Benzo[a]pyrene enhances lipid peroxidation induced DNA damage in aorta of apolipoprotein E knockout mice

The genotoxic compound benzo[a]pyrene (B[a]P) enhances atherosclerotic plaque progression, possibly by inducing oxidative stress and subsequent lipid peroxidation (LPO). Since LPO plays a key role in atherosclerosis, stable LPO derived DNA modifications such as 1,N6-ethenodeoxy-adenosine (epsilondA) and 3,N4-ethenodeoxy-cytidine (epsilondC) may be useful biomarkers for in vivo oxidative stress. In this study, benzo[a]pyrene-diol-epoxide (BPDE)-DNA, epsilondA and epsilondC were determined by 32P-postlabelling in apolipoprotein E knockout (ApoE-KO) mice treated with 5mg/kg B[a]P by gavage. After 4 days, BPDE-DNA adduct levels were higher in aorta (10.8 +/- 1.4 adducts/10(8) nucleotides) than in lung (3.3 +/- 0.7, P < 0.05), which is a known target organ for B[a]P. Levels of epsilondA were higher in aorta of B[a]P-exposed animals than in unexposed controls (8.1 +/- 4.4 vs 3.4 +/- 2.1 adducts per 10(8) parent nucleotides, P < 0.05). On the other hand, epsilondC levels were not affected by B[a]P exposure. Serum low density lipoprotein (LDL) levels were lower in B[a]P-exposed mice than in controls (9.3 +/- 3.7 and 13.3 +/- 4.0mmol/l, respectively), whereas high density lipoprotein (HDL) levels were higher (1.4 +/- 1.6 and 0.4 +/- 0.3mmol/l, respectively). Consequently, a three-fold difference in the LDL/HDL ratio was observed (P = 0.001). epsilondA levels were positively related with plasma HDL concentrations (R = 0.68, P = 0.02), suggesting that the HDL mediated protection of the vessel wall against reactive lipid peroxides was reduced in B[a]P-exposed apoE-KO mice. Our observations show that direct as well as lipid peroxidation induced DNA damage is formed by B[a]P in aorta of apoE-KO mice, which may be involved in atherosclerotic plaque progression. This study further indicates that etheno-DNA adducts are useful biomarkers for in vivo oxidative stress in atherosclerosis.     

The relationship between biomarkers of oxidative DNA damage, polycyclic aromatic hydrocarbon DNA adducts, antioxidant status and genetic susceptibility following exposure to environmental air pollution in humans

Polycyclic aromatic hydrocarbons (PAHs) appear to be significant contributors to the genotoxicity and carcinogenicity of air pollution present in the urban environment for humans. Populations exposed to environmental air pollution show increased levels of PAH DNA adducts and it has been postulated that another contributing cause of carcinogenicity by environmental air pollution may be the production of reactive oxygen species following oxidative stress leading to oxidative DNA damage. The antioxidant status as well as the genetic profile of an individual should in theory govern the amount of protection afforded against the deleterious effects associated with exposure to environmental air pollution. In this study we investigated the formation of total PAH (bulky) and B[a]P DNA adducts following exposure of individuals to environmental air pollution in three metropolitan cities and the effect on endogenously derived oxidative DNA damage. Furthermore, the influence of antioxidant status (vitamin levels) and genetic susceptibility of individuals with regard to DNA damage was also investigated. There was no significant correlation for individuals between the levels of vitamin A, vitamin E, vitamin C and folate with M₁dG and 8-oxodG adducts as well as M₁dG adducts with total PAH (bulky) or B[a]P DNA adducts. The interesting finding from this study was the significant negative correlation between the level of 8-oxodG adducts and the level of total PAH (bulky) and B[a]P DNA adducts implying that the repair of oxidative DNA damage may be enhanced. This correlation was most significant for those individuals that were non smokers or those unexposed to environmental air pollution. Furthermore the significant inverse correlation between 8-oxodG and B[a]P DNA adducts was confined to individuals carrying the wild type genotype for both the GSTM1 and the GSTT1 gene (separately and interacting). This effect was not observed for individuals carrying the null variant.     

Alternative pathways of polycyclic aromatic hydrocarbons activation: the formation of polar DNA adducts.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants, and some are potent carcinogens in rodents. Carcinogenic PAHs are activated in the cells to metabolites that react with DNA to form covalent adducts. For most PAHs the reactive, electrophilic species which bind to DNA, are bay-region diol-epoxides. Application of 32P-postlabeling to PAH-DNA adducts analysis revealed that for some PAHs the adduct profiles generated in model systems are more complex and include adducts which are more polar than those formed by classic bay-region diol-epoxides. This minireview summaries the information gained on typical representatives of polar PAH-DNA adducts. Formation of triol-epoxide-DNA adducts was proposed for chrysene and a non-alterant PAH, benzo[b]fluoranthene (B[b]F). 5-OH-B[b]F, the precursor of B[b]F triol-epoxide, was found to be a potent tumor initiator in mouse skin. For planar PAHs such as dibenzanthracenes the possibility of bis-diol epoxide-DNA adducts formation was suggested. The most comprehensive data were obtained for dibenz[a,j]anthracene (DB[a,j]A). This hydrocarbon when applied to SENCAR mouse skin forms up to 23 species of adducts, most of which are polar. Among these polar adducts seven were identified as derived from DB[aj]A-3,4-10,11-bis-diol. Analysis of tumor-initiating activity showed, however, that this proximate metabolite was inactive in this respect. In contrast, an excellent correlation was observed between levels of less polar DNA adducts (i.e. those derived from bay-region diolepoxides) and skin tumor initiating activity of DB[a,j]A. Thus, while triol-epoxides seems to be involved in tumor initiating activity of the parent compound, non alterant B[b]F, the significance of bis-diol epoxide-DNA adducts, at least those derived from DB[aj]A, is minor.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part I: acellular assay

Acellular assay of calf thymus DNA ± rat liver microsomal S9 fraction coupled with ³²P-postlabelling was used to study the genotoxic potential of organic compounds bound onto PM10 particles collected in three European cities—Prague (CZ), Kosice (SK) and Sofia (BG) during summer and winter periods. B[a]P alone induced DNA adduct levels ranging from 4.8 to 768 adducts/10⁸ nucleotides in the concentration dependent manner. However, a mixture of 8 c-PAHs with equimolar doses of B[a]P induced 3.7–757 adducts/10⁸ nucleotides, thus suggesting the inhibition of DNA adduct forming activity by interaction among various PAHs. Comparison of DNA adduct levels induced by various EOMs indicates higher variability among seasons than among localities. DNA adduct levels for Prague collection site varied from 19 to 166 adducts/10⁸ nucleotides, for Kosice from 22 to 85 and for Sofia from 6 to 144 adducts/10⁸ nucleotides. Bioactivation with S9 microsomal fraction caused 2- to 7-fold increase in DNA adduct levels compared to −S9 samples, suggesting a crucial role of indirectly acting genotoxic EOM components, such as PAHs. We have demonstrated for the first time a significant positive correlation between B[a]P content in EOMs and total DNA adduct levels detected in the EOM treated samples (R = 0.83; p = 0.04). These results suggest that B[a]P content in EOM is an important factor for the total genotoxic potential of EOM and/or B[a]P is a good indicator of the presence of other genotoxic compounds causing DNA adducts. Even stronger correlation between the content of genotoxic compounds in EOMs and total DNA adduct levels detected (R = 0.94; p = 0.005) was found when eight c-PAHs were taken into the consideration. Our findings support a hypothesis that a relatively limited number of EOM components is responsible for a major part of its genotoxicity detectable as DNA adducts by ³²P-postlabelling.     

In vitro genotoxicity of PAH mixtures and organic extract from urban air particles part II: human cell lines

Principal aims of this study were at first, to find a relevant human derived cell line to investigate the genotoxic potential of PAH-containing complex mixtures and second, to use this cell system for the analysis of DNA adduct forming activity of organic compounds bound onto PM10 particles. Particles were collected by high volume air samplers during summer and winter periods in three European cities (Prague, Kosice, and Sofia), representing different levels of air pollution. The genotoxic potential of extractable organic matter (EOM) was compared with the genotoxic potential of individual carcinogenic polycyclic aromatic hydrocarbons (c-PAHs) as well as their artificial mixtures. Metabolically competent human hepatoma HepG2 cells, confluent cultures of human diploid lung fibroblasts (HEL), and the human monocytic leukemia cell line THP-1 were used as models. DNA adducts were analyzed by ³²P-postlabeling. The total DNA adduct levels induced in HepG2 cells after exposure to EOMs were higher than in HEL cells treated under the same conditions (15–190 versus 2–15 adducts/10⁸ nucleotides, in HepG2 and HEL cells, respectively). THP-1 cells exhibited the lowest DNA adduct forming activity induced by EOMs (1.5–3.7 adducts/10⁸ nucleotides). A direct correlation between total DNA adduct levels and c-PAH content in EOM was found for all EOMs in HepG2 cells incubated with 50 μg EOM/ml (R = 0.88; p = 0.0192). This correlation was even slightly stronger when B[a]P content in EOMs and B[a]P-like adduct spots were analyzed (R = 0.90; p = 0.016). As THP-1 cells possess a limited metabolic capacity for most c-PAHs to form DNA reactive intermediates and are also more susceptible to toxic effects of PAHs and various EOM components, this cell line seemed to be an inappropriate system for genotoxicity studies of PAH-containing complex mixtures. The seasonal variability of genotoxic potential of extracts was stronger than variability among the three localities studied. In HepG2 cells, the highest DNA adduct levels were induced by EOM collected in Prague in the winter period, followed by Sofia and Kosice. However, in the summer sampling period, the order was quite opposite: Kosice > Sofia > Prague. When the EOM content per m³ of air was taken into consideration in order to compare real exposures of humans to genotoxic compounds in all three localities, extracts from respirable dust particles collected in Sofia exhibited the highest genotoxicity regardless of the sampling period. The results indicate that most of DNA adducts detected in cells incubated with EOMs have their origin in low concentrations of c-PAHs representing 0.03–0.17% of EOM total mass. Finally, our results suggest that HepG2 cells have a metabolic capacity for PAHs similar to human hepatocytes and represent therefore the best in vitro model for investigating the genotoxic potential of complex mixtures containing PAHs among the three cell lines tested in this study.     

Differential repair of polycyclic aromatic hydrocarbon DNA adducts from an actively transcribed gene

Polycyclic aromatic hydrocarbons (PAHs) are carcinogens with varying potencies. These compounds are metabolized to diol epoxides that react to form DNA adducts. Nucleotide excision repair is a critical cellular defense against these bulky DNA adducts which, if not repaired, can lead to mutations and the initiation of cancer. The structural features of the PAH-adducts play a role in differential repair of these adducts by the global genomic repair subpathway of nucleotide excision repair. DNA adducts derived from the PAHs containing bay-regions are repaired more rapidly than adducts derived from PAHs containing fjord-regions. We have employed the host cell reactivation assay to examine the rate of repair of these adducts in an actively transcribing gene. The pGL3 plasmid containing a luciferase gene was damaged with diol epoxides of benzo[a]pyrene (B[a]P-DE), dibenzo[a,l]pyrene (DB[a,l]P-DE), benzo[g]chrysene (B[g]Ch-DE), and benzo[c]phenanthrene (B[c]Ph-DE). The plasmids were transfected into B-lymphocytes with normal repair capacity as well as lymphocytes derived from patients with the XP-A, XP-C and CS-B syndromes. We found that XPA cells were able to transcribe slowly past B[g]Ch-adducts but not the other PAHs. Using the amount of luciferase produced as a measure of DNA repair, we found that the relative rates of repair in the actively transcribing luciferase gene was B[a]P-DE > DB[a,l]P-DE, B[g]Ch-DE, >B[c]Ph-DE in repair proficient and XP-C cells. These results indicate that the abilities to transcribe past and to repair the PAH adducts are dependent on different structural features of the DNA adducts.     

H2AX phosphorylation in A549 cells induced by the bulky and stable DNA adducts of benzo[a]pyrene and dibenzo[a,l]pyrene diol epoxides

Early events in the cellular response to DNA damage, such as double strand breaks, rely on lesion recognition and activation of proteins involved in maintenance of genomic stability. One important component of this process is the phosphorylation of the histone variant H2AX. To investigate factors explaining the variation in carcinogenic potency between different categories of polycyclic aromatic hydrocarbons (PAHs), we have studied the phosphorylation of H2AX (H2AXγ). A549 cells were exposed to benzo[a]pyrene diol epoxide [(+)-anti-BPDE] (a bay-region PAH) and dibenzo[a,l]pyrene diol epoxide [(−)-anti-DBPDE] (a fjord-region PAH) and H2AXγ was studied using immunocytochemistry and Western blot. Hydrogen peroxide (H₂O₂) was used to induce oxidative DNA damage and strand breaks. As showed with single cell gel electrophoresis, neither of the diol epoxides resulted in DNA strand breaks relative to H₂O₂. Visualisation of H2AXγ formation demonstrated that the proportion of cells exhibiting H2AXγ staining at 1 h differed between BPDE, 40% followed by a decline, and DBPDE, <10% followed by an increase. With H₂O₂ treatment, almost all cells demonstrated H2AXγ at 1 h. Western blot analysis of the H2AXγ formation also showed concentration and time-dependent response patterns. The kinetics of H2AXγ formation correlated with the previously observed kinetics of elimination of BPDE and DBPDE adducts. Thus, the extent of H2AXγ formation and persistence was related to both the number of adducts and their structural features.     

Determinants of anti-benzo[a]pyrene diol epoxide-DNA adduct formation in lymphomonocytes of the general population

We evaluated determinants of anti-benzo[a]pyrenediolepoxide-(B[a]PDE)-DNA adduct formation (adduct induced by the ultimate carcinogenic metabolite of B[a]P) in lymphomonocytes of subjects environmentally exposed to low doses of polycyclic aromatic hydrocarbons (PAHs) (B[a]P). Our study population consisted of 585 Caucasian subjects, all municipal workers living in North-East Italy and recruited during their periodic check-ups after informed consent. PAH (B[a]P) exposure was assessed by questionnaire. Anti-B[a]PDE-DNA levels were measured by HPLC fluorescence analysis. We found that cigarette smoking (smokers (22%) versus non-smokers, p<0.0001), dietary intake of PAH-rich meals (> or =52 (38%) versus <52 times/year, p<0.0001), and outdoor exposure (> or =4 (19%) versus <4h/day; p=0.0115) significantly influenced adduct levels. Indoor exposure significantly increased the frequency of positive subjects (> or =0.5 adducts/10(8) nucleotides; chi(2) for linear trend, p=0.051). In linear multiple regression analysis the major determinants of increased DNA adduct levels (ln values) were smoking (t=6.362, p<0.0001) and diet (t=4.035, p<0.0001). In this statistical analysis, indoor and outdoor exposure like other factors of PAH exposure had no influence. In non-smokers, the influence of diet (p<0.0001) and high indoor exposure (p=0.016) on anti-B[a]PDE-DNA adduct formation became more evident, but not that of outdoor exposure, as was confirmed by linear multiple regression analysis (diet, t=3.997, p<0.0001 and high indoor exposure, t=2.522, p=0.012). This study indicates that anti-B[a]PDE-DNA adducts can be detected in the general population and are modulated by PAH (B[a]P) exposure not only with smoking - information already known from studies with limited number of subjects - but also with dietary habits and high indoor exposure. In non-smokers, these two factors are the principal determinants of DNA adduct formation. The information provided here seems to be important, since DNA adduct formation in surrogate tissue is an index of genotoxic exposure also in target organs (e.g., lung) and their increase may also be predictive of higher risk for PAH-related cancers.     

The effect of dibenzo[a,1]pyrene and benzo[a]pyrene on human diploid lung fibroblasts: the induction of DNA adducts, expression of p53 and p21(WAF1) proteins and cell cycle distribution

Polycyclic aromatic hydrocarbons (PAHs) present in ambient air are considered as potential human carcinogens, but the detailed mechanism of action is still unknown. Our aim was to study the in vitro effect of exposure to dibenzo[a,l]pyrene (DB[a,l]P), the most potent carcinogenic PAH ever tested, and benzo[a]pyrene (B[a]P) in a normal human diploid lung fibroblast cells (HEL) using multiple endpoints. DNA adduct levels were measured by 32P-postlabelling, the expression of p53 and p21(WAF1) proteins by western blotting and the cell cycle distribution by flow cytometry. For both PAHs, the DNA adduct formation was proportional to the time of exposure and dependent on the stage of cell growth in culture. DNA binding was detectable even at the lowest concentration used (24h exposure, 0.01 microM for both PAHs). The highest DNA adduct levels were observed after 24h of exposure in near-confluent cells (>90% of cells at G0/G1 phase), but DNA damage induced by DB[a,l]P was approximately 8-10 times higher at a concentration one order of magnitude lower as compared with B[a]P (for B[a]P at 1 microM and for DB[a,l]P at 0.1 microM: 237+/-107 and 2360+/-798 adducts/10(8) nucleotides, respectively). The induction of p53 and p21(WAF1) protein occurred subsequent to the induction of DNA adducts. The DNA adduct levels correlated with both p53 (R=0.832, P<0.001 and R=0.859, P<0.001, for DB[a,l]P and B[a]P, respectively) and p21(WAF1) levels (R=0.808, P<0.001 and R=0.797, P=0.001, for DB[a,l]P and B[a]P, respectively), regardless of the PAH exposure and the phase of cell growth. The results showed that a detectable increase of p53 and p21(WAF1) proteins (> or = 1.5-fold as compared with controls) requires a minimal DNA adduct level of approximately 200-250 adducts/10(8) nucleotides for both PAHs tested and suggest that the level of adducts rather than their structure triggers the p53 and p21(WAF1) responses. The cell cycle was altered after 12-16h of treatment, and after 24h of exposure to 0.1 microM DB[a,l]P in growing cells, there was approximately 24% increase in S phase cells accompanied by a decrease in G1 and G2/mitosis (G2/M) cells. Cell treatment with 1.0 microM B[a]P resulted in more subtle alterations. We conclude that DB[a,l]P, and to a lesser degree B[a]P, are able to induce DNA adducts as well as p53 and p21(WAF1) without eliciting G1 or G2/M arrests but rather an S phase delay/arrest. Whether the S phase delay observed in our study is beneficial for the survival of the cells remains to be further established.     

Comparison of the genotoxic activities of the K-region dihydrodiol of benzo[a]pyrene with benzo[a]pyrene in mammalian cells: morphological cell transformation; DNA damage; and stable covalent DNA adducts

Benzo[a]pyrene (B[a]P) is the most thoroughly studied polycyclic aromatic hydrocarbon (PAH). Many mechanisms have been suggested to explain its carcinogenic activity, yet many questions still remain. K-region dihydrodiols of PAHs are metabolic intermediates depending on the specific cytochrome P450 and had been thought to be detoxification products. However, K-region dihydrodiols of several PAHs have recently been shown to morphologically transform mouse embryo C3H10T1/2CL8 cells (C3H10T1/2 cells). Because K-region dihydrodiols are not metabolically formed from PAHs by C3H10T1/2 cells, these cells provide a useful tool to independently study the mechanisms of action of PAHs and their K-region dihydrodiols. Here, we compare the morphological cell transforming, DNA damaging, and DNA adducting activities of the K-region dihydrodiol of B[a]P, trans-B[a]P-4,5-diol with B[a]P. Both trans-B[a]P-4,5-diol and B[a]P morphologically transformed C3H10T1/2 cells by producing both Types II and III transformed foci. The morphological cell transforming and cytotoxicity dose response curves for trans-B[a]P-4,5-diol and B[a]P were indistinguishable. Since morphological cell transformation is strongly associated with mutation and/or larger scale DNA damage in C3H10T1/2 cells, the identification of DNA damage induced in these cells by trans-B[a]P-4,5-diol was sought. Both trans-B[a]P-4,5-diol and B[a]P exhibited significant DNA damaging activity without significant concurrent cytotoxicity using the comet assay, but with different dose responses and comet tail distributions. DNA adduct patterns from C3H10T1/2 cells were examined after trans-B[a]P-4,5-diol or B[a]P treatment using 32P-postlabeling techniques and improved TLC elution systems designed to separate polar DNA adducts. While B[a]P treatment produced one major DNA adduct identified as anti-trans-B[a]P-7,8-diol-9,10-epoxide-deoxyguanosine, no stable covalent DNA adducts were detected in the DNA of trans-B[a]P-4,5-diol-treated cells. In summary, this study provides evidence for the DNA damaging and morphological cell transforming activities of the K-region dihydrodiol of B[a]P, in the absence of covalent stable DNA adducts. While trans-B[a]P-4,5-diol and B[a]P both induce morphological cell transformation, their activities as DNA damaging agents differ, both qualitatively and quantitatively. In concert with the morphological cell transformation activities of other K-region dihydrodiols of PAHs, these data suggest a new mechanism/pathway for the morphological cell transforming activities of B[a]P and its metabolites.     

Survival and mutagenesis of bacterial plasmids with localized carcinogen adducts

Induction of 6-thioguanine (TG) resistance by chemical mutagens was examined in a line of cells derived from a human epithelial teratocarcinoma cell clone. The cells, designated as P3 cells, have a stable diploid karyotype with 46(XX) chromosomes, including a translocation between chromosomes 15 and 20. Efficient recovery of TG-resistant mutants induced by the direct-acting mutagens: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG); 7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10 -tetrahydrobenzo[a]pyrene (BPDE); and benzo[a]pyrene (B[a]P); activated in a cell-mediated assay, required an expression time of 7 days and a saturation density of 2 X 10(4) cells/60-mm petri dish. The TG-resistant mutant cells induced by MNNG and BPDE maintained their resistant phenotype 4-6 weeks after isolation. This mutant phenotype was associated with a more than 10-fold reduction in hypoxanthine-guanine phosphoribosyl transferase (HGPRT) activity relative to that of the parental P3 cell line, which was shown to catalyze the formation of 4.6 pmoles inosine-5'-monophosphate (IMP)/min/microgram protein. Induction of TG resistance was also observed in P3 cells cocultivated in a cell-mediated assay with human breast carcinoma cells, which are capable of polycyclic aromatic hydrocarbon (PAH) metabolism, after treatment with the carcinogenic PAHs: B[a]P, chrysene, 7,12-dimethylbenz[a]anthracene (DMBA), and 3-methylcholanthrene (MCA). The degree of mutant induction in this assay was related to the carcinogenic potency of these PAHs in experimental animals. The most potent mutagen was DMBA, followed in decreasing order by MCA, B[a]P, and chrysene. DMBA, at 0.4 microM, increased the frequency of mutants for TG resistance from 2 for the control to about 200 TG-resistant mutants/10(6) colony-forming cells (CFC). Benzo[e]pyrene (B[e]P) and pyrene, which are not carcinogenic, were not effective in the assay. None of the PAHs was mutagenic in the P3 cells cultivated in the absence of the PAH-metabolizing cells. These results indicate that the P3 cells can be useful for the study of mutagenesis at the HGPRT locus by direct-acting chemical mutagens, as well as by chemicals activated in a cell-mediated assay.     

A comparison of mutational specificity of mutations induced by S9-activated B[a]P and benzo[a]pyrene-7,8-diol-9,10-epoxide at the endogenous aprt gene in CHO cells

We have determined the mutational specificity of S9-activated benzo[a]pyrene (B[a]P) at the endogenous aprt locus in a hemizygous Chinese hamster ovary cell line. The aprt gene of recovered mutants was amplified using the polymerase chain reaction (PCR) and directly sequenced. This spectrum was then compared to mutations recovered following treatment with the B[a]P metabolite, benzo[a]pyrene diol-epoxide (BPDE). No significant difference between the two spectra in the types of mutations produced, or their distribution was observed. This observation supports the hypothesis that BPDE is the reactive metabolite of B[a]P, responsible for the significant biological effects caused by this ubiquitous polycyclic aromatic hydrocarbon. The major mutation recovered was the G:C-->T:A transversion, and mutations were primarily localized within runs of guanines. We also confirmed our previous finding that mutation by B[a]P is non-random, targeting events in runs of guanines flanked by adenine residues. This same target hotspot region is found in codon 61 of the human c-Ha-ras1 proto-oncogene. This may help explain the selective activation of this codon by BPDE.     

Benzo(a)pyrene diolepoxide-haemoglobin and albumin adducts at low levels of benzo(a)pyrene exposure

A biomonitoring study was conducted to simultaneously measure individual benzo(a)pyrene (BaP) exposure in 50 office employees, not occupationally exposed to polycyclic aromatic hydrocarbons (PAH), using personal samplers and the formation of (+) r-7, t-8-dihyroxy-t-9,t-10-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BPDE) adducts to haemoglobin (BPDE–Hb) and serum albumin (BPDE–SA). The population enrolled was exposed to an average of 0.58 ± 0.46 ng BaP m^?3 (mean ± SD). The concentration of BaP collected from smokers' samples was double that from non-smokers (P = 0.007). BPDE adducts to Hb and SA were quantified as BaP tetrols released from hydrolysis of macromolecules and measured by high-resolution gas chromatography–negative ion chemical ionization–mass spectrometry. BPDE–Hb adducts were detected in 16% of the population and BPDE–SA adducts in 28%. Smoking did not affect adduct formation. When BaP personal monitoring data were used as the criterion of exposure, no correlation was found with the presence and the levels of BPDE–Hb and BPDE–SA adducts. Undetected sources of PAH, such as the diet, might markedly alter the exposure profile depicted by individual air sampling and affect the frequency and levels of protein biomarkers. This is the first comparative analysis of BPDE–Hb and BPDE–SA adducts, providing reference values for these biomarkers in a general urban population. However it is difficult to establish which biomarkers would be the more relevant in assessing low BaP exposure, due to undetectable factors such as dietary PAHs, that might have influenced the results to some degree.     

Cytosine methylation in a CpG sequence leads to enhanced reactivity with Benzo[a]pyrene diol epoxide that correlates with a conformational change.

Benzo[a]pyrene (B[a]P) is a widespread environmental carcinogen that must be activated by cellular metabolism to a diol epoxide form (BPDE) before it reacts with DNA. It has recently been shown that BPDE preferentially modifies the guanine in methylated 5'-CpG-3' sequences in the human p53 gene, providing one explanation for why these sites are mutational hot spots. Using purified duplex oligonucleotides containing identical methylated and unmethylated CpG sequences, we show here that BPDE preferentially modified the guanine in hemimethylated or fully methylated CpG sequences, producing between 3- and 8-fold more modification at this site. Analysis of this reaction using shorter duplex oligonucleotides indicated that it was the level of the (+)-trans isomer that was specifically increased. To determine if there were conformational differences between the methylated and unmethylated B[a]P-modified DNA sequences that may be responsible for this enhanced reactivity, a native polyacrylamide gel electrophoresis analysis was carried out using DNA containing isomerically pure B[a]P-DNA adducts. These experiments showed that each adduct resulted in an altered gel mobility in duplex DNA but that only the presence of a (+)-trans isomer and a methylated C 5' to the adduct resulted in a significant gel mobility shift compared with the unmethylated case.     

Both replication bypass fidelity and repair efficiency influence the yield of mutations per target dose in intact mammalian cells induced by benzo[a]pyrene-diol-epoxide and dibenzo[a,l]pyrene-diol-epoxide

Mutations induced by polycyclic aromatic hydrocarbons (PAH) are expected to be produced when error-prone DNA replication occurs across unrepaired DNA lesions formed by reactive PAH metabolites such as diol epoxides. The mutagenicity of the two PAH-diol epoxides (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) and (+/-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DBPDE) was compared in nucleotide excision repair (NER) proficient and deficient hamster cell lines. We applied the (32)P-postlabelling assay to analyze adduct levels and the hprt gene mutation assay for monitoring mutations. It was found that the mutagenicity per target dose was 4 times higher for DBPDE compared to BPDE in NER proficient cells while in NER deficient cells, the mutagenicity per target dose was 1.4 times higher for BPDE. In order to investigate to what extent the mutagenicity of the different adducts in NER proficient cells was influenced by repair or replication bypass, we measured the overall NER incision rate, the rate of adduct removal, the rate of replication bypass and the frequency of induced recombination in the hprt gene. The results suggest that NER of BPDE lesions are 5 times more efficient than for DBPDE lesions, in NER proficient cells. However, DBPDE adducts block replication more efficiently and also induce 6 times more recombination events in the hprt gene than adducts of BPDE, suggesting that DBPDE adducts are, to a larger extent, bypassed by homologous recombination. The results obtained here indicate that the mutagenicity of PAH is influenced not only by NER, but also by replication bypass fidelity. This has been postulated earlier based on results using in vitro enzyme assays, but is now also being recognized in terms of forward mutations in intact mammalian cells.     

Short Communication: The effect of CYP1A1 induction on the formation of benzo a pyrene adducts in liver and lung DNA and plasma albumin in rats exposed to benzo a pyrene:adduct quantitation by immunoassay and an HPLC method

Induction of cytochrome P450 enzymes by exposure to polycyclic aromatic hydrocarbons (PAH) can result in both decreased or increased PAH adduct levels. The lung is a main target site for PAH-carcinogenesis. By HPLC determination of B[ a]P-r-7, t-8-dihydrodiol, t-9, 10-epoxide (BPDE-I)-DNA adducts in rat, the level of the ultimate carcinogenic B[a]P-metabolite was higher in lungs than in liver. However, measured by immunoassay, the total benzo[a]pyrene (B[a]P)-DNA adduct levels were higher in liver than in lungs. Induction of CYP1A1 in vivo in rat by repeated i.p. doses of methylcholanthrene (MC) prior to a single dose of B[a]P resulted in a 2.4 times increase in CYP1A1 activity in liver tissue and 1.5 times higher levelsof total B[a]P-DNA adducts in lung and liver compared with controls which only received B[a]P. Increased levels of BPDE-I-DNA adducts were significantly correlated to increased CYP1A1 activity in induced lung tissue but not in liver. The times to reach maximum adduct levels were similar for both controls and MC-induced rats in both lung and liver,and plasma albumin. The BPDE-I-albumin adducts reached a maximum level around 1 day after B[a]P exposure and could not be used as a reliable marker of the short term PAH exposure in this study.     

Short Communication: The effect of CYP1A1 induction on the formation of benzo a pyrene adducts in liver and lung DNA and plasma albumin in rats exposed to benzo a pyrene:adduct quantitation by immunoassay and an HPLC method

Induction of cytochrome P450 enzymes by exposure to polycyclic aromatic hydrocarbons (PAH) can result in both decreased or increased PAH adduct levels. The lung is a main target site for PAH-carcinogenesis. By HPLC determination of B [a]P-r-7, t-8-dihydrodiol, t-9, 10-epoxide (BPDE-I)-DNA adducts in rat, the level of the ultimate carcinogenic B[a]P-metabolite was higher in lungs than in liver. However, measured by immunoassay, the total benzo[a]pyrene (B[a]P)-DNA adduct levels were higher in liver than in lungs. Induction of CYP1A1 in vivo in rat by repeated i.p. doses of methylcholanthrene (MC) prior to a single dose of B[a]P resulted in a 2.4 times increase in CYP1A1 activity in liver tissue and 1.5 times higher levelsof total B[a]P-DNA adducts in lung and liver compared with controls which only received B[a]P. Increased levels of BPDE-I-DNA adducts were significantly correlated to increased CYP1A1 activity in induced lung tissue but not in liver. The times to reach maximum adduct levels were similar for both controls and MC-induced rats in both lung and liver,and plasma albumin. The BPDE-I-albumin adducts reached a maximum level around 1 day after B[a]P exposure and could not be used as a reliable marker of the short term PAH exposure in this study.     

Repair of DNA lesions: mechanisms and relative repair efficiencies

DNA is frequently damaged by endogenous agents inside the cells. Some exogenous agents such as polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment and may thus contribute to the 'background' DNA damage in humans. DNA lesions are normally removed by various repair mechanisms. The major repair mechanisms for various DNA lesions are summarized. In contrast to the extensively studied repair mechanisms, much less is known about the relative repair efficiencies of various DNA lesions. Since DNA repair is a crucial defense against carcinogenesis, it may constitute an important factor affecting the carcinogenicity of DNA damaging agents. We have adopted a human cell-free system for measuring relative DNA repair efficiencies based on the concept of repair competition between acetylaminofluorene adducts and other DNA lesions of interest. Using this in vitro system, we determined the relative repair efficiencies of PAH adducts induced by: anti-(+/-)-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide (BPDE), anti-(+/-)-benz[a]anthracene-trans-3,4-dihydrodiol-1,2-epoxide (BADE-I), anti-(+/-)-benz[a]anthracene-trans-8,9-dihydrodiol-10, 11-epoxide (BADE-II), anti-(+/-)-benzo[b]fluoranthene-trans-9, 10-dihydrodiol-11,12-epoxide (BFDE), anti-(+/-)-chrysene-trans-1, 2-dihydrodiol-3,4-epoxide (CDE), and anti-(+/-)-dibenzo[a, l]pyrene-trans-11,12-dihydrodiol-13,14-epoxide (DBPDE). While damage by BPDE, DBPDE, CDE, and BFDE were repaired by nucleotide excision repair as efficiently as AAF adducts, the repair of BADE-I and BADE-II adducts were significantly slower in human cell extracts. Damage by DBPDE at 3 microM in vitro yielded approximately 5-fold higher DNA adducts than BPDE as determined by quantitative PCR. This potent DNA reactivity may account in part for the potent carcinogenicity of dibenzo[a,l]pyrene. The correlation of these results to the carcinogenic properties of the PAH compounds is discussed. Furthermore, we show that NER plays a role in AP site repair in vivo in the eukaryotic model organism yeast.