PDE-DNA Adducts Research Articles

The effects of everyday-life exposure to polycyclic aromatic hydrocarbons on biological age indicators

BackgroundFurther knowledge on modifiable aging risk factors is required to mitigate the increasing burden of age-related diseases in a rapidly growing global demographic of elderly individuals. We explored the effect of everyday exposure to polycyclic aromatic hydrocarbons (PAHs), which are fundamental constituents of air pollution, on cellular biological aging. This was determined via the analysis of leukocyte telomere length (LTL), mitochondrial DNA copy number (LmtDNAcn), and by the formation of anti-benzo[a]pyrene diolepoxide (B[a]PDE–DNA) adducts.MethodsThe study population consisted of 585 individuals living in North-East Italy. PAH exposure (diet, indoor activities, outdoor activities, traffic, and residential exposure) and smoking behavior were assessed by questionnaire and anti-B[a]PDE–DNA by high-performance-liquid-chromatography. LTL, LmtDNAcn and genetic polymorphisms [glutathione S-transferase M1 and T1 (GSTM1; GSTT1)] were measured by polymerase chain reaction. Structural equation modelling analysis evaluated these complex relationships.ResultsAnti-B[a]PDE–DNA enhanced with PAH exposure (p = 0.005) and active smoking (p = 0.0001), whereas decreased with detoxifying GSTM1 (p = 0.021) and in females (p = 0.0001). Subsequently, LTL and LmtDNAcn reduced with anti-B[a]PDE–DNA (p = 0.028 and p = 0.018), particularly in males (p = 0.006 and p = 0.0001). Only LTL shortened with age (p = 0.001) while elongated with active smoking (p = 0.0001). Besides this, the most significant determinants of PAH exposure that raised anti-B[a]PDE–DNA were indoor and diet (p = 0.0001), the least was outdoor (p = 0.003).ConclusionNew findings stemming from our study suggest that certain preventable everyday life exposures to PAHs reduce LTL and LmtDNAcn. In particular, the clear association with indoor activities, diet, and gender opens new perspectives for tailored preventive measures in age-related diseases.CapsuleEveryday life exposure to polycyclic aromatic hydrocarbons reduces leukocyte telomere length and mitochondrial DNA copy number through anti-B[a]PDE-DNA adduct formation.

Abstract 230: The inhibitory effects of an anthocyanin enriched fraction of black raspberry (BRB), protocatechuic acid and ferulic acid on DB[a,l]P-induced DNA adduct formation in mouse oral tissues

Abstract Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region; in the USA, over 45,000 cases and about 11,000 deaths from the disease occur annually. Progress in the prevention and control of OSCC has been hampered by the lack of appropriate animal models that would reflect human exposure. Tobacco smoking is considered a major etiological factor in the development of oral cancer. Previous studies have demonstrated the ability of diets containing 5-10% freeze-dried black raspberry (BRB) powder to inhibit the development of chemically-induced cancers in multiple organ sites in rodents including 7,12-dimethylbenz(a)anthracene (DMBA) induced squamous cell carcinomas (SCC) in the hamster cheek pouch. However, DMBA is not present in the environment and the hamster cheek pouch model may not be applicable to humans. We had previously reported the carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, in mouse oral cavity. Recently, we demonstrated that administration of BRB at 5% in the diet can significantly reduce DNA adducts resulted from the administration of DB[a,l]P. It has also been shown that anthocyanins are the most abundant compounds in BRBs that can account for much of their antioxidant activity. In the current study, under identical conditions we compare the inhibitory effects of an anthocyanin enriched fraction extracted from black raspberry (BRBE), protocatechuic acid (PA, a major metabolite of anthocyanins) or ferulic acid (FA, a major phenolic acid in raspberries shown to reduce oral carcinogenesis) with BRB on DNA adducts induced by DB[a,l]P in mouse oral cavity. Levels of DB[a,l]PDE-DNA adducts were quantified by a LC-MS/MS method. We demonstrated that the administration of BRBE (1.6 %), protocatechuic acid (0.2%) or ferulic acid (0.05%) in the diet, starting 2 weeks before DB[a,l]P (24 nmol, 3 times a week for 5 weeks) significantly resulted in 29.6%, 23.0% or 25.3% reduction of the level of (-)-anti-trans-DB[a,l]PDE-dA in murine oral tissues, respectively, compared to a 19.2% reduction of BRB. Our results clearly showed that the anthocyanin components of BRB and its metabolite, PA, are responsible for the inhibitory effects of BRB on the DB[a,l]P- induced DNA adducts formation; in addition, BRB and its related components can inhibit the metabolic activation of DB[a,l]P, and may prevent the subsequent mutagenesis and carcinogenesis resulting from exposure to DB[a,l]P. Supported by NIH grant #CA173465. Citation Format: Kun-Ming Chen, Shangmin Zhang, Yuan-Wan Sun, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The inhibitory effects of an anthocyanin enriched fraction of black raspberry (BRB), protocatechuic acid and ferulic acid on DB[a,l]P-induced DNA adduct formation in mouse oral tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 230. doi:10.1158/1538-7445.AM2014-230

Abstract 3602: Effect of alcohol on DB[a,l]P, an environmental pollutant and a tobacco smoke constituent, on DNA adduct formation in mouse oral tissues.

Abstract Alcohol acts synergistically with tobacco smoke to greatly increase the risk of oral squamous cell carcinoma (OSCC) up to 20-fold; the mechanisms of this synergism remain unknown. Research in this area has been hampered by the lack of appropriate animal models that would mimic chronic simultaneous human exposure to alcohol and representative carcinogens in tobacco or tobacco smoke as a whole. Recently, we developed a more relevant animal model (Int. J. Cancer, 2012, 2783-90) and showed that dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, induces SCC in oral tissues of mice. We also showed that its diol epoxide metabolite, (±)-anti-DB[a,l]PDE, is a potent and specific carcinogen in tongue and other oral tissues in mice. We hypothesize that chronic alcohol consumption will increase the carcinogenic potency of DB[a,l]P in the mouse oral cavity by multiple mechanisms including the depletion of glutathione (GSH) and increasing levels of covalent DB[a,l]PDE-DNA adducts. As an initial study to test our hypothesis, mice were fed Isocaloric Lieber-DeCarli-type liquid diets formulated by BioServ (Frenchtown, N.J.) with or without ethanol (35% of total calories, increased gradually from 0, 11, 22 to 35%). After 3 weeks, mice were treated with 2 daily doses of DB[a,l]P (240 nmol each) and/or tobacco smoke particulates (TSP, 2R4F, 2.5 mg/mouse). Mice were sacrificed 24 hrs later and DB[a,l]PDE-DNA adduct levels were quantified by our recently developed LC-MS/MS method (Chem. Res. Toxicol. 2011, 1297-303). We showed that alcohol consumption significantly increased the levels of (-)-anti-trans-DB[a,l]P-dA in the absence of TSP (25%) or in the presence of TSP (75%). We also demonstrated that GSH levels in buccal mucosa were decreased (38%) in mice fed with liquid diet (35% calories from ethanol) compared with mice fed with control diet (0.89±0.13 μmol/g vs 1.23±0.17 μmol/g). Collectively, our preliminary results support our hypothesis that alcohol consumption will increase the carcinogenic potency of DB[a,l]P by altering the metabolic detoxification via depleting GSH and increasing DNA damage in oral tissues of mice. Citation Format: Shang-Min Zhang, Kun-Ming Chen, John R. Richie, Ana Calcagnotto, Cesar Aliaga, Arthur Berg, Yuan-Wan Sun, Arun K. Sharma, Shantu Amin, Karam El-Bayoumy. Effect of alcohol on DB[a,l]P, an environmental pollutant and a tobacco smoke constituent, on DNA adduct formation in mouse oral tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3602. doi:10.1158/1538-7445.AM2013-3602

Abstract 3708: Effects of black raspberry and related components on DB[a,l]P-induced DNA adduct formation in mouse oral tissues.

Abstract Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region; in the USA, over 45,000 cases and about 11,000 deaths from the disease occur annually. Progress in the prevention and control of OSCC has been hampered by the lack of appropriate animal models that would reflect human exposure. Tobacco smoking is considered a major etiological factor in the development of oral cancer. Previous studies have demonstrated the ability of diets containing 5-10% freeze-dried black raspberry (BRB) powder to inhibit the development of chemically-induced cancers in multiple organ sites in rodents including 7,12-dimethylbenz(a)anthracene (DMBA) induced squamous cell carcinomas (SCC) in the hamster cheek pouch. DMBA, however, is not present in the environment and the hamster cheek pouch model may not be applicable to humans. Recently, we developed a relevant animal model (Int. J. Cancer, 2012, 2783-90) and showed that dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, induces SCC in oral tissues of mice. In the current study, we evaluated the effects of BRB and related components including kaempferol and resveratrol on DNA adducts induced by DB[a,l]P in mouse oral cavity as an initial step to explore the potential preventive activity of BRB and its related components on DB[a,l]P-induced oral cancer. The DB[a,l]PDE-DNA adduct levels were quantified by our recently developed LC-MS/MS method (Chem. Res. Toxicol. 2011, 1297-303). We demonstrated that the administration of BRB (5%), kaempferol (0.025%) or resveratrol (0.002%) in the diet, starting 2 weeks before DB[a,l]P (24 nmol, 3 times a week for 5 weeks) resulted in 27%, 36% or 65% reduction of the level of (-)-anti-trans-DB[a,l]PDE-dA in murine oral tissues, respectively. In conclusion, our results support that dietary consumption of BRB and its related components can inhibit the metabolic activation of DB[a,l]P, and may prevent the subsequent mutagenesis and carcinogenesis resulting from exposure to DB[a,l]P. Citation Format: Kun-Ming Chen, Shang-Min Zhang, Cesar Aliaga, Yuan-Wan Sun, Arthur Berg, Arun Sharma, Shantu Amin, Gary Stoner, Karam El-Bayoumy. Effects of black raspberry and related components on DB[a,l]P-induced DNA adduct formation in mouse oral tissues. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3708. doi:10.1158/1538-7445.AM2013-3708

Abstract 1333: Carcinogenicity and formation of DNA adducts induced by dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, in the ovary of mouse

Abstract Ovarian cancer ranks second among gynecologic cancers, and it is the fifth leading cause of cancer deaths among women; 13,850 deaths are expected in 2010. There is usually no obvious symptoms of early stage ovarian cancer; therefore only 15% of ovarian cancers are diagnosed in the localized stage, and the five-year survival rate for ovarian cancer patients is 46%. A positive relation of mucinous ovarian tumors to tobacco smoking had been reported; furthermore, an association was shown between mucinous ovarian cancer with ex-smokers as well. The mechanisms that can account for the role of tobacco smoking on this disease remain unknown. Currently, there is no preclinical animal model that can accurately reflect the ovarian cancer resulting from chronic exposure to chemical carcinogens present in cigarette smoke. A previous report showed that dibenzo[a,l]pyrene (DB[a,l]P) can induce ovarian cancer in mice. Therefore, in this study we examined the carcinogenicity and DNA adducts formation in the ovary of mice treated with DB[a,l]P. In carcinogenesis study, ovarian tumors in B6C3F1 mice were induced by administering DB[a,l]P into the oral cavity of mice. Several groups of B6C3F1 mice (20/group) received various doses of DB[a,l]P (24, 12, 6, and 3 nmol/mouse), and additional groups received DMSO 3 times a week for 38 weeks or were left untreated as controls. Mice were sacrificed 4 weeks after the last carcinogen administration. A short-term bioassay was also conducted to detect and quantify DNA-adducts induced by DB[a,l]P as a function of time in the ovary; mice were administered orally (24 nmol) 3 times a week for 5 weeks. Ovaries were removed from mice 48 h, 1, 2 and 4 weeks after the last dose. DNA were isolated and hydrolyzed enzymatically, and the DB[a,l]PDE-DNA adducts were analyzed by our newly developed LC-MS/MS method. At the dose of 6 nmol in carcinogenesis study, we observed the highest total ovarian tumor incidence (79%), but the incidence of malignancy was only 15%. However, at the dose of 12 nmol, the total tumor incidence was 75%, and the incidence of malignant granulosa cell tumor was 65%. In addition to ovarian tumors, we also observed lesions in sites distal from the ovaries including the skin, mammary, lung, and oral tissues at the dose of 24 nmol, which were rare in other dosing groups. Treatment of DB[a,l]P in mice resulted in the formation of (−)-anti-cis-DB[a,l]PDE-dA and (−)-anti-trans-DB[a,l]PDE-dA adducts, which were 1.6 and 3.1 fmol/µg DNA respectively in ovaries of mice within 48 hours, and the level of adducts decreased dramatically over a week. Our results indicated that DB[a,l]P can be activated to form (−)-anti-DB[a,l]PDE; the latter may account for DB[a,l]P-induced ovarian cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1333. doi:10.1158/1538-7445.AM2011-1333

Micronuclei Related toAnti–B[a]PDE-DNA Adduct in Peripheral Blood Lymphocytes of Heavily Polycyclic Aromatic Hydrocarbon–Exposed Nonsmoking Coke-Oven Workers and Controls

Micronuclei (MN) frequency associated to biologically effective dose of polycyclic aromatic hydrocarbons [PAH; anti-benzo[a]pyrene diolepoxide (B[a]PDE)-DNA] within the same subjects' peripheral blood lymphocytes (PBL) was evaluated. Study subjects were nonsmoking male Polish coke-oven workers (n=49) and matched controls (n=45) verified for PAH exposure by urinary 1-pyrenol. We found that coke-oven workers, heavily exposed to PAHs (80% workers exceeded the urinary 1-pyrenol biological exposure index value), presented significantly higher MN frequency in PBLs than controls (P<0.01). Substantial difference was also found for adduct levels in PBLs (P<0.01). Increase in MN levels was significantly related to anti-B[a]PDE-DNA formation, key adduct of the ultimate carcinogenic metabolite of B[a]P (n=94; r=0.47; P<0.001). The dose-response relationship was improved when subjects with adduct levels above the 3rd tertile (>or=4.35 adducts/10(8) nucleotides) were excluded (n=61; r=0.69; P<0.001). Saturation of adduct/MN formation at high levels may disturb the underlying relationship. Linear multiple regression analysis, without subjects of 3rd tertile adduct level (n=61), revealed that adduct formation (t=4.61; P<0.001), but not 1-pyrenol, was the significant determinant in increasing MN. In conclusion, the increase in MN frequency is mainly related to the specific anti-B[a]PDE-DNA formation within PBLs of the same subject. Our results substantiate, with the use of an early indicator of biological effect as well, that workers are at higher cancer risk than controls.

Influence of GSTM1 null and low repair XPC PAT+ on anti-B[a]PDE-DNA adduct in mononuclear white blood cells of subjects low exposed to PAHs through smoking and diet

The influence of low-activity NER genotypes ( XPC PAT−/ +, XPA- A23G, XPD Asp312Asn, XPD Lys751Gln) and GSTM1 ( active or null) was evaluated on anti-benzo[ a]pyrene diol epoxide-(B[ a]PDE)-DNA adduct formed in the lymphocyte plus monocyte fraction (LMF). The sample population consisted of 291 healthy subjects with low exposure to polycyclic aromatic hydrocarbons (PAHs) (B[ a]P) through their smoking ( n = 126 smokers) or dietary habits ( n = 165 non-smokers with high (≥52 times/year) consumption of charcoaled meat or pizza). The bulky anti-B[ a]PDE-DNA adduct levels were detected by HPLC/fluorescence analysis and genotypes by PCR. Anti-B[ a]PDE-DNA was present (≥0.5 adducts/10 8 nucleotides) in 163 (56%) subjects (median (range) 0.77 (0.125–32.0) adducts/10 8 nucleotides), with smokers showing a significantly higher adduct level than non-smokers with high consumption of PAH-rich meals ( P < 0.01). Our exposed-sample population with unfavourable XPC PAT+/− or +/+ and GSTM1 null genotypes has the significantly highest adduct level ( P < 0.01). Taking into account tobacco smoke and diet as sources of exposure to B[ a]P, low-activity XPC PAT+ shows a major role in smokers ( P < 0.05) and GSTM1 null in non-smokers with frequent consumption of PAH-rich meals ( P < 0.01). The modulation of anti-B[ a]PDE-DNA adduct in the LMF by GSTM1 null and low-activity XPC PAT+ polymorphisms may be considered as potential genetic susceptibility factors that can modify individual responses to low PAH (B[ a]P) genotoxic exposure, with the consequent risk of cancer in the general population.

The characterization of (±)- anti-benzo[ a]pyrene diolepoxide–DNA adducts and (±)- anti-dibenzo[ a,l]pyrene diolepoxide–DNA adducts in the same DNA sample using solid-matrix phosphorescence

Solid-matrix phosphorescence (SMP) spectra and lifetimes were used to characterize the (±)- anti-benzo[ a]pyrene diolepoxide [(±)-anti-B[ a]PDE] and (±)- anti-dibenzo[ a,l]pyrene diolepoxide [(±)- anti-DB[ a,l]PDE] bonded to the same sample of DNA. SMP spectra and lifetimes were also acquired for two samples of DNA that had only (±)-anti-B[ a]PDE or (±)- anti-DB[ a,l]PDE bonded to the individual samples of DNA. A detailed comparison of the SMP properties was made among the three samples of DNA. The SMP excitation spectra for the (±)-anti-B[ a]PDE–DNA and the (±)- anti-DB[ a,l]PDE–DNA adducts were very similar. However, the SMP emission spectra of the two DNA adduct systems were very dissimilar with a major emission band for the (±)-anti-B[ a]PDE–DNA adducts appearing at 613 nm and for the (±)- anti-DB[ a,l]PDE–DNA adducts a major band was at 558 nm. It was possible to selectively use SMP emission wavelengths and obtain a SMP excitation of spectrum of the (±)- anti-DB[ a,l]PDE–DNA adducts in the dual adducted DNA sample without the (±)-anti-B[ a]PDE–DNA adducts emitting SMP. In addition, it was shown that the SMP emission spectrum of the dual adducted DNA sample could be used to detect both adduct systems in the modified DNA sample. It was demonstrated that the SMP lifetimes could be effectively employed to characterize the dual adducted DNA sample. For example, the SMP decay curve for the (±)- anti-DB[ a,l]PDE–DNA adducts could be acquired without any SMP emission from the (±)-anti-B[ a]PDE–DNA adducts. Also, ln(SMP intensity) versus time plots were very useful in characterizing the dual adducted DNA sample.

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 (≥52 (38%) versus <52 times/year, p < 0.0001), and outdoor exposure (≥4 (19%) versus <4 h/day; p = 0.0115) significantly influenced adduct levels. Indoor exposure significantly increased the frequency of positive subjects (≥0.5 adducts/10 8 nucleotides; χ 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 solid-matrix phosphorescence of (±)- anti-dibenzo[ a, l]pyrene diol epoxide-DNA adducts and benzo[ e]pyrene

New solid-matrix phosphorescence (SMP) methods for (±)- anti-DB[ a, l]PDE-DNA adducts and B[ e]P were developed. The methods can be used to detect and characterize (±)- anti-DB[ a, l]PDE-DNA adducts and B[ e]P by employing SMP spectra, intensities, and lifetimes acquired with the heavy-atom salt, TlNO 3, on Whatman 1PS paper. With the SMP data, a number of photophysical parameters were calculated such as biexponential SMP decay curves, pre-exponential factors, and fractional contribution to SMP decay curves. The SMP results were compared with earlier SMP data for (±)- anti-BPDE-DNA adducts and tetrol I-1. The SMP results show that small molecular-weight compounds like B[ e]P can be readily detected and characterized by SMP. For example, the limit of detection for B[ e]P was 0.60 pmol. Comparison of the SMP properties of the (±)- anti-DB[ a, l]PDE-DNA adducts with earlier SMP data for the (±)- anti-BPDE-DNA adducts showed major differences in the SMP spectra, intensities, and lifetimes. The methods developed are important for the comparison of the SMP properties of different diol epoxides of PAH bonded to DNA.

Solid-Matrix Fluorescence Quenching of Benzo[E]Pyrene and (±)-Anti-Dibenzo[a,l]Pyrene Diolepoxide-DNA Adducts

The solid-matrix fluorescence (SMF) quenching of benzo[e]pyrene and (+/-)-anti-dibenzo[a, l]pyrene-11,12-diol-13,14-epoxide ((+/-)-antiDB[a, l]PDE)-DNA adducts with thallium nitrate (TlNO(3)) and sodium iodide (NaI) was examined and several SMF quenching models were developed. The SMF quenching data for B[e]P with either TlNO(3) or NaI fit a two-independent-binding-site model. However, the SMF quenching of (+/-)-anti-DB[a, l]PDE-DNA adducts with TlNO(3) fits a sphere of action model, but quenching with NaI was modeled with the two-independent-binding-site model. The data were compared with earlier SMF quenching data for 7,8,9,10-tetrahydroxytetrahydro-benzo[a]pyrene (tetrol I-1) and (+/-)-anti-benzo[a]pyrene-trans-7,8-dihydrodiol-9,10-epoxide ((+/-)-anti-BPDE)DNA adducts. The interpretation of the SMF quenching data for the (+/-)-anti-DB[a, l]PDE-DNA adducts was distinctively different than the interpretation of the SMF quenching data for the (+/-)-antiBPDE-DNA adducts. This initial study shows that SMF quenching has the potential to characterize polycyclic aromatic hydrocarbonDNA adducts with different numbers of aromatic rings. In addition, the data indicated that external and intercalated DNA adducts interacted with heavy-atom salts in dissimilar fashions. The new SMF methodology developed is useful for the characterization of both polycyclic aromatic hydrocarbon-DNA adducts and metabolites from polycyclic aromatic hydrocarbons.

DNA Damage, Repair, and Mutation Induction by (+)- Syn and (−)- Anti -Dibenzo[ a,l ]Pyrene-11,12-Diol-13,14-Epoxides in Mouse Cells

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental carcinogens. PAHs are classified into bay and fjord region compounds according to structural differences in the molecule region where enzymatic epoxidation occurs. Dibenzo[a,l]pyrene (DB[a,l]P), one of the fjord region compounds, has been demonstrated to be the most carcinogenic PAH known to date. DB[a,l]P is activated to fjord region (+)-syn and (-)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene (DB[a,l]PDE) metabolites. In this study, we analyzed mutagenesis induced by (+)-syn- and (-)-anti-DB[a,l]PDE at the cII transgene in Big-Blue mouse cells. The mutant frequency of untreated cells (background level) was 6.53 x 10(-5). This level increased 3.7-fold for 20 nmol/L, 5.3-fold for 50 nmol/L, and 7.9-fold for 100 nmol/L (+)-syn-DB[a,l]PDE, respectively. In the case of (-)-anti-DB[a,l]PDE it increased 4.5-fold for 20 nmol/L, 6.7-fold for 50 nmol/L, and 10.6-fold for 100 nmol/L, respectively, indicating that (-)-anti-DB[a,l]PDE is slightly more mutagenic than (+)-syn-DB[a,l]PDE. The mutational spectra of (+)-syn- and (-)-anti-DB[a,l]PDE were quite similar except for several hotspots, specific for either (+)-syn-DB[a,l]PDE or (-)-anti-DB[a,l]PDE. The most frequently induced mutations were A to T transversions, which were 43.9% for (+)-syn- and 38.8% for (-)-anti-DB[a,l]PDE. In addition, G to T transversions were induced significantly, at frequencies of 18.5% by (+)-syn- and 18.1% by (-)-anti-DB[a,l]PDE. Using UvrABC cleavage and ligation-mediated PCR or the terminal transferase-dependent PCR method, we have determined DB[a,l]PDE-DNA adduct formation sites and repair rates in carcinogen-exposed cells. The mutation hotspots coincided with sites of strong adduct formation, but not all of the adduct hotspots were mutational hotspots. Slow adduct removal occurred for both (+)-syn- and (-)-anti-DB[a,l]PDE adducts over a time period of up to 72 hours. The data suggest that, although the (-)-anti-isomer is slightly more mutagenic, DNA adducts of both DB[a,l]PDE stereoisomers may have similar biological properties. We discuss the implications of these findings for human cancer mutagenesis.

Reduced nucleotide excision repair and GSTM1-null genotypes influence anti-B[a]PDE-DNA adduct levels in mononuclear white blood cells of highly PAH-exposed coke oven workers.

It is important to identify the potential genetic-susceptible factors that are able to modulate individual responses to exposure to carcinogenic polycyclic aromatic hydrocarbons (PAHs). In the present study we evaluated the influence of four polymorphisms of nucleotide excision repair (NER) genes [xeroderma pigmentosum-C (XPC)-PAT +/-, xeroderma pigmentosum-A (XPA) 5' non-coding region-A23G, XPD-exon 23 A35931C Lys751Gln, xeroderma pigmentosum-D (XPD)-exon 10 G23591A Asp312Asn] and that of glutathione S-transferase mu1 (GSTM1-active or -null) on benzo[a]pyrene diol epoxide (B[a]PDE)-DNA adduct levels from the lympho-monocyte fraction (LMF) of highly PAH benzo[a]pyrene (B[a]P)-exposed Polish coke oven workers (n = 67, 67% current smokers) with individual urinary post-shift excretion of 1-pyrenol exceeding the proposed biological exposure index (BEI) (2.28 micromol/mol creatinine). The bulky (+/-)-r-7,t-8-dihydroxy-t-9,10-oxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-B[a]PDE)-DNA adduct levels were detected by high-performance liquid chromatography (HPLC)/fluorescence analysis and genotypes by polymerase chain reaction. We found that workers with the low DNA repair capacity of XPC-PAT+/+ and XPA-A23A genotypes had significantly increased anti-B[a]PDE-DNA adduct levels (Mann-Whitney U-test, z = 2.24, P = 0.02 and z = 2.65, P = 0.01). Moreover, DNA adducts were also raised in workers without GSTM1 activity (GSTM1-null genotype) (Mann-Whitney U-test, z = 2.25, P = 0.0246). Workers with unfavourable XPC-PAT+/+ and XPA-A23A NER genotypes, alone (approximately 65% of workers) or combined with GSTM1-null genotype (approximately 75% of workers) were in the tertile with the highest adduct level, i.e. >4.11 adducts/10(8) nt (chi2 = 5.85, P = 0.0156 and chi2 = 5.40, P = 0.01). The increase in anti-B[a]PDE-DNA adduct levels (ln values) was significantly related in a multiple linear regression analysis to PAH exposure (i.e. urinary post-shift excretion of 1-pyrenol) (t = 2.61, P = 0.0115), lack of GSTM1 activity (t = 2.41, P = 0.0192) and to low DNA repair capacity of the XPC-PAT+/+ genotype (t = 2.34, P = 0.0226). The influence of the XPA-A23A genotype was not evident in this statistical analysis, and no associations with XPD polymorphisms, dietary habits or tobacco smoking were found. The modulation of anti-B[a]PDE-DNA adducts in the LMF by GSTM1-null and some low-activity NER genotypes may be considered as a potential genetic susceptibility factor capable of modulating individual responses to PAH (B[a]P) genotoxic exposure and the consequent risk of cancer in coke oven workers.

Cytochrome P450 1B1 determines susceptibility to dibenzo[a,l]pyrene-induced tumor formation.

Metabolic activation, DNA binding, and tumorigenicity of the carcinogenic polycyclic aromatic hydrocarbon dibenzo[a,l]pyrene (DB[a,l]P) catalyzed by murine cytochrome P450 (P450) enzymes were investigated. DNA binding of DB[a,l]P in human mammary carcinoma MCF-7 and human P450-expressing Chinese hamster V79 cell lines was previously shown to occur preferentially with metabolically generated fjord region DB[a,l]P-11,12-dihydrodiol 13,14-epoxides (DB[a,l]PDE). To elucidate different capabilities of murine P450 1A1 and 1B1 for metabolic activation of DB[a,l]P, V79 cell cultures stably expressing P450s 1A1 or 1B1 from mice were exposed to 10 or 100 nM DB[a,l]P. Both cell lines transformed DB[a,l]P to DNA binding intermediates. As with V79 cells expressing the corresponding human P450 enzyme [Luch et al. (1998) Chem. Res. Toxicol. 11, 686-695], murine P450 1B1-catalyzed metabolism and DNA binding proceeded exclusively through generation of fjord region DB[a,l]PDE. In addition, only DB[a,l]PDE-derived DNA adducts were found in V79 cells expressing P450 1A1 from mice. This is in contrast to our recent findings with V79 cells expressing P450 1A1 from humans or rats which catalyzed the formation of both highly polar DNA adducts as well as nonpolar DB[a,l]PDE-DNA adducts. To establish the role of P450 1B1 in DB[a,l]P-induced tumor formation in vivo, we treated P450 1B1-null and wild-type mice intragastrically and monitored survival rates and appearance of neoplasias in various organs. All wild-type mice (n = 17) used in this study developed at least one tumor at one site (tumor rate of 100%). In contrast, 5 of 13 P450 1B1-null mice were observed to be free from any tumor (tumor rate of 62%). The organ sites of tumor formation and the dignity of tumors were different between wild-type and P450 1B1-null mice. Wild-type mice were diagnosed with both benign and malignant tumors of the ovaries, lymphoid tissues, as well as with skin and endometrial hyperplasias, whereas P450 1B1-null mice developed only lung adenomas and endometrial hyperplasias. DNA binding studies using embryonic fibroblasts isolated from these animals provided further evidence that P450 1B1-catalyzed formation of fjord region DB[a,l]PDE-DNA adducts is the critical step in DB[a,l]P-mediated carcinogenesis in mice, and probably also in man.

The Role of Cytochrome P450 1B1 in Dibenzo[ a,l ]pyrene-induced Carcinogenesis

In human cells, the most carcinogenic polycyclic aromatic hydrocarbon dibenzo[ a,l ]pyrene (DB[ a,l ]P) forms high levels of DNA adducts through formation of the ( m )- anti -(11 R ,12 S )-diol (13 S ,14 R )-epoxide (DB[ a,l ]PDE) and its metabolic precursor, the ( m )-(11 R ,12 R )-diol. Generation of these adducts results from the catalytic activity of cytochrome P450 (P450) 1A1 and 1B1. Additional adducts such as (+)- syn -DB[ a,l ]PDE-DNA or more polar DNA adducts were detected only after increasing exposure doses of the parent compound or in cells that express P450 1A1. At low concentrations (·;100 nM) exclusively ( m )- anti -DB[ a,l ]PDE-DNA adducts were formed by P450 1B1, which is constitutively expressed in many mammalian tissues. Measurement of DNA binding and mutagenicity of DB[ a,l ]P in V79 cells expressing human P450 enzymes revealed a higher activity of P450 1B1 compared to 1A1 at low concentrations. Treatment of P450 1B1 knockout mice and DNA binding studies with fibroblasts isolated from these animals provided further evidence for the central role of P450 1B1-catalyzed formation of ( m )- anti -DB[ a,l ]PDE-DNA adducts in DB[ a,l ]P-induced carcinogenesis.

The Role of Cytochrome P450 1B1 in Dibenzo[ a,l ]pyrene-induced Carcinogenesis

In human cells, the most carcinogenic polycyclic aromatic hydrocarbon dibenzo[ a,l ]pyrene (DB[ a,l ]P) forms high levels of DNA adducts through formation of the ( m )- anti -(11 R ,12 S )-diol (13 S ,14 R )-epoxide (DB[ a,l ]PDE) and its metabolic precursor, the ( m )-(11 R ,12 R )-diol. Generation of these adducts results from the catalytic activity of cytochrome P450 (P450) 1A1 and 1B1. Additional adducts such as (+)- syn -DB[ a,l ]PDE-DNA or more polar DNA adducts were detected only after increasing exposure doses of the parent compound or in cells that express P450 1A1. At low concentrations (·;100 nM) exclusively ( m )- anti -DB[ a,l ]PDE-DNA adducts were formed by P450 1B1, which is constitutively expressed in many mammalian tissues. Measurement of DNA binding and mutagenicity of DB[ a,l ]P in V79 cells expressing human P450 enzymes revealed a higher activity of P450 1B1 compared to 1A1 at low concentrations. Treatment of P450 1B1 knockout mice and DNA binding studies with fibroblasts isolated from these animals provided further evidence for the central role of P450 1B1-catalyzed formation of ( m )- anti -DB[ a,l ]PDE-DNA adducts in DB[ a,l ]P-induced carcinogenesis.

A quantitative comparison of dibenzo[a,l]pyrene‐DNA adduct formation by recombinant human cytochrome P450 microsomes

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord-region dihydrodiol, trans-DB[a,l]P-11,12-diol, to syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides (syn- and anti-DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using 32P postlabeling, thin-layer chromatography, and high-pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE-DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a,l]PDE-DNA adducts and unidentified polar adducts at rates threefold and 17-fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti- and syn-DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti-DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn-DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74–82, 1999. Published 1999 Wiley-Liss, Inc.

A quantitative comparison of dibenzo[a,l]pyrene-DNA adduct formation by recombinant human cytochrome P450 microsomes

Dibenzo[a,l]pyrene (DB[a,l]P), an extremely potent environmental carcinogen, is metabolically activated in mammalian cells and microsomes through the fjord-region dihydrodiol, trans-DB[a,l]P-11, 12-diol, to syn- and anti-DB[a,l]P-11,12-diol-13,14-epoxides (syn- and anti-DB[a,l]PDEs). The role of seven individual recombinant human cytochrome P450s (1A1, 1A2, 1B1, 2B6, 2C9, 2E1, and 3A4) in the metabolic activation of DB[a,l]P and formation of DNA adducts was examined by using (32)P postlabeling, thin-layer chromatography, and high-pressure liquid chromatography. We found that, in the presence of epoxide hydrolase, only P450 1A1 and P450 1B1 catalyzed the formation of DB[a,l]PDE-DNA adducts and several unidentified polar adducts. Human P450 1A1 catalyzed the formation of DB[a, l]PDE-DNA adducts and unidentified polar adducts at rates threefold and 17-fold greater than did human P450 1B1 (256 fmol/h/nmol P450 versus 90 fmol/h/nmol P450 and 132 fmol/h/nmol P450 versus 8 fmol/h/nmol P450, respectively). P450 1A1 DNA adducts were derived from both anti- and syn-DB[a,l]PDE at rates of 73 fmol/h/nmol P450 and 51 fmol/h/nmol P450, respectively. P450 1B1 produced adducts derived from anti-DB[a,l]PDE at a rate of 82 fmol/h/nmol, whereas only a small number of adducts were derived from syn-DB[a,l]PDE (0.4 fmol/h/nmol). These results demonstrated the potential of human P450 1A1 and P450 1B1 to contribute to the metabolic activation and carcinogenicity of DB[a,l]P and provided additional evidence that human P450 1A1 and 1B1 differ in their stereospecific activation of DB[a,l]P. Mol. Carcinog. 26:74-82, 1999. Published 1999 Wiley-Liss, Inc.

Preparation, isolation, and characterization of Dibenzo[a,l]pyrene diol epoxide-deoxyribonucleoside monophosphate adducts by HPLC and fluorescence line-narrowing spectroscopy.

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon that has been identified in the environment. Earlier studies in our laboratory indicated that more than 80% of the DB[a,l]P-DNA adducts formed in vitro were depurinating adducts and that most of the stable adducts were formed from diol epoxide intermediates. To complete the profile of both stable and depurinating adducts of DB[a,l]P, we have synthesized standard adducts by reacting 3'-dAMP or 3'-dGMP with either (+/-)-anti- or (+/-)-syn-dibenzo[a,l]pyrene 11,12-dihydrodiol 13, 14-epoxide (DB[a,l]PDE). The adducts were separated by HPLC with an ion-pair column and were identified by fluorescence line-narrowing spectroscopy (FLNS). A total of six pairs of stereoisomers along with another stable DB[a,l]PDE-DNA adduct were successfully isolated and identified. Pairs of (+/-)-trans and (+/-)-cis isomers were expected to be formed from the reaction of anti-DB[a,l]PDE with either dAMP or dGMP. While we were able to identify two pairs of stereoisomeric (+/-)-syn-DB[a,l]PDE-dAMP (cis and trans) and two pairs of stereoisomeric (+/-)-anti-DB[a,l]PDE-dAMP (cis and trans) adducts, identification of all the stereoisomers of dGMP adducts proved to be impossible. A pair of (+/-)-syn-trans-DB[a,l]PDE-dGMP adducts, a pair of (+/-)-anti-cis-DB[a,l]PDE-dGMP adducts, and one syn-cis-DB[a,l]PDE-dGMP adduct were conclusively identified by FLNS. These standard adducts will be used to identify the stable DNA adducts formed by DB[a,l]P and DB[a,l]PDE in vitro and in vivo.

The level of DNA modification by (+)-syn-(11S,12R,13S,14R)- and (-)-anti-(11R,12S,13S,14R)-dihydrodiol epoxides of dibenzo[a,l]pyrene determined the effect on the proteins p53 and p21WAF1 in the human mammary carcinoma cell line MCF-7.

The polycyclic aromatic hydrocarbon (PAH) dibenzo[a,l]pyrene (DB[a,l]P), the most carcinogenic PAH tested in rodent bioassays, exerts its pathobiological activity via metabolic formation of electrophilically reactive DNA-binding fjord region (+)-syn-(11S,12R,13S,14R)- or (-)-anti-(11R,12S,13S,14R)-DB[a,l]P-dihydrodiol epoxides (DB[a,l]-PDEs). DB[a,l]P is metabolized to these DB[a,l]PDEs which bind to DNA in human mammary carcinoma MCF-7 cells. The molecular response of MCF-7 cells to DNA damage caused by DB[a,l]PDEs was investigated by analyzing effects on the expression of the tumor suppressor protein p53 and one of its target gene products, the cyclin-dependent kinase inhibitor p21WAF1. Treatment of MCF-7 cells with (+)-syn- and (-)-anti-DB[a,l]PDE at a concentration range of 0.001-0.1 microM resulted in DB[a,l]PDE-DNA adduct levels between 2 and 30, and 3 and 80 pmol/mg DNA, respectively, 8 h after exposure. (-)-anti-DB[a,l]PDE exhibited a higher binding efficiency that correlated with a significantly stronger p53 response at low concentrations of the dihydrodiol epoxides. The level of p53 increased by 6-8 h after treatment. The p21WAF1 protein amount exceeded control levels by 12 h and remained elevated for 96 h. At a dose of 0.01 microM (+)-syn-DB[a,l]PDE, an increase in p21WAF1 was observed in the absence of a detectable change in p53 levels. The results indicate that the increase in p53 induced by DB[a,l]PDEs in MCF-7 cells requires an adduct level of approximately 15 pmot/mg DNA and suggest that the level of adducts rather than the specific structure of the DB[a,l]PDE-DNA adduct formed triggers the p53 response. The PAH-DNA adduct level formed may determine whether p53 and p21VAF1 pathways respond, resulting in cell-cycle arrest, or fail to respond and increase the risk of mutation induction by these DNA lesions.