Recoveries of DNA adducts of polycyclic aromatic hydrocarbons in the 32P-postlabelling assay

The ³²P-postlabelling assay for analysis of DNA adducts of chemicalcarcinogens has been applied in a large number of experimentalanimal and human studies. Most human studies have dealt withoccupational and environmental exposures to polycyclic aromatichydrocarbons (PAHs). The postlabelling assay does not allowdirect chemical identification, and most studies with this methodhave not been performed in a quantitative way. Very little istherefore known about the identity and absolute levels of adducts,which are important contributors to the process of risk identificationand quantitation. In the present study it was, therefore, decidedto test some parameters suspected to affect recoveries of adductsin the phosphorylation step of the assay. For this purpose 12different PAHs were reacted individually and in a mixture withDNA in the presence of a rat liver S9 metabolizing system. Differentconcentrations of ATP, calcium chloride and polynucleotide kinasewere tested using the nuclease P1 enhancement. We found thateach factor contributed to adduct recovery and that optimalconditions could be defined. Diluting the modified DNA samplesup to 1000 times had little influence on the recoveries of adducts.Comparing the nuclease P1 and the butanol extraction proceduresfor adduct purification showed that both methods gave similarpaterns and levels of major adducts. The absolute recoveriesin postlabelling, based on 3H-binding of radiolabelled compounds,were for most of the tested compounds relatively low. The factthat the nuclease P1 and the butanol extraction procedures gavesimilar recoveries points towards common factor(s) involvedin the reduction of the recovered adduct levels. Based on theobserved recoveries the conclusion can be drawn that when postlabellingrelated adducts in human samples the true total adduct levelscan be considerably underestimated, even if optimal conditionsare used.     

DNA adducts in urothelial cells: Relationship with biomarkers of exposure to arylamines and polycyclic aromatic hydrocarbons from tobacco smoke

Markers of exposure to polycyclic aromatic hydrocarbons (urinary 1-hydroxypyrene-glucuronide) and aromatic amines (4-aminobiphenyl-hemoglobin adducts), as well as urinary mutagenicity, were measured in 47 healthy smokers and 50 non-smokers. DNA adducts were determined by P32-postlabeling in the exfoliated bladder cells of 39 healthy subjects. Both 1-hydroxypyrene-glucuronide (1-OHPG) and 4-aminobiphenyl adducts (4-ABP-Hb) were associated with smoking habits, but only 4-ABP-Hb adducts were associated with consumption of black, air-cured tobacco. The levels of 2 DNA adducts (numbers 2 and 4) in urothelial cells were clearly associated with 4-ABP-Hb adducts, in all subjects and in smokers. Levels of one of these DNA adducts (number 2) were also associated with 1-hydroxypyrene-glucuronide in urines, but in smokers the association was not statistically significant. Overall, these observations constitute further evidence of a role of arylamines in tobacco-induced bladder cancer. © 1996 Wiley-Liss, Inc.     

32P-HPLC suitable for characterization of DNA adducts formed in vitro by polycyclic aromatic hydrocarbons and derivatives

Analysis of DNA adducts demands both high sensitivity and goodresolution. A high-performance liquid chromato-graphy methodfor ³²P-postlabeled DNA adducts (³²P-HPLC) was used to investigateDNA adduct formation from 38 polycyclic hydrocarbons and biphenylsin vitro. The ³²P-HPLC method proved to be useful for separation,detection and characterization of DNA adducts from most of thesubstances. The in vitro method used to form the DNA adducts,with calf thymus DNA, nucleotide 3'-phosphates and metabolicactivation through S-9 liver homo-genate, gave poor quantitativereproducibility. However, the results showed that the ³²P-HPLCmethod was suitable for characterizing DNA adducts from manysubstances. From 35 of the tested substances 365 DNA and nucleotide3'-phospate adducts were detected and characterized concerningretention times. Of the adducts, 171 were detected in DNA and39 of them from five substances were characterized concerningtarget nucleotides. The retention time library built can beused in future analyses of DNA with complex patterns of DNAadducts.     

Carcinogen--DNA adducts and gene mutation in foundry workers with low-level exposure to polycyclic aromatic hydrocarbons

Carcinogen-DNA adducts and somatic gene mutation at the hypoxanthineguanine phosphoribosyl transferase (HPRT) locus were evaluatedin peripheral leukocytes of workers in an iron foundry withexposure to benzo[a]pyrene (B[a]P) and other polycyclic aromatichydrocarbons (PAHs). During the two year study period, B[     

DNA adducts and personal air monitoring of carcinogenic polycyclic aromatic hydrocarbons in an environmentally exposed population

The effect of personal exposure to air pollution on DNA adductsin humans was analyzed in a group (n = 30) of women workingoutdoors uo to 8 h/day) as postal workers or gardeners in thecity of Teplice, Czech Republic (Northern Bohemia), where winterinversions may result in high levels of air pollution from coalcombustion. Ten of these women were followed up during the nextwinter season by repeated personal exposure monitoring and analysisof the DNA adducts in their white blood cells (in four samplingperiods). Personal exposure monitoring for respirable particles(<2.5 µm) was conducted for the 24 h period prior tocollection of blood and urine. Particle extracts were analyzedfor carcinogenic polycyclic aromatic hydrocatbons (PAH). Urinesamples were collected for cotinine analysis to control forexposure to tobacco smoke. DNA isolated from white blood cellswas analyzed by ³²P-postlabeling with the butanol enrichmentprocedure. There were 21 non-smokers and nine light smokersin the pilot study (November 1992) and only non-smokers in thefollow-up study (winter season 1993/94). In both studies highpersonal exposure variability between the individuals sampledon the same day was observed. In the pilot study we found asignificantly higher (P < 0.05), level of DNA adducts inthe 14 non-smoking women sampled on November 24, when theirexposure to carcinogenic PAH was also significantly higher (P< 0.05), compared with seven non-smoking women sampled onNovember 26. We also found a significant correlation (r = 0.541,P < 0.016) between individual exposure to carcinogenic PAHand DNA adducts for the group of non-smoking (n = 21). No significantdifference in DNA adduct levels was found between non-smokersand smokers. In the follow-up study, during one sampling periodthe ambient and personal air monitors exhibited a significantlyelevated exposure to respirable particles and carcinogenic PAH.Analyzing data from the follow-up study, a significant effectof personal exposure on DNA adduct levels and their relationshipwith short-term exposure to carcinogenic PAH was found. Theresults suggest that DNA adduct levels in white blood cellsreflect a short-term environmental exposure.     

Determination of polycyclic aromatic hydrocarbons in the urine, benzo(a)pyrene diol epoxide-DNA adducts in lymphocyte DNA, and antibodies to the adducts in sera from coke oven workers exposed to measured amounts of polycyclic aromatic hydrocarbons in the work atmosphere

Workers in coke oven plants have a higher incidence of lung cancer than the general population. They are exposed to a variety of chemicals, in particular the polycyclic aromatic hydrocarbons (PAH), including benzo(a)pyrene. To evaluate the genotoxic effects of PAH exposure, air samples and urine samples were analyzed for PAH by capillary gas chromatography and high-performance liquid chromatography, respectively. Since benzo(a)pyrene is activated to 7 beta,8 alpha-dihydroxy-(9 alpha,10 alpha)-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) and binds to DNA, we have used ultrasensitive enzymatic radioimmunoassay and synchronous fluorescence spectrophotometry to measure BPDE-DNA adducts in lymphocyte DNA. The results show that workers were exposed to high concentrations of atmospheric PAH. However, the mean PAH exposure levels are reduced 60% when the workers wore masks during work. When compared to exposure levels, the urinary excretion of PAH was relatively low. Approximately one-third of the workers had detectable putative BPDE-DNA adducts in lymphocytes by ultrasensitive enzymatic radioimmunoassay, and 10% of the samples had emission peaks at 379 nm by synchronous fluorescence spectrophotometry. The four most positive samples were the same in both of the assays. Antibodies to an epitope(s) on BPDE-DNA were found in the sera of approximately one-third of the workers. Detection of DNA adducts and antibodies to these adducts are internal indicators of exposure to benzo(a)pyrene.     

Albumin adducts of naphthalene metabolites as biomarkers of exposure to polycyclic aromatic hydrocarbons.

We investigated the utility of adducts formed by the reaction of the naphthalene metabolites naphthalene-1,2-oxide, 1,2-naphthoquinone (1,2-NPQ), and 1,4-naphthoquinone (1,4-NPQ) with serum albumin (Alb) as biomarkers of exposure to polycyclic aromatic hydrocarbons. Cysteinyl serum Alb adducts of 1,2- and 1,4-NPQ (1,2-NPQ-Alb and 1,4-NPQ-Alb, respectively) but not of naphthalene-1,2-oxide were detected in 28 coke oven workers and 22 controls from the steel industry of northern China. The median level of 1,2-NPQ-Alb in coke oven workers (76.6 pmol/g) was significantly higher than that observed in controls (44.9 pmol/g; P = 0.0027). However, the median level of 1,4-NPQ-Alb in exposed subjects was not significantly different from that of controls (48.6 versus 44.2 pmol/g; P = 0.296). Levels of 1,2-NPQ-Alb were significantly correlated with exposure category (controls, side and bottom workers, and top-of-oven workers) as well as with previously measured levels of urinary naphthalene, 1- and 2-naphthol, and 1-pyrenol in these subjects. Multiple linear regression analysis revealed that 35% of the variation in 1,2-NPQ-Alb could be explained by the work category and age. A negative relationship between 1,2-NPQ-Alb and age was observed, suggesting that cytochrome P450 c metabolism diminished with age at approximately 3%/year of life.     

Assessment of carcinogenicity, using PAH-DNA adducts, in workers exposed to polycyclic aromatic hydrocarbons

Introduction

Polycyclic aromatic hydrocarbons (PAH) are environmental contaminants that have been of interest in cancer research for a considerable length of time. DNA adduct formation is considered a marker and indicator for exposure to PAH. The aim of this study was to determine PAH-DNA adduct levels in peripheral blood lymphocytes and urine obtained from workers exposed to PAH, and to evaluate tobacco use, GSTM1 and GSTT1 as possible contributory risk factors.

Material and methods

Our study included a random sample of 66 workers exposed to PAH and 49 non-exposed workers.

Results

PAH-DNA adduct levels of exposed workers were lower than that of the non-exposed group (p<0.05). However, current smoking, GSTM1-negatives, and current smoking with GSTM1-negatives were more frequent in the non-exposed group. In addition, non-exposed workers reported exposure to PAH in their current jobs, as compared with the exposed group (p<0.001). Linear regression analysis identified the levels of benzo-[b]-fluoranthene in the working area as the only significant DNA adduct-forming risk factor (p=0.025).

Conclusion

Further research, with an appropriately large sample size, is highly recommended in measuring PAH-DNA adduct levels and evaluating their relationship with the different types of PAH.     

CARCINOGENSIS: Formation of DNA adducts by the co-mutagen norharman with aromatic amines

Norharman, widely distributed in our environment, is alone notmutagenic to Salmonella typhimurium TA98 and TA100 either withor without S9 mix, but becomes mutagenic to S. Typhimurium TA98with S9 mix when non-mutagenic aromatic amines like anilineor o- or m-toluidine are added. Thus norharman has been calleda ‘co-mutagen’. In the present study we examinedwhether or not DNA adducts are formed in DNA of S.typhimuriumTA98 by treatment with norharman and aromatic amines using ³²P-post-labelinganalysis under modified adduct intensification conditions. Whena sample of norharman (8 mg) and aniline (4 mg) was incubatedwith 4 ml of overnight culture of S.typhimurium TA98 in thepresence of 20 ml S9 mix for 6 h at 37°C, three adduct spotswere detected at a total relative adduct labeling (RAL) of 10.8± 2.27/10⁸ nucleotides. Under the same conditions, amixture of norharman (8 mg) and o-toluidine (4 mg) yielded threeadduct spots at a RAL of 3.74 ± 1.71/10⁸ nucleotides.With a combination of norharman and m-toluidine, a single adductspot was seen at a RAL of 0.04 ± 0.01/10⁸ nucleotides.In contrast, norharman with p-toluidine did not produce adductspots. Furthermore, neither norharman nor the aromatic aminesthemselves gave any evidence of adducts. Thus DNA adduct formationby norharman with aromatic amines correlates with the co-mutagenicaction of norharman in S.typhimurium TA98.     

Differential ability of Ambystoma tigrinum hepatic microsomes to produce mutagenic metabolites from polycyclic aromatic hydrocarbons and aromatic amines

A number of carcinogenic aromatic amines when activated by liver microsomes from a salamander, Ambystoma tigrinum, are mutagenic for Salmonella tester strains sensitive to frameshift mutagens. However, 2 polycyclic aromatic hydrocarbons (PAH) (benzo[a]pyrene (BaP) and perylene) that are rendered mutagenic by mammalian microsomes are not activated by Ambystoma mixed-function oxidases. BaP was chosen for study because it is a well-known environmental carcinogen; perylene, an isomer of BaP, has been implicated as an etiological agent in cutaneous neoplasia in Ambystoma. These results support the observation that amphibians are quite resistant to PAH carcinogenesis and suggest that aromatic amines may be more appropriate model carcinogens.     

Cancer initiation by polycyclic aromatic hydrocarbons results from formation of stable DNA adducts rather than apurinic sites.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants with high carcinogenic potencies that have been linked to the etiology of human cancers through their presence in cigarette smoke and environmental mixtures. They are metabolically activated in cells by cytochrome P450 enzymes and/or peroxidases to reactive intermediates that damage DNA. One pathway of activation forms dihydrodiol epoxides that covalently bind to exocyclic amino groups of purines in DNA to form stable adducts. Another pathway involves formation of radical cations that bind to the N7 or C8 of purines to form unstable adducts that depurinate to leave apurinic (AP) sites in DNA. In the present study the proportions of stable DNA adducts and AP sites formed by the carcinogenic PAHs dibenzo[a,l]-pyrene (DB[a,l]P), 7,12-dimethylbenz[a]anthracene (DMBA), and benzo[a]pyrene (B[a]P) have been investigated in a target tissue for carcinogenesis, mouse epidermis. After topical application of the PAHs on the skin of female SENCAR mice epidermal DNA was isolated and the formation of stable DNA adducts was measured by (33)P-postlabeling and HPLC analysis. AP sites in DNA were measured with an aldehyde reactive probe in a slot-blot assay. At both 4 and 24 h after exposure, DB[a,l]P formed significantly higher amounts of stable DNA adducts than DMBA, and B[a]P exhibited the lowest level of binding. In contrast, the number of AP sites present in mice treated with these PAHs was in the order: DMBA > B[a]P > DB[a,l]P. The level of AP sites was significantly lower than the level of stable adducts for each PAH. The most potent carcinogen, DB[a,l]P, induced the highest level of stable adducts and the lowest level of AP sites in epidermal DNA. These results indicate that stable DNA adducts rather than AP sites are responsible for tumor initiation by carcinogenic PAHs.     

Polyclonal antibodies to DNA modified with 4-nitroquinoline 1-oxide: application for the detection of 4-nitroquinoline 1-oxide-DNA adducts in vivo

Antibodies against 4-nitroquinoline 1-oxide (4NQO) adducts were elicited in rabbits immunized with 4NQO-modified DNA complexed with methylated bovine serum albumin. In enzyme-linked immunosorbent assay (ELISA), the antibodies could recognize either denatured or native 4NQO-modified DNA, but not unmodified DNA, DNA modified with other carcinogens or free 4NQO derivative. Modification levels as low as 5 mumol of adduct per one mole DNA nucleotide (5 adducts/10(6) nucleotides) can be easily detected by the competitive ELISA. Indirect immunofluorescence staining by anti 4NQO-DNA antibody indicated that the antibodies bound specifically to the nuclei of normal human skin fibroblast cells treated with 4NQO. The intensity of fluorescence was proportional to the dose of 4NQO used to treat the cells, and the fluorescence-positive cells could be detected after treatment with 0.25 microM 4NQO (which resulted in the formation of 10(4) adducts per cell). Applying the competitive ELISA to the quantitation of DNA-adducts in rats treated with 4NQO, it was confirmed that the sensitivity of immunochemical assays was equivalent to that of isotopic assays. These methods should be helpful in studies on the formation of adducts and their removal in cells and tissues.     

Dosimeters of human exposure to carcinogens: polycyclic aromatic hydrocarbon-macromolecular adducts

The metabolic activation of polycyclic aromatic hydrocarbons (PAH), for example benzo[a]pyrene, leads to the formation of carcinogen-macromolecular adducts. Methods that make it possible to detect low levels of these adducts in human peripheral blood samples should be useful in the dosimetry of human exposure to carcinogens. We demonstrated previously the usefulness of enzyme immunoassays and of synchronous fluorescence spectroscopy (SFS) for detecting and characterizing low levels of PAH-macromolecular adducts present in synthetic adduct mixtures. These methods have now been refined and applied to the analysis of samples of peripheral blood collected from occupationally exposed individuals (coke-oven workers) and from people attending smoking cessation clinics. The results of both immunoassays and SFS show the presence of benzo[a]pyrene diol epoxide (BPDE)-DNA, BPDE-haemoglobin and other putative PAH-macromolecular adducts in peripheral blood samples from certain individuals.     

Detection of antibodies to the benzo(a)pyrene diol epoxide-DNA adducts in sera from individuals exposed to low doses of polycyclic aromatic hydrocarbons.

The exposure to DNA reactive carcinogens is known to elicit a specific humoral immunological response, with the production of antibodies toward the carcinogen adducts. Consequently, the presence of circulating anti-carcinogen antibodies has been proposed as a marker of carcinogen exposure, and as a potential modulating factor in chemical carcinogenesis. In this work, the presence of serum antibodies to 7beta,8alpha-dihydroxy-9alpha10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-DNA (BPDE-DNA) adducts was determined in two groups of workers occupationally exposed to low doses of polycyclic aromatic hydrocarbons (PAHs), i.e. policemen (194 subjects) and workers in the aluminum industry (105 subjects). Specific anti BPDE-DNA antibodies were detected in 5.7% (11/194) of policemen and 13.3% (14/105) of aluminium industry workers. Among policemen, a small, not significant (p=0.09), prevalence of positives was observed in traffic wardens compared to office workers. A borderline significant (p=0.052) prevalence of positives was also observed in heavy smokers compared to light smokers among aluminium industry workers. These results basically support previous findings on the association between chronic exposure to polycyclic aromatic hydrocarbons and formation of anti-BPDE-DNA antibodies, even though such association appears to be weak, possibly biased by individual factors which are still largely unidentified.     

Synchronous fluorescence spectrophotometry of benzo[a]pyrene diol epoxide-DNA adducts in workers exposed to polycyclic aromatic hydrocarbons

Synchronous fluorescence spectrophotometry (SFS) for benzo[a]pyrene diol epoxide (BPDE)-DNA adducts detects adducts in lymphocyte DNA in some but not all individuals exposed occupationally to polycyclic aromatic hydrocarbons (PAHs). Individual differences in exposure, activation and/or DNA repair can be detected by SFS. Several in vitro and in vivo studies are currently being done to clarify the specificity and usability of SFS as a biomonitoring method for PAH exposure in the work environment.     

Haemoglobin adducts of aromatic amines in people exposed to cigarette smoke

In a population-based study in Turin, Italy, smokers of blond tobacco showed 4-aminobiphenyl (4-ABP) adduct levels some three times higher than nonsmoking subjects, and smokers of black tobacco showed levels about five times greater than nonsmokers. A dose-response relationship between the number of cigarettes smoked per day and 4-ABP adduct level was observed, but did not account for the higher adduct levels observed in smokers of black tobacco. Smoking-related increases in haemoglobin adducts were also observed for o-toluidine, p-toluidine, 2,4-dimethylaniline and 2-ethylaniline. Smoking subjects showed 3-aminobiphenyl adduct levels about 12 times greater than those of nonsmokers, who rarely showed a detectable level. This may indicate that there are fewer sources of 3-aminobiphenyl exposure not related to tobacco smoke. Smokers of black tobacco showed higher adduct levels than smokers of blond tobacco for 4-ABP, p-toluidine and 2,4-dimethylaniline.     

DNA adduct formation in human and mouse skin by mixtures of polycyclic aromatic hydrocarbons

32P-Postlabelling analysis has been used to detect the formation in vivo of DNA adducts by components of complex mixtures of polycyclic aromatic hydrocarbons (PAHs) in coal-tar, creosote, bitumen, juniper tar, used engine oils and fuel exhaust condensates. The presence of DNA adducts derived from these agents has been investigated in mouse skin, in human skin explants maintained in short-term organ culture and in human skin in vivo, and the formation of many different PAH-DNA adducts was observed. Similar levels and patterns of adducts were found in DNA from human skin to those in mouse skin, which is known to be susceptible to the carcinogenic activity of PAH mixtures, thus demonstrating the potential hazard to man of epidermal contact with these materials.     

Metabolism and DNA binding of polycyclic aromatic hydrocarbons by human diploid fibroblasts.

The metabolism of three carcinogenic polycyclic aromatic hydrocarbons was measured in mid-(population doubling levels of 20–39) and late-(population doubling levels > 55) passage cultures of WI-38 human diploid fibroblasts. Cultures of mid-and late-passage cells metabolized these hydrocarbons to both water- and organic solvent-soluble derivatives. In stationary cultures, the same amounts of benzo(a)pyrene (BP) and 3-methyl-cholanthrene were metabolized per cell, but late-passage cells metabolized only one-third as much 7,12-dimethylbenz(a)anthracene per cell as mid-passage cells. The DNA adducts formed from BP in WI-38 cells were examined by chromatography of enzyme-degraded DNA samples on Sephadex LH20 columns. On columns eluted with methanol-water gradients, the [³H]BP-deoxyribonucleoside adducts eluted in the same volume as the product peak isolated from DNA that had been reacted with 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydroBP. On columns eluted with borate-containing methanol-water gradients, 62% of the cellular products eluted in the same volume as the product peak isolated from DNA that had been reacted with (±) 7α,8β-dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydroBP (the anti-isomer) and 35% eluted in the same volume as a product peak isolated from DNA that had been reacted with (±) 7α,8β-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydroBP (the syn-isomer). Thus, more than one type of DNA adduct is formed in these cells, possibly through the reacticn of different isomers of this BP-diol-epoxide with DNA.