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.     

Addiction and imaging of the living human brain

The CYP1A1, CYP2D6 and GSTM1 genes encode biotransforming enzymes involved in activation and detoxification of xenobiotics. Metabolically activated chemical compounds may interact with DNA and form adducts. In this study, the effect of the GSTM1, CYP1A1 exon 7 and CYP2D6 polymorphisms on DNA adduct levels was studied in 170 healthy volunteers. DNA adducts levels were measured by 32P-postlabelling in mononuclear white blood cells (WBC, lymphocytes and monocytes) and granulocytes collected in summer and winter. The influence of the genotype on the level of DNA adducts in both types of WBCs was observed only in summer samples. Individuals with GSTM1 deficient (null) genotype had significantly elevated level of adducts in mononuclear WBCs (p = 0.045) and granulocytes (p = 0.031) compared to GSTM1 positives. Higher adduct levels in carriers of combined GSTM1(null)/CYP1A1-Ile/Val genotype were found in both types of WBCs when compared to GSTM1(+)/CYP1A1-Ile/Ile genotype carriers (p = 0.046 in granulocytes, p = 0.092 in mononuclear WBCs). CYP2D6 wild-type homozygotes (EMs) and heterozygotes (HEMs) were shown to have significantly higher mononuclear WBC DNA adduct levels than mutant homozygotes (PMs) (p = 0.037 and p = 0.014). When confounding factors associated with PAH exposure were taken into account a statistically significant effect of CYP1A1 exon 7 polymorphism on DNA adduct levels was found (p = 0.012 in mononuclear WBCs, p = 0.043 in granulocytes). In a subgroup of current smokers (n = 95) high DNA adduct levels in granulocytes were associated with GSTM1(null) genotype, and increased adduct levels in mononuclear WBCs correlated with CYP2D6 EM and HEM genotypes. In winter samples the association between the genotype and DNA adduct levels was not observed.     

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.     

Trace elements in human scalp hair and soil in Irian Jaya

Benzo[a]pyrene (BaP) and other polycyclic aromatic hydrocarbons (PAHs) which are present in cigarette smoke, are common air and food genotoxic contaminants and possible human carcinogens. We measured the following PAH levels: benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, BaP, dibenzo[a,h]anthracene, benzo[g,h,i]perylene as well as (+/-) syn and anti BaP diol-epoxide (BPDE) DNA adducts in autopsy samples from the lungs of non-smokers, ex-smokers and smokers who had lived in Florence, Italy. PAH levels in lung tissue were similar in all groups, with the exception of dibenzo[a,h]anthracene (DBA), which was higher in lung samples from smokers (n = 10, 0.18+/-0.17 ng/g d.w, mean +/- S.D.) compared to non-smokers (n = 15, 0.046+/-0.025 ng/g d.w) (P < 0.05), whereas ex-smokers (n = 5), had intermediate levels (0.07+/-0.03 ng/g d.w). The average level of total BPDE-DNA adducts was 4.46+/-5.76 per 10(8) bases in smokers, 4.04+/-2.37 per 10(8) in ex-smokers and 1.76+/-1.69 per 10(8) in non-smokers. The levels of non-smokers were significantly different (P < 0.05) from the levels of the smokers and ex-smokers combined. Total BPDE-DNA adducts were correlated with BaP levels in the lung samples in which both determinations were obtained (r = 0.63). Our results demonstrate that the biological load of PAHs due to environmental pollution is similar in individuals who smoke and those who do not, but BPDE-DNA adducts are higher in smokers and ex-smokers compared to non-smokers. This study further confirms the usefulness of BPDE-DNA adduct levels determination in the lungs from autopsy samples for monitoring long-term human exposure to BaP, a representative PAH.     

Impact of inherited polymorphisms in glutathione S-transferase M1, microsomal epoxide hydrolase, cytochrome P450 enzymes on DNA, and blood protein adducts of benzo(a)pyrene-diolepoxide

The benzo(a)pyrene (BaP) metabolite benzo(a)pyrenediolepoxide (BPDE) is strongly implicated as a causative agent of lung cancer. To assess the risk of exposure to BaP, we made a combined analysis of levels of BPDE adducts to hemoglobin (Hb), serum albumin (SA), and lymphocyte DNA in 44 patients with incident lung cancer, as a prototype of a population mainly exposed to tobacco-derived BaP. We also investigated whether genetic polymorphisms of cytochrome P450IA1 (CYPIA1), microsomal epoxide hydrolase (mEH), and glutathione S-transferase M1 (GSTM1), which are involved in BaP metabolism, can be determinants of adduct formation. BPDE-Hb, BPDE-SA, and BPDE-DNA adducts were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography-negative ion chemical ionization-mass spectrometry to achieve high specificity and sensitivity. Individuals with detectable Hb adducts were positive for SA adducts but not vice versa, suggesting that BPDE-Hb adducts are less informative indicators of BaP exposure. Using PCR methods on DNA, we characterized GSTM1 deletion, CYPIA1 MspI and exon 7 valine variants, and mEH polymorphisms at amino acid positions 113 (EH3) and 139 (EH4). Levels of BPDE adducts were no different among CYPIA1, mEH, and GSTM1 genotypes. However, individuals with measurable BPDE-SA adducts were CYPIA1 variant carriers more frequently (P = 0.03). There was a slightly higher percentage of DNA detectable adducts in subjects with CYPIA1 exon 7 valine polymorphism. When subjects were classified by both polymorphisms on the mEH gene, those with two slow alleles (EH3 homozygous mutated) and no fast alleles (EH4 homozygous wild type) had a lower frequency of BPDE-SA adducts and no DNA adducts (P = 0.06). These results are based on a small number of observations thus far, but this exploratory study suggests that CYPIA1 and mEH variants might have an impact on BPDE exposure markers such as BPDE-SA adducts. Chemical specificity in adduct measurements is important to identify the biomarkers that reflect BaP exposure more accurately.     

Using 3'' untranslated sequences to identify differentially expressed genes in Leishmania

Because garbage collectors work in the street, they are exposed to polycyclic aromatic hydrocarbons (PAHs) in motor vehicle exhaust gas as they work. Urinary 1-hydroxypyrene (1-OH-pyrene) began to be used as a biological monitoring index for human exposure to high concentrations of PAHs. The objective of this study was to examine the applicability of urinary 1-OH-pyrene as a biological monitoring index for human low-level PAH exposure, such as the PAH exposure experienced while working in the street. The subjects were fifteen male garbage collectors. We measured individual exposure to PAHs, urinary 1-OH-pyrene concentrations and urinary cotinine concentrations. Individual air samplers were attached to the collar of the clothing of five workers to capture PAHs. Urine samples were collected before work, around noon and after finishing the day's work. In all, five PAH samples and 45 urine samples were collected. As control data, we analyzed the urinary 1-OH-pyrene and urinary cotinine levels of six smoking and four non-smoking control subjects who were not occupationally exposed to PAHs. The benzo[a]pyrene level in the air sampled for 5-6 h was 2.5-10.5 ng/m3, and the pyrene level as 10.3-70.3 ng/m3. These levels were similar to those in the vicinity of streets in Japan. A positive correlation between total PAH levels and the pyrene levels was observed. The average urinary 1-OH-pyrene level of the smokers was 0.21 +/- 0.13 mumol/mol creatinine, vs. 0.15 +/- 0.11 mumol/mol creatinine in the non-smokers. The urinary 1-OH-pyrene level obtained in this study was slightly higher than in the control group. No correlation was found between pyrene exposure and the urinary 1-OH-pyrene level of the five workers who wore the personal samplers. A significant positive correlation was observed between the urinary 1-OH-pyrene level and urinary cotinine level of the smokers. A significant positive correlation was also observed between the urinary 1-OH-pyrene and urinary cotinine levels of the control group smokers. In conclusion, urinary 1-OH-pyrene is not applicable for biological monitoring of extremely low levels of exposure to PAHs, as in the case of working in the street. Caution is required to exclude the effects of smoking when evaluating PAH exposure.     

In vivo formation of mutagens by intraperitoneal administration of polycyclic aromatic hydrocarbons in animals during exposure to nitrogen dioxide

Consumption of fossil fuels has increased indoor and outdoor concentrations of polycyclic aromatic hydrocarbons (PAHs) and nitrogen dioxide (NO2). To study the combined effect of PAH administration and NO2 exposure on mutagenicity of urine from animals we injected 400 mg/kg body wt i.p. one of five kinds of PAH (pyrene, fluoranthene, fluorene, anthracene and chrysene) into ICR mice, Wistar rats, Syrian golden hamsters or Hartley guinea pigs after exposure to 20 p.p.m. NO2 gas for 24 h and then exposed the animals to NO2 gas for an additional 24 h. During the latter 24 h we collected the urine and assayed its mutagenicity with the Ames Salmonella strains after treatment with beta-glucuronidase and arylsulfatase and extraction with dichloromethane. The urine from mice treated with both PAH and NO2 showed high mutagenicity for Salmonella typhimurium strains TA98 and TA100, whereas the urine from mice treated with PAH and air showed almost no mutagenic activity. The mutagenicity was decreased in nitroreductase- and acetyltransferase-deficient strains TA98NR and TA98/1,8-DNP6 respectively. Treatment with a mixture of 20% of each of the five kinds of PAH and NO2 augmented the urinary mutagenicity of mice 1.5-fold. The urine from hamsters treated with pyrene or fluoranthene and NO2 was also highly mutagenic, but that from rats or guinea pigs was not very mutagenic. The mutagenicity was also decreased in strains TA98NR and TA98/1,8-DNP6. These results suggest that the urine contains nitro compounds and that the nitration of PAHs occurs in the body of animals under exposure to NO2 gas. Actually, the nitrated metabolites of pyrene, 1-nitro-6/8-hydroxypyrene and 1-nitro-3-hydroxypyrene, were detected in the urine from mice treated with pyrene under exposure to NO2 gas. To elucidate the mechanism of in vivo nitration, NO2 (20 p.p.m.) was bubbled through 50 mM Tris-HCl buffer (pH 7.4) or dichloromethane solution containing pyrene or 1-hydroxypyrene (10 microg/ml). Pyrene was not nitrated by NO2 in either aqueous or organic solutions. However, 1-hydroxypyrene was changed to nitrohydroxypyrenes by NO2 in the Tris-HCl buffer, but not in the organic solution. Ascorbic acid, alpha-tocopherol, glutathione oleic acid and hemoglobin were found to inhibit the nitration of 1-hydroxypyrene in aqueous solution. The urinary mutagenicity of mice treated with both pyrene and NO2 was also decreased by oral administration of ascorbic acid and alpha-tocopherol. These results suggest that 1-hydroxypyrene is nitrated by an ionic reaction in the animal body after hydroxylation of pyrene in the liver.     

Changes in birth weight, birth length and head circumference of Hungarian children in the county Baranya between 1968 and 1979-1981

Wildland (forest) firefighters are exposed to a wide range of carcinogenic polycyclic aromatic hydrocarbons (PAH) in forest fire smoke. PAH undergo metabolic activation and can subsequently bind to DNA. In this study, we investigated the association between occupational and dietary PAH exposures and the formation of WBC PAH-DNA adducts in a population of wildland firefighters. An enzyme-linked immunosorbent assay using an antiserum elicited against benzo(a)pyrene-modified DNA was used to measure PAH-DNA adducts in WBC obtained from 47 California firefighters at two time points, early and late in the 1988 forest fire season. PAH-DNA adduct levels were not associated with cumulative hours of recent firefighting activity. However, firefighters who consumed charbroiled food within the previous week had elevated PAH-DNA adduct levels, which were related to frequency of charbroiled food intake. These findings suggest that dietary sources of PAH contribute to PAH-DNA adduct levels in peripheral WBC and should be evaluated when using this assay to assess occupational and environmental PAH exposure.     

Coal tar residues produce both DNA adducts and oxidative DNA damage in human mammary epithelial cells

In the present study we compare the metabolic activation of coal tar, as measured by the production of both DNA adducts and oxidative DNA damage, with that of a single carcinogen that is a constituent of this complex mixture in human mammary epithelial cells (HMEC). We find that a significant level of DNA adducts, detected by 32P-postlabeling, are formed in HMEC following exposure to coal tar residues. This treatment also results in the generation of high levels of oxidative DNA damage, as measured by the production of one type of oxidative base modification, thymine glycols. The amounts of both DNA adducts and thymine varied considerably between the various coal tar residues and did not correlate with either the total amount of polycyclic aromatic hydrocarbons (PAH) or the amount of benzo[a]pyrene (B[a]P) present in the residue. Fractionating the residue from one of the sites by sequential extraction with organic solvents indicated that while the ability to produce both types of DNA damage was contained mostly in a hexane-soluble fraction, a benzene-soluble fraction produced high levels of reactive oxygens relative to the number of total DNA adducts. We find that the total amount of PAH or B[a]P present in the coal tars from the various sites was not a predictor of the level of total DNA damage formed.     

Aromatic DNA adducts in human white blood cells and skin after dermal application of coal tar

A group of eczema patients topically treated with coal tar (CT) ointments was used as a model population to examine the applicability of DNA adducts in WBC subpopulations as a measure of dermal exposure to polycyclic aromatic hydrocarbons (PAHs). Aromatic DNA adducts were examined by 32P-postlabeling in exposed skin and WBC subsets, and urinary excretion of PAH metabolites was determined to assess the whole-body burden. The median urinary excretion of 1-hydroxypyrene and 3-hydroxybenzo(a)pyrene was 0.39 (range, 0.12-1.57 micromol/mol creatinine) and 0.01 micromol/mol creatinine (range, <0.01-0.04 micromol/mol creatinine), respectively, before the dermal application of CT ointments. After treatment for 1 week, these levels increased to 139.7 (range, 26.0-510.5 micromol/mol creatinine) and 1.18 micromol/mol creatinine (range, <0.01-2.14 micromol/mol creatinine), respectively, indicating that considerable amounts of PAHs were absorbed. Median aromatic DNA adduct levels were significantly increased in skin from 2.9 adducts/10(8) nucleotides (nt; range, 0.7-10.0 adducts/10(8) nt) before treatment to 63.3 adducts/10(8) nt (range, 10.9-276.2 adducts/10(8) nt) after treatment with CT, in monocytes from 0.28 (range, 0.25-0.81 adducts/10(8) nt) to 0.86 adducts/10(8) nt (range, 0.56-1.90 adducts/10(8) nt), in lymphocytes from 0.33 (range, 0.25-0.89 adducts/10(8) nt) to 0.89 adducts/10(8) nt (range, 0.25-3.01 adducts/10(8) nt), and in granulocytes from 0.28 (range, 0.25-0.67 adducts/10(8) nt) to 0.54 adducts/10(8) nt (range, 0.25-1.58 adducts/10(8) nt). A week after stopping the CT treatment, the DNA adduct levels in monocytes and granulocytes were reduced to 0.38 (range, 0.25-0.71 adducts/10(8) nt) and 0.38 adducts/10(8) nt (range, 0.25-1.01 adducts/10(8) nt), respectively, whereas the adduct levels in lymphocytes remained enhanced [1.59 adducts/10(8) nt (range, 0.25-2.40 adducts/10(8) nt)]. Although the adduct profiles in skin and WBC subsets were not identical, and the adduct levels in WBCs were significantly lower as compared with those in skin, the total DNA adduct levels in skin correlated significantly with the adduct levels in monocytes and lymphocytes, but not with those in granulocytes. Excretion of urinary metabolites during the first week of treatment was correlated with the percentage of the skin surface treated with CT ointment and decreased to background levels within a week after the cessation of treatment. 3-Hydroxybenzo(a)pyrene excretion, but not that of 1-hydroxypyrene, correlated significantly with the levels of DNA adducts in skin that comigrated with benzo(a)pyrene-diol-epoxide-DNA. This study indicates that the DNA adduct levels in mononuclear WBCs can possibly be used as a surrogate for skin DNA after dermal exposure to PAHs.     

Anti-benzo[a]pyrene diolepoxide--DNA adduct levels in peripheral mononuclear cells from coke oven workers and the enhancing effect of smoking

The level of (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) bound to DNA of lymphocytes plus monocytes in 39 coke oven workers exposed to polycyclic aromatic hydrocarbons (PAH) and 39 non-exposed persons (controls) were investigated, each of the groups consisting of smokers and non-smokers. The adduct level was measured by an improved HPLC/fluorescence method (Rojas, M., Alexandrov, K., van Schooten, F. J., Hillebrand, M., Kriek, E. and Bartsch, H., Carcinogenesis, 15, 557-560, 1994) through the release of the corresponding benzo[a]pyrene (B[a]P) tetrols. The anti-BPDE-DNA adduct was detected in 51% of coke oven workers exposed to PAH and in 18% of the non-exposed (control) subjects. The mean level of anti-BPDE-DNA adducts/10(8) nucleotides in coke oven workers (15.7 +/- 37.8) was approximately 8 times higher than in non-exposed subjects (2.0 +/- 8.7). The interindividual variation of adduct levels was approximately 100-fold in coke oven workers and approximately 50-fold in controls respectively. Smokers in the exposed group had 3.5 times more DNA adducts than non-smokers. With the exception of one non-smoker with very high adduct levels (52.8 adducts/10(8)), the control subjects showed the presence of barely detectable adducts in only 16% of the samples examined. The increased in vivo formation in some smokers and high variability of anti-BPDE-DNA adducts in coke oven workers suggests variations in genetically controlled activation/inactivation reactions of PAH metabolism.     

Inhibition of CYP1A1-dependent activity by the polynuclear aromatic hydrocarbon (PAH) fluoranthene

Polynuclear aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants, and recently bioassay-based induction studies have been used to determine exposures to complex mixtures of PAHs. Induction of CYP1A1-dependent activity in H4IIE rat hepatoma cells has been used extensively as a bioassay for halogenated aromatic hydrocarbons and more recently for PAHs. Fluoranthene (FL) is a prevalent PAH contaminant in diverse environmental samples, and FL did not induce CYP1A1-dependent ethoxyresorufin O-deethylase (EROD) activity significantly in H4IIE cells. However, in cells cotreated with 2 x 10(-5) M FL plus the potent inducers 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[k]fluoranthene (BkF) (2 x 10(-8) M), there was a significant decrease in EROD activities. Furthermore, treatment of TCDD-induced rat microsomes with FL caused an 80% decrease in EROD activity. Studies showed that FL did not affect induction of CYP1A1 protein or mRNA levels in H4IIE cells, and analysis of enzyme inhibition data using microsomal CYP1A1 indicated that FL noncompetitively inhibited CYP1A1-dependent activity. 32P-Postlabeling revealed no significant FL-DNA adduct formation in H4IIE cells treated with FL. However, in cells cotreated with FL plus BkF or benzo[a]pyrene (BaP), certain PAH-DNA adducts were induced 2-fold. This study demonstrated that FL is an inhibitor of CYP1A1-dependent enzyme activity in rat hepatoma H4IIE cells and that the genotoxic potency of some carcinogenic PAHs may be modulated by FL in mixtures containing relatively high levels of this compound.     

Comparison of immunoaffinity chromatography enrichment and nuclease P1 procedures for 32P-postlabelling analysis of PAH-DNA adducts

32P-postlabelling analysis for detecting DNA adducts formed by polycyclic aromatic compounds is one of the most widely used techniques for assessing genotoxicity associated with these compounds. In cases where the formation of adducts is extremely low, a crucial step in the analysis is an enrichment procedure for adducts prior to the radiolabelling step. The nuclease P1 enhancement procedure is the most established and frequently used of these methods. An immunoaffinity procedure developed for class specific recognition for polycyclic aromatic hydrocarbon (PAH)-DNA adducts has therefore been compared with the nuclease P1 method for a range of DNA adducts formed by PAHs. The evaluation was carried out with skin DNA from mice treated topically with benzo[a]pyrene, 7,12-dimethylbenz[a]anthracene, 5-methylchrysene or chrysene. The immobilised antibody had the highest affinity for adducts structurally similar to the BPDE-I-deoxyguanosine adduct ([+/-]-N2-(7r,8t,9r-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene-1 0t-yl)-2'-deoxyguanosine) against which the antibody had been raised. Of the PAH-modified DNAs evaluated, the maximum adduct recovery was obtained for DNA containing the BPDE I-deoxyguanosine adduct. With DMBA-modified DNA, the profiles of adducts recovered from the column were similar when the column material was treated either with a digest of DMBA-modified DNA or with 32P-labelled DMBA adducts. I-compounds (endogenous adducts in tissue DNA of unexposed animals), which had similar chromatographic properties to PAH-DNA adducts, were not enriched by the immunoaffinity procedure. Compared to the simple nuclease P1 enhancement procedure, the immunoaffinity methods were lengthier and more labour intensive. Advantages of the immunoaffinity procedure include: specificity, allowing the selective detection of a certain class of adducts: efficient adduct enrichment, providing a viable alternative to other enrichment procedures; adequate sensitivity for model studies and the potential to purify adducts for further characterisation. However, as a general screen for detecting the formation of DNA adducts, the nuclease P1 procedure was viewed as the initial method of choice since it was capable of detecting a wider range of PAH-DNA adducts.     

Polycyclic aromatic hydrocarbon-DNA adducts in human placenta and modulation by CYP1A1 induction and genotype

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.     

Absence of the glutathione S-transferase M1 gene increases cytochrome P4501A2 activity among frequent consumers of cruciferous vegetables in a Caucasian population

The cancer protective effect of cruciferous vegetables has been attributed to induction of phase II enzymes. But cruciferous vegetables also induce cytochrome P4501A2 (CYP1A2), which catalyzes the metabolic activation of various procarcinogens, including aromatic amines in tobacco. Thus, frequent intake of cruciferous vegetables could also result in cancer-enhancing effects. GSTM1 is involved in the detoxification of various carcinogens, but it also enhances the excretion of isothiocyanates and possibly other enzyme inducers in cruciferous vegetables. We, therefore, hypothesized that GSTM1 null genotype might be associated with increased CYP1A2 activity among frequent consumers of cruciferous vegetables because the excretion of CYP1A2 inducers contained in these vegetables may be partially inhibited in the absence of the GSTM1 enzyme. Three hundred twenty-eight non-Hispanic white residents of Los Angeles County (265 males and 63 females) were genotyped for the presence or absence of GSTM1 alleles and phenotyped for CYP1A2 activity. Information on usual dietary habits was obtained from these subjects through in-person interviews. Among frequent (at least once a week) consumers of broccoli, GSTM1 null individuals exhibited a 21% higher geometric mean level of CYP1A2 activity relative to GSTM1 non-null individuals (5.24 versus 4.32, two-sided P = 0.01). No such difference was observed in subjects who consumed broccoli less frequently (two-sided P = 0.39). This interactive effect of GSTM1 genotype and vegetable intake on CYP1A2 activity also was observed when overall intake of the five cruciferous vegetables under study (broccoli, cabbage, cauliflower, Brussels sprouts, and mustard greens) was examined. Among weekly consumers of cruciferous vegetables, GSTM1 null individuals showed a 16% higher geometric mean level of CYP1A2 activity relative to GSTM1 non-null individuals (5.03 versus 4.33, two-sided P = 0.02), whereas no difference was evident among those who consumed cruciferous vegetables less frequently (two-sided P = 0.35). Our results suggest that cruciferous vegetables contain CYP1A2 inducers, which are deactivated in the presence of GSTM1.     

Effect of polycyclic aromatic hydrocarbons on the elimination kinetics of pyrene and the urinary excretion profile of 1-hydroxypyrene in the rat

Pyrene was chosen as a noncarcinogen model of polycyclic aromatic hydrocarbons (PAHs). Groups of male Wistar rats were dosed with pyrene and with mixture of pyrene and fluoranthene, pyrene and benz[a]anthracene, or pyrene, fluoranthene, and benz[a]anthracene at 20 mg/kg by intravenous or oral routes. Blood samples were taken at 0.25, 0.5, 1, 2, 3, 4, and 5 h after administration. The concentration of pyrene was determined by gas chromatography. The toxicokinetic parameters for pyrene were determined from the time course of blood concentration. A significant increase in the bioavailability of pyrene after treatment with other PAHs was observed. Urinary 1-hydroxypyrene excretion was analyzed after pretreatment with acenaphthene, naphthalene, chrysene, phenanthrene, benz[a]anthracene, and benzo[a]pyrene. The urine from rats was collected for 3 d and the concentration of 1-hydroxypyrene was determined using high-performance liquid chromatography (HPLC). Most compounds examined caused a decrease in the urinary excretion of the metabolite of pyrene.     

Effect of polycyclic aromatic hydrocarbons on somatic chromosomes of coal tar workers

The genotoxic effect of polycyclic aromatic hydrocarbons (PAHs) on somatic human chromosomes obtained from lymphocytes of 49 coal tar workers exposed to 26 micrograms/m3 benzo(a)pyrene, 16 mg/m3 benzene and 0.04 mg/m3 H2S in the ambient air, compared to equal numbers of matched controls breathing air containing 1 microgram/m3 benzo(a)pyrene, 1.5 mg/m3 benzene and 0.02 mg/m3 H2S, was investigated. The mitotic index (MI), chromosome aberrations (CAs), sister chromatid exchanges (SCEs) and satellite associations (SAs) were analysed. All the parameters showed a significant increase (p < 0.01 and 0.05) in the exposed individuals compared with the controls: viz MI, 4.59-7.92; CAs, 0.77-3.0; SCEs, 5.89-6.80; and SAs, 8.18-14.26. The occurrence of the DG type of satellite associations were highest and the 3D type lowest. The frequency of SCEs was highest in coal tar workers with an exposure period of 6-10 years. It is suggested that these results show PAH is genotoxic for humans.     

Exposure to polycyclic aromatic hydrocarbons in occupational versus urban environmental air

OBJECTIVES: To evaluate the balance between occupational and environmental exposure to suspended particulate matter (SPM) and polycyclic aromatic hydrocarbons (PAHs), comparison measurements were performed in a coal-fired power plant and the urban atmosphere from the town nearby. METHODS: The analysis of SPM for PAH content was done according to a high-performance liquid chromatography (HPLC)-based method. The microscopic assessment was performed using scanning electron microscopy (SEM) by silver coverage of the samples derived by air filter. RESULTS: Contrary to expectations, the results showed low levels of particle-bound PAHs in the occupational environment (< 1 ng benzo(a)pyrene/m3 air) and high levels in urban air (range 80-1250 ng benzo(a)pyrene/m3). The SPM collected from the power plant exhibited non-respirable characteristics (particles larger than 10 microm), whereas urban SPM almost exclusively contained respirable airborne particles (<3 microm). CONCLUSIONS: The PAH burden, combined with the enhanced probability of respiratory absorption, confers a much greater hazard potential to the urban SPM. Under these conditions, in areas or countries in which old technologies remain in use, occupational exposure to SPM containing PAHs might represent a severe underestimation of the total risk as it does not take into account the background air pollution.     

Growth and cuticular synthesis in Ascaris suum larvae during development from third to fourth stage in vitro

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.     

Genotoxicity of coke-oven and urban air particulate matter in in vitro acellular assays coupled with 32P-postlabeling and HPLC analysis of DNA adducts

This study is an in vitro part of the ongoing biomarker studies with population from a polluted region of Northern Bohemia and coke-oven workers from Czech and Slovak Republics. The aim of this study is to compare DNA adduct forming ability of chemical compound classes from both the urban and coke-oven extractable organic mass (EOM) of airborne particles. The crude extracts were fractionated into seven fractions by acid-base partitioning and silica gel column chromatography. In in vitro acellular assays we used calf thymus DNA (CT DNA) with oxidative (+S9) and reductive activation mediated by xanthine oxidase (+XO) under anaerobic conditions. Both the butanol and nuclease P1 versions of 32P-postlabeling for detection of bulky aromatic and/or hydrophobic adducts were used. The results showed that the spectra of major DNA adducts resulting from both the in vitro assays are within the fractions similar for both the urban and coke-oven samples. The highest DNA adduct levels with S9-activation were detected for the neutral aromatic fraction, followed by slightly polar and acidic fractions for both samples. With XO-mediated metabolism, the highest DNA adduct levels were detected for both the acidic fractions. Assuming additivity of compound activities, then the acidic fraction, which in the urban sample comprises a major portion of EOM mass (28%), may contain the greatest activity in both in vitro assays (39 and 69%, +S9 and +XO, respectively). In contrast, the aromatic fraction constituting only 8% of total urban EOM mass may account for comparable activity (34%) with organic acids. The highest DNA adduct forming activity of the coke-oven sample accounts for the aromatic fraction (82 and 63%, +S9 and +XO, respectively) that also contains the greatest portion of the total EOM (48%). To characterize some of the specific DNA adducts formed, we coupled TLC on 20x20 cm plates with HPLC analysis of 32P-postlabeled adducts. In both S9-treated samples of the aromatic fraction, we tentatively identified DNA adducts presumably diolepoxide-derived from: 9-hydroxy-benzo[a]pyrene (9-OH-B[a]P), benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide[+/-] (anti-BPDE), benzo[b,j,k]fluoranthenes (B[b]F, B[j]F, B[k]F), chrysene (CHRY), benz[a]-anthracene (B[a]A) and indeno[cd]pyrene (I[cd]P). These DNA adducts accounted for about 57% of total DNA adducts detected in both S9-treated samples of the aromatic fraction. DNA adducts of XO-treated samples were sensitive to nuclease P1 and HPLC profiles of the major adducts were markedly different from the major adducts of S9-treated samples. However, the combination of TLC and HPLC did not confirm the presence of DNA adducts derived from 1-nitropyrene (1 NP), 9-nitroanthracene (9 NA) and 3-nitrofluoranthene (3 NF) that were detected by GC-MS in the slightly polar fraction. We concluded that the chemical fractionation procedure facilitates the assessing of DNA adduct forming ability of different chemical compound classes. However, based on the results obtained with the whole extracts, it does not fulfil a task of the actual contribution of individual fractions within the activity of the whole extracts. Our results are the first in detecting of DNA adducts derived from urban air and coke-oven particulate matter.