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Maternal diet can contribute to carcinogenic exposures and also modify effects of environmental exposures on maternal and fetal genetic stability. In this study, associations between maternal diet and the levels of dioxin-like plasma activity, bulky DNA adducts in white blood cells and micronuclei (MN) in lymphocytes from mother to newborns were examined. From 98 pregnant women living in the greater area of Copenhagen, Denmark in 2006-2007, maternal peripheral blood and umbilical cord blood were collected, together with information on health, environmental exposure and lifestyle. Maternal diet was estimated on the basis of maternal food frequency questionnaire (FFQ) completed by the end of pregnancy. Biomarkers were detected in paired blood samples through the dioxin-responsive chemical-activated luciferase expression (CALUX)(®) bioassay, (32)P-postlabelling technique and cytokinesis-block MN assay. Maternal preference for meats with dark surface were significantly associated with higher bulky DNA adducts in both maternal (β 95%CI; 0.46 (0.08, 0.84)) and cord blood (β 95%CI; 0.46 (0.05, 0.86)) before and after adjustment for potential confounders. No other significant associations between the 18 dietary variables and the biomarkers measured in maternal and fetal samples were identified. The present study suggests that maternal intake of meats with dark surface contributes to the bulky DNA adduct levels in maternal and umbilical cord blood. Relationship between food preparation and bulky DNA adducts appear to be captured by a FFQ while potential associations for other biomarkers might be more complex or need larger sample size., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The gastrointestinal route of exposure to particulate matter is important because particles are ingested via contaminated foods and inhaled particles are swallowed when removed from the airways by the mucociliary clearance system. We investigated the effect of an intragastric administration by oral gavage of diesel exhaust particles (DEP) in terms of DNA damage, oxidative stress and DNA repair in colon epithelial cells, liver, and lung of rats. Eight rats per group were exposed to Standard Reference Material 2975 at 0.064 or 0.64 mg/kg bodyweight for 6 and 24 h. Increased levels of 8-oxo-7,8-dihydro-2'-deoxyguanosine lesions were observed at the highest dose after 6 and 24 h in all three organs. 8-Oxo-7,8-dihydro-2'-deoxyguanosine is repaired by oxoguanine DNA glycosylase 1 (OGG1); upregulation of this repair system was observed as elevated pulmonary OGG1 mRNA levels after 24 h at both doses of DEP, but not in the colon and liver. A general response of the antioxidant defence system is further indicated by elevated levels of heme oxygenase 1 mRNA in the liver and lung 24 h after administration. The level of bulky DNA adducts was increased in liver and lung at both doses after 6 and 24h (DNA adducts in colon epithelium were not investigated). In summary, DEP administered via the gastrointestinal tract at low doses relative to ambient exposure generates DNA damage and increase the expression of defence mechanisms in organs such as the lung and liver. The oral exposure route should be taken into account in risk assessment of particulate matter.

Exposure to benzene can cause DNA damage and the subsequent development of cancer. In this study, study subjects were 31 laboratory workers at a petrochemical factory and 31 gasoline service attendants. Control subjects were 34 workers from a mail sorting service center. Occupational exposures to benzene were assessed using biomarkers of exposure in blood and urine. Induction of DNA-repair capacity was assessed as a biomarker of early effect. The effects of polymorphisms in a metabolizing gene (CYP2E1), in detoxification genes (NQO1 and GSTT1), and in a DNA-repair gene (XRCC1, codon 399) on biomarker levels were evaluated. The mean individual benzene exposure of laboratory workers (24.40+/-5.82 ppb) and that of gasoline service attendants (112.41+/-13.92 ppb) were significantly higher than in controls (1.39+/-0.17 ppb, p<0.001). Blood benzene levels of laboratory workers (169.12+/-30.60 ppt) and gasoline service attendants (483.46+/-59.62 ppt) were significantly higher than those of the controls (43.30+/-4.89 ppt, p<0.001). Trans,trans-muconic acid levels in post-shift urine samples collected from laboratory workers (0.14+/-0.02 mg/g creatinine) and gasoline service attendants (0.20+/-0.02 mg/g creatinine) were significantly higher than in urine samples of controls (0.04+/-0.01 mg/g creatinine, p<0.001). The level of benzene exposure was correlated with blood benzene levels (R2=0.65, p<0.01) and post-shift urinary trans,trans-muconic acid concentrations (R2=0.49, p<0.01). As a biomarker of early effect, DNA-repair capacity was assessed by use of the cytogenetic challenge assay, i.e., chromosomal aberrations in peripheral lymphocytes were assessed after challenging blood cultures with 1 Gy gamma radiation. A significantly lower DNA-repair capacity--determined as dicentrics in laboratory workers (0.17 per metaphase cell) and in gasoline service attendants (0.19 per metaphase cell) compared with controls (0.12 per metaphase cell, p<0.001)--was observed. The frequency of deletions in laboratory workers (0.22 per metaphase cell) and gasoline service attendants (0.39 per metaphase cell) were significantly higher than in control workers (0.16 per metaphase cell, p<0.01 and p<0.001, respectively). An increase in radiation-induced dicentrics and deletions indicate a lower DNA-repair capacity in benzene-exposed workers. The influence of genetic polymorphisms on the biomarkers was assessed. Benzene-exposed workers who carried CYP2E1*1/*5 or *5/*5 genotypes excreted slightly higher levels of trans,trans-muconic acid than workers who carried the CYP2E1*1/*1 genotype. In this study, NQO1 and GSTT1 genotypes did not have any effect on the levels of trans,trans-muconic acid. In the case of XRCC1, laboratory workers with 399Arg/Gln or Gln/Gln had a lower DNA-repair capacity--measured as radiation-induced frequency of dicentrics and deletions--than those with the 399Arg/Arg genotype (p<0.01). Our results show that biomarkers of internal dose and early biological effect in people occupationally exposed to benzene are influenced by genetic polymorphisms in susceptibility genes.

Several large prospective investigations are under way or are planned in different parts of the world, aiming at the investigation of gene-environment interactions for chronic diseases. Technical, practical and ethical issues are raised by such large investigations. Here we describe how such issues were approached within a case-control study nested in EPIC, a large European cohort, and the kind of validation studies that have been set up. The GenAir investigation aimed at measuring the effects of air pollution and environmental tobacco smoke on human health in EPIC with a nested design and with biological measures. Validation studies included (a) comparisons between cotinine measurements, hemoglobin adducts and questionnaire data; (b) an analysis of the determinants of DNA adduct concentration; (c) comparison among different genotyping methods; (d) an analysis of the determinants of plasma DNA amounts. We also describe how the ethical issues were dealt with in our investigation.

This study was performed in an Estonian shale-oil mine with the purpose to develop and apply a number of biomarkers for occupational diesel-exhaust exposure monitoring. Increased breathing-zone exposures to exhaust from operators of diesel-powered trucks in the mine was confirmed in the environmental monitoring part of the study, showing a 7.5-fold higher exposure to particle-associated 1-nitropyrene (1-NP) in 50 underground workers compared with 42 surface workers [P.T.J. Scheepers, D. Coggon, L.E. Knudsen, R. Anzion, H. Autrup, S. Bogovski, R.P. Bos, D. Dahmann, P. Farmer, E.A. Martin, V. Micka, V. Muzyka, H.-G. Neumann, J. Poole, A. Schmidt-Ott, F. Seiler, J. Volf, I. Zwirner-Baier, Biomarkers for occupational diesel exhaust exposure monitoring (BIOMODEM)-a study in underground mining, Toxicol. Lett. 134 (2002) 305-317; P.T.J. Scheepers, V. Micka, V. Muzyka, R. Anzion, D. Dahmann, J. Poole, R.P. Bos, Exposure to dust and particle-associated 1-nitropyrene of drivers of diesel-powered equipment in underground mining, Ann. Occp. Hyg. 47 (2003) 379-388]. Analysis of DNA damage by the Comet assay on frozen blood samples was performed on the total study group and showed significantly higher levels (p=0.003) in underground workers (smokers) driving diesel-powered excavation machines (median 155 on a scale from 0 to 400, among 47 persons), compared with surface workers who smoked (median of 90, among 46 persons). The level of DNA damage in underground smokers was significantly higher (p=0.04) than in non-smokers. Samples from 2 of the 3 sampling weeks had significantly lower DNA damage compared with the third week, probably due to timely processing and freezing. These samples also showed significant differences (p<0.001) between underground workers (median 145, among 41 persons) and surface workers (median 60, among 30 persons). An HPLC method was developed for the analysis of (32)P-postlabelled 1-NP-DNA-adducts, and was applied to a sub-sample of 20 workers. No significant differences between surface and underground workers were found in this sub-sample with respect to the minor, unidentified adducts that had similar chromatographic properties to 1-NP adducts, and smoking did not have any effect on adduct levels. No significant effects of the genotypes of GSTM1, GSTP1 and GSTT1 on DNA-adducts and on DNA damage as measured by the Comet assay were found in the total study group. The study confirms an increased level of DNA damage in workers exposed to exhaust from truck-driving in the mine. However, the results of the environmental and biological monitoring of 1-NP did not correlate, suggesting that inhalation exposure to diesel exhaust is not reflected by an increase in 1-NP-DNA-adduct levels and/or that factors other than occupational exposure to diesel exhaust are primary determinants of these DNA-adduct levels.

Sucrose-rich diets have repeatedly been observed to have co-carcinogenic actions in colon and liver of rats and to increase the number of 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) induced aberrant crypt foci in rat colon. To investigate a possible interaction between sucrose and IQ on the genotoxicity in rat liver and colon, we gave Big Blue rats a diet containing sucrose (0%, 3.45% or 13.4% w/w) and/or IQ (70 ppm) for a period of 3 weeks. Sucrose and IQ increased the mutation frequency in the colon. The effect of combined treatments with IQ and sucrose on the mutation frequencies was additive indicating that sucrose and IQ act independently. This was supported by the mutation spectra where sucrose expands the background mutations in the colon, whereas IQ, in other studies, more specifically has induced G:C --> T:A transversions. In the liver IQ increased the mutation frequency, whereas addition of sucrose reduced the effect of IQ in a dose-dependent manner. The level of bulky DNA adducts in liver and colon was increased in animals exposed to either sucrose or IQ. In animals exposed to IQ, addition of sucrose had marginal effects on the level of bulky DNA adducts. Markers of oxidative damage and DNA repair were generally unaffected by the treatments. In conclusion, sucrose and IQ in the diet induced mutations in the colon by independent mechanisms, whereas an interaction was observed in liver leading to a decrease in mutations by the combined treatment.

Recent studies have identified an indirect genotoxicity pathway involving inflammation as one of the mechanisms underlying the carcinogenic effects of air pollution/diesel exhaust particles (DEP). We investigated the short-term effects of DEP on markers of inflammation and genotoxicity in vitro and in vivo. DEP induced an increase in the mRNA level of pro-inflammatory cytokines and a higher level of DNA strand breaks in the human lung epithelial cell line A549 in vitro. For the in vivo study, mice were exposed by inhalation to 20 or 80 mg/m3 DEP either as a single 90-min exposure or as four repeated 90-min exposures (5 or 20 mg/m3) and the effects in broncho-alveolar lavage (BAL) cells and/or lung tissue were characterized. Inhalation of DEP induced a dose-dependent inflammatory response with infiltration of macrophages and neutrophils and elevated gene expression of IL-6 in the lungs of mice. The inflammatory response was accompanied by DNA strand breaks in BAL cells and oxidative DNA damage and increased levels of bulky DNA adducts in lung tissue, the latter indicative of direct genotoxicity. The effect of a large single dose of DEP was more pronounced and sustained on IL-6 expression and oxidative DNA damage in the lung tissue than the effect of the same dose administered over four days, whereas the reverse pattern was seen in BAL cells. Our results suggest that the effects of DEP depend on the rate of delivery of the particle dose. The mutation frequency (MF), after DEP exposure, was determined using the transgenic Muta Mouse and a similar exposure regimen. No increase was observed in MF in lung tissue 28-days after exposure. In conclusion, short-term exposure to DEP resulted in DNA strand breaks in BAL cells, oxidative DNA damage and DNA adducts in lungs; and suggested that DNA damage in part is a consequence of inflammatory processes. The response was not associated with increased MF, indicating that the host defence mechanisms were sufficient to counteract the adverse effects of inflammation. Thus, there may be thresholds for the inflammation-associated genotoxic effects of DEP inhalation.

Background: Previous studies in Denmark have shown that bus drivers and tramway employees were at an increased risk for developing several types of cancer and that bus drives from central Copenhagen have high levels of biomarkers of DNA damage., Aims: The present study evaluates 1-hydroxypyrene concentrations and mutagenic activity in urine as biomarkers of exposure in non-smoking bus drivers in city and rural areas on a work day and a day off and in non-smoking mail carriers working outdoors (in the streets) and indoors (in the office)., Methods: Twenty-four hour urine samples were collected on a working day and a day off from 60 non-smoking bus drivers in city and rural areas and from 88 non-smoking mail carriers working outdoors (in the streets) and indoors (in the office). The concentration of 1-hydroxypyrene was measured by means of HPLC and the mutagenic activity was assessed by the Ames assay with Salmonella tester strain YG1021 and S9 mix. The N-acetyltransferase (NAT2) phenotype was used as a biomarker for susceptibility to mutagenic/carcinogenic compounds., Results: Bus drivers excreted more 1-hydroxypyrene in urine than did mail carriers. The differences were slightly smaller when NAT2 phenotype, cooking at home, exposure to vehicle exhaust, and performing physical exercise after work were included. The NAT2 slow acetylators had 29% (1.29 [CI: 1.15-1.98]) higher 1-hydroxypyrene concentrations in urine than the fast acetylators. Male bus drivers had 0.92 revertants/mol creatinine [CI: 0.37-1.47] and female bus drivers 1.90 revertants/mol creatinine [CI: 1.01-2.79] higher mutagenic activity in urine than mail carriers., Conclusion: The present study indicates that bus drivers are more exposed to polycyclic aromatic hydrocarbons (PAH) and mutagens than mail carriers. Mail carriers who worked outdoors had higher urinary concentration of 1-hydroxypyrene, a marker of exposure to PAH, than those working indoors. The individual levels of urinary mutagenic activity were not correlated to excretion of 1-hydroxypyrene. This might be due to the fact that the most potent mutagenic compounds in diesel exhaust are not PAH but dinitro-pyrenes. Among bus drivers, fast NAT2 acetylators had higher mutagenic activity in urine than slow NAT2 acetylators and female bus drivers had higher mutagenic activity than male bus drivers.

Exposure to ambient air pollution has been associated with cancer. Ambient air contains a complex mixture of toxics, including particulate matter (PM) and benzene. Carcinogenic effects of PM may relate both to the content of PAH and to oxidative DNA damage generated by transition metals, benzene, metabolism and inflammation. By means of personal monitoring and biomarkers of internal dose, biologically effective dose and susceptibility, it should be possible to characterize individual exposure and identify air pollution sources with relevant biological effects. In a series of studies, individual exposure to PM(2.5), nitrogen dioxide (NO(2)) and benzene has been measured in groups of 40-50 subjects. Measured biomarkers included 1-hydroxypyrene, benzene metabolites (phenylmercapturic acid (PMA) and trans-trans-muconic acid (ttMA)), 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) in urine, DNA strand breaks, base oxidation, 8-oxodG and PAH bulky adducts in lymphocytes, markers of oxidative stress in plasma and genotypes of glutathione transferases (GSTs) and NADPH:quinone reductase (NQO1). With respect to benzene, the main result indicates that DNA base oxidation is correlated with PMA excretion. With respect to exposure to PM, biomarkers of oxidative damage showed significant positive association with the individual exposure. Thus, 8-oxodG in lymphocyte DNA and markers of oxidative damage to lipids and protein in plasma associated with PM(2.5) exposure. Several types of DNA damage showed seasonal variation. PAH adduct levels, DNA strand breaks and 8-oxodG in lymphocytes increased significantly in the summer period, requiring control of confounders. Similar seasonal effects on strand breaks and expression of the relevant DNA repair genes ERCC1 and OGG1 have been reported. In the present setting, biological effects of air pollutants appear mainly related to oxidative stress via personal exposure and not to urban background levels. Future developments include personal time-resolved monitors for exposure to ultrafine PM and PM(2.5,) use of GPS, as well as genomics and proteomics based biomarkers.

Earlier studies have indicated that sucrose increases 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)-induced aberrant crypt foci in the colon. In this study, we investigated the role of sucrose in IQ-induced genotoxicity of the colon mucosa and liver. Big Blue rats were fed with IQ (20 ppm in feed) and/or sucrose (3.45 or 6.85 wt.% in feed) for 3 weeks. IQ increased DNA strand breaks in the colon, whereas the mutation frequency was increased in the liver. The level of IQ-induced DNA adducts was elevated in both colon mucosa cells and liver. In the liver, high sucrose intake increased the level of DNA adducts above that of IQ and low sucrose intake. Oxidative DNA damage detected in terms of 7-hydro-8-oxo-2'-deoxyguanosine by HPLC-EC, or endonuclease III or formamidopyrimidine DNA glycosylase sensitive sites were unaltered in the colon and liver. Expression of ERCC1 and OGG1 mRNA levels were unaffected by IQ or sucrose feeding. Biomarkers of oxidative stress, including Vitamin C, malondialdehyde and protein oxidations (gamma-glutamyl semialdehyde and 2-amino adipic semialdehyde) were unaltered in plasma and in liver. In conclusion, sucrose feeding increases IQ-induced genotoxicity in liver but not in colon, suggesting different mechanisms for sucrose and IQ in colon mutagenesis.

Many genetic polymorphisms in metabolism enzymes are important for the risk of cancer as shown in a large number of case-control studies. The relative risk estimates have shown large variations between such population studies. However, in most studies the relative risk estimates are in the range of 2. Some polymorphisms are effect modifiers, i.e. without exposure they have no consequence and the effect of exposure can appear independent of the genotype. Genetic polymorphisms in metabolism of environmental toxicants plays a significant role in exposures to traffic generated air pollution in Copenhagen, revealing statistically significant higher levels of chromosomal aberrations in non-smoking bus drivers with glutathion-S-transferase M1, GSTM1 null and N-acetyltransferase 2, NAT2 slow genotypes. Combined with cohort studies showing positive associations between high chromosomal levels and increased cancer risk, such results indicate effect modification regarding cancer risk. In risk assessment the safety 'factor' of 10 is generally accepted to allow for variation in individual susceptibility. Reviewing the literature justifies the factor of 10 when considering single polymorphisms. However in an individual with several susceptible metabolism genotypes as well as other determinants of susceptibility, e.g. defective DNA repair, poor-nutritional state, etc. the risk may increase far above a safety of 10.Historically, genetic polymorphisms have been taken into consideration in employment and currently the application in insurance situations is criticised.

Epidemiologic studies indicate that prolonged exposure to high pollution levels is associated with increased risk of cancer, especially lung cancer. However, under conditions of moderate or low air pollution, epidemiologic evidence does not permit reliable conclusions. Biomarker-based population studies may serve as complementary tools providing a better understanding of the relative contribution of ambient atmospheric pollution to the overall genotoxic burden suffered by city dwellers. However, past efforts to apply biomarkers to studies of low levels exposure to urban air pollution have given inconclusive results, partly because of the absence of adequate data on personal exposure, covering a time-window which is appropriate for the biomarkers being examined, as well as a battery of biomarkers reflecting different stages of the carcinogenic process. In the present paper, the potential of biomarker-based population studies to aid the assessment of the genotoxic and carcinogenic effects of urban air pollution is reviewed by reference to the achievements and limitations of earlier reported studies. The design and methodology adopted in a recently completed large-scale population study, carried out in the context of the European Union Environment and Climate Programme, known by the short name of AULIS project, is discussed and descriptive statistics of the main findings of the project are presented. These findings indicate that for cohorts suffering moderate-to-low exposures to airborne particulate-bound polycyclic aromatic hydrocarbons (PAHs), no simple correlation with biomarkers of genotoxicity existed and suggest that additional factors made a significant contribution to the overall genotoxic burden.

The dorsal skin of C3H/Tif/hr hairless mice was painted with coal tar, pharmacological grade. Epidermal cells and hepatocytes were isolated after 4, 24, 48 and 96 h and DNA strand breaks were determined as tail moment by the alkaline comet assay. The tail moment of epidermal cells was significantly greater at the time points 4, 24, 48 and 96 h after exposure compared to the controls, with the most DNA strand breaks at 24 h. The DNA strand breaks in epidermal cells increased linearly with the dose of coal tar. In hepatocytes, no difference in DNA strand breaks was found between exposed animals and controls. DNA adducts were determined by the 32P-postlabeling assay. For epidermal cells, the mean DNA adduct level was 12-fold greater in coal tar painted mice after 24 h than in controls. Again, a linear dose/response relationship was seen 24 h after painting. For liver DNA, the mean DNA adduct level was 3-fold greater than for controls. The mutation frequency in epidermal and liver cells was examined in lambdalacZ transgenic mice (MutaMouse). Thirty-two days after painting, the mutation frequency in epidermal cells was 16-fold greater in coal tar treated mice compared to controls. No effect was detected in hepatocytes. We found that a single painting of coal tar resulted in strong genotoxic effects in the murine epidermis, evidenced by induction of DNA strand breaks and DNA adducts in hairless mice and lambdalacZ mutations in the MutaMouse. This demonstrates that it is possible to detect genotoxic effects of mixtures with high sensitivity in mouse skin by these end-points.

Biotransformation plays an important role in the carcinogenic activity and organ specificity of environmental carcinogens. Large interindividual variation in the biotransformation has been reported, and genetic polymorphisms in some xenobiotica metabolizing enzymes can in part explain some of these differences. The concentration of the ultimate carcinogen, that will react with DNA, is determined by the rate of activation and detoxification. Individuals with a decreased rate of detoxification, i.e., lacking the glutathione S-transferase M1 gene, have a slightly higher level of bulky carcinogen-DNA adduct in some tissues, and do also have an increased level of chromosomal aberrations. In addition, the genotype may also influence the type of mutations, e.g., in tumor suppressor gene, transversion being predominant in the GSTM1 null group. People with slow N-acetyltransferase activity do generally have a higher adduct level of aromatic amines in bladder tissues. Genetic polymorphism in either CYP1A1 or glutathione S-transferase is linked to an increased risk of smoking related cancers, while N-acetyltransferase activity is related to cancers in which aromatic amines are the main risk factor. Combination of the high risk genotypes for activating and detoxification enzymes, e.g., CYP1A MspI/GSTM1 null is not only associated with an increased risk of cancer development, but also an increased level of markers of the biological active dose and early markers of effect. Additional studies on the role of genetic variants of xenobiotica metabolizing enzymes and combinations thereof at relevant low levels of exposure are important in order to establish guidance values for toxic compounds.

Polycyclic aromatic hydrocarbon (PAH)-DNA adducts were studied in human lung from 39 lung cancer patients by synchronous fluorescence spectrophotometric (SFS) and 32P-postlabeling assays. Regression analysis of the samples failed to detect any correlation between benzo[a]pyrene-diolepoxide (BPDE)-DNA adducts detected by SFS and the BPDE co-migrating spot detected by 32P-postlabeling. We have also analyzed the relationship between adduct levels and TP53 mutations. By postlabeling diagonal radioactive zone (DRZ) adducts were detected in 37 of 39 (95%) lung tissues from lung cancer patients and the adduct level ranged from 6.81 to 108.50 adducts/10(8) nucleotide. Thirty-three of 39 (85%) had detectable levels of BPDE-DNA adducts (> 1 adduct/10(9) nucleotide). Current heavy smokers (> 20 cigarettes/day) have significantly higher DRZ adduct levels compared to individuals smoking less than 20 cigarettes/day. By SFS combined with immunoaffinity column (IAC), 11 of 39 (28%) samples had detectable adduct levels, and 6 of 11 (55%) were detectable by SFS following purification of benzo[a]pyrene (BP)-tetrols by high pressure liquid chromatography (HPLC). Six of 33 (18%) samples were positive for BPDE-DNA adducts by both postlabeling and HPLC/SFS. No correlation was observed between the SFS and 32P-postlabeling assays for the detection of BPDE-DNA adducts. However, there was a good correlation between adduct levels detected by IAC/SFS and HPLC/SFS. We found a weak association between total PAH-DNA adduct levels in lung tissue and TP53 mutations.