Metabolism and distribution of benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione) in human lung cells by liquid chromatography tandem mass spectrometry: detection of an adenine B[a]P-7,8-dione adduct.

Benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione) is produced in human lung cells by the oxidation of (±)-B[a]P-7,8-trans-dihydrodiol, which is catalyzed by aldo-keto reductases (AKRs). However, information relevant to the cell-based metabolism of B[a]P-7,8-dione is lacking. We studied the metabolic fate of 2 µM 1,3-[(3)H(2)]-B[a]P-7,8-dione in human lung adenocarcinoma A549 cells, human bronchoalveolar H358 cells, and immortalized human bronchial epithelial HBEC-KT cells. In these three cell lines, 1,3-[(3)H(2)]-B[a]P-7,8-dione was rapidly consumed, and radioactivity was distributed between the organic and aqueous phase of ethyl acetate-extracted media, as well as in the cell lysate pellets. After acidification of the media, several metabolites of 1,3-[(3)H(2)]-B[a]P-7,8-dione were detected in the organic phase of the media by high performance liquid chromatography-ultraviolet-radioactivity monitoring (HPLC-UV-RAM). The structures of B[a]P-7,8-dione metabolites varied in the cell lines and were identified as B[a]P-7,8-dione conjugates with glutathione (GSH) and N-acetyl-l-cysteine (NAC), 8-O-monomethylated-catechol, catechol monosulfate, and monoglucuronide, and monohydroxylated-B[a]P-7,8-dione by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We also obtained evidence for the first time for the formation of an adenine adduct of B[a]P-7,8-dione. Among these metabolites, the identity of the GSH-B[a]P-7,8-dione and the NAC-B[a]P-7,8-dione was further validated by comparison to authentic synthesized standards. The pathways of B[a]P-7,8-dione metabolism in the three human lung cell lines are formation of GSH and NAC conjugates, reduction to the catechol followed by phase II conjugation reactions leading to its detoxification, monohydroxylation, as well as formation of the adenine adduct.

Synthesis of 13C4-labelled oxidized metabolites of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene.

Polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BaP), are ubiquitous environmental contaminants that are implicated in causing lung cancer. BaP is a component of tobacco smoke that is transformed enzymatically to active forms that interact with DNA. We reported previously development of a sensitive stable isotope dilution LC/MS method for analysis of BaP metabolites. We now report efficient syntheses of 13C4-BaP and the complete set of its 13C4-labelled oxidized metabolites needed as internal standards They include the metabolites not involved in carcinogenesis (Group A) and the metabolites implicated in initiation of cancer (Group B). The synthetic approach is novel, entailing use of Pd-catalyzed Suzuki, Sonogashira, and Hartwig cross-coupling reactions combined with PtCl2-catalyzed cyclization of acetylenic compounds. This synthetic method requires fewer steps, employs milder conditions, and product isolation is simpler than conventional methods of PAH synthesis. The syntheses of 13C4-BaP and 13C4-BaP-8-ol each require only four steps, and the 13C-atoms are all introduced in a single step. 13C4-BaP-8-ol serves as the synthetic precursor of all the oxidized metabolites of 13C-BaP implicated in initiation of cancer. The isotopic purities of the synthetic 13C4-BaP metabolites were estimated to be ≥99.9%.

Quantitation of benzo[a]pyrene metabolic profiles in human bronchoalveolar (H358) cells by stable isotope dilution liquid chromatography-atmospheric pressure chemical ionization mass spectrometry.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and are carcinogenic in multiple organs and species. Benzo[a]pyrene (B[a]P) is a representative PAH and has been studied extensively for its carcinogenicity and toxicity. B[a]P itself is chemically inert and requires metabolic activation to exhibit its toxicity and carcinogenicity. Three major metabolic pathways have been well documented. The signature metabolites generated from the radical cation (peroxidase or monooxygenase mediated) pathway are B[a]P-1,6-dione and B[a]P-3,6-dione, the signature metabolite generated from the diol-epoxide (P450 mediated) pathway is B[a]P-r-7,t-8,t-9,c-10-tetrahydrotetrol (B[a]P-tetrol-1), and the signature metabolite generated from the o-quinone (aldo-keto reductase mediated) pathway is B[a]P-7,8-dione. The contributions of these different metabolic pathways to cancer initiation and the exploitation of this information for cancer prevention are still under debate. With the availability of a library of [(13)C(4)]-labeled B[a]P metabolite internal standards, we developed a sensitive stable isotope dilution atmospheric pressure chemical ionization tandem mass spectrometry method to address this issue by quantitating B[a]P metabolites from each metabolic pathway in human lung cells. This analytical method represents a 500-fold increased sensitivity compared with that of a method using HPLC-radiometric detection. The limit of quantitation (LOQ) was determined to be 6 fmol on column for 3-hydroxybenzo[a]pyrene (3-OH-B[a]P), the generally accepted biomarker for B[a]P exposure. This high level of sensitivity and robustness of the method was demonstrated in a study of B[a]P metabolic profiles in human bronchoalveolar H358 cells induced or uninduced with the AhR ligand, 2,3,7,8-tetrachlorodibenzodioxin (TCDD). All the signature metabolites were detected and successfully quantitated. Our results suggest that all three metabolic pathways contribute equally in the overall metabolism of B[a]P in H358 cells with or without TCDD induction. The sensitivity of the method should permit the identification of cell-type differences in B[a]P activation and detoxication and could also be used for biomonitoring human exposure to PAH.

Specificity of human aldo-keto reductases, NAD(P)H:quinone oxidoreductase, and carbonyl reductases to redox-cycle polycyclic aromatic hydrocarbon diones and 4-hydroxyequilenin-o-quinone.

Polycyclic aromatic hydrocarbons (PAHs) are suspect human lung carcinogens and can be metabolically activated to remote quinones, for example, benzo[a]pyrene-1,6-dione (B[a]P-1,6-dione) and B[a]P-3,6-dione by the action of either P450 monooxygenase or peroxidases, and to non-K region o-quinones, for example B[a]P-7,8-dione, by the action of aldo keto reductases (AKRs). B[a]P-7,8-dione also structurally resembles 4-hydroxyequilenin o-quinone. These three classes of quinones can redox cycle, generate reactive oxygen species (ROS), and produce the mutagenic lesion 8-oxo-dGuo and may contribute to PAH- and estrogen-induced carcinogenesis. We compared the ability of a complete panel of human recombinant AKRs to catalyze the reduction of PAH o-quinones in the phenanthrene, chrysene, pyrene, and anthracene series. The specific activities for NADPH-dependent quinone reduction were often 100-1000 times greater than the ability of the same AKR isoform to oxidize the cognate PAH-trans-dihydrodiol. However, the AKR with the highest quinone reductase activity for a particular PAH o-quinone was not always identical to the AKR isoform with the highest dihydrodiol dehydrogenase activity for the respective PAH-trans-dihydrodiol. Discrete AKRs also catalyzed the reduction of B[a]P-1,6-dione, B[a]P-3,6-dione, and 4-hydroxyequilenin o-quinone. Concurrent measurements of oxygen consumption, superoxide anion, and hydrogen peroxide formation established that ROS were produced as a result of the redox cycling. When compared with human recombinant NAD(P)H:quinone oxidoreductase (NQO1) and carbonyl reductases (CBR1 and CBR3), NQO1 was a superior catalyst of these reactions followed by AKRs and last CBR1 and CBR3. In A549 cells, two-electron reduction of PAH o-quinones causes intracellular ROS formation. ROS formation was unaffected by the addition of dicumarol, suggesting that NQO1 is not responsible for the two-electron reduction observed and does not offer protection against ROS formation from PAH o-quinones.

Detoxication of structurally diverse polycyclic aromatic hydrocarbon (PAH) o-quinones by human recombinant catechol-O-methyltransferase (COMT) via O-methylation of PAH catechols.

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Metabolic activation of intermediate PAH trans-dihydrodiols by aldo-keto reductases (AKRs) leads to the formation of electrophilic and redox-active o-quinones. We investigated whether O-methylation by human recombinant soluble catechol-O-methyltransferase (S-COMT) is a feasible detoxication step for a panel of structurally diverse PAH-catechols produced during the redox-cycling process. Classes of PAH non-K-region o-quinones (bay region, methylated bay region, and fjord region o-quinones) produced by AKRs were employed in the studies. PAH o-quinones were reduced to the corresponding catechols by dithiothreitol under anaerobic conditions and then further O-methylated by human S-COMT in the presence of S-[³H]adenosyl-l-methionine as a methyl group donor. The formation of the O-methylated catechols was detected by HPLC-UV coupled with in-line radiometric detection, and unlabeled products were also characterized by LC-MS/MS. Human S-COMT was able to catalyze O-methylation of all of the PAH-catechols and generated two isomeric metabolites in different proportions. LC-MS/MS showed that each isomer was a mono-O-methylated metabolite. ¹H NMR was used to assign the predominant positional isomer of benzo[a]pyrene-7,8-catechol as the O-8-monomethylated catechol. The catalytic efficiency (k(cat)/K(m)) varied among different classes of PAH-catechols by 500-fold. The ability of S-COMT to produce two isomeric products from PAH-catechols was rationalized using the crystal structure of the enzyme. We provide evidence that O-8-monomethylated benzo[a]pyrene-7,8-catechol is formed in three different human lung cell lines. It is concluded that human S-COMT may play a critical role in the detoxication of PAH o-quinones generated by AKRs.

Regulation of benzo[a]pyrene-mediated DNA- and glutathione-adduct formation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in human lung cells.

Environmental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), require metabolic activation to DNA-reactive metabolites in order to exert their tumorigenic effects. Benzo[a]pyrene (B[a]P), a prototypic PAH, is metabolized by cytochrome P450 (P450) 1A1/1B1 and epoxide hydrolase to (-)-B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol). B[a]P-7,8-dihydrodiol then undergoes further P4501A1/1B1-mediated metabolism to the ultimate carcinogen, (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE), which forms DNA-adducts primarily with 2'-deoxyguanosine (dGuo) to form (+)-anti-trans-B[a]PDE-N(2)-dGuo (B[a]PDE-dGuo) in DNA. Pretreatment of cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to induce P4501A1/1B1 mRNA expression through the aryl hydrocarbon receptor (AhR) pathway. This causes increased B[a]PDE-dGuo formation in liver cells. In contrast, TCDD induction of H358 lung cells surprisingly caused a decrease in (-)-B[a]P-7,8-dihydrodiol-mediated (+)-B[a]PDE-dGuo adduct formation when compared with the non-TCDD-induced cells. Furthermore, treatment of the TCDD-induced cells with (±)-B[a]PDE also resulted in decreased (+)-B[a]PDE-dGuo adduct formation when compared with the non-TCDD-induced cells. These data suggested that it was a detoxification pathway that had been up-regulated rather than an activation pathway that had been down-regulated. LC-MS was used to analyze B[a]PDE-dGuo and B[a]PDE-GSH-adducts in H358 lung and HepG2 liver cells. There was a significant increase in the (-)-B[a]PDE-GSH-adduct with high enantiomeric excess after treatment of the TCDD-induced H358 cells with (±)-B[a]PDE when compared with the noninduced cells. This could explain why increased activation of (-)-B[a]P-7,8-dihydrodiol through TCDD up-regulation of P4501A1/1B1 did not lead to increased (+)-B[a]PDE-dGuo adducts in the H358 lung cells. In addition, TCDD did not induce B[a]PDE-GSH-adduct formation in HepG2 liver cells. (±)-B[a]PDE-GSH-adducts were formed at much lower levels in both TCDD-induced and noninduced HepG2 cells when compared with (-)-B[a]PDE-GSH-adducts in the H358 lung cells. Therefore, our study has revealed that there is a subtle balance between activation and detoxification of B[a]P in lung-derived compared with liver-derived cells and that this determines how much DNA damage occurs.

Regiospecific oxidation of polycyclic aromatic phenols to quinones by hypervalent iodine reagents.

The hypervalent iodine reagents o-iodoxybenzoic acid (IBX) and bis(trifluoro-acetoxy)iodobenzene (BTI) are shown to be general reagents for regio-controlled oxidation of polycyclic aromatic phenols (PAPs) to specific isomers (ortho, para, or remote) of polycyclic aromatic quinones (PAQs). The oxidations of a series of PAPs with IBX take place under mild conditions to furnish the corresponding ortho-PAQs. In contrast, oxidations of the same series of PAPs with BTI exhibit variable regiospecificity, affording para-PAQs where structurally feasible and ortho-PAQs or remote PAQ isomers in other cases. The structures of the specific PAQ isomers formed are predictable on the basis of the inherent regioselectivities of the hypervalent iodine reagents in combination with the structural requirements of the phenol precursors. IBX and BTI are recommended as the preferred reagents for regio-controlled oxidation of PAPs to PAQs.

Aryl hydrocarbon receptor facilitates DNA strand breaks and 8-oxo-2'-deoxyguanosine formation by the aldo-keto reductase product benzo[a]pyrene-7,8-dione.

Polycyclic aromatic hydrocarbon (PAH) o-quinones produced by aldo-keto reductases are ligands for the aryl hydrocarbon receptor (AhR) (Burczynski, M. E., and Penning, T. M. (2000) Cancer Res. 60, 908-915). They induce oxidative DNA lesions (reactive oxygen species-mediated DNA strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) formation) in human lung cells. We tested whether the AhR enhances PAH o-quinone-mediated oxidative DNA damage by translocating these ligands to the nucleus. Using the single cell gel electrophoresis (comet) assay to detect DNA strand breaks in murine hepatoma Hepa1c1c7 cells and its AhR- and aryl hydrocarbon receptor nuclear translocator-deficient variants, benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione) produced fewer DNA strand breaks in AhR-deficient cells compared with aryl hydrocarbon receptor nuclear translocator-deficient and wild type Hepa1c1c7 cells. Decreased DNA strand breaks were also observed in human bronchoalveolar H358 cells in which the AhR was silenced by siRNA. The antioxidant alpha-tocopherol and the iron chelator/antioxidant desferal decreased the formation of B[a]P-7,8-dione-mediated DNA strand breaks indicating that they were reactive oxygen species-dependent. By coupling the comet assay to 8-oxoguanine glycosylase (hOGG1), which excises 8-oxo-Gua, strand breaks dependent upon this lesion were measured. hOGG1 treatment produced more DNA single strand breaks in B[a]P-7,8-dione-treated Hepa cells and H358 cells than in its absence. The levels of hOGG1-dependent DNA strand breaks mediated by B[a]P-7,8-dione were lower in AhR-deficient Hepa and AhR knockdown H358 cells. The AhR antagonist alpha-naphthoflavone also attenuated B[a]P-7,8-dione-mediated DNA strand breaks. The decrease in 8-oxo-dGuo levels in AhR-deficient Hepa cells and AhR knockdown H358 cells was validated by immunoaffinity capture stable isotope dilution ([(15)N(5)]8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry. We conclude that the AhR shuttles PAH o-quinone genotoxins to the nucleus and enhances oxidative DNA damage.

Synthesis of phenol and quinone metabolites of benzo[a]pyrene, a carcinogenic component of tobacco smoke implicated in lung cancer.

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants produced in the combustion of organic matter. PAHs are present in automobile exhaust and tobacco smoke, and they have recently been designated as human carcinogens. Current evidence indicates that PAHs are activated enzymatically to mutagenic metabolites that interact with DNA. There is evidence for three pathways of activation: the diol epoxide path, the radical-cation path, and the quinone path. The relative importance of these paths for human lung cancer has not been established. We now report syntheses of the principal phenol and quinone isomers of the prototype PAH carcinogen benzo[a]pyrene (BP) that are known or are suspected to be formed as metabolites of BP in human bronchoalveolar cells. The methods of synthesis were designed to be adaptable to the preparation of the (13)C-labeled analogues of the BP metabolites. These compounds are needed as standards for sensitive LC-MS/MS methods for analysis of BP metabolites formed in lung cells. Efficient novel syntheses of the 1-, 3-, 6-, 9-, and 12-BP phenols and the BP 1,6-, 3,6-, 6,12-, and 9,10-quinones are now reported. The syntheses of the BP phenols (except 6-HO-BP) involve the key steps of Pd-catalyzed Suzuki-Miyaura cross-coupling of a naphthalene boronate ester with a substituted aryl bromide or triflate ester. The BP quinones were synthesized from the corresponding BP phenols by direct oxidation with the hypervalent iodine reagents IBX or TBI. These reagents exhibited different regiospecificities. IBX oxidation of the 7- and 9-BP phenols provided the ortho-quinone isomers (BP 7,8- and 9,10-diones, respectively), whereas TBI oxidation of the 1-, 3-, and 12-BP phenols furnished BP quinone isomers with carbonyl functions in separate rings (BP 1,6-, 3,6-, and 6,12-diones, respectively).

Oxidation of PAH trans-dihydrodiols by human aldo-keto reductase AKR1B10.

AKR1B10 has been identified as a potential biomarker for human nonsmall cell lung carcinoma and as a tobacco exposure and response gene. AKR1B10 functions as an efficient retinal reductase in vitro and may regulate retinoic acid homeostasis. However, the possibility that this enzyme is able to activate polycyclic aromatic hydrocarbon (PAH) trans-dihydrodiols to form reactive and redox-active o-quinones has not been investigated to date. AKR1B10 was found to oxidize a wide range of PAH trans-dihydrodiol substrates in vitro to yield PAH o-quinones. Reactions of AKR1B10 proceeded with improper stereochemistry, since it was specific for the minor (+)-benzo[a]pyrene-7S,8S-dihydrodiol diastereomer formed in vivo. However, AKR1B10 displayed reasonable activity in the oxidation of both the (-)-R,R and (+)-S,S stereoisomers of benzo[g]chrysene-11,12-dihydrodiol and oxidized the potentially relevant, albeit minor, (+)-benz[a]anthracene-3S,4S-dihydrodiol metabolite. We find that AKR1B10 is therefore likely to play a contributing role in the activation of PAH trans-dihydrodiols in human lung. AKR1B10 retinal reductase activity was confirmed in vitro and found to be 5- to 150-fold greater than the oxidation of PAH trans-dihydrodiols examined. AKR1B10 was highly expressed at the mRNA and protein levels in human lung adenocarcinoma A549 cells, and robust retinal reductase activity was measured in lysates of these cells. The much greater catalytic efficiency of retinal reduction compared to PAH trans-dihydrodiol metabolism suggests AKR1B10 may play a greater role in lung carcinogenesis through dysregulation of retinoic acid homeostasis than through oxidation of PAH trans-dihydrodiols.

Evidence for the aldo-keto reductase pathway of polycyclic aromatic trans-dihydrodiol activation in human lung A549 cells.

Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (+/-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-trans-dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione), a redox-active o-quinone. We show that B[a]P-7,8-trans-dihydrodiol (AKR substrate) and B[a]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (+/-)-trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene or the diol-epoxide, (+/-)-anti-7,8-dihydroxy-9alpha,10beta-epoxy-7,8,9,10-tetrahydro-B[a]P. B[a]P-7,8-trans-dihydrodiol and B[a]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP(+)/NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([(15)N(5)]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[a]P-7,8-trans-dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O-methyl transferase (COMT) inhibitor, suggesting that COMT protects against o-quinone-mediated redox cycling. We conclude that activation of PAH-trans-dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.

Fjord-region benzo[g]chrysene-11,12-dihydrodiol and benzo[c]phenanthrene-3,4-dihydrodiol as substrates for rat liver dihydrodiol dehydrogenase (AKR1C9): structural basis for stereochemical preference.

This study demonstrates that benzo[g]chrysene-11,12-dihydrodiol (B[g]C-11,12-dihydrodiol) derived from the fjord-region parent hydrocarbon B[g]C is oxidized by rat AKR1C9 with a k c a t/ K m 100 times greater than that observed with the commonly studied bay-region benzo[ a]pyrene-7,8-dihydrodiol (B[a]P-7,8-dihydrodiol). Conversely, despite its strikingly similar structure to B[ g]C-11,12-dihydrodiol, benzo[ c]phenanthrene-3,4-dihydrodiol (B[ c]Ph-3,4-dihydrodiol) is consumed by AKR1C9 at sluggish rates comparable to those observed with B[ a]P-7,8-dihydrodiol. CD spectroscopy revealed that only the (+)-B[ g]C-11,12-dihydrodiol stereoisomer was oxidized, while AKR1C9 oxidized both stereoisomers of B[a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol. The (+)- S, S- and (-)- R, R-stereoisomers of B[g]C-11,12-dihydrodiol were purified by chiral RP-HPLC. The 11 S,12 S-stereoisomer was oxidized at the same rate as the racemate. The 11 R,12 R-stereoisomer did not act as an inhibitor to AKR1C9, indicating that the (-)- R, R-stereoisomer was excluded from the active site. To understand the basis of stereochemical preference, we screened alanine-scanning mutants of active site residues of AKR1C9. These studies revealed that in comparison to the wild type, F129A, W227A, and Y310A enabled the oxidation of both the B[g]C-11 S,12 S-dihydrodiol and the B[g]C-11 R,12 R-dihydrodiol. Molecular modeling revealed that unlike B[a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol, B[g]C-11,12-dihydrodiol enantiomers are significantly bent out of plane. As a consequence, the (-)- R, R-stereoisomer was prevented from binding to the active site because of unfavorable interactions with F129, W227, or Y310. Additionally, LC/MS validated that the product of the reaction of B[g]C-11,12-dihydrodiol oxidation catalyzed by AKR1C9 was B[g]C-11,12-dione, which was trapped in vitro with the nucleophile 2-mercaptoethanol. The similarity between rates of trans-dihydrodiol oxidation by the rat and human liver specific AKRs (AKR1C9 and AKR1C4) implicate these enzymes in hepatocarcinogenesis in rats observed with the fjord-region PAH.

Synthesis of dibenzo[def,p]chrysene, its active metabolites, and their 13C-labeled analogues.

Dibenzo[def,p]chrysene (DBC) is a highly carcinogenic polycyclic aromatic hydrocarbon suspected to be involved in initiation of lung cancer in smokers. Efficient new syntheses of DBC, its active metabolites [DBC diol (1), DBC dione (2), DBC diol epoxide (3)], and their previously unknown 13C2-labeled analogues are reported. The 13C2-labeled analogues are required as standards for sensitive methods of analysis of their DNA adducts in human cells using stable isotope dilution liquid chromatography/tandem mass spectrometry.

Strategies for synthesis of adducts of omicron-quinone metabolites of carcinogenic polycyclic aromatic hydrocarbons with 2'-deoxyribonucleosides.

Polycyclic aromatic hydrocarbons (PAHs) are major environmental carcinogens produced in the combustion of fossil fuels, tobacco, and other organic matter. Current evidence indicates that PAHs are transformed enzymatically to active metabolites that react with DNA to form adducts that result in mutations. Three activation pathways have been proposed: the diol epoxide path, the radical-cation path, and the quinone path. The latter involves aldo-keto reductase mediated oxidation of PAH dihydrodiol metabolites to catechols that enter into redox cycles with quinones. This results in generation of reactive oxygen species (ROS) that attack DNA, and the PAH quinones also react with DNA to form adducts. Several strategies for synthesis of the stable adducts formed by the o-quinone metabolites of carcinogenic PAHs with 2'-deoxyribonucleosides were investigated and compared. The PAH quinones studied were benz[a]anthracene-3,4-dione and its 7-methyl- and 7,12-dimethyl- derivatives. The parent PAHs represent a range of carcinogenicity from inactive to highly potent. Two synthetic methods were devised that differ in the catalyst employed, Pd(OAc)(2) or CuI. The Pd-mediated method involved coupling a protected amino-catechol PAH derivative with a halo-2'-deoxyribonucleoside. The copper-mediated method entailed reaction of a halo-PAH catechol derivative with a 2'-deoxyribonucleoside. Adducts of benz[a]anthracene-3,4-dione (and its 7-methyl- and 7,12-dimethyl- derivatives) with 2'-deoxyadenosine and 2'-deoxyguanosine were prepared by these methods. Availability of adducts of these types through synthesis makes possible for the first time biological studies to determine the role of these adducts in tumorigenesis. The copper-mediated method offers advantages of economy, adaptability to large-scale preparation, utility for synthesis of (13)C- or (15)N-labeled analogues, and nonformation of bis-adducts as secondary products.

Synthesis of 13C2-Benzo[a]pyrene and its 7,8-Dihydrodiol and 7,8-Dione Implicated as Carcinogenic Metabolites.

Synthesis of the 13C2-labelled analogues of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene and its active metabolites are described. The method entails Pd-catalyzed Suzuki-Miyaura coupling of a naphthalene boronic acid with 2-bromobenzene-1,3-dialdehyde followed by Wittig reaction of the product with 13CH2=PPh3.

Substituent effects in benz[a]anthracene carbocations: a stable ion, electrophilic substitution (nitration, bromination), and DFT study.

A series of novel carbocations were generated from isomeric monoalkylated and dialkylated benz[a]anthracenes (BAs) by low-temperature protonation in FSO(3)H/SO(2)ClF. With the monoalkyl derivatives (5-methyl, 6-methyl, 7-methyl, and 7-ethyl) as well as the D-ring methylated analogues (9-methyl, 10-methyl, and 11-methyl), the C-7 or the C-12 protonated carbocations were observed (as the sole or major carbocation) in all cases. Protonation of the 12-methyl derivative (9) gave the C-7 protonated carbocation (9H+) as the kinetic species and the ipso-protonated carbocation (9aH+) as the thermodynamic cation. With the 12-ethyl derivative (10), relief of steric strain in the bay-region greatly favors ipso-protonation (10aH+). With 3,9-dimethyl (14), C-7 protonation (14H+) is strongly favored (with <10% protonation at C-12), and with 1,12-dimethyl (15) the sole species observed is the C-7 protonated carbocation (15H+). For 7-methyl-12-ethyl, 7-ethyl-12-methyl, and 7,12-diethyl derivatives (16, 17, and 18), two ipso-protonated carbocations were initially formed (C-7/C-12), rearranging in time to give the C-12 protonated carbocations exclusively (16aH+, 17aH+, and 18aH+). Protonation outcomes are compared with the computed relative energies by DFT. Charge delocalization paths in the resulting carbocations were deduced based on the magnitude of Deltadelta13C values. For the thermodynamically more stable C-12 protonated carbocations, the charge delocalization path is analogous to those derived based on computed NPA charges for the benzylic carbocations formed by 1,2-epoxide (bay-region) and 5,6-epoxide (K-region) ring opening. Nitration (and bromination) of the 4-methyl, 7-methyl, 7-ethyl, 3,9-dimethyl, and 1,12-dimethyl derivatives resulted in isolation and characterization of several novel derivatives. Excellent agreement is found between low-temperature protonation selectivities and the regioselectivities observed in model substitution reactions.

Metabolism of benzo[a]pyrene in human bronchoalveolar H358 cells using liquid chromatography-mass spectrometry.

Benzo[ a]pyrene (B[ a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways: by peroxidases (e.g., cytochrome P450 peroxidase) to yield radical cations, by P4501A1/1B1 monooxygenation and epoxide hydrolase to yield diol epoxides, and by P4501A1/1B1 monooxygenation, epoxide hydrolase, and aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung; however, the profile of B[ a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[ a]P, and metabolites generated by peroxidase (B[ a]P-1,6- and B[ a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation [3-hydroxy-B[ a]P, B[ a]P-7,8- and 9,10- trans-dihydrodiols, and B[ a]P- r-7, t-8, t-9, c-10-tetrahydrotetrol (B[ a]P-tetraol-1)], and from AKRs (B[ a]P-7,8-dione) were detected and quantified by RP-HPLC, with in-line photo-diode array and radiometric detection, and identified by liquid chromatography-mass spectrometry (LC-MS). Progress curves showed a lag phase in the formation of 3-hydroxy-B[ a]P, B[ a]P-7,8- trans-dihydrodiol, B[ a]P-tetraol-1, and B[ a]P-7,8-dione over 24 h. Northern blot analysis showed that B[ a]P induced P4501B1 and AKR1C isoforms in H358 cells in a time-dependent manner, providing an explanation for the lag phase. Pretreatment of H358 cells with 10 nM 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) eliminated this lag phase but did not alter the levels of the individual metabolites observed, suggesting that both B[ a]P and TCDD induction ultimately yield the same B[ a]P metabolic profile. The one exception was B[ a]P-3,6-dione which was formed without a lag phase in the absence and presence of TCDD, suggesting that the peroxidase responsible for its formation was neither P4501A1 nor 1B1. Candidate peroxidases that remain include PGH synthases and uninduced P450 isoforms. This study shows that the P4501A1/1B1 and AKR pathways are inducible in human lung cells and that the peroxidase pathway was not. It also provides evidence that each of the pathways of PAH activation yields their distinctive metabolites in H358 human lung cells and that each pathway may contribute to the carcinogenic process.

Efficient syntheses of C(8)-aryl adducts of adenine and guanine formed by reaction of radical cation metabolites of carcinogenic polycyclic aromatic hydrocarbons with DNA.

The synthesis of the C(8)-aryl adducts of adenine and guanine formed by reaction of the radical cation metabolites of carcinogenic polycyclic aromatic hydrocarbons (PAHs), such as benzo[a]pyrene (BP) and dibenzo[def,p]chrysene (DBC), with DNA is reported. The synthetic approach involves in the key step direct reaction of a PAH aldehyde with a di- or triamine precursor of a purine. The method is operationally simple, affords good yields of adducts, and is broad in its scope. The C(8)-aryl adducts of adenine and guanine derived from BP (6-BP-8-Ade and 6-BP-8-Gua) and DBC (10-DBC-8-Ade and 10-DBC-8-Gua) were synthesized in good yields by this method. Analogous C(8)-aryl adenine and guanine derivatives of other PAHs (anthracene, benz[a]anthracene, and chrysene) were also readily prepared via this approach. This method of synthesis is superior to the only method that is currently available. It entails direct reaction of short-lived PAH radical cations (generated electrochemically or chemically) with 2'-deoxyribonucleosides or the corresponding purine bases. It provides the adducts in low yields accompanied by complex mixtures of secondary products. An alternative synthesis that involves Pd-catalyzed Suzuki-Miyaura coupling of arylboronic acids with 8-bromopurine nucleosides was also investigated. Although the C(8)-purine adducts of PAHs, such as naphthalene, phenanthrene, pyrene, and chrysene, could be prepared by this method, analogous adducts of carcinogenic PAHs and other structurally related PAHs, e.g., anthracene, benz[a]anthracene, benzo[a]pyrene, and dibenzo[def,p]chrysene, could not be obtained. This difference was shown to be a consequence of the facility of competing hydrolytic deboronation of the corresponding arylboronic acids.

Aldo-keto reductase- and cytochrome P450-dependent formation of benzo[a]pyrene-derived DNA adducts in human bronchoalveolar cells.

There is substantial evidence to suggest that polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) induce lung cancer through metabolic activation. As part of a program to delineate the routes of PAH activation, we have examined DNA adducts that are formed in human lung cells. A stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry method was used to quantify eight anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE)-derived DNA adducts in four H358 human bronchoalveolar cell lines with different phenotypes. In P450 1A1/P450 1B1-induced H358 cells exposed to (+/-)-B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), (+)-anti-trans-B[a]PDE-N2-2'-deoxyguanosine [(+)-anti-trans-B[a]PDE-N2-dGuo] was the major DNA adduct, and it formed with no lag phase. In AKR1A1-transfected H358 cells, (+)-anti-trans-B[a]PDE-N2-dGuo was also the major adduct with a 3 h lag phase before significant adduct formation was detected. In AKR1A1-transfected H358 cells with induced P450 1A1/P450 1B1, (+)-anti-trans-B[a]PDE-N2-dGuo was formed with no lag phase in amounts similar to those in the H358 cells with up-regulated P450 1A1/P450 1B1. Surprisingly, the greatest amount of (+)-anti-trans-B[a]PDE-N2-dGuo was formed in the control H358 cells. Furthermore, (+)-anti-trans-B[a]PDE-N2-dGuo formation was 2-fold higher in (-)-B[a]P-7,8-dihydrodiol-exposed H358 cells when compared with (+/-)-B[a]P-7,8-dihydrodiol-exposed cells. The P450 1A1/1B1 inhibitor 2,4,3',5'-tetramethoxystilbene did not attenuate DNA adduct formation in the control H358 cells, suggesting that another P450 was responsible. These data raise the intriguing possibility that P450 1A1/P450 1B1 and AKR1A1 may be protective against (+)-B[a]PDE-mediated DNA damage.

Polycyclic aromatic hydrocarbon (PAH) o-quinones produced by the aldo-keto-reductases (AKRs) generate abasic sites, oxidized pyrimidines, and 8-oxo-dGuo via reactive oxygen species.

Reactive and redox-active polycyclic aromatic hydrocarbon (PAH) o-quinones produced by Aldo-Keto Reductases (AKRs) have the potential to cause depurinating adducts leading to the formation of abasic sites and oxidative base lesions. The aldehyde reactive probe (ARP) was used to detect these lesions in calf thymus DNA treated with three PAH o-quinones (BP-7,8-dione, 7,12-DMBA-3,4-dione, and BA-3,4-dione) in the absence and presence of redox-cycling conditions. In the absence of redox-cycling, a modest amount of abasic sites were detected indicating the formation of a low level of covalent o-quinone depurinating adducts (>3.2 x 10(6) dNs). In the presence of NADPH and CuCl2, the three PAH o-quinones increased the formation of abasic sites due to ROS-derived lesions destabilizing the N-glycosidic bond. The predominant source of AP sites, however, was revealed by coupling the assay with human 8-oxoguanine glycosylase (hOGG1) treatment, showing that 8-oxo-dGuo was the major lesion caused by PAH o-quinones. The levels of 8-oxo-dGuo formation were independently validated by HPLC-ECD analysis. Apyrimidinic sites were also revealed by coupling the assay with Escherichia coli (Endo III) treatment showing that oxidized pyrimidines were formed, but to a lesser extent. Different mechanisms were responsible for the formation of the oxidative lesions depending on whether Cu(II) or Fe(III) was used in the redox-cycling conditions. In the presence of Cu(II)-mediated PAH o-quinone redox-cycling, catalase completely suppressed the formation of the lesions, but mannitol and sodium benzoate were without effect. By contrast, sodium azide, which acts as a *OH and 1O2 scavenger, inhibited the formation of all oxidative lesions, suggesting that the ROS responsible was 1O2. However, in the presence of Fe(III)-mediated PAH o-quinone redox-cycling, the *OH radical scavengers and sodium azide consistently attenuated their formation, indicating that the ROS responsible was *OH.