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Background: To examine the molecular benefits of the government action to close the local coal burning power plant in Tongliang County, Chongqing Municipality, we compared biologic markers and health outcomes in two successive birth cohorts enrolled before and after the plant was shut down. In this city, polycyclic aromatic hydrocarbons (PAH) were primarily emitted by the coal burning facility. We previously reported that cord blood levels of PAH-DNA adducts (a biomarker of exposure) and various adverse health outcomes were reduced in the second cohort, whereas levels of brain-derived neurotrophic factor/BDNF (a protein involved in neuronal growth) were increased. Here we assessed telomere length (TL), which has been associated with risk of certain chronic diseases, early mortality, aging and cognitive decline in adults., Objectives: The goals of the present study were to determine whether TL differed between the two cohorts and whether prenatal PAH exposure, estimated by PAH-DNA adducts in cord white blood cells of newborns in China, were predictive of shorter TL in cord blood, suggesting the potential accrual of risk of certain chronic diseases during the prenatal period. We explored relationships of TL with BDNF and neurodevelopmental outcomes, each previously associated with PAH-DNA adducts in these cohorts, as well as the potential mediating role of TL in the associations between adducts and neurodevelopmental outcomes., Methods: We analyzed TL in cord blood of 255 newborns who also had data on PAH-DNA adducts, BDNF, and relevant covariates. Multiple regression analysis was carried out to test associations between adducts and TL and between TL and BDNF, adjusting for relevant covariates. In the subset with developmental quotient (DQ) scores from Gesell testing at age 2 (N = 210), we explored whether TL was a mediator of the relationship between PAH-DNA adducts and DQ scores by first examining the associations between cord adducts and DQ, cord adducts and TL, and TL and DQ, adjusting for the same covariates., Results: As hypothesized, the mean TL was significantly higher in the second cohort compared to the first cohort. Overall, PAH-DNA cord adducts were significantly and inversely correlated with TL. Multiple regression analysis showed a significant association between adducts and TL, after adjusting for key covariates: β (effect size per standard deviation adducts) = -0.019, p = .003. The regression coefficient of TL on (Ln) BDNF was also significant (β = 0.167, p < .001). Exploratory analysis, regressing TL on Gesell developmental scores, showed generally inverse, but not significant associations. TL was not, therefore, deemed to be a potential mediator of the association between adducts and developmental scores at age two., Conclusion: This study provides the first evidence that prenatal PAH exposure from coal burning may adversely affect TL, with potential implications for future risk of chronic diseases including cardiovascular disease. The improvement in TL in the second cohort and the observed correlation between increased TL and higher levels of BDNF indicate direct benefits to the health and development of children resulting from the government's closure of the power plant., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Polycyclic aromatic hydrocarbons (PAH) are carcinogenic, neurotoxic environmental pollutants generated during incomplete combustion of fossil fuel and other organic material. PAH exposure has been associated with adverse fetal development and epigenetic alterations in cord blood. Several molecular epidemiology studies have established PAH-DNA adducts as biomarkers of PAH exposure., Objectives: We investigated the relationship between LINE1 DNA methylation and PAH-DNA adduct levels in cord blood, and with neurodevelopmental outcomes., Methods: In Tongliang County, China, the current study enrolled two population-based cohorts of nonsmoking pregnant women before (2002) and after (2005) the closure of a local coal-fired power plant in May 2004. We analyzed cord blood samples collected from mothers in the two cohorts (n=110 from 2002 cohort and n=107 from 2005 cohort) for PAH-DNA adducts and genomic LINE1 DNA methylation. Neurodevelopmental data on children were collected using the Gesell Developmental Scales (GDS) at age 2 and using the Wechsler Intelligence Scale for Children (WISC) at age 5., Results: A significant inverse relationship was observed between PAH-DNA adducts and LINE1 DNA methylation (β=-0.010, p<0.038). A significant, positive association between LINE1 methylation and scores on WISC full scale and verbal (β=85.31, p<0.005; β=94.36, p<0.003) but not on the GDS. Mediation analysis did not find LINE1 to be a direct mediator between PAH-DNA adducts and IQ score., Conclusion: LINE1 methylation in cord blood DNA was a positive predictor of IQ at age 5 and was decreased at higher levels of prenatal PAH exposure measured by PAH-DNA adducts in cord blood. However, the adverse effects of prenatal exposure to PAH on IQ scores did not appear to be directly mediated by altered LINE1 methylation., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Polycyclic aromatic hydrocarbons (PAH) are a class of chemicals common in the environment. Certain PAH are carcinogenic, although the degree to which genetic variation influences susceptibility to carcinogenic PAH remains unclear. Also unknown is the influence of genetic variation on the procarcinogenic effect of in utero exposures to PAH. Benzo[a]pyrene (B[a]P) is a well-studied PAH that is classified as a probable human carcinogen. Within our New York City-based cohort, we explored interactions between maternal exposure to airborne PAH during pregnancy and maternal and newborn haplotypes (and in one case, a single-nucleotide polymorphism) in key B[a]P metabolism genes on B[a]P-DNA adducts in paired cord blood samples. The study subjects included non-smoking African-American (n = 132) and Dominican (n = 235) women with available data on maternal PAH exposure, paired cord adducts and genetic data who resided in the Washington Heights, Central Harlem and South Bronx neighborhoods of New York City. We selected seven maternal and newborn genes related to B[a]P metabolism, detoxification and repair for our analyses: CYP1A1, CYP1A2, CYP1B1, GSTM3, GSTT2, NQO1 and XRCC1. We found significant interactions between maternal PAH exposure and haplotype on cord B[a]P-DNA adducts in the following genes: maternal CYP1B1, XRCC1 and GSTM3, and newborn CYP1A2 and XRCC1 in African-Americans; and maternal XRCC1 and newborn NQO1 in Dominicans. These novel findings highlight differences in maternal and newborn genetic contributions to B[a]P-DNA adduct formation, as well as ethnic differences in gene-environment interactions, and have the potential to identify at-risk subpopulations who are susceptible to the carcinogenic potential of B[a]P.

Background: Bulky DNA adducts are markers of exposure to genotoxic aromatic compounds, which reflect the ability of an individual to metabolically activate carcinogens and to repair DNA damage. Polycyclic aromatic hydrocarbons (PAHs) represent a major class of carcinogens that are capable of forming such adducts. Factors that have been reported to be related to DNA adduct levels include smoking, diet, body mass index (BMI), genetic polymorphisms, the season of collection of biologic material, and air pollutants., Methods: We pooled 11 studies (3,600 subjects) in which bulky DNA adducts were measured in human white blood cells with similar (32)P-postlabeling techniques and for which a similar set of variables was available, including individual data on age, gender, ethnicity, batch, smoking habits, BMI, and season of blood collection, and a limited set of gene variants., Results: Lowest DNA adduct levels (P = 0.006) were observed in the spring (median = 0.50 adducts per 10(8) nucleotides), followed by summer (0.64), autumn (0.70), and winter (0.85). The same pattern emerged in multivariate analysis but only among never smokers (P = 0.02). Adduct levels were significantly lower (P = 0.001) in northern Europe (the Netherlands and Denmark; mean = 0.60, median = 0.40) than in southern Europe (Italy, Spain, France, and Greece; mean = 0.79, median = 0.60)., Conclusions: In this large pooled analysis, we have found only weak associations between bulky DNA adducts and exposure variables. Seasonality (with higher adducts levels in winter) and air pollution may partly explain some of the interarea differences (north vs. south Europe), but most inter-area and interindividual variations in adduct levels still remain unexplained., Impact: Our study describes the largest pooled analysis of bulky DNA adducts so far, showing that interindividual variation is still largely unexplained, though seasonality seems to play a role., (©2010 AACR.)

Bulky DNA adducts are biomarkers of exposure to aromatic compounds and of the ability of the individual to metabolically activate carcinogens and to repair DNA damage. Their ability to predict cancer onset is uncertain. We have performed a pooled analysis of three prospective studies on cancer risk in which bulky DNA adducts have been measured in blood samples collected from healthy subjects (N = 1947; average follow-up 51-137 months). In addition, we have performed a meta-analysis by identifying all articles on the same subject published up to the end of 2006, including case-control studies. In the pooled analysis, a weakly statistically significant increase in the risk of lung cancer was apparent (14% per unit standard deviation change in adduct levels, 95% confidence interval 1-28%; using the weighted mean difference method, 0.15 SD, units higher adducts in cases than in controls). The association was evident only in current smokers and was absent in former smokers. Also the meta-analysis, which included both lung and bladder cancers, showed a statistically significant association in current smokers, whereas the results in never smokers were equivocal; in former smokers, no association was detected. The results of our pooled and meta-analyses suggest that bulky DNA adducts are associated with lung cancer arising in current smokers after a follow-up of several years.

Prior work suggests that body size and fat content may influence carcinogen-DNA adduct levels measured in white blood cells. Here we consider energy balance more broadly by assessing the impact of body mass index (BMI), physical activity and calorie intake on the presence of benzo[a]pyrene-DNA (BP-DNA) adducts in white blood cell DNA. Our cross-sectional study employed subjects from a separately conducted intervention trial. Physical activity and food intake data were collected at 12 and 15 months of follow-up, respectively. BP-DNA adducts were measured by high-performance liquid chromatography (HPLC) in white blood cell samples collected at 12 months of follow-up. Complete data on all variables were available from 143 subjects. Logistic regression showed that BMI was inversely associated with the presence of detectable adducts (OR = 0.90, p = 0.02), and that hours of moderate-intensity physical activity were positively associated with the presence of detectable adducts (OR = 1.04, p = 0.04). These results provide further evidence that body fat content influences carcinogen-DNA adduct levels, probably by altering the distribution of the lipophilic parent compound.

Polycyclic aromatic hydrocarbons (PAHs) are an important class of toxic pollutants released by fossil fuel combustion. Other pollutants include metals and particulate matter. PAH-DNA adducts, or benzo[a]pyrene (BaP) adducts as their proxy, provide a chemical-specific measure of individual biologically effective doses that have been associated with increased risk of cancer and adverse birth outcomes. In the present study we examined the relationship between prenatal PAH exposure and fetal and child growth and development in Tongliang, China, where a seasonally operated coal-fired power plant was the major pollution source. In a cohort of 150 nonsmoking women and their newborns enrolled between 4 March 2002 and 19 June 2002, BaP-DNA adducts were measured in maternal and umbilical cord blood obtained at delivery. The number of gestational months occurring during the period of power plant operation provided a second, more general measure of exposure to plant emissions, in terms of duration. High PAH-DNA adduct levels (above the median of detectable adduct level) were associated with decreased birth head circumference (p=0.057) and reduced children's weight at 18 months, 24 months, and 30 months of age (p<0.05), after controlling for potential confounders. In addition, in separate models, longer duration of prenatal exposure was associated with reduced birth length (p=0.033) and reduced children's height at 18 (p=0.001), 24 (p<0.001), and 30 months of age (p<0.001). The findings suggest that exposure to elevated levels of PAHs, with the Tongliang power plant being a significant source, is associated with reduced fetal and child growth in this population.

Polycyclic aromatic hydrocarbons (PAH), of which benzo[a]pyrene is a representative member, are combustion-related environmental pollutants and include known carcinogens. Laboratory animal studies indicate that the dose of PAHs to the fetus is on the order of a 10th that to the mother and that there is heightened susceptibility to PAH-induced carcinogenesis during the fetal and infancy periods. Carcinogen-DNA adducts, a measure of procarcinogenic genetic damage, are considered a biomarker of increased cancer risk. Here we compare the levels of benzo[a]pyrene-DNA adducts as a proxy for PAH-DNA damage measured in maternal blood and newborn cord blood obtained at delivery in four different populations of mothers (total of 867) and newborns (total of 822), representing a 30-fold range of exposure to ambient PAHs. The populations include residents in Northern Manhattan, participants in a study of the effects of the World Trade Center disaster, residents in Krakow, Poland, and residents in Tongliang, China. Mean adduct concentrations in both maternal and cord blood and the proportion of samples with detectable adducts, increased across the populations [Northern Manhattan < World Trade Center (WTC) < Krakow < Tongliang], consistent with the trend in estimated ambient exposure to PAHs (P < 0.001). For mothers, the means in the respective populations were Northern Manhattan (0.21 adducts per 10(8) nucleotides), WTC (0.23 adducts per 10(8) nucleotides), Krakow (0.28 adducts per 10(8) nucleotides), Tongliang (0.31 adducts per 10(8) nucleotides); the corresponding means in the newborns were Northern Manhattan (0.23), WTC (0.24), Krakow (0.29), Tongliang (0.31). The percentage of mothers with detectable levels of adducts in the respective populations were Northern Manhattan (36.8%), WTC (57.5%), Krakow (72.9%), Tongliang (73.4%); the corresponding percentages among the newborns were Northern Manhattan (42.4%), WTC (60.6%), Krakow (71.1%), Tongliang (79.5%). Despite the estimated 10-fold lower PAH dose to the fetus based on laboratory animal experiments, the adduct levels in the newborns were similar to or higher than in the mothers. This study suggests that the fetus may be 10-fold more susceptible to DNA damage than the mother and that in utero exposure to polycyclic aromatic hydrocarbons may disproportionately increase carcinogenic risk. The data support preventive policies to limit PAH exposure to pregnant women and children.

We investigated the association between alcohol consumption, GSTM1 genotype, and polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels in breast tissue. Women referred for breast surgery were enrolled prior to surgery, responded to an interview, and gave a blood sample. Women diagnosed with ductal carcinoma in situ and invasive ductal or lobular cancer were defined as cases, and women with benign conditions without atypia were defined as controls. Paraffin-embedded tumor and nontumor tissue from cases and benign tissue from controls were retrieved from the pathology samples. GSTM1 genotype status was determined by PCR using WBC DNA, and PAH-DNA adduct levels were measured in breast tissue using immunohistochemistry. In tumor and nontumor tissue from cases, the GSTM1-null genotype was associated with increased adduct levels among current alcohol consumers but not among nondrinkers. In nontumor tissue, the interaction between genotype and alcohol consumption was significant (P=0.02), but in tumor tissue, the interaction did not achieve statistical significance (P=0.10). In benign tissue from controls, there was no association between genotype and adducts, regardless of drinking status. Among subjects with the null genotype who drank alcohol, adduct levels were significantly higher in tumor and nontumor tissue from cases than in benign tissue from controls. These results indicate the presence of a novel gene-lifestyle interaction that influences PAH-DNA adduct levels in breast tissue from cases but not controls. This apparent difference in PAH metabolism in response to alcohol may be an important clue as to how alcohol influences breast cancer risk.

Gene-environment interactions are hypothesized to be major contributors to susceptibility to environmental carcinogens and interindividual variability in cancer risk. We present findings on associations between genetic susceptibility due to inherited polymorphisms of the Phase II detoxification enzyme sulfotransferase 1A1 (SULT1A1), breast cancer risk, and polycyclic aromatic hydrocarbon (PAH)-DNA adducts. A hospital based case-control study was conducted at the New York-Presbyterian Medical Center (NYPMC). The study utilized two control groups: one comprised of women with benign breast disease (BBD) and the other comprised of women visiting NYPMC for routine gynecologic checkups (healthy controls). Blood samples were collected from cases and controls; and breast tissue from pathology blocks was collected from cases (tumor and non-tumor tissue) and BBD controls (benign tissue). PAH-DNA adduct levels were measured by immunohistochemistry in breast tissue samples, and the SULT1A1 (Arg/His) polymorphism at codon 213 was determined by PCR RFLP analyses using DNA from white blood cells. Increasing number of His alleles was modestly associated with breast cancer case-control status, when cases were compared to healthy controls (p for trend = 0.08), when cases were compared to BBD controls (p for trend = 0.08) and when cases were compared to both control groups combined (p for trend = 0.07). Contrary to our hypothesis PAH-DNA adduct levels in breast tissue were not associated with SULT1A1 genotype. Our findings are consistent with a prior report that the Arg/His polymorphism in SULT1A1 is associated with breast cancer risk.

Transplacental exposure to carcinogenic air pollutants from the combustion of fossil fuels is a growing health concern, given evidence of the heightened susceptibility of the fetus. These mutagenic/carcinogenic pollutants include aromatic compounds such as polycyclic aromatic hydrocarbons that bind to DNA, forming chemical-DNA adducts. We have investigated the genotoxic effects of transplacental exposure in humans by analyzing aromatic-DNA adducts and the frequency of gene mutations at the hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus in umbilical cord and maternal blood samples. Here we show, in a cross-sectional study of 67 mothers and 64 newborns from the Krakow Region of Poland, that aromatic-DNA adducts measured by (32)P-postlabeling are positively associated with HPRT mutant frequency in the newborns (beta = 0.56, P = 0.03) after controlling for exposure to tobacco smoke, diet, and socioeconomic status. In contrast to the fetus, HPRT mutations and DNA adducts do not reflect similar exposure periods in the mother, and the maternal biomarkers were not correlated. Adducts were higher in the newborn than the mother, indicating differential susceptibility of the fetus to DNA damage; but HPRT mutation frequency was 4-fold lower, consistent with the long lifetime of the biomarker. These results provide the first demonstration of a molecular link between somatic mutation in the newborn and transplacental exposure to common air pollutants, a finding that is relevant to cancer risk assessment.

We review our studies on the role of polycyclic aromatic hydrocarbon (PAH)-DNA adducts in breast cancer. Additionally we report on analyses of the reliability of the scoring procedures used with immunohistochemical assay for PAH-DNA adducts and of potential bias arising from the use of benign breast disease (BBD) controls. We conducted a case-control study utilizing two control groups: BBD controls who donated tissue and blood samples, and healthy controls who donated blood samples. In comparisons of tumor tissue from cases and benign tissue from BBD controls, increasing adduct levels were significantly associated with case-control status [odds ratio (OR) = 2.40, 95% confidence interval (CI) 1.18-4.92], whereas in comparisons of nontumor tissue from cases and benign tissue from BBD controls the association was nonsignificant (OR = 1.97, 95% CI 0.94-4.17). We also show among cases, but not among BBD controls, that the GSTM1 null genotype is associated with increased adduct levels in breast tissue. Our reliability study found the scoring procedures used with the immunohistochemical assay to have high reliability, 0.93 in nontumor, 0.82 in tumor, and 0.74 in benign tissues. However, we found that the technician significantly contributed to the total variability of a series of data. Finally, we did not find a consistent bias to the null associated with the use of BBD controls; however, BBD controls may overestimate the prevalence of family history of breast cancer compared to that of healthy controls (18% vs.14%). We hypothesize that the higher prevalence results from a referral bias and discuss how this may influence our results., (Copyright 2002 Wiley-Liss, Inc.)

In this matched case-control study nested within the prospective Physicians' Health Study, we evaluated whether DNA damage in blood samples collected at enrollment significantly predicted risk, consistent with our hypothesis that cases have greater biological susceptibility to polycyclic aromatic hydrocarbons and other aromatic tobacco carcinogens. The subjects were 89 cases of primary lung cancer and 173 controls, all males, matched on smoking, age, and duration of follow-up. Aromatic-DNA adducts were measured in WBCs by the nuclease P1-enhanced (32)P-postlabeling method that primarily detects smoking-related adducts. Among current smokers, but not former or nonsmokers, there was a significant increase in mean adduct levels of cases compared with controls (11.04 versus 5.63; P = 0.03). "Healthy" current smokers who had elevated levels of aromatic DNA adducts in WBCs were approximately three times more likely to be diagnosed with lung cancer 1-13 years later than current smokers with lower adduct concentrations (odds ratio, 2.98; 95% confidence interval, 1.05-8.42; P = 0.04). We were not able to discern case-control differences in former smokers and nonsmokers. The findings are of interest because they suggest that individuals who become cases have greater biological susceptibility to tobacco carcinogens, a biological difference, which manifests most clearly while exposure is ongoing.

Human and experimental evidence indicates that the developing fetus may be more susceptible than the adult to the effects of certain carcinogens, including some polycyclic aromatic hydrocarbons (PAHs). Factors that can modulate susceptibility include proliferation rates, detoxification capabilities, and DNA repair capacity. Biomarkers can facilitate quantification of age-related susceptibility among human populations. In this study, we report on three biomarkers measured in paired blood samples collected at birth from 160 Polish mothers and newborns: 70 pairs from Krakow (a city with high air pollution including PAHs) and 90 pairs from Limanowa (an area with lower ambient pollution but greater indoor coal use). Biomarkers were: WBC aromatic-DNA adducts by (32)P-postlabeling and PAH-DNA adducts by ELISA (as indicators of DNA damage from PAHs and other aromatics) and plasma cotinine (as an internal dosimeter of cigarette smoke). Correlations were assessed by Spearman's rank test, and differences in biomarker levels were assessed by the Wilcoxon signed-ranks test. A significant correlation between paired newborn/maternal samples was seen for aromatic-DNA adduct levels (r = 0.3; P < 0.001) and plasma cotinine (r = 0.8; P < 0.001) but not PAH-DNA adduct levels (r = 0.14; P = 0.13). Among the total cohort, levels of the three biomarkers were higher in newborn samples compared with paired maternal samples. The difference was significant for aromatic-DNA adduct levels (16.6 +/- 12.5 versus 14.21 +/- 15.4/10(8) nucleotides; P = 0.002) and plasma cotinine (14.2 +/- 35.5 versus 8.3 +/- 24.5 ng/ml; P < 0.001) but not for PAH-DNA adduct levels (7.9 +/- 9.9 versus 5.9 +/- 8.2/10(8) nucleotides; P = 0.13). When analyses were restricted to the 80 mother/newborn pairs from whom the blood sample was drawn concurrently (within 1 h of each other), levels of all of the three biomarkers were significantly higher in the newborn compared with paired maternal blood samples (P < 0.05). Results suggest reduced detoxification capabilities and increased susceptibility of the fetus to DNA damage, especially in light of experimental evidence that transplacental exposures to PAHs are 10-fold lower than paired maternal exposures. The results have implications for risk assessment, which currently does not adequately account for sensitive subsets of the population.

Although the etiology of the majority of human breast cancers is unknown, environmental carcinogens are suspected to play a role. In this study, we investigated polycyclic aromatic hydrocarbon-DNA adducts in 78 breast cancer patients and benign breast disease patients with lifetime environmental exposure to polycyclic aromatic hydrocarbon (PAH) compounds. Adducts were detected in paraffin sections by immunoperoxidase method using polyclonal antiserum and were quantitated by the image-analyzing system. A significantly higher level of adducts was found in benign breast disease as compared to cancer patients (P < .001; Mann-Whitney U test). Neither smoking nor genetic polymorphisms in glutathione S-transferase and cytochrome P450 influenced the level of adducts. This exploratory study demonstrates the usefulness of the immunoperoxidase method to detect PAH-DNA adducts in stored breast tissue and suggests further research on a larger population, including patients from both high- and low-pollution environments.

A major goal in molecular epidemiology is to identify preventable environmental risk factors and susceptible subpopulations. In a hospital-based molecular epidemiological case-control study of breast cancer, we investigated the relationship between DNA damage from exposure to polycyclic aromatic hydrocarbons (PAHs) and susceptibility attributable to inherited deletion of the xenobiotic detoxifying gene, glutathione S-transferase M1 (GSTM1). Prior to breast surgery, women (n = 227) were enrolled and interviewed and donated a blood sample. PAH-DNA adduct levels were measured by immunohistochemistry in breast tissue samples retrieved from pathology blocks, and GSTM1 genotype was determined by PCR using WBC DNA. The GSTM1 analysis included 95 cases and 87 benign breast disease controls. GSTM1 genotype was not associated with breast cancer case-control status (odds ratio = 0.73; 95% confidence interval, 0.37-1.44). However, the GSTM1 null genotype predicted PAH-DNA adduct levels in malignant (beta = 0.407; P = 0.003) and nonmalignant (beta = 0.243; P = 0.05) breast tissue from cases. This relationship was not seen in tissue from controls (beta = 0.095; P = 0.341). When tissue from controls was compared with tumor tissue from cases, there was a significant case-control difference in PAH-DNA adduct levels among women who were GSTM1 null. There was no such case-control difference among women who were homozygous or heterozygous for GSTM1. There was an interaction between GSTM1 and case-control status on adduct levels in breast tissue (P = 0.002). The results suggest that genetic susceptibility to the formation of PAH-DNA adducts in breast tissue may play a role in breast cancer development.

A number of polycyclic aromatic hydrocarbons (PAH) are widespread environmental contaminants that cause mammary cancer experimentally. We investigated whether exposure and susceptibility to PAH, as measured by PAH-DNA adducts in breast tissue, are associated with human breast cancer. We carried out a hospital-based case-control study using immunohistochemical methods to analyze PAH-DNA adducts in tumor and nontumor breast tissue from cases and benign breast tissue from controls. The subjects were white, African-American and Latina women without prior cancer or treatment, including 119 women with breast cancer and 108 with benign breast disease without atypia. PAH-DNA adducts measured in breast tumor tissue of 100 cases and in normal tissue from 105 controls were significantly associated with breast cancer (OR=4.43, 96% CI 1.09-18.01) after controlling for known breast cancer risk factors and current active and passive smoking, and dietary PAH. There was substantial interindividual (17-fold) variability in adducts overall, with 27% of cases and 13% of controls having elevated adducts. The odds ratio for elevated adducts in tumor tissue compared with control tissue was 2.56 (1. 05-6.24), after controlling for potential confounders. Adduct levels in tumor tissue did not vary by stage or tumor size. Among 86 cases with paired tumor and nontumor tissue, adducts levels in these two tissues were highly correlated (r=0.56, P<0.001). However, the corresponding associations between case-control status and adducts measured in nontumor tissue from 90 cases and in normal tissue from 105 controls were positive but not statistically significant. Overall, neither active nor passive smoking, or dietary PAH were significantly associated with PAH-DNA adducts or breast cancer case-control status. These results suggest that genetic damage reflecting individual exposure and susceptibility to PAH may play a role in breast cancer; but more research is needed to determine whether the findings are relevant to causation or progression of breast cancer.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants; a number are carcinogenic. Metabolic polymorphisms may modulate susceptibility to PAH-induced DNA damage and carcinogenesis. This study investigates the relationship between PAH-DNA adduct levels (in maternal and newborn WBCs) and two polymorphisms: (a) an MspI RFLP in the 3' noncoding region of cytochrome P4501A1 (CYP1A1); and (b) an A-->G transition in nucleotide 313 of glutathione S-transferase P1 (GSTP1), resulting in an ile105val substitution. CYP1A1 catalyzes the bioactivation of PAH; the CYP1A1 MspI RFLP has been associated with cancer of the lung. GSTP1 catalyzes the detoxification of PAH; the val allele has greater catalytic efficiency toward PAH diol epoxides. The study involves 160 mothers and their newborns from Poland. Regression models controlled for maternal smoking and other confounders. No association was seen between maternal adduct levels and either polymorphism, separately or combined. However, adduct levels were higher among newborns with the CYP1A1 MspI restriction site (heterozygotes and homozygotes combined) compared with newborns lacking the restriction site (P = 0.06). Adducts were higher among GSTP1 ile/val and ile/ile newborns compared with GSTP1 val/val newborns (P = 0.08). Adduct levels were 4-fold higher among GSTP1 ile/ile newborns having the CYP1A1 restriction site compared with GSTP1 val/val newborns who lacked the CYP1A1 restriction site (P = 0.04). This study demonstrates a significant combined effect of phase I and phase II polymorphisms on DNA damage from PAHs in fetal tissues. It illustrates the importance of considering interindividual variation in assessing risks of transplacental exposure to PAHs.

This study investigated the relationship in human placenta between polycyclic aromatic hydrocabon (PAH)-DNA adduct levels and two biomarkers of cytochrome P4501A1 (CYP1A1): gene induction evidenced by CYP1A1 mRNA, and a genetic polymorphism, the CYP1A1 MspI RFLP. CYP1A1 codes for an inducible enzyme system that catalyzes the bioactivation of PAHs. Prior research found a high correlation in human lung tissue between CYP1A1 activity and DNA damage from PAHs. The CYP1A1 Mspi RFLP has been linked in some studies to risk of lung cancer. The relationships in human placenta between DNA damage, CYP1A1 activity and genotype have not been well characterized and may be relevant to risks from transplacental PAH exposure. The study cohort consisted of 70 newborns from Krakow, Poland, a city with elevated air pollution, and 90 newborns from nearby Limanowa, an area with lower air pollution but greater indoor coal use. Contrary to results seen previously in lung tissue, CYP1A1 mRNA was not significantly correlated with PAH-DNA adduct levels in the placenta. Smoking (self-reported maternal and infant plasma cotinine) was significantly associated with CYP1A1 mRNA levels (P < 0.01), but not with PAH-DNA adduct levels. Placental PAH-DNA adduct levels were significantly higher in infants with the CYP1A1 MspI restriction site compared with infants without the restriction site (P < 0.01), implicating a genetic factor in inter-individual variation in DNA damage in human placenta. Further studies are needed to determine the relevance of this finding to risk of transplacental carcinogenesis.

Biomarkers such as DNA adducts have significant potential to improve quantitative risk assessment by characterizing individual differences in metabolism of genotoxins and DNA repair and accounting for some of the factors that could affect interindividual variation in cancer risk. Inherent uncertainty in laboratory measurements and within-person variability of DNA adduct levels over time are putatively unrelated to cancer risk and should be subtracted from observed variation to better estimate interindividual variability of response to carcinogen exposure. A total of 41 volunteers, both smokers and nonsmokers, were asked to provide a peripheral blood sample every 3 weeks for several months in order to specifically assess intraindividual variability of polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels. The intraindividual variance in PAH-DNA adduct levels, together with measurement uncertainty (laboratory variability and unaccounted for differences in exposure), constituted roughly 30% of the overall variance. An estimated 70% of the total variance was contributed by interindividual variability and is probably representative of the true biologic variability of response to carcinogenic exposure in lymphocytes. The estimated interindividual variability in DNA damage after subtracting intraindividual variability and measurement uncertainty was 24-fold. Inter-individual variance was higher (52-fold) in persons who constitutively lack the Glutathione S-Transferase M1 (GSTM1) gene which is important in the detoxification pathway of PAH. Risk assessment models that do not consider the variability of susceptibility to DNA damage following carcinogen exposure may underestimate risks to the general population, especially for those people who are most vulnerable.

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.

This study is part of an ongoing investigation of biomarkers in iron foundry workers exposed to polycyclic aromatic compounds. Foundry workers with the highest exposures had elevated levels of DNA adducts in their white blood cells in previous studies. The purpose of this study was to characterize the nature of DNA reactive chemicals in foundry air samples through incubating the foundry filter extract with DNA and activation enzymes. Calf thymus DNA was incubated with foundry filter extract and activated by either rat liver activation mixture (S9 mix) or xanthine oxidase. A complex pattern of adducts was observed on thin-layer chromatography (TLC) by the 32P-postlabeling assay. Two selected polycyclic aromatic hydrocarbons (PAHs)--1-NP-and anti(+/-)benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide [anti(+/-) BPDE]-DNA adducts--were used as marker compounds in characterizing the postlabeled DNA adducts by TLC combined with high-performance liquid chromatography (HPLC). After an initial separation of DNA adducts by TLC, individual spots were isolated and separated further on HPLC. HPLC analysis and spiking with anti(+/-)BPDE-DNA standard confirmed the co-migration of the anti(+/-)BPDE-DNA standard with one PAH adduct formed by the S9 mix-activated DCM extract in calf thymus DNA.

The formation of polycyclic aromatic hydrocarbon-DNA adducts was studied in peripheral blood lymphocytes obtained from men with occupational and environmental exposure. Subjects included coke factory workers, residents from the vicinity of the cokery, and rural region inhabitants (16 individuals in each exposure group). Adducts were determined by immunohistochemical analysis using a polyclonal antiserum recognizing benzo(a)pyrene and related polycyclic aromatic hydrocarbon diol epoxide-DNA adducts, a biotinylated secondary antiserum, and streptavidin-conjugated FITC. Propidium iodide was used to quantitate nuclear DNA. Dual fluorescence intensities were simultaneously measured with a Zeiss Axiovert microscope and a Bio-Rad MRC-600 argon laser scanning confocal attachment. Adducts were significantly elevated (P < 0.001) in both occupational and environmental groups, as compared to the rural control group by Mann-Whitney U test. The distribution of the data indicated the existence of cells with relatively higher adduct levels. The percentages of these so called "higher adduct-level cells" were 13.6, 11.5, and 3.7 in cokery workers, environmentally exposed individuals, and rural controls, respectively. The immunohistochemical method allows visualization and relative quantitation of polycyclic aromatic hydrocarbon-DNA adducts in individual lymphocytes. It requires a much smaller amount of blood than the previously used 32P-postlabeling and ELISA methods, which used isolated bulk DNA. It can also be used for adduct quantitation in biopsy material. The results of this pilot study indicate that this technique is a promising addition to biomonitoring studies.

Coal tar treated psoriasis patients were used as a model population to evaluate a panel of immunoassays for monitoring exposure to benzo[a]pyrene (BP) and related polycyclic aromatic hydrocarbons (PAH). The assays included measurement of PAH diol epoxide-DNA adducts in white blood cells by competitive enzyme-linked immunosorbent assay (ELISA) with fluorescence endpoint detection, PAH-albumin adducts by competitive ELISA with color endpoint detection and serum levels of antibodies recognizing BP diol epoxide-DNA adducts by noncompetitive color ELISA. PAH-DNA adducts by ELISA were elevated in patients (mean 6.77 +/- 12.05/10(8)) compared to controls (4.90 +/- 8.81/10(8), p = 0.12). There was no difference in PAH-albumin adducts between patients (mean 0.61 +/- 0.31 fmol/micrograms) and controls (0.63 +/- 0.30 fmol/micrograms). Glutathione S-transferase M1 genotype was also determined but no relationship was found between presence of the gene and either DNA or protein adduct levels. About 30% of both patients and controls had measurable titer of antibodies recognizing BPDE-I-DNA adducts. Measurement of white blood cell DNA adducts by ELISA was the most sensitive method for detecting PAH exposure in coal tar-treated psoriasis patients.

Breast cancer is the second leading cause of cancer death among American women. Known risk factors account for only approximately one-third of the 182,000 new cases diagnosed each year in the United States. There is both concern and debate over the contribution of environmental exposures related to lifestyle, occupation, and ambient pollution, particularly in high risk areas such as Long Island, NY and the rest of the northeastern United States. Biomarkers such as carcinogen-DNA adducts can help to explore the role of environmental risk factors for breast cancer by documenting DNA damage from specific carcinogens directly in human tissue. In this pilot study, a total of 31 breast tissue samples were analyzed by the 32P-postlabeling method for carcinogen-DNA adducts characteristic of complex mixtures of aromatic compounds (such as polycyclic aromatic hydrocarbons) and tobacco smoke. The samples included tumor and tumor-adjacent tissues from 15 women with breast cancer and normal tissue samples from 4 women undergoing breast reduction. Among the breast cancer cases, the mean aromatic/hydrophobic-DNA adduct level in all tissues assayed was 5.3 +/- 2.4 (SD) adducts/10(8) nucleotides compared to 2.3 +/- 1.5 among the samples from the noncancer patients. Breast tissue (tumor and/or nontumor) from 30% (5 of 15) of women with breast cancer displayed a pattern of adducts (referred to as a diagonal zone of radioactivity) associated previously, in studies of other tissues, with exposure to tobacco smoke. The 5 positive samples were from current smokers; tissue samples from the 8 nonsmoking cases did not show this characteristic pattern (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinogen-DNA adducts and somatic gene mutation at the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus were evaluated in peripheral leukocytes of workers in an iron foundry with exposure to benzo[a]pyrene (B[a]P) and other polycyclic aromatic hydrocarbons (PAHs). During the two year study period, B[a]P exposure declined by approximately 40%, from a maximum of 60 ng/m3 in the first year to < 36 ng/m3 1 year later. A total of 64 persons were sampled in November/December of the two successive study years; 24 of them gave two samples one year apart. The biomarkers included carcinogen-DNA adducts in leukocytes (PAH-DNA measured by an immunoassay, aromatic-DNA by the 32P-postlabeling method) and HPRT mutation in lymphocytes. After adjusting for smoking, levels of PAH-DNA, aromatic-DNA and HPRT mutation frequency (Mf) increased with exposure among the 64 workers sampled during the 2 year period (P < or = 0.05). However, the markers showed a differential response to the change in exposure, consistent with their individual biology. For example, among the 24 workers sampled in both years, carcinogen-DNA adducts (which have a half-life on the order of several months) were markedly reduced from the first to the second year (PAH-DNA, 6.2 versus 2.3/10(8); aromatic-DNA, 2.5 versus 1.4/(8); P < 0.01). HPRT Mf (a longer-lived marker) was somewhat less affected by the decline in exposure (1.3 versus 0.8, P < or = 0.05). Moreover, in the second year several long-term workers had low levels of adducts, but elevated HPRT Mf. Thus, PAH-DNA and HPRT Mf were highly correlated in the first year (n = 17; r = 0.67; P < 0.01), but not in the second year or in the two years combined. However, when analysis was restricted to workers with detectable levels of adducts (who included the more highly exposed workers) the correlation was significant between PAH-DNA and HPRT (n = 17; r = 0.65; P = 0.005). In contrast, aromatic-DNA adducts and HPRT were not correlated in either year. These results suggest a molecular link between somatic gene mutation and PAHs; and they highlight the need in such molecular epidemiologic studies to consider the varying lifetimes of the individual markers.

Sixty-three male cigarette smokers were entered into a cross-sectional study to determine whether inverse associations existed between polycyclic aromatic hydrocarbon (PAH)-DNA adduct levels and intake/serum levels of vitamin A, vitamin C and vitamin E. Associations between PAH-DNA adducts and intakes of carotene, as well as serum levels of beta-carotene, were also determined. Fasting blood samples were collected for assays of PAH-DNA adducts in circulating mononuclear cells, plasma cotinine and serum levels of vitamin A, beta-carotene, vitamin C and vitamin E. Since genetic deficiency in the detoxifying enzyme glutathione S-transferase M1 (GSTM1) has been associated with increased risk of lung cancer, GSTM1 genotype was also determined. Analysis of PAH-DNA adducts by competitive enzyme-linked immunosorbent assay (ELISA) indicated that 70% of the subjects had detectable adducts, with a mean of 4.38 adducts/10(8) nucleotides (range 1.00-24.1/10(8)). Pearson's method was utilized to determine whether any associations existed between the various host variables and PAH-DNA adducts. Previously, no significant associations were found between PAH-DNA adducts and cigarettes smoked/day, pack-years, daily/life-time tar exposures or plasma cotinine levels (Santella et al., Carcinogenesis, 13, 2041-2045, 1992). PAH-DNA adducts were inversely associated with serum cholesterol-adjusted vitamin E levels (r = -0.25, P < or = 0.05) and with smoking-adjusted vitamin C serum levels (r = -0.22, P < or = 0.09). Stratification by GSTM1 genotype indicated that these associations were limited to subjects with the null genotype. The relationship between adducts and serum cholesterol-adjusted vitamin E was significant in those of the null genotype (r = -0.38, P < or = 0.04), but not in those with the gene present (r = -0.12, P = 0.5). Similarly, for smoking-adjusted vitamin C, the relationship with adducts was stronger in subjects with the null genotype (r = -0.35, P < or = 0.06) than in those with GSTM1 present (r = -0.05, P = 0.77). These results are consistent with findings of prior epidemiological studies identifying significant inverse associations between anti-oxidant micronutrient status or GSTM1 genotype and the incidence of lung cancer. Additional studies should be conducted to confirm a possible role for vitamin E in PAH-DNA adduct formation and to explore further the possible roles of vitamin A, beta-carotene and vitamin C in modulating adduct formation and lung cancer risk.

Blood samples were obtained from volunteers working in a Finnish iron foundry who were occupationally exposed to polycyclic aromatic hydrocarbons (PAH) and from control subjects not known to be occupationally exposed to this class of chemical carcinogens. Foundry workers were classified as belonging to high, medium or low exposure groups according to their exposure to airborne benzo[a]pyrene: high, greater than 0.2: medium, 0.05-0.2: low, less than 0.05 micrograms benzo[a]pyrene/m3 air). Aromatic adducts were found to be present in white blood cell DNA from most of the exposed workers using the enzyme-linked immunosorbent assay (ELISA) to detect aromatic DNA adducts and the 32P-postlabelling technique. There was a dose-response relationship between the estimated exposure and adduct levels by both methods, and a reasonable correlation between the results of the immunoassay and postlabelling carried out in two laboratories. The levels of adducts found in the samples from the high and medium exposure groups by ELISA ranged up to five adducts in 10(7) nucleotides: the aromatic adducts detected by the postlabeling assay were at a level of two adducts/10(8) nucleotides in the high and medium exposure categories. No effect due to age, sex or the smoking habits of the subjects was observed. The results indicate that DNA extracted from white blood cells of highly exposed workers is more likely to contain aromatic DNA adducts than that from workers without occupational exposure to PAH, but large interindividual variations were evident. This study suggests that the antibody and 32P-postlabelling assays may be useful in monitoring human exposure to known and previously unidentified environmental genotoxic agents.

A new tool for the study of occupational carcinogenesis is "molecular dosimetry" or biomonitoring to establish the biologically effective dose of carcinogens in workers. Human monitoring of biologically effective dose and preclinical response has the potential to flag the need for protective measures and/or surveillance. Comparable biologically effective dose and preclinical response data in humans and laboratory animals for whom tumor incidence is known can also enhance risk extrapolation between species. This paper will provide a brief overview of biomonitoring methods now under development, including advantages, limitations, applications to date, and research needs. Application to the monitoring of worker populations requires careful thought about the use to which monitoring data will be put.

Iron foundry workers, exposed to high levels of polycyclic aromatic hydrocarbons (PAHs), silica, and metal fumes and dusts, are at elevated risk of lung cancer. Benzo(a)pyrene and a number of structurally related PAHs are metabolically activated to diol epoxides (e.g., 7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a) pyrene) which are mutagenic, carcinogenic in experimental animals, and form covalent adducts with DNA. The levels of these adducts were measured in an enzyme-linked immunosorbent assay using a polyclonal anti-benzo(a)pyrene diol epoxide-I-DNA antibody which cross-reacts with DNA modified by diol epoxides of structurally related PAHs. DNA was analyzed from peripheral blood cells of 35 Finnish foundry workers and 10 controls. Workers were classified as having low (less than 0.05 micrograms/m3), medium (0.05-0.2 micrograms/m3), or high (greater than 0.2 micrograms/m3) exposure to benzo(a)pyrene (as an indicator of PAH). When adjustment was made for cigarette smoking and time since vacation, benzo(a)pyrene exposure was significantly related to adduct levels (P = 0.0001). Each of the three exposure groups had significantly elevated adduct levels compared to controls. Among the exposed workers, the low group differed significantly from the high and medium categories. This study supports the usefulness of monitoring adduct formation in a population occupationally exposed to carcinogens.

An interlaboratory comparison of immunoassays using antisera elicited against benzo[a]pyrene-diol-epoxide-modified DNA (BPDE-I-DNA) was carried out resulting in standardization of antisera, competitors and assay conditions. The assays used included competitive enzyme-linked immunosorbent assays (ELISA) with color and fluorescence endpoint detection and an ultrasensitive enzyme radioimmunoassay (USERIA) with a radioactive endpoint. Three different antisera were compared, two of which were obtained from different rabbits immunized with the same BPDE-I-DNA and a third from an animal immunized with another BPDE-I-DNA sample. Samples of standardized BPDE-I-DNA with high (36 pmol adduct/microgram DNA; 1.2 adducts/10(2) nucleotides) and low (4.5 fmol/microgram DNA; 1.5 adducts/10(6) nucleotides) modification levels were prepared and used in each laboratory. The antisera were all elicited against DNAs modified to a high extent, and it was therefore not surprising that they detected adducts in a slightly modified DNA sample with lower efficiency than those in highly modified DNA samples. The discrepancy of antibody recognition between the highly and slightly modified samples varied between 1.4- and 11.2-fold depending on the antiserum and assay. To ascertain the quantitative capability of the immunoassays, the modification level of DNA isolated from mouse keratinocytes treated with [3H]benzo[a]pyrene was determined by radioactivity and immunoassay. These results indicated that when a biological sample is assayed against a BPDE-I-DNA standard modified in the same range as the biological samples (4.5 fmol/microgram), quantitative recovery of adducts is achieved by immunoassay. These studies resulted in the realization that interlaboratory differences in immunoassay procedure can have significant consequences for data comparison and that where possible it is preferable for laboratories to use the same antisera and modified DNA standards.

In order to validate markers of internal dose and biologically effective dose of carcinogens, a battery of measurements was made on blood samples from 22 smokers and 24 nonsmokers. The markers included immunoreactivity in an enzyme-linked immunosorbent assay (ELISA) quantified in white blood cells with the use of a polyclonal anti-benzo[a]pyrene diol epoxide-I-DNA antibody, 4-aminobiphenyl hemoglobin (4-ABP-Hb) adducts measured by negative chemical ionization mass spectrometry, sister chromatid exchange (SCE) in cultured lymphocytes, and cotinine in plasma measured by radioimmunoassay. Several blood samples were drawn from each subject. In blood samples 1 and 3 having detectable levels of DNA adducts, mean femtomole-per-microgram levels were consistently higher among smokers compared to nonsmokers. The borderline significance of this difference may be attributable to the small numbers of subjects. Consistently higher adduct levels were seen in females compared to males. In sample 3, adduct levels were significantly correlated with measurements of active smoking in smokers and with passive smoking in nonsmokers. By contrast to the ELISA data, which may reflect cumulative exposure from multiple background sources, the 4-ABP-Hb assay was able to distinguish clearly between smokers and nonsmokers. SCEs were significantly elevated in the smokers compared to nonsmokers. Also observed were significant correlations between 4-ABP-Hb and both cotinine and SCEs, as well as a positive correlation between the 4-ABP-Hb and DNA adduct levels (sample 3) that was highly significant. The correlation between DNA and 4-ABP-Hb adducts was significant in smokers but not nonsmokers (sample 3). These results support the need for batteries of markers to detect and to quantify the carcinogenic dose to humans resulting from both specific and "background" environmental exposures.