Genotoxic effects of benzo[a]pyrene and dibenzo[a,l]pyrene in a human lung cell line

Several studies have shown that polycyclic aromatic hydrocarbons (PAHs) produce genotoxic effects in assays performed in vivo and in vitro. This study was undertaken to investigate the ability of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) to induce DNA damage in a human lung fibroblast cell line (MRC-5), using sister-chromatid exchanges test (SCEs), the comet assay, and evaluating point mutations in codon 12 of the K-ras protooncogene by polymerase chain reaction-single-strand conformation polymorphisms (PCR-SSCPs) and restriction fragment length polymorphisms (RFLP)-enriched PCR methods. Sister-chromatid exchanges frequencies were significantly increased in cells exposed to benzo[a]pyrene and dibenzo[a,l]pyrene in relation to controls (p < .001). Using the standard alkaline comet assay, significant differences between groups were found for the variable comet moment (CM) when cells were exposed to BP (p < .001) and DBP (p < .001). Nevertheless, PCR-SSCP and RFLP-enriched PCR methods did not show any association between treatments with BP and DBP and K-ras point mutations. The data presented in this study indicated that BP and DBP induced both DNA strand breaks and sister-chromatid exchanges but not significant point mutations at codon 12 of K-ras gene in the MRC-5 cell line.     

Identification and quantification of DNA adducts in the oral tissues of mice treated with the environmental carcinogen dibenzo[a,l]pyrene by HPLC-MS/MS

Tobacco smoking is one of the leading causes for oral cancer. Dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke constituent, is the most carcinogenic polycyclic aromatic hydrocarbon (PAH) tested to date in several animal models (target organs: skin, lung, ovary, and mammary tissues). We have recently demonstrated that DB[a,l]P is also capable of inducing oral cancer in mice; however, its metabolic activation to the ultimate genotoxic metabolite dibenzo[a,l]pyrene-11,12-dihydrodiol-13,14-epoxide (DB[a,l]PDE) in mouse oral cavity has not been examined. Here we developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify (±)-anti-DB[a,l]PDE-dA adducts in oral tissues of mice treated with DB[a,l]P. [(15)N(5)]-(±)-anti-DB[a,l]PDE-N(6)-dA adducts were synthesized as internal standards. The stereoisomeric adducts were characterized by MS, NMR, and CD analysis. The detection limit of the method is 8 fmol with 100 μg of digested DNA as the matrix. Two adducts were detected and identified as (-)-anti-cis and (-)-anti-trans-DB[a,l]PDE-dA in the oral tissues of mice following the direct application of DB[a,l]P (240 nmol per day, for 2 days) into the oral cavity, indicating that DB[a,l]P is predominantly metabolized into (-)-anti-DB[a,l]PDE in this target organ. We also compared the formation and removal of adducts as a function of time, following the direct application of DB[a,l]P (24 nmol, 3 times per week for 5 weeks) into the oral cavity of mice. Adducts were quantified at 48 h, 1, 2, and 4 weeks after the last dose. Maximal levels of adducts occurred at 48 h, followed by a gradual decrease. The levels (fmol/μg DNA) of (-)-anti-trans adducts (4.03 ± 0.27 to 1.77 ± 0.25) are significantly higher than (-)-anti-cis-DB[a,l]PDE-dA adduct (1.63 ± 0.42 to 0.72 ± 0.04) at each time point (p < 0.005). The results presented here indicate that the formation and persistence of (-)-anti-DB[a,l]PDE-dA adducts may, in part, contribute to the initiation of DB[a,l]P-induced oral carcinogenesis.     

Proteomic analysis of MCF-7 cells treated with benzo[a]pyrene, dibenzo[a,l]pyrene, coal tar extract, and diesel exhaust extract

Polycyclic aromatic hydrocarbon (PAH) DNA adducts have been associated with carcinogenesis, which is accompanied by multiple alterations in gene expression. We used two-dimensional electrophoresis to distinguish protein expression changes induced in MCF-7 cells by individual PAH (B[a]P and DB[a,l]P) and PAH mixtures (coal tar extract [SRM 1597] and diesel exhaust extract [SRM 1975]). Spots of interest were identified by MALDI-TOF-TOF. Our results have shown alterations in the expression of heat-shock proteins, cytoskeletal proteins, DNA associated proteins, and glycolytic and mitochondrial proteins. The proteins that were universally altered in expression were actin cytoplasmic 1, tubulin alpha and myosin light chain alkali, cyclophilin B, and heterogeneous ribonucleoprotein B1 (a protein involved in access to telomerase and mRNA maturation). Additional proteins with altered expression include histone H2A.1, heat-shock protein 70-2, galectin-3, nucleoside diphosphate kinase, ATP synthase, and electron transfer flavoprotein. While sharing similarities, each PAH treatment exhibited a unique proteomic fingerprint.     

Catalytic activities of human alpha class glutathione transferases toward carcinogenic dibenzo[a,l]pyrene diol epoxides

In this study, human glutathione transferases (GSTs) of alpha class have been assayed with the ultimate carcinogenic (-)-anti- and (+)-syn-diol epoxides (DEs) derived from the nonplanar dibenzo[a,l]pyrene (DBPDE) and the (+)-anti-diol epoxide of the planar benzo[a]pyrene [(+)-anti-BPDE] in the presence of glutathione (GSH). In all DEs, the benzylic oxirane carbon reacting with GSH, possess R-absolute configuration. GSTA1-1 demonstrated activity with all DEs tested whereas A2-2 and A3-3 only were active with the DBPDE enantiomers. With GSTA4-4, no detectable activity was observed. GSTA1-1 was found to be the most efficient enzyme and demonstrated a catalytic efficiency (k(cat)/K(m)) of 464 mM(-)(1) s(-)(1) with (+)-syn-DBPDE. This activity was about 7-fold higher than that observed with (-)-anti-DBPDE and more than 65-fold higher than previously observed with less complex fjord-region DEs. GSTA3-3 also demonstrated high k(cat)/K(m) with the DEs of DBP and a high preference for the (+)-syn-DBPDE enantiomer [190 vs 16.2 mM(-)(1) s(-)(1) for (-)-anti-DBPDE]. Lowest k(cat)/K(m) value of the active enzymes was observed with GSTA2-2. In this case, 30.4 mM(-)(1) s(-)(1) was estimated for (+)-syn-DBPDE and 3.4 mM(-)(1) s(-)(1) with (-)-anti-DBPDE. Comparing the activity of the alpha class GSTs with (-)-anti-DBPDE and (+)-anti-BPDE revealed that GSTA1-1 was considerable more active with the former substrate (about 25-fold). Molecular modeling studies showed that the H-site of GSTA1-1 is deeper and wider than that of GSTA4-4. This is mainly due to the changes of Ser212-->Tyr212 and Ala216-->Val216, which cause a shallower active site, which cannot accommodate large substrates such as DBPDE. The higher activity of GSTA1-1 with (+)-syn-DBPDE relative to (-)-anti-DBPDE is explained by the formation of more favorable interactions between the substrate and the enzyme-GSH complex. The presence of GSTA1-1 in significant amounts in human lung, a primary target tissue for PAH carcinogenesis, may be an important factor for the protection against the harmful action of this type of potent carcinogenic intermediates.     

Inhibition of the mutagenicity and metabolism of 6-methyl-benzo[a]pyrene and 6-hydroxymethyl-benzo[a]pyrene

Previously reported inhibitors of benzo[a]pyrene (BaP) mutagenicity in Salmonella typhimurium strain TA98 were tested for their effectiveness against the mutagenicity of 6-methyl-benzo[a]pyrene (6-CH3-BaP), 6-hydroxymethyl-benzo[a]pyrene (6-CH2OH-BaP) and 6-acetoxymethyl-benzo[a]pyrene (6-CH3COOCH2-BaP). Dose-response curves obtained for phenothiazine (PTH), 2-chlorophenothiazine (2Cl-PTH), phenylisothiocyanate (PHN), phenethylisothiocyanate (PNE), trans-retinol (TR) and disulfiram (TETD) showed a variety of degrees of inhibition of mutagenicity. Additionally, glutathione (GSH) was found to inhibit the mutagenicity of 6-CH3COOCH2-BaP, and the mutagenicity of 6-CH2OH-BaP was enhanced by the addition of supplemental ATP, Na2SO4 and EDTA. Only 2Cl-PTH was equally as good an inhibitor of 6-CH3-BaP and BaP, reducing revertant colonies to less than 50% of control at 10 X BaP concentration. To probe the mechanism of inhibition, the effect of 2Cl-PTH on the binding of BaP and the 6-substituted benzo[a]pyrenes to cytochrome P-450 was investigated by difference spectroscopy. Also, the effect of 2Cl-PTH on the subsequent metabolism of 6-CH3-BaP and 6-CH2OH-BaP was investigated by rapid scan difference spectroscopy and high-performance liquid chromatographic separation of products. The results are consistent with a major mechanism of inhibition for 2Cl-PTH involving a competition for the cytochrome P-450 binding site.     

Glutathione conjugation and DNA adduct formation of dibenzo[a,l]pyrene and benzo[a]pyrene diol epoxides in V79 cells stably expressing different human glutathione transferases

Mammalian V79 cells stably expressing human glutathione transferase (GST) A1-1, M1-1, and P1-1 (the allelic variant with Val105 and Ala114) have been constructed and characterized. The cells have been used to study the capacity of individual GST isoenzymes in conjunction with GSH to detoxify diol epoxides from dibenzo[a,l]pyrene (DBPDE), the most carcinogenic polycyclic aromatic hydrocarbon (PAH) identified so far, and diol epoxides from benzo[a]pyrene (BPDE). The relationship between GSH-conjugation and DNA adduct-formation has been investigated as well as factors governing the accessibility of lipophilic diol epoxide substrates for the soluble GSTs in the cells. Relative to control cells, those expressing GSTA1-1 showed the highest rate (about 50-fold increase) to perform GSH-conjugation of (-)-anti-DBPDE (R-absolute configuration at the benzylic oxirane carbon in the fjord-region) followed by GSTM1-1 (25-fold increase) and GSTP1-1 (10-fold increase). GSTA1-1 was found to be strongly inhibited when expressed in cells (10% of fully functional protein). Taking this factor into account, the rates of conjugation found in the cells fairly well reflected the order of catalytic efficiencies (k(cat)/K(m)) obtained with the pure enzymes. Increased GSH conjugation of (-)-anti-DBPDE was associated with a reduction in DNA adduct formation. GSTA1-1 inhibited the formation of adducts more than 6-fold and GSTM1-1 and GSTP1-1 about 2-fold. With (+)-anti-BPDE, GSTP1-1-expressing cells demonstrated a substantially higher rate of GSH-conjugate formation than cells with GSTA1-1 and GSTM1-1 cells (33- and 10-fold increase, respectively). Relative to control cells, GSTM1-1 was found to inhibit DNA adduct formation of (+)-anti-BPDE most effectively followed by GSTP1-1 and GSTA1-1 (12-, 4-, and 3-fold, respectively). Values of k(cat)/K(m) and estimated oil/water partition coefficients of DBPDE and BPDE were used to calculate the concentration of free diol epoxides in solution and expected rates of GSH conjugate formation in cells, and these theoretical results were compared with the observed ones. With the highly reactive (+)-anti-BPDE, 1-2% of the expected activity was observed, whereas the corresponding values for the less reactive (-)-anti-DBPDE were up to 13%. The most obvious explanations for the low observed rate with (+)-anti-BPDE are rapid and competing reactions such as hydrolysis and/or more unspecific chemical and physical reactions with cellular constituents (proteins, lipids, nucleic acids, etc.). In addition, the difference between the theoretical and observed rates may also reflect participation of factors such as macromolecular crowding and reduced rates of diffusion, factors expected to further restrict the accessibility of GST and the diol epoxides in the intact cell.     

Mechanisms by which benzo[a]pyrene, an environmental carcinogen, suppresses B cell lymphopoiesis.

The capacity for polycyclic aromatic hydrocarbons (PAH) to suppress immune cell function has been well documented. Nevertheless, mechanisms responsible for PAH immunosuppression and potential effects of PAH on lymphocyte development (lymphopoeisis) remain poorly defined. Murine bone marrow cultures were used in the present studies to determine if and by what mechanism(s) benzo[a]pyrene (B[a]P), a prototypic and highly carcinogenic PAH, suppresses B cell lymphopoiesis. Emphasis was placed on similarities between the processes leading to transformation and immunosuppression and on a possible role for programmed cell death (apoptosis) in B[a]P lymphotoxicity. Data presented herein indicate that: (1) B[a]P suppresses B cell lymphopoiesis in bone marrow cultures at extremely low concentrations (10(-8) M); (2) benzo[e]pyrene, the relatively noncarcinogenic congener of B[a]P, is approximately 1000 times less potent than B[a]P in suppressing B cell lymphopoiesis; (3) bone marrow cells from PAH-resistant DBA/2 mice are less sensitive to B[a]P than cells from C57BL/6 mice; (4) B[a]P induces preB cell apoptosis; and (5) alpha-naphthaflavone, an inhibitor of Ah-receptor dependent, P450 isoenzyme activity, blocks B[a]P-mediated preB cell apoptosis and inhibits B[a]P-dependent suppression of lymphopoiesis. The results support the hypothesis that B[a]P suppression of B cell lymphopoiesis is mediated at least in part by the induction of programmed cell death and that the Ah receptor and/or P450 isoenzymes are involved in this process. The results suggest the potential for PAH to affect development of the B lymphocyte repertoire.     

Characterization of dibenzo[a,l]pyrene-trans-11,12-diol (dibenzo[def,p]chrysene) glucuronidation by UDP-glucuronosyltransferases

Dibenzo[a,l]pyrene (DB[a,l]P) (dibenzo[def,p]chrysene) is a highly carcinogenic polycyclic aromatic hydrocarbon (PAH) that has been identified in tobacco smoke and is found in our environment due to incomplete combustion of organic matter. Its metabolites are known to form stable DNA adducts in bacteria and mammalian cells, and can lead to tumors in animal models. Glucuronidation of major metabolites of DB[a,l]P by the uridine-5'-diphosphate glucuronosyltransferase (UGT) family of enzymes is an important route of detoxification of this pro-carcinogen. The focus of the current study was to characterize the glucuronidation of the pro-carcinogenic enantiomers DB[a,l]P-(+)-trans-11S,12S-diol and DB[a,l]P-(-)-trans-11R,12R-diol. Glucuronidation assays with HEK293 cell lines overexpressing individual human UGT enzymes demonstrated that UGTs 1A1, 1A4, 1A7, 1A8, 1A9, 1A10, and 2B7 glucuronidated one or both DB[a,l]P-trans-11,12-diol enantiomers. Three glucuronide conjugates were observed in activity assays with UGTs 1A1 and 1A10, while two glucuronides were formed by UGTs 1A7, 1A8, and 1A9, and one glucuronide was made by UGT1A4 and UGT2B7. Enzyme kinetic analysis indicated that UGT1A9 was the most efficient UGT at forming both the (+)-DB[a,l]P-11-Gluc and (-)-DB[a,l]P-11-Gluc products, while UGTs 1A1 and 1A10 were the most efficient at forming the (+)-DB[a,l]P-12-Gluc product (as determined by k(cat)/K(M)). Incubations with human liver microsomes showed the formation of three diastereomeric glucuronide products: (+)-DB[a,l]P-11-Gluc, (+)-DB[a,l]P-12-Gluc, and (-)-DB[a,l]P-11-Gluc, with an average overall ratio of 31:32:37 in four liver specimens. Human bronchus and trachea tissue homogenates demonstrated glucuronidation activity against both DB[a,l]P-trans-11,12-diol enantiomers, with both tissues producing the (+)-DB[a,l]P-11-Gluc and (+)-DB[a,l]P-12-Gluc with little or no formation of (-)-DB[a,l]P-11-Gluc. These results indicate that multiple UGTs are involved in the stereospecific glucuronidation of DB[a,l]P-trans-11,12-diol in a pattern consistent with their expression in respiratory tract tissues and that glucuronidation may be an important first-line detoxification mechanism of DB[a,l]P metabolites.     

Identification and quantification of the depurinating DNA adducts formed in mouse skin treated with dibenzo[a,l]pyrene (DB[a,l]P) or its metabolites and in rat mammary gland treated with DB[a,l]P

Dibenzo[a,l]pyrene (DB[a,l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon and has been identified in the environment. Comparative tumorigenicity studies in mouse skin and rat mammary gland indicate that DB[a,l]P is slightly more potent than DB[a,l]P-11,12-dihydrodiol and much more potent than (+/-)-syn-DB[a,l]P-11,12-dihydrodiol-13,14-epoxide {(+/-)-syn-DB[a,l]PDE} and (+/-)-anti-DB[a,l]PDE. We report here the identification and quantification of the depurinating adducts formed in mouse skin treated with DB[a,l]P, DB[a,l]P-11,12-dihydrodiol, (+/-)-syn-DB[a,l]PDE, or (+/-)-anti-DB[a,l]PDE and rat mammary gland treated with DB[a,l]P. The biologically formed adducts were compared with standard adducts by their retention times on HPLC and their spectra obtained by fluorescence line-narrowing spectroscopy at low temperature. In mouse skin treated with DB[a,l]P, depurinating adducts comprised 99% of the total adducts. Most of the depurinating adducts were formed by one-electron oxidation, with 63% at Ade and 12% at Gua. The remainder were formed by the diol epoxide, with 18% at Ade and 6% at Gua. When mouse skin was treated with DB[a,l]P-11,12-dihydrodiol, depurinating adducts comprised 80% of the total, and the predominant one was with Ade (69%). Treatment of skin with (+/-)-syn-DB[a,l]PDE resulted in 32% depurinating adducts, primarily at Ade (25%), whereas treatment with (+/-)-anti-DB[a,l]PDE produced 97% stable adducts. The formation of depurinating adducts following treatment of rat mammary gland with DB[a,l]P resulted in approximately 98% depurinating adducts, with the major adducts formed by one-electron oxidation. Only one depurinating diol epoxide adduct was formed. Tumorigenicity, mutations, and DNA adduct data suggest that depurinating Ade adducts play a major role in the initiation of tumors by DB[a,l]P.     

Preliminary physiologically based pharmacokinetic models for benzo[a]pyrene and dibenzo[def,p]chrysene in rodents

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated as byproducts of natural and anthropogenic combustion processes. Despite significant public health concern, physiologically based pharmacokinetic (PBPK) modeling efforts for PAHs have so far been limited to naphthalene, plus simpler PK models for pyrene, nitropyrene, and benzo[a]pyrene (B[a]P). The dearth of published models is due in part to the high lipophilicity, low volatility, and myriad metabolic pathways for PAHs, all of which present analytical and experimental challenges. Our research efforts have focused upon experimental approaches and initial development of PBPK models for the prototypic PAH, B[a]P, and the more potent, albeit less studied transplacental carcinogen, dibenzo[def,p]chrysene (DBC). For both compounds, model compartments included arterial and venous blood, flow limited lung, liver, richly perfused and poorly perfused tissues, diffusion limited fat, and a two compartment theoretical gut (for oral exposures). Hepatic and pulmonary metabolism was described for both compounds, as were fractional binding in blood and fecal clearance. Partition coefficients for parent PAH along with their diol and tetraol metabolites were estimated using published algorithms and verified experimentally for the hydroxylated metabolites. The preliminary PBPK models were able to describe many, but not all, of the available data sets, comprising multiple routes of exposure (oral, intravenous) and nominal doses spanning several orders of magnitude. Supported by Award Number P42 ES016465 from the National Institute of Environmental Health Sciences.     

Metabolism and excretion of benzo[a]pyrene in the rabbit

1. Following i.v. administration of [14C]benzo[a]pyrene (3 mumol/kg) to rabbits, 30% of the 14C dose appeared in bile and 12% in urine, within six hours. 2. Biliary and urinary metabolites were mainly conjugated; less than 12% of the 14C was extractable with ethyl acetate, but after treatment with beta-glucuronidase or aryl sulphatase 30-40% became extractable. 3. H.p.l.c. analysis of the extracts indicated that the major non-polar metabolite was benzo[a]pyrene, 9,10-diol (18% of 14C in bile and 24% of 14C in urine, mainly conjugated with glucuronic acid). Smaller amounts of the 4,5-diol, the 3,6-quinone, and the 9-hydroxy- and 3-hydroxybenzo[a]pyrene were also found in bile (total less than 10%), together with 9-hydroxybenzo[a]pyrene and two unknown metabolites (X and Y) in urine (total less than 4%). 4. The proximate carcinogen, the 7,8-diol, was not detected in any extract. 5. After intraduodenal administration of biliary metabolites of [14C]benzo[a]pyrene (approx. 0 X 3 mumol), 14C was excreted in the bile (21% dose) and urine (14%) within 23 h, indicating that metabolites can undergo enterohepatic circulation in the rabbit.     

Metabolism and excretion of benzo[a]pyrene in the rabbit

1. Following i.v. administration of [¹⁴C]benzo[a]pyrene (3 μmol/kg) to rabbits, 30% of the ¹⁴C dose appeared in bile and 12% in urine, within six hours.

2. Biliary and urinary metabolites were mainly conjugated; <12% of the ¹⁴C was extractable with ethyl acetate, but after treatment with β-glucuronidase or aryl sulphatase 30–40% became extractable.

3. H.p.l.c. analysis of the extracts indicated that the major non-polar metabolite was benzo[a]pyrene, 9,10-diol (18% of ¹⁴C in bile and 24% of ¹⁴C in urine, mainly conjugated with glucuronic acid). Smaller amounts of the 4,5-diol, the 3,6-quinone, and the 9-hydroxy- and 3-hydroxybenzo[a]pyrene were also found in bile (total <10%), together with 9-hydroxybenzo[a]pyrene and two unknown metabolites (X and Y) in urine (total <4%).

4. The proximate carcinogen, the 7,8-diol, was not detected in any extract.

5. After intraduodenal administration of biliary metabolites of [¹⁴C]benzo[a]pyrene (approx. 0·3 μmol), ¹⁴C was excreted in the bile (21% dose) and urine (14%) within 23?h, indicating that metabolites can undergo enterohepatic circulation in the rabbit.     

Mdm2 as a Sensitive and Mechanistically Informative Marker for Genotoxicity Induced by Benzo[a]pyrene and Dibenzo[a,l]pyrene

Mdm2 is an oncoprotein interacting with p53 and maintaininglow p53 levels in unstressed cells. Here we investigated theeffect of genotoxic compounds on Mdm2 phosphorylation levels.Employing the Mdm2 2A10 antibody and phosphatase treatment wefound that Mdm2 accumulated in HepG2 cells when exposed to lowconcentrations of genotoxic compounds such as mitomycin C, etoposide,5-fluorouracil, and benzo[a]pyrene (BP). The low-dose responseswere not accompanied by p53 accumulation and the effect of lowconcentrations of BP on Mdm2 was not affected by small interferingRNA for p53. In human lymphoblasts 10nM BP induced an Mdm2 response.Low concentrations of BP also induced binding of Mdm2 to chromatinin HepG2 cells, but no p53 binding or H2AX phosphorylation.The more mutagenic dibenzo[a,l]pyrene as well as higher BP concentrationsinstead induced H2AX and p53 Ser15 association with chromatin.Acrolein potentiated the effect of BP on p53 stabilization andchromatin binding. Taken together, these data suggest that (1)Mdm2 is a sensitive biomarker for certain types of genotoxicity,and (2) that polycyclic aromatic hydrocarbons-induced Mdm2 bindingto chromatin reflects repairable damage, whereas chromatin bindingof p53 Ser15 and H2AX indicates more persistent DNA damage.The analysis of Mdm2 and related endpoints might be useful forevaluating mutagenic potentials of DNA damages. It is suggestedthat patterns documented here can be used for separating BPdoses that induce readily repaired DNA adducts from doses thatoverwhelm this capacity.     

Absence of mutagenicity of coralyne and related antileukemic agents: structural comparison with the potent carcinogen 7,12-dimethylbenz[a]anthracene.

The structural similarity between antileukemic alkaloid coralyne and the carcinogenic and antineoplastic hydrocarbon 7, 12-dimethylbenz[a]anthracene, as well as the similarity between the antileukemic alkaloid nitidine and the carcinogenic hydrocarbon 5-methylchrysene, prompted a mutagenicity evaluation of coralyne sulfoacetate, nitidine chloride, the 8-ethyl homolog of coralyne, nitidine methosulfate, and the tetramethoxy analog of nitidine by the Ames method against the histidine-auxotroph strains of Salmonella typhimurium TA-1537, TA-1538, TA-98, and TA-100; 7,12-dimethylbenz[a]anthracene was used as a reference standard. The mutagenicity of these antileukemic compounds was either completely eliminated or drastically reduced, but the mutagenic response was generally high for 7,12-dimethylbenz[a]anthracene. The results suggest that the presence of a quaternary nitrogen atom and alkoxy groups could be important in alleviating the mutagenicity of the parent mutagenic and carcinogenic hydrocarbons.     

Role of activated oxygen species on the mutagenicity of benzo[a]pyrene

Different scavengers of active oxygen species (superoxide dismutase, catalase, mannitol and dimethylfuran) were tested in the Ames Salmonella assay to determine the role of the reactive oxygen species in the benzo[a]pyrene (B[a]P) mutagenesis process. Exogenously added superoxide dismutase or catalase at 10-100 micrograms ml-1 top agar, or 3-12 mM mannitol showed no effect on B[a]P mutagenicity in the presence of S9 mix. However, dimethylfuran (DMF), a singlet oxygen scavenger, inhibited in a dose-related manner the mutagenic response of B[a]P in the presence of the microsomal fraction. DMF at 3 and 6 mM inhibited the number of revertants by 69 and 93% for strain TA 100, and 76 and 78% for TA98, respectively. DMF at these levels was neither toxic nor mutagenic to the bacteria. The result indicates that singlet oxygen may play an important role in promoting B[a]P mutagenicity.     

Conformations of benzene- and dibenzo[a,l]pyrene diol epoxides studied by density functional theory: ground states,transition states,dynamics, and solvent effects

The (-)-anti- and (+)-syn-diol epoxides of dibenzo[a,l]pyrene (DBPDE, 11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a,l]pyrene) and the stereochemically corresponding benzene diol epoxides (BDE, 1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydrobenzene) have been studied by density functional theory (DFT) to determine the structures, energies, dynamics, thermal properties, and solvent effects on the different conformers. The smaller BDE is used as a model compound for studies of transitions between diequatorial and diaxial conformations of the hydroxyl groups. It was found that DBPDE is distorted due to overcrowding in the fjord region and that the arene oxide prefers to be on the same side of the saturated ring as the distal ring ("in") in most stereoisomeric states. For the anti-diastereomer, a diequatorial orientation of the hydroxyl groups is preferred, while the orientation preference in the syn-diastereomer seems to depend on the solvent and the in/out conformation. Transition states for the interconversions between in and out conformations of DBPDE as well as between diequatorial and diaxial conformations on BDE have been found, and transition rates have been estimated by transition state theory. The barriers are found to be moderate, the highest being 9.6 kcal/mol. Solvent effects as well as zero-point vibrational energy and thermal effects were included and found to be significant in some cases. The results presented here are in agreement with previous experimental studies.     

A novel method for the isolation and identification of stable DNA adducts formed by Dibenzo[a,l]pyrene and Dibenzo[a,l]pyrene 11, 12-dihydrodiol 13,14-epoxides in vitro.

Our laboratory previously reported the identification and quantification of depurinating DNA adducts of dibenzo[a,l]pyrene (DB[a,l]P) in vitro, which comprise about 84% of all the DNA adducts that are formed [Li, K.-M., et al. (1995) Biochemistry 34, 8043-8049]. To determine a complete adduct profile and identify both stable and depurinating DNA adducts, we have developed a relatively simple, nonradioactive method for the identification of stable DNA adducts by combining enzymatic digestion, HPLC, and fluorescence line-narrowing spectroscopy (FLNS) techniques. Calf thymus DNA, bound to either (+/-)-anti- or (+/-)-syn-DB[a,l]PDE or rat liver microsome-activated DB[a,l]P, was first digested to 3'-mononucleotides with micrococcal nuclease and spleen phosphodiesterase. The adducts were then separated by HPLC with an ion-pair column and identified by FLNS by using the spectra of standards for comparison. In reactions with (+/-)-anti-DB[a,l]PDE, three adducts, an anti-cis-DB[a,l]PDE-dGMP, an anti-trans-DB[a, l]PDE-dAMP, and an anti-cis-DB[a,l]PDE-dAMP, were identified by HPLC and FLNS. In reactions with (+/-)-syn-DB[a,l]PDE, a pair of syn-trans-DB[a,l]PDE-dGMP adducts as well as a syn-cis-DB[a, l]PDE-dGMP, a syn-cis-DB[a,l]PDE-dAMP, and a pair of syn-trans-DB[a, l]PDE-dAMP adducts were identified. From the digest of microsome-activated DB[a,l]P-bound DNA, a syn-trans-DB[a,l]PDE-dGMP, an anti-cis-DB[a,l]PDE-dGMP, a syn-trans-DB[a,l]PDE-dAMP, and a syn-cis-DB[a,l]PDE-dAMP adduct were identified. An anti-cis-DB[a, l]PDE-dAMP adduct was identified only by (32)P-postlabeling. A total of five of the stable adducts formed by DB[a,l]P and nine of the stable adducts formed by DB[a,l]PDE in vitro have been identified. These adducts were also correlated to adduct spots in the (32)P-postlabeling method by cochromatography with standards. Approximately 93% of the stable adducts formed in reactions with (+/-)-anti-DB[a,l]PDE, 90% of adducts with (+/-)-syn-DB[a,l]PDE, and 85% of adducts formed with microsome-activated DB[a,l]P have been identified as Gua or Ade adducts. Equal amounts of stable Gua and Ade adducts were observed in the microsome-catalyzed binding of DB[a, l]P to calf thymus DNA, while 1.4 times more Gua adducts than Ade adducts were obtained in reactions with (+/-)-anti- or (+/-)-syn-DB[a,l]PDE.     

In vitro metabolism of benzo[a]pyrene and dibenzo[def,p]chrysene in rodent and human hepatic microsomes

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and often carcinogenic contaminants released into the environment during natural and anthropogenic combustion processes. Benzo[a]pyrene (B[a]P) is the prototypical carcinogenic PAH, and dibenzo[def,p]chrysene (DBC) is a less prevalent, but highly potent transplacental carcinogenic PAH. Both are metabolically activated by isoforms of the cytochrome P450 enzyme superfamily to form reactive carcinogenic and cytotoxic metabolites. Metabolism of B[a]P and DBC was studied in hepatic microsomes of male Sprague-Dawley rats, naïve and pregnant female B6129SF1/J mice, and female humans, corresponding to available pharmacokinetic data. Michaelis–Menten saturation kinetic parameters including maximum rates of metabolism (V_MAX, nmol/min/mg microsomal protein), affinity constants (K_M, μM), and rates of intrinsic clearance (CL_INT, ml/min/kg body weight) were calculated from substrate depletion data. CL_INT was also estimated from substrate depletion data using the alternative in vitro half-life method. V_MAX and CL_INT were higher for B[a]P than DBC, regardless of species. Clearance for both B[a]P and DBC was highest in naïve female mice and lowest in female humans. Clearance rates of B[a]P and DBC in male rat were more similar to female human than to female mice. Clearance of DBC in liver microsomes from pregnant mice was reduced compared to naïve mice, consistent with reduced active P450 protein levels and elevated tissue concentrations and residence times for DBC observed in previous in vivo pharmacokinetic studies. These findings suggest that rats are a more appropriate model organism for human PAH metabolism, and that pregnancy's effects on metabolism should be further explored.