PAH) exposure in the garages, as compared to the general environment. In a Hungarian vulcanizing plant, DNA adduct determinations and 1-OH-PY did not show significantly elevated exposure levels as compared to controls. The glycophorin A (GPA) somatic mutation assay was also negative for this occupational exposure. The results support previous observations of a lack of correlation between DNA adducts detectable by 32P-postlabelling and those measured by the PAH-DNA immunoassay in the same DNA sample. These studies also demonstrate a lack of close correlation between levels of DNA adducts and urinary 1-OH-PY in the same individual.     

Exposure-dependent accumulation of N-(2-hydroxypropyl)valine in hemoglobin of F344 rats exposed to propylene oxide by the inhalation route

The detection of hemoglobin adducts by mass spectrometry is a very sensitive and specific measurement of the extent of covalent binding of electrophilic chemicals. The exposure-dependent accumulation of N-(2-hydroxypropyl)valine (N-HPVal) in globin of rats exposed to propylene oxide (PO) (0, 5, 25, 50, 300 or 500 ppm) by the inhalation route was measured to assess the utility of Hb adducts as biomarkers of exposure. Analysis of N-HPVal by gas-chromatography tandem mass spectrometry showed a linear exposure-dependent response for adduct accumulation in globin of rats exposed to PO for 3 days (6 h/day). After 20 days of exposure (6 h/day; 5 days/week), the exposure-response curve was slightly sub-linear. DNA adducts had been measured in several organs of the same animals in a companion study. The dose-response for accumulation of DNA adducts was similar to that obtained for Hb adducts. However, the number of DNA adducts varied by 17-fold between different tissues. The highest number of DNA adducts was found in respiratory nasal tissue, followed by lung and then liver. These data demonstrate that hemoglobin adducts provide a sensitive dosimeter for systemic exposure, but cannot be used to predict the extent of DNA binding in individual tissues. Furthermore, the exposure-response curve for both hemoglobin and DNA adduct accumulation does not reflect the tumor incidence curve for PO, providing evidence that the assessment of risk to cancer is more complex than simple biomarker measurements. When the present rat data were compared with recent N-HPVal measurements in humans, similar binding was found.     

Specific plant DNA adducts as molecular biomarkers of genotoxic atmospheric environments

The general purpose of this study was to determine whether the formation of DNA addition products ('adducts') in plants could be a valuable biomarker of genotoxic air pollution. Plants from several species were exposed to ambient atmosphere at urban and suburban sites representative of different environmental conditions. The levels of NO2 and of the quantitatively major genotoxic air pollutants benzene, toluene, and xylene were monitored in parallel with plant exposure. DNA adducts were measured in bean (Phaseolus vulgaris), rye-grass (Lolium perenne), and tobacco (Nicotiana tabacum) seedlings by means of the [32P]-postlabeling method. Whereas, no correlation was found between the levels of the major genotoxic air pollutants and the total amounts of DNA adducts, individual analyses revealed site-specific and plant species-specific adduct responses, both at the qualitative and quantitative level. Among these, the amount of a specific rye-grass DNA adduct (rgs1) correlated with benzene/toluene/xylene levels above a threshold. For further characterization, rye-grass seedlings were treated in controlled conditions with benzene, toluene, xylene or their derivatives. On the other hand, in vitro DNA adduct formation assays were developed involving benzene, toluene, xylene, or their derivatives, and plant microsomes or purified peroxidase. Although in some cases, these approaches produced specific adduct responses, they failed to generate the rgs1 DNA adduct, which appeared to be characteristic for on-site test-plant exposure. Our studies have thus identified an interesting candidate for further analysis of environmental biomarkers of genotoxicity.     

Studies on biomarkers in cancer etiology and prevention: a summary and challenge of 20 years of interdisciplinary research

Sensitive, specific methods have been developed that allow quantitative measurements of the metabolites of carcinogen metabolites and of DNA and protein adducts in humans exposed occupationally, environmentally and endogenously to genotoxic agents. The interrelationship between exposure to carcinogens, host risk factors and the responses of biomarkers has been examined in cross-sectional, ecological and case-control studies which provided new insights into the causes of cancer and the mechanisms of carcinogenesis. The identification of hitherto unknown DNA-reactive chemicals formed in the human body from dietary precursors and of carcinogenic components of complex mixtures has increased the possibility of establishing causal relationships in etiology. The identification of individuals and subgroups heavily exposed to carcinogens has led to the development of measures for avoiding or decreasing exposure to carcinogenic risk factors. New, ultrasensitive methods for measuring DNA adducts allow the quantification and structural elucidation of specific DNA damage in humans arising from oxidative stress and lipid peroxidation (LPO), which have been found to be the driving forces in several human malignancies. Background DNA damage in "unexposed" individuals has been shown unequivocally to be due to LPO products, and a significant interindividual variation in adduct levels has been shown in individuals with comparable exposure to carcinogens. Thus, pharmacogenetic variants with higher susceptibility to carcinogenic insults, due to genetic polymorphism in xenobiotic-metabolizing enzymes, have been characterized by a combination of genotyping and measurements of macromolecular adducts. Dosimetry has been used in human studies to evaluate the efficacy of interventions with chemopreventive agents like ascorbic acid, dietary phenols and green tea. Advances in the application of selected biomarkers in human studies are reviewed and illustrated by examples from the author's research conducted during the past two decades.