Aromatic DNA Adducts Research Articles

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.

Early age at smoking initiation and tobacco carcinogen DNA damage in the lung.

DNA adducts formed as a consequence of exposure to tobacco smoke may be involved in carcinogenesis, and their presence may indicate a high risk of lung cancer. To determine whether DNA adducts can be used as a "dosimeter" for cancer risk, we measured the adduct levels in nontumorous lung tissue and blood mononuclear cells from patients with lung cancer, and we collected data from the patients on their history of smoking. We used the 32P-postlabeling assay to measure aromatic hydrophobic DNA adducts in nontumorous lung tissue from 143 patients and in blood mononuclear cells from 54 of these patients. From the smoking histories, we identified exposure variables associated with increased DNA adduct levels by use of multivariate analyses with negative binomial regression models. RESULTS/ CONCLUSIONS: We found statistically significant interactions for variables of current and former smoking and for other smoking variables (e.g., pack-years [number of packs smoked per day x years of smoking] or years smoked), indicating that the impact of smoking variables on DNA adduct levels may be different in current and former smokers. Consequently, our analyses indicate that models for current and former smokers should be considered separately. In current smokers, recent smoking intensity (cigarettes smoked per day) was the most important variable. In former smokers, age at smoking initiation was inversely associated with DNA adduct levels. A highly statistically significant correlation (r=.77 [Spearman's correlation]; two sided P<.001) was observed between DNA adduct levels in blood mononuclear cells and lung tissue. Our results in former smokers suggest that smoking during adolescence may produce physiologic changes that lead to increased DNA adduct persistence or that young smokers may be markedly susceptible to DNA adduct formation and have higher adduct burdens after they quit smoking than those who started smoking later in life.

Hprt mutant frequency and aromatic DNA adduct level in non-smoking and smoking lung cancer patients and population controls.

T cell cloning and 32P-post-labelling methods were used to study the mutant frequency (MF) at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus and the aromatic DNA adduct level (AL) in peripheral lymphocytes of newly diagnosed lung cancer patients (92 ever-smokers and 87 never-smokers) and matched population controls (82 ever-smokers and 79 never-smokers). Overall, the MF (total mean 20.6 x 10(-6)) and AL (4.1 x 10(-8)) were similar in cases and controls with the same smoking status, indicating that the disease has limited effect on the two endpoints. When cases and controls were combined, the AL was significantly higher in current smokers than in former or never-smokers (P = 0.0003) and the MF was significantly higher in ever-smokers than in never-smokers (P = 0.004). Age affected the MF significantly in ever-smokers (1.6%/year, 95% CI 0.6-2.5, adjusted for pack-years and years since last smoking), especially among cases (2.1%/year, 95% CI 0.5-3.7). An increase of AL with age was observed in currently smoking cases only (2.3%/year, 95% CI 0.3-4.2, adjusted for smoking dose). For currently smoking cases, there was also a more pronounced effect of smoking dose on both endpoints and a significant correlation between AL and MF (r = 0.52, P = 0.04) was observed among those with the highest dose. Our data also provide additional evidence for the different turnover times of smoking-induced DNA adducts and hprt mutations. The stronger increase of MF and AL with age and dose in currently smoking patients compared with controls is consistent with an interaction between smoking and genetic host factors.

Relationship between tissue concentrations of polycyclic aromatic hydrocarbons and DNA adducts in green-lipped mussels (Perna viridis)

Green-lipped mussels (Perna viridis) were collected from a site in Hong Kong which is relatively free from polycyclic aromatic hydrocarbon (PAH) contamination, and maintained in situ at this and three other sites with different degrees of PAH contamination. The transplanted mussels were retrieved after a 30-day field exposure. DNA adducts in the gill tissues were quantified, and tissue concentrations of benzo[a]pyrene as well as total PAHs (with potential carcinogenicity) determined for individual mussels. Results indicate that (1) tissue concentration of PAHs and adduct levels in mussels collected from a single site can be highly variable; and (2) adduct levels were related to tissue concentrations of benzo[a]pyrene as well as total PAHs of individual animals.

Exposure of marine mammals to genotoxic environmental contaminants : Application of the 32P-postlabeling assay for measuring DNA-xenobiotic adducts

There is little information on exposure of marine mammals to genotoxic environmental contaminants. The 32P-postlabeling assay has been successfully used to assess exposure to genotoxic polycyclic aromatic compounds in fish and humans. In the present study, a preliminary investigation showed that polycyclic aromatic compound-like DNA adducts were present in hepatic tissues of harbor seals (Phoca vitulina richardsi) exposed to petroleum following the Exxon Valdez oil spill. However, for marine mammals, effects from changes in tissue condition on DNA recovery and quality is of concern, because tissue samples are often collected from animals that have been dead for unknown periods of time. To assess the effects of postmortem thermal history on DNA recovery from tissue and on DNA adduct quantitation, samples of harbor porpoise (Phocoena phocoena) hepatic tissue were incubated for up to 10 d at 4 and 30 °C. Only traces (<4 μg) of hepatic DNA were recovered from 200 mg of tissue after incubation at 30 °C for 36 h. At 4 °C, DNA (50–130 μg) was recovered from tissue incubated for up to 6 d; whereas DNA recovery at 10 d was minimal. Chromatograms of 32P-labeled DNA digests of liver tissue held at 4 and 30 °C and salmon sperm DNA held at 30 °C for 2 d had comparable profiles, suggesting that alteration of DNA bases had occurred during incubation of porpoise liver tissue. Moreover, the chromatograms of DNA extracted from liver tissues of harbor porpoises caught incidentally in a northwest Atlantic fishery, packed in ice and sampled several days later also exhibited similar altered DNA structures. Although, altered DNA structures that can interfere with the DNA adduct quantitation were present in autolyzed tissue, changes in the 32P-postlabeling chromatography conditions can decrease the interference. Moreover, in a study with tissues taken from California sea lions (Zalophus californianus) immediately postmortem and stored at –80 °C until processing, DNA structures associated with tissue breakdown were not observed. The DNA from sea lions, however, had putative age-dependent hepatic DNA modifications, which have a distinctive profile, and must be considered when evaluating exposure of marine mammals to polycyclic aromatic compounds. Overall, the findings showed that with attention to the postmortem thermal history of the tissue samples hepatic DNA adducts, as measured by 32P-postlabeling, have the potential to serve as a biological indicator of exposure of marine mammals to environmental genotoxic compounds.

The p53 codon 249 mutational hotspot in hepatocellular carcinoma is not related to selective formation or persistence of aflatoxin B1 adducts.

Sequence-dependent formation and lack of repair of polycyclic aromatic hydrocarbon-induced DNA adducts correlates well with the positions of p53 mutational hotspots in smoking-related lung cancers (Denissenko et al, 1996, 1998). The mycotoxin aflatoxin B1 (AFB1) is considered to be a major causative agent in hepatocellular carcinoma (HCC) in regions with presumed high food contamination by AFB1. A unique mutational hotspot, a G to T transversion at the third base of codon 249 of the p53 gene is observed in these tumors. To test whether a selectivity of AFB1 adduct formation is related to this peculiar mutational spectrum, we have mapped AFB1-DNA adducts at nucleotide resolution using ligation-mediated PCR and terminal transferase-dependent PCR. Human HepG2 cells were exposed to AFB1 metabolically activated in the presence of rat liver microsomes. Significant adduct formation was seen at the third base of codon 249. However, this was not the major site of AFB1 adducts and strong adduction was also observed at codons 226, 243, 244, 245 and 248 in exon 7 of the p53 gene and at several codons in exon 8. The damage at codon 249 does not consist of a unique abasic site or ring-opened aflatoxin B1 adduct but rather is consistent with the principal N7-guanine adduct of AFB1. Time course experiments indicate that, under the conditions used, AFB1 adducts are not removed in a strand-selective manner and adduct removal from the third base of codon 249 proceeds at a relatively fast rate (50% in 7 h). The incomplete correspondence between sites of persistent AFB1 damage and the specific codon 249 mutation suggests that AFB1 may not be involved in mutation of this site or that additional mechanisms such as parallel infection with hepatitis B virus may be required for selection of codon 249 mutants in HCC.

A study of multiple biomarkers in coke oven workers--a cross-sectional study in China.

We conducted a cross-sectional molecular epidemiological study of coke oven workers exposed to the established carcinogen polycyclic aromatic hydrocarbons (PAHs) to evaluate the relationships between both traditional 'exposure markers' and a series of biomarkers, including urinary 1-hydroxypyrene as a marker of internal dose, leukocyte aromatic DNA adducts as markers of biologically effective dose, serum p53 protein as a response marker and genetic polymorphisms of cytochrome P4501A1 and glutathione S-transferase MI as susceptibility markers. Twenty-five male subjects each were randomly selected from the top, middle and bottom work areas of the oven, and the control plant. They were matched for age and smoking status. The mean levels of PAH exposure, monitored by stationary and personal samplers, and of worker urinary 1-hydroxypyrene differed significantly between the top, middle and bottom of the oven and control work areas. The highest stationary and personal PAH concentrations and 1-hydroxypyrene levels were demonstrated at the top work area. Good correlations were found between the stationary PAH levels, personal PAH levels and urinary 1-hydroxypyrene levels. No positive correlations were demonstrated between aromatic DNA adduct levels and current or cumulative PAH exposure dose. In the presence of genetic polymorphisms of cytochrome P4501A1, a positive correlation was demonstrated between aromatic DNA adducts and urinary 1-hydroxypyrene levels. There was also a significant correlation between serum p53 protein levels and the cumulated benzo[a]pyrene exposure dose. Although these biomarkers have certain limitations, they are applicable to cancer epidemiology, and may contribute to our understanding of the mechanisms of carcinogenesis.

Differences in aromatic-DNA adduct levels between alveolar macrophages and subpopulations of white blood cells from smokers.

The 32P-post-labelling assay for DNA adduct quantification gives the opportunity to examine endogenous exposure to DNA reactive compounds. Most human biomonitoring studies applied white blood cells (WBC) or cells obtained by broncho-alveolar lavages (BAL) as source of DNA, but still it is not clear what cell type represents the most reliable indicator for exposure to cigarette smoke-associated genotoxins. At first, we examined DNA adduct levels by means of nuclease P1 (NP1) enriched 32P-post-labelling in separated WBC subpopulations after in vitro incubations for 18 h with 10 microM benzo[a]pyrene (B[a]P). DNA adduct levels were highest in monocytes (10.7 +/- 2.9 adducts/10(8) nucleotides, n = 8), followed by lymphocytes (5.9 +/- 1.7, n = 8), and granulocytes (0.5 +/- 0.2, n = 8). Secondly, aromatic-DNA adduct levels were determined in BAL cells and WBC-subsets from (non-)smoking volunteers. In smoking individuals, adduct levels were in the ranking order: BAL cells (3.7 +/- 1.0, n = 5) > monocytes (2.0 +/- 0.5, n = 8) > or = lymphocytes (1.6 +/- 0.4, n = 8) > granulocytes (0.8 +/- 0.2, n = 8) by NP1-enrichment and monocytes (9.0 +/- 3.2, n = 5) > or = lymphocytes (8.0 +/- 2.1, n = 6) > granulocytes (2.1 +/- 0.3, n = 7) by butanol-enriched 32P-post-labelling. Aromatic-DNA adduct levels were significantly higher in WBC-subsets of smokers as compared with non-smokers, except for DNA adducts in granulocytes using butanol enrichment. Thirdly, dose-response relationships were investigated in mononuclear white blood cells (MNC, i.e. monocytes plus lymphocytes) and BAL-cells of a larger group of smoking individuals (n = 78). Adduct levels in MNC were related to daily exposure to cigarette-tar (r = 0.31, P < 0.01). Adduct levels in BAL cells seemed to be correlated with pack-years, but after correction for age this relationship was lost. Butanol extraction resulted in 5-6-fold higher DNA adduct levels in MNC, whereas butanol extraction of BAL-DNA of the same individuals yielded only 2-fold higher adduct levels. The two enrichment procedures of 32P-post-labelling were correlated in BAL cells (r = 0.86, P < 0.001, n = 12). We conclude that particularly MNC are good surrogates for the detection of smoking-related DNA adducts.

DNA damage as an intermediate biomarker in intervention studies.

The development of sensitive assays for measurement of DNA damage in humans has great potential for enhancing intervention studies. Methods for DNA adduct measurement include immunoassays, [32p] postlabeling, high-performance liquid chromatography with fluorescence or electrochemical detection, and gas chromatography/mass spectroscopy. It is now well established that DNA adducts are a marker of exposure to various environmental, lifestyle, or occupational chemical carcinogens. Our own studies concentrate on immunologic detection of adducts by enzyme-linked immunosorbent assay (ELISA) of isolated DNA or quantitative immunohistochemical analysis of intact cells. Polycyclic aromatic hydrocarbon (PAH)-DNA adducts are elevated in blood cells of foundry and coke oven workers, individuals with high levels of exposure to environmental air pollution, and smokers. The study in smokers also found an inverse relationship between serum antioxidants and PAH-DNA, and is the basis for an ongoing antioxidant intervention. DNA adducts of PAH and 4-aminobiphenyl and oxidative DNA damage (8-oxo-deoxyguanosine) are being measured in blood mononuclear cells and exfoliated oral and bladder cells from subjects on antioxidants or placebo. Data on published intervention studies investigating oxidative damage and general aromatic DNA adducts measured by postlabeling are also summarized. These studies have already demonstrated that DNA adducts can be modulated by interventions and suggest that they can provide important mechanistic information in support of larger scale studies.

32P-Postlabelling of aromatic DNA adducts in white blood cells and alveolar macrophages of smokers: saturation at high exposures

DNA adducts may serve as a molecular dosimeter of exposure to cigarette smoke-associated carcinogens such as polycyclic aromatic hydrocarbons (PAH). Target tissues for cigarette smoke-induced carcinogenesis are rarely accessible; therefore, peripheral blood cells or cells obtained by bronchoalveolar lavage (BAL) may be used as surrogate sources of exposed DNA. However, the relationship between cigarette smoke exposure and aromatic–DNA adducts in white blood cells and BAL cells is still unclear. In this study, we examined DNA adduct formation in lymphocytes and BAL cells in several populations of smoking individuals by means of 32P-postlabelling. Significant correlations between the amount of cigarettes smoked per day and the level of aromatic–DNA adducts were found in lymphocytes. In BAL cells, DNA adduct levels were associated with age ( p=0.05) and gender ( p=0.10) after adjustment for smoking behaviour. Adduct formation levelled off at higher exposure levels, suggesting less efficient adduct formation; decreases in the formation of adducts per unit of exposure were found in lymphocytes ( r s=−0.80, p<0.001) and BAL cells ( r s=−0.72, p<0.001). To assess intra-individual variation in adduct levels at constant smoking behaviour, sampling was repeated after a period of 2 and 6 months. In lymphocytes, repeated measurements with an interval of 2 months were highly correlated ( r=0.84, p=0.009, n=8), whereas repeated measurements with an interval of 6 months showed no correlation ( r=0.30, p=0.27, n=16). Repeated measurements in BAL cells showed a significant correlation after 6 months ( r=0.68, p=0.03, n=10). Furthermore, in a group of occupationally exposed aluminium workers, adduct levels in total white blood cells were correlated with the average concentrations of PAH in the ambient air of workers who smoked cigarettes, whereas in non-smokers, no such relationship was found. We conclude that cigarette smoking may directly or indirectly influence DNA adduct levels and saturation of DNA adduct formation may occur, leading to non-linear dose–response relationships.

729 Aromatic DNA adduct levels in alveolar macrophages and lymphocytes of smoking volunteers; Effects of N-acetylcysteine (NAC) supplementation

729 Aromatic DNA adduct levels in alveolar macrophages and lymphocytes of smoking volunteers; Effects of N-acetylcysteine (NAC) supplementation

Correlation of mutagenic potencies of various petroleum oils and oil coal tar mixtures with DNA adduct levels in vitro

An in vitro system was utilized to measure DNA adduct-forming ability of petroleum oils and oil coal tar mixtures to define correlations between DNA adduct levels and their mutagenic potencies. The system consisted of reaction of dimethyl sulfoxide extracts of oils with calf thymus DNA in the presence of Aroclor-induced hamster liver microsomes for 30 min. Following DNA extraction, DNA adducts were measured by the nuclease P1-enhanced postlabeling assay coupled with two-dimensional polyethyleneimine (PEI)-cellulose TLC. Thin layer plates showed putative aromatic DNA adducts, with levels ranging from 60 to 1400 adducts per 10 9 DNA nucleotides. TLC mobilities suggested adducts to be aromatic compounds containing 4 or more rings. A good correlation (coefficient of correlation=0.91) was observed between DNA adduct levels and Salmonella mutagenicity for 19 oils. All 19 samples tested produced DNA adducts. To expedite the TLC procedure, adducts were resolved by one-dimensional TLC and the radioactivity measured using a mechanical scanner. Results were comparable to those obtained by two-dimensional TLC and quantification after scraping. Our data show that the in vitro incubation system coupled with the postlabeling adduct assay is a useful screening method to identify mutagenic and potentially carcinogenic oils.

Methods for routine biological monitoring of carcinogenic PAH-mixtures

The ability of a biomarker to provide an assessment of the integrated individual dose following uptake through multiple routes is especially valuable for mixtures of polycyclic aromatic hydrocarbons (PAH), due to methodological and practical difficulties of collecting and analysing samples from the various environmental compartments like air, water and soil and various media such as diet, cigarette smoke and workroom air. Since 1980, a large variety of novel approaches and techniques have been suggested and tested, e.g. urinary thioethers, mutagenicity in urine, levels of PAH or PAH-metabolites in blood and urine and methods for determination of adducts in DNA and proteins. Two approaches are more frequently reported: PAH-DNA-adduct monitoring in blood cells and urinary 1-hydroxypyrene monitoring. A large research effort has been made to use the extent of binding of PAH to DNA as a biomarker of exposure. The 32P-post-labeling assay detects the total of aromatic DNA-adducts and the adduct level in white blood cells is claimed to be an indicator of the biological effect of the PAH-mixture. However, the levels of aromatic DNA-adducts may be subject to appreciable analytical and biological variation. The present technical complexity of the method makes it more convenient for research applications than for routine application in occupational health practice. Pyrene is a dominant compound in the PAH mixture and is mainly metabolised to the intermediary 1-hydroxypyrene to form 1-hydroxypyrene-glucuronide, which is excreted in urine. Since the introduction of the determination of 1-hydroxypyrene in urine as a biomarker for human exposure assessment in 1985, many reports from different countries from Europe, Asia and America confirmed the potential of this novel approach. The conclusion of the first international workshop on 1-hydroxypyrene in 1993 was that urinary 1-hydroxypyrene is a solid biological exposure indicator of PAH. Studies with a comparison of several biomarkers confirmed that 1-hydroxypyrene in urine is a valid and sensitive indicator of exposure. Periodical monitoring of 1-hydroxypyrene appears to be a powerful method in controlling occupational PAH-exposure in industries. The reference level and the biological exposure limit of 1-hydroxypyrene in urine are discussed.

Aromatic amine DNA adduct formation in chronically-exposed mice: considerations for human comparison

Lifetime chronic exposure of mice to the aromatic amines 4-aminobiphenyl (ABP) and 2-acetylaminofluorene (AAF) produces liver and urinary bladder tumors. In parallel experiments, DNA adduct levels in target tissues reach a steady-state (a balance between adduct formation and removal) after about four weeks of either AAF or ABP ingestion. For these and other carcinogens, steady-state DNA adduct levels most frequently increase linearly with dose, but the formation of tumors also depends upon a variety of factors, including the proliferative capacity of the target tissue, the sex of the animal, genotoxic properties of the specific adducts formed, and other unknown events. Chronic dosing experiments in animal models are of interest for human risk assessment because human exposure is typically intermittent, involving repeated exposures. However, it is to be expected that in a genetically-diverse human population, where the lifetime averages >70 years, the relationship between tumorigenesis and DNA adduct formation will be relatively more complex than that observed in mice. From our studies of chronic ABP exposure in male mice, we have obtained the daily dose of ABP and the steady-state level of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP) adduct associated with a 50% mouse bladder tumor incidence. Our attempt at a human extrapolation for adducts and urinary bladder cancer in smoking males (20–40 cigarettes/day) is based on the ABP dose per cigarette, values for the dG-C8-ABP adduct in bladder biopsies of lifetime heavy smokers at age ∼70, and the smoking-related bladder tumor incidence (absolute lifetime risk) for Caucasian males in the United States aged 65–84 years. The extrapolation has produced two major predictions, one related to adduct formation and the other related to tumorigenesis. First, the observed level of smoking-related dG-C8-ABP in DNA of human bladder epithelium, expressed as a function of daily ABP intake, is about 3500-times higher than similar data for mice, which suggests that humans may perform the biotransformation of ABP more efficiently than mice. Second, at a similar bladder tumor incidence, mouse bladder contained adduct concentrations that were much higher than those observed in human bladder; for example, at a 2.6% tumor incidence, mouse bladder contained an average of 55.5 fmol dG-C8-ABP/μg DNA (1850 adducts/10 8 nucleotides), while bladders from Caucasian male smokers contained an average of 0.036 fmol dG-C8-ABP/μg DNA (1.2 adducts/10 8 nucleotides). This suggests that factors other than ABP–DNA adducts, such as adducts of other carcinogens, the influence of promoters, and synergistic effects of all of these factors contribute substantially to smoking-related bladder cancer in humans.

Aromatic DNA adducts in foundry workers in relation to exposure, life style and CYP1A1 and glutathione transferase M1 genotype.

Levels of aromatic DNA adducts in foundry workers and controls were followed at four annual samplings. During this time exposure to polycyclic aromatic hydrocarbons (PAH) decreased and the level of DNA adducts decreased accordingly. In the total group exposure was related to the level of adducts. Adduct levels correlated with urinary 1-hydroxypyrene (LOGU1OH), air benzo[a]pyrene, weekly working hours and daily cigarette consumption. In a multivariate model 1-hydroxypyrene had a consistent effect. Neither glutathione transferase M1 (GSTM1) nor cytochrome P450 1A1 (CYP1A1) genotypes had clear effects. Yet the individuals lacking GSTM1 had a stronger effect of LOGU1OH and some effect by other sources of PAH, such as charcoal broiled food, although all these variables were not significant in the multivariate model. The rare individuals with a CYP1A1 polymorphism MspI containing an amino acid change at isoleucine had an increased level of adducts. The results showed that the postlabelling method used was able to detect an increase in aromatic DNA adducts in leukocytes when exposure to benzo[a]pyrene in air was approximately 5 ng/m3. At such low levels smoking and charcoal broiled food may be important contributors to adducts.

A romatic DNA adduct levels in human peripheral blood lymphocytes and total white blood cells by 32P postlabelling: need for validation

Long lived lymphocytes tend to have higher 32P postlabelling measured levels of adducts than short lived granulocytes in environmental and life style associated i.e. smoking exposures. With the aim of investigating this issue for occupational exposure to PAH and contributing to further validation of some technical aspects of the 32P postlabelling assay, two Italian laboratories analysed PAH-DNA adducts from lymphocytes and total white blood cells WBC. Seventy seven blood samples from coke oven workers employed at a steel plant located in Taranto, Southern Italy, and 14 samples from control subjects were collected. At the University of Padua, DNA was purified from peripheral blood lymphocytes PBL. Two years later, at the University of Bari, white blood cells WBC were isolated from replicate blood samples stored at- 80 C and DNA purified by the same method. In both cases, the nuclease P1 modified postlabelling assay was used to determine aromatic DNA adduct levels. The mean adduct levels were 5.13 3.37 Padua and 2.48 1.27 Bari per 108 nucleotides. Both laboratories observed large inter individual variations of adduct levels ranging from 0.09 to 18.93 per 108 nucleotides. Both the correlation and the agreement of the two sets of data were assessed. Slight correlation r = 0.39; p 0.01 and a poor level of agreement were found, the intra class correlation coefficient being equal to 0.05. Better correlation coefficient r = 0.54, p 0.01 and intra class correlation coefficient r = 0.50 were observed comparing only the adduct levels determined on the diagonal zone DRZ. Our findings seem to confirm the same divergence reported in the literature on DNA adduct levels between lymphocytes and granulocytes.

Biomarkers of marine pollution observed in species of mullet living in two eastern Mediterranean harbours

The activities of enzymes associated with xenobiotic metabolism and or oxidative processes, and the levels of aromatic DNA adducts, have been determined in the livers of grey mullet Oedalechilus labeo and Lisa ramada living in two eastern Mediterranean harbours. Glutathione peroxidase GSH P activity was 2.5 times higher 9 IU g-1 liver and glutathione reductase GSSG R activity was twice as high 2.5 IU g-1 liver in fish from the more polluted harbour at Mersin than in the harbour near Erdemli. Superoxide dismutase SOD activity was 25 lower 4.3 IU g-1 liver in the more polluted harbour. The concentrations of glutathione and malondialdehyde varied both with species and environment by a factor of 2.5-3. DNA adducts in liver were determined by 32P postlabelling. In Oedalechilus labeo in the more polluted harbour, adduct levels were 258 21 adducts per 108 nucleotides mean SE; two groups of Lisa ramada were distinguished having 261 48 and 30 6 adducts per 108 nucleotides, respectively. The average adduct level in a group of mullet of mixed species in the less polluted harbour was 3.3 2.3 adducts per 108 nucleotides. The results illuminate the ability of mullet to live in contaminated marine environments, and show that enzyme activities and liver DNA adduct levels can serve as indicators of marine pollution.

Capillary Electrophoresis-Fluorescence Line-Narrowing (CE-FLN) System for DNA Adduct Characterization

Abstract Capillary electrophoresis (CE) interfaced with low-temperature (4.2 K) fluorescence line-narrowing (FLN) spectroscopy is used for separation and structural characterization of polycyclic aromatic hydrocarbon-derived DNA adducts. The CE-FLN system is applied to the separation and identification of mixtures of benzo[a]pyrene and dibenzo[a,l]pyrene DNA adducts. Interfacing CE with FLN spectroscopy allows on-line separation and high-resolution spectroscopic identification of CE-separated DNA adducts, providing a new powerful tool for DNA adduct structure characterization.

DNA Adducts inMytilus GalloprovincialisandZosterisessor OphiocephalusCollected from PAC-Polluted and Reference Sites of the Venice Lagoon

Abstract Mediterranean mussels (Mytilus galloprovincialis) and bottom-dwelling grass gobies (Zosterisessor ophiocephalus) were collected from the Venice lagoon both in front of the open sea and from the industrial area of Porto Marghera. DNA adducts were measured by means of the nuclease P1-enhanced 32P-DNA postlabelling assay. Autoradiograms of gill DNA from animals of the industrial area showed a tract of unresolved adducts along the diagonal radioactive zone. Such adducts were detected at low but significant levels in different sampling periods only in mussels and fish collected at Porto Marghera. The presence of bulky aromatic DNA adducts in native mussels is consistent with previous results on the appearance of a weak adduct in gill DNA of Mytilus galloprovincialis treated with benzo[a]pyrene. However, the mechanisms leading to DNA damage and the fate of the initial lesions in marine invertebrates exposed to genotoxic agents are still poorly understood.

Detection of Polynuclear Aromatic Hydrocarbon Diol Epoxide-Derived DNA and Globin Adducts in Humans by Gas Chromatography-Mass Spectrometry

Abstract Gas chromatography-negative ion chemical ionization-mass spectrometry-selected ion monitoring (GC-NICI-MS-SIM) was employed to detect tetramethyl ether derivatives of tetraols released upon hydrolysis of DNA and globin adducts derived from diol epoxides of polynuclear aromatic hydrocarbons (PAH). The methodology involves the following steps: 1) isolation of DNA or globin; 2) mild acid hydrolysis under vacuum; 3) isolation of the resulting tetraols and derivatization to the corresponding tetramethyl ethers using CH3I; 4) HPLC purification followed by GC-NICI-MS-SIM analysis. Analysis of human globin samples by this method indicates the presence of adducts which release chrysene-1,2,3,4-tetraol and benzo[a]pyrene-7,8,9,10-tetraol in smokers and nonsmokers. The level of BP-diol epoxide globin adducts in smokers was significantly higher than that in nonsmokers (2.75 vs 0.96 fmol/mg globin). The concentration of chrysene diol-epoxide globin adducts in the same subjects was about two orders of magnitud...