Bioalkylation of benz[a]anthracene, 7-methylbenz[a]anthracene, and 12-methylbenz[a]anthracene in rat lung cytosol preparations

Benz[a]anthracene (BA) and the monomethyl meso-anthracenic or L-region derivatives 7-methylbenz[a]anthracene (7-methylBA) and 12-methylbenz[a]anthracene (12-methylBA) underwent a bioalkylation substitution reaction in rat lung ctyosol preparations, fortified with S-adenosyl-L-methionine to form the more potent carcinogen 7,12-dimethylbenz[a]anthracene. The methyl groups of the highly reactive L-region methylated metabolites also underwent enzymatic hydroxylation in rat lung cytosol preparations to yield the corresponding hydroxymethyl derivatives, 7-hydroxymethylbenz[a]anthracene, 7-hydroxymethyl-12-methylbenz[a]anthracene, and 7,12-dihydroxymethylbenz[a]anthracene. The biooxidation reaction took place enzymatically, and exclusively, or nearly so, at the reactive methyl groups attached to the meso positions or L-region of the hydrocarbon. Bioalkylation and biooxidation reactions did not occur when the hydrocarbons were incubated with a boiled cytosol preparation, indicating the need for enzymatic activation of the L-region methyl groups. Also, the bioalkylation reaction did not occur in the absence of S-adenosyl-L-methionine. Furthermore, the S-adenosyl-L-methionine-dependent reaction was inhibited by S-adenosyl-L-homocysteine, suggesting that the reaction is catalyzed by a cytosolic S-adenosyl-L-methionine-dependent methyltransferase.     

Chromatographic Characterization of Hemoglobin Benzo[a]pyrene-7,8-diol-9,10-epoxide Adducts

The formation of hemoglobin-carcinogen adducts has been detectedin carcinogen-treated animals and in human populations. Althoughpolynuclear aromatic hydrocarbons are ubiquitous in the humanenvironment and DNA-aromatic hydrocarbon adducts have been detectedin human tissue, the occurrence of hemoglobin-polynuclear aromatichydrocarbon adducts in humans has not been thoroughly described.In this study we examined the effects of reaction conditionson the extent of in vitro reaction of human hemoglobin and (+)[³H]benzo[a]pyrene-7,8-dol-9,10-epoxide (anti)(BPDE), a metabolitethought to be largely responsible for the carcinogenic effectof benzo[a]pyrene. The chromatographic properties of the resultinghemoglobin-BPDE adducts were examined by conventional DEAE-celluloseion exchange liquid chromatography and by reversed phase highperformance liquid chromatography. Several adducts were formedwhich were chromatographically resolved from hemoglobin andfrom the individual globins. Some adducts were basic and someacidic relative to unaltered hemoglobin, suggesting adduct formationby reaction at carboxyl and basic nitrogen groups, respectively.Alteration of the ion - chromatographic properties of the adductsby an ionic sulfhydryl reagent, together with only a moderateeffect of pH on the extent of adduct formation, indicated thatthe adducts were not formed via reaction with the ß₉₃cysteine sulfhydryl group. The chromatographic techniques employedmay be applicable for the characterization and analysis of otherhemoglobin-carcinogen adducts.     

Glutathione conjugates as metabolites of benz[a]anthracene.

1. [3H]Benz[a]anthracene is converted into water-soluble metabolites by microsomal plus soluble fractions of rat-liver in the presence of NADPH and glutathione. Chromatography on Sephadex G25 gave four radioactive peaks; the first contained hydrocarbon or hydrocarbon derivatives bound to soluble protein while the other three peaks contained glutathione conjugates of hydrocarbon metabolites. 2. Conjugates formed when either of the benz[a]anthracene metabolites, 5,6-dihydro-5,6-dihydroxybenz[a]anthracene or 8,9-dihydro-8,9-dihydroxybenz[a]anthracene, were similarly incubated were probably S-(5,6,8,9-tetrahydro-5,6,9-trihydroxybenz[a]anthracen-8-yl)glutathione and S-(5,6,8,9-tetrahydro-6,8,9-trihydroxybena[a]anthracen-5-yl)glutathione respectively. The corresponding peak obtained in the metabolism of benz[a]anthracene probably contains a mixture of these two isomers. 3. The third peak contained the conjugate, S-(5,6-dihydro-l-hydroxybenz-[a]anthracen-k-yl)glutathione, also formed by the conjugation of the "K-region" epoxide of benz[a]anthracene with glutathione. This was not formed in the metabolism of the dihydrodiols. 4. The fourth peak contained a new type of conjugate that is probably S-(8,9,10,11-tetrahydro-8,9,10-trihydroxybenz[a]anthracen-11-yl)glutathione. This conjugate is chromatographically similar to a product obtained from incubation of the 8,9-dihydrodiol, and is probably formed by microsomal oxidation of the 10,11-bond of the dihydrodiol, followed by conjugation of the resulting diol-epoxide with glutathione.     

Synthese von 2,2a,3,4-Tetrahydro-azeto[1,2-d]benz[b]-1,4-oxazin-2,4-dionen

Synthesis of 2,2a,3,4-Tetrahydroazeto[1,2-d]benzo[b]-1,4-oxazine-2,4-diones The 1-(2-hydroxyaryl)-4-oxoazetidine-2-carboxylic acids 7c, 7d and 10 react with dicyclohexylcarbodiimide to give the title compounds 11, 12 and 13 . Compound 13 can also be obtained by nitration of 12 . The starting materials 7c , 7d and 10 are synthesized from the l-aryl-4-oxoazetidine-2,2-dicarboxylates 4a and 4b , which are available from 1 by a method analogous to that described by Sheehan²⁾ or via 8 and 9 .     

TPX2 in malignantly transformed human bronchial epithelial cells by anti-benzo[a]pyrene-7,8-diol-9,10-epoxide

In order to elucidate the function of the targeting protein for Xenopus kinesin-like protein 2 (Xklp2) (TPX2) in the malignant transformation of human bronchial epithelial cells induced by anti-benzo[a]pyrene-trans-7, 8-dihydrodiol-9, 10-epoxide (anti-BPDE), TPX2 was characterized in cells at both the gene and the protein levels. TPX2 was present at higher levels in 16HBE-C cells than in 16HBE cells as demonstrated by two-dimensional gel electrophoresis, immunocytochemistry, Western blot analysis and RT-PCR. TPX2 was also detected in lung squamous-cell carcinoma tissues by immunohistochemistry, but not in normal lung tissues. Depression of TPX2 by RNA interference in 16HBE-C cells led to a decrease in cell proliferation, S-phase cell cycle arrest and cell apoptosis. Abnormal TPX2 tyrosine phosphorylation was detected in 16HBE-C cells, and this could be inhibited, to different degrees, by tyrosine kinase inhibitors. Inhibiting tyrosine phosphorylation in 16HBE-C cells by three selected tyrosine protein kinase inhibitors, tyrphostin 47, AG112 and AG555, caused G(0)/G(1)-phase cell cycle arrest. Our results suggest that anti-BPDE can cause the over-expression of TPX2 and its aberrant tyrosine phosphorylation. Misregulation of TPX2 affects the cell cycle state, proliferation rates and apoptosis.     

Characterization of DNA adducts derived from (+/- )-trans-3,4-dihydroxy-anti-1,2-epoxy-1,2,3,4- tetrahydrodibenz[a,j]anthracene and (+/- )-7-methyl-trans-3,4-dihydroxy- anti-1,2-epoxy-1,2,3,4-tetrahydrodibenz[a,j]anthracene

Structural characterizations of the DNA adducts derived from reaction of the racemic bay region anti-diol epoxides of dibenz[a,j]anthracene and 7-methyldibenz[a,j]anthracene with calf thymus DNA are presented. Quantities of adducts necessary for spectroscopic characterization were obtained from reactions of the respective diol epoxides with individual deoxyribonucleotides. Both hydrocarbon diol epoxides showed similar adduct profiles upon reaction with calf thymus DNA in vitro which were composed mainly of three deoxyguanosine and four deoxyadenosine adducts. No significant modification of pyrimidine bases in DNA was detected with either of the diol epoxides. Approximately 3 times more deoxyguanosine than deoxyadenosine residues in the DNA were found to be modified by both diol epoxides. The DNA reactions showed very similar stereo- and enantioselectivities with both diol epoxides. The stereochemistries of addition of the purine bases to the diol epoxides were determined from analysis of the NMR spectra of individual adducts. The predominant adducts formed were products of trans addition of the exocyclic amino group of purines to the diol epoxides. The enantiomeric nature of the various adducts was determined from reaction of the individual deoxyribonucleotides with the pure (+)-anti-diol epoxide of dibenz[a,j]anthracene. The major deoxyguanosine and deoxyadenosine adducts from reactions with DNA were found to arise from the (+)-enantiomer of both hydrocarbon diol epoxides. The high reactivities of both diol epoxides (24-38%) with DNA in solution are consistent with the high tumor-initiating activity exhibited by the diol epoxide of dibenz[a,j]anthracene relative to the parent hydrocarbon.     

Pulmonary carcinogenesis in mice with a single intratracheal instillation of 9, 10-dimethyl benz[a]anthracene

A single intratracheal instillation of 9,10-dimethyl benz(a)anthracene (DMBA) at 3 different doses of 5, 10, and 20 mg/kg body weight to Balb/c mice for 12 weeks had caused a significant incidence of pulmonary tumors along with inflammatory changes. The number of macrophages in the broncho-alveolar lavage (BAL) fluid increased significantly, while the neutrophil and lymphocyte count as well as the protein content in the BAL fluid remained unchanged. A marked elevation in the lipid peroxidation product as well as the antioxidative enzymes were noted in the DMBA-treated group. The BAL fluid, which contains the surfactant membrane, was tested for rotational diffusion of the small hydrocarbon fluorophore, diphenyl hexatriene, and resulted in an enhanced fluorescence polarization and anisotropy value as well as the order parameter. DMBA treatment also altered the toxicity parameters, such as the lipid peroxidation, catalase, total protein, reduced glutathione, and alanine and amino transferase activities in the liver and kidney tissues. The results suggest that DMBA-induced lung tumor development in Balb/c mice could be an important model for the study of pathophysiology of BAL-fluid-associated surfactant and offers to test a variety of promising chemopreventive/chemotherapeutic agents.     

Synthese von 2,2a,3,4-Tetrahydro[1,2-d]benz[b]-1,4-oxazin-2,4-dionen, 2. Mitt.

Synthesis of 2,2a,3,4,-Tetrahydro[1,2d]benz[b]-1,4-oxazine-2,4-diones, II The diasteromeric 3-amino-β-lactams 7 and 8 were obtained from the azides 5 and 6 and the phthalimido derivatives 9 and 12 . Compound 9 was synthesized directly from 2 by means of phthalimidoacetyl chloride/triethylamine, whereas 12 is accessible from 11 only by the Mitsunobu reaction. Acylation of 7 or 8 (to 13 or 14 ) is followed by hydrolysis of the ester function (to 15 or 16 ) and debenzylation to yield 17 or 18 which are lactonized to 19 or 20 by the action of DCC.     

Synthesis of the tumorigenic 3,4-dihydrodiol metabolites of dibenz[a,j]anthracene and 7,14-dimethyldibenz[a,j]anthracene

Syntheses are described of the trans-3,4-dihydrodiol derivatives (2a and 2b) of dibenz[a,j]anthracene and 7,14-dimethyldibenz[a,j]anthracene (1a and 1b), implicated as their proximate carcinogenic metabolites. Conversion of 2a to the bay region anti-diol epoxide derivative 3a, its putative ultimate carcinogenic metabolite, is also reported. The related diol epoxide derivative of 2b could not be prepared due to its chemical instability. Tumorigenicity assays confirm that 1b and 2b are potent carcinogens on mouse skin, while 1a and 2a are only relatively weakly active. The diol epoxide 3a exhibited significantly higher tumorigenicity than its dihydrodiol precursor 2a. These findings are consistent with the hypothesis that the bay region diol epoxide metabolites are the active carcinogenic forms of these hydrocarbons. They also support the generalization that methyl substitution in bay regions enhances the carcinogenic activity of polycyclic aromatic hydrocarbons.     

Oxidative DNA damage induced by benz[a]anthracene dihydrodiols in the presence of dihydrodiol dehydrogenase

Tobacco smoke and polluted air are risk factors for lung cancer and contain many kinds of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) and benz[a]anthracene (BA). BA, as well as B[a]P, is assessed as probably carcinogenic to humans (IARC group 2A). BA is metabolized to several dihydrodiols. Dihydrodiol dehydrogenase (DD), a member of the aldo-keto reductase superfamily, catalyzes NAD(P)+-linked oxidation of dihydrodiols of aromatic hydrocarbons to corresponding catechols. To clarify the role of DD on PAH carcinogenesis, we examined oxidative DNA damage induced by trans-dihydrodiols of BA and B[a]P treated with DD using 32P-5'-end-labeled DNA fragments obtained from the human p53 tumor suppressor gene. In addition, we investigated the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG), an indicator of oxidative DNA damage, in calf thymus DNA by using HPLC with an electrochemical detector. DD-catalyzed BA-1,2-dihydrodiol caused Cu(II)-mediated DNA damage including 8-oxodG formation in the presence of NAD+. BA-1,2-dihydrodiol induced a Fpg sensitive and piperidine labile G lesion at the 5'-ACG-3' sequence complementary to codon 273 of the human p53 tumor suppressor gene, which is known as a hotspot. DNA damage was inhibited by catalase and bathocuproine, suggesting the involvement of H2O2 and Cu(I). The observation of NADH production by UV-visible spectroscopy suggested that DD catalyzed BA-1,2-dihydrodiol most efficiently to the corresponding catechol among the PAH-dihydrodiols tested. A time-of-flight mass spectroscopic study showed that the catechol form of BA-1,2-dihydrodiol formed after DD treatment. In conclusion, BA-1,2-dihydrodiol can induce DNA damage more efficiently than B[a]P-7,8-dihydrodiol and other BA-dihydrodiols in the presence of DD. The reaction mechanism on oxidative DNA damage may be explained by theoretical calculations with an enthalpy change of dihydrodiols and oxidation potential of their catechol forms. DD may play an important role in BA carcinogenesis via oxidative DNA damage.     

DNA binding and mutagenicity of 7-substituted derivatives of benz[A]anthracene.

The binding of carcinogenic 7-hydroxymethylbenz[a]anthracene (7-HOCH2-B[a]A) and 7-acetoxymethylbenz[a]anthracene (7-AcOCH2-B[a]A) to calf thymus DNA was studied in the presence or absence of microsomal enzymes or nucleoside phosphates. In the absence of microsomes or ATP little or no binding was detected for either hydrocarbon (HC). Microsomal enzymes significantly enhanced the binding of both HC's to DNA when compared to control as measured by radioactivity bound to DNA. When 7-HOCH2-B[a]A was incubated at 37 degrees with ATP there was a linear increase in binding over a six hr period. Of the nucleoside phosphates tested, ATP, and to a lesser extent ADP and CTP, mediated binding of 7-HOCH2-B[a]A suggesting formation of a reactive phosphate ester. Mutagenicity studies with 7-acetoxymethyl, 7-hydroxymethyl, 7-formyl-, 7-methyl-, and 7-methoxymethyl-B[a]A were conducted using the Salmonella reverse mutation assay. All compounds exhibited mutagenic activity in the presence of S-9; only 7-AcOCH2-B[a]A was active without S-9 indicating this compound to be an ultimate mutagen.     

Evaluation of chlorinated benz[a]anthracene on hepatic toxicity in rats and mutagenic activity in Salmonella typhimurium

Chlorinated benz[a]anthracenes (Cl‐BaA) are halogenated aromatic compounds (typified by dioxins) found in the environment at relatively high concentrations. Fischer 344 rats were intragastrically administered 0, 1, or 10 mg of Cl‐BaA or its parent compound benz[a]anthracene (BaA) per kg of body weight for 14 consecutive days. Both chemicals at 10 mg/kg/day inhibited the gain in body weight, and consequent increase in relative liver weight. Hepatic gene expression of cytochrome P450 (CYP) 1A1, 1A2, and 1B1 was significantly stimulated by administration of BaA (10 mg/kg/day) compared with the control. After administration of Cl‐BaA, only the CYP1A2 gene was significantly induced, even at the lower dosage; CYP1A1 and 1B1 mRNA levels remained unchanged in Cl‐BaA‐treated rats compared with controls. To elucidate the role of such Cl‐BaA exposure and induced CYPs at toxicity onset, we investigated the mutagenicity of BaA and Cl‐BaA using Salmonella typhimurium TA98 and TA100. BaA and Cl‐BaA at 10 μg/plate produced positive results in both strains in the presence of rat S‐9. Incubation of Cl‐BaA with recombinant rat CYP1A2 produced a significantly higher number of revertant colonies in TA98 and TA100 than in controls, but no such change was observed for BaA. In conclusion, BaA changes its own physiological and toxicological actions by its chlorination; (1) daily exposure to Cl‐BaA selectively induces hepatic CYP1A2 in rats and (2) Cl‐BaA induces frameshift mutations in the presence of CYP1A2, although BaA does not exert mutagenicity. This indicates that CYP1A2 may metabolize Cl‐BaA to active forms. © 2011 Wiley Periodicals, Inc. Environ Toxicol 2013.     

Morphological and functional disorders of the immature rat uterus after postnatal exposure to benz[a]anthracene and benzo[k]fluoranthene

We investigated the effects of postnatal exposure to benz[a]anthracene (B[a]A) and benzo[k]fluoranthene (B[k]F) on the development of the rat uterus. Neonates were injected on each postnatal days 1-14 with B[a]A (0.1, 1.0 or 10.0mg/kg), B[k]F (0.1, 1.0 or 5.0mg/kg), ethynylestradiol (EE; 1.0μg/kg) or a vehicle. The rats were killed on day 23. All doses of B[a]A and B[k]F induced a reduction of the uterine weight, a reduction of the estrogen receptor α expression in the luminal epithelium, glandular epithelium and stromal cells. Moreover, the uterotrophic response to EE (3-day administration of 1.0μg/kg on postnatal days 20-22) in rats exposed to B[a]A, B[k]F and EE was lower than in controls. The study showed that postnatal exposure to B[a]A and B[k]F resultes in morphological and functional disorders of the immature rat uterus.     

Oxidative DNA damage induced by benz[a]anthracene metabolites via redox cycles of quinone and unique non-quinone

Benz[a]anthracene (BA) is one of the most abundant polycyclic aromatic hydrocarbons (PAHs) that are ubiquitous environmental pollutants. PAH carcinogenesis is explained by DNA adduct formation by PAH diol epoxide and oxidative DNA damage by PAH o-quinone. Benz[a]anthracene-trans-3,4-dihydrodiol (BA-3,4-dihydrodiol) is a minor metabolite but shows higher mutagenicity and tumorigenicity than parent BA. We confirmed that a BA o-quinone type metabolite, benz[a]anthracene-3,4-dione (BA-3,4-dione), induced oxidative DNA damage in the presence of cytochrome P450 reductase. Interestingly, we found that BA-3,4-dihydrodiol nonenzymatically caused Cu(II)-mediated DNA damage including 8-oxo-7,8-dihydro-2'-deoxyguanosine formation and the addition of NADH enhanced DNA damage. BA-3,4-dihydrodiol induced a double-base lesion of C and G at the 5'-ACG-3' sequence complementary to codon 273 of the human p53 tumor suppressor gene, which is known as a hotspot. The DNA damage was inhibited by catalase and bathocuproine, indicating the involvement of H(2)O(2) and Cu(I). Time-of-flight mass spectroscopic study suggested that BA-3,4-dihydrodiol undergoes Cu(II)-mediated autoxidation leading to the formation of its hydroxylated form of BA-3,4-dihydrodiol, capable of causing oxidative DNA damage. It is noteworthy that BA-3,4-dihydrodiol can nonenzymatically induce DNA damage more efficiently than BA-3,4-dione with metabolic activation. In conclusion, oxidative DNA damage induced by BA-3,4-dihydrodiol not only via quinone-type redox cycle but also via a new type of redox cycle participates in the expression of carcinogenicity of BA and BA-3,4-dihydrodiol.