Identification and quantitation of benzo[a]pyrene-DNA adducts formed by rat liver microsomes in vitro (original) (raw)
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Identification and quantitation of benzo[a]pyrene-DNA adducts formed in mouse skin
2009
The DNA adducts of benzo[a]pyrene (BP) formed in vitro were previously identified and quantitated. In this paper, we report the identification and quantitation of the depurination adducts of BP, 8-(benzo[a]pyrene-6-yl)guanine (BP-6-C8Gua), BP-6-N7Gua, and BP-6-N7Ade, formed in mouse skin by one-electron oxidation, as well as the major stable adduct formed via the diolepoxide pathway, BP diolepoxide bound at C-10 to the
Repair of DNA damaged by mutagenic metabolites of benzo(a)pyrene in human cells
Chemico-Biological Interactions, 1978
The repair of human DNA after damage by known and potential metabolites of benzo(a)pyrene has been examined utilizing the bromodeoxyuridine photolysis assay. Repair was characterized as either ultraviolet ("long") or ionizing radiation type ("short") repair utilizing normal cells and cells deficient in ultraviolet-type repair endonuclease from a patient with xeroderma pigmentosum (XP). We have found that only (+-)-7~,8a~iihydroxy-9~,-10~poxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 1) and its disastereomer, (+-)-7~,Sa,~lihydroxy-9a,10~ ~poxy-7,8,9,10-tetrahydrobenzo(a)pyrene (BP diol epoxide 2) elicit damage to DNA which is recognizable by the ultraviolet excision repair system in normal human cells. Benzo(a)pyrene 4,5-, 9,10-, ll,12~xides do not elicit damage which is repairable by this repair system. The 1,2<liol-3,4~poxides from naphthalene have no measurable activity in our assay. These results indicate that both the benzo(a)pyrene ring structure and the diol epoxide groups are important in causing the damage to DNA which is repairable by the ultraviolet excision repair system. These results parallel the reported high mutagenic activity of these compounds and support the concept that benzo(a)pyrene 7,8~liol-9,10epoxides may be the ultimate, metabolically activated forms of benzo(a)pyrene.
Biochemistry, 1994
A metabolite of benzo[a] pyrene, 9-r, 1O-t-dihydroxy-7,8-c-oxy-7,8,9,1O-tetrahydrobenzo[a]pyrene (BPDE-111), that is not thought to be involved in carcinogenesis has nevertheless been shown to bind extensively to DNA in vitro. The adducts formed by this non-bay-region diol epoxide in Chinese hamster ovary cells are much less mutagenic than those formed by an isomeric diol epoxide that is carcinogenic. We have isolated and characterized three major adducts formed by in vitro reaction of BPDE-I11 with DNA. The major adduct, accounting for over half of the total is formed by reaction of BPDE-I11 with the N 7 position of dGuo and is recovered after enzymatic digestion as an N7-Gua adduct. A second major adduct involves the N 2 position of dGuo, while the third adduct is tentatively identified as a C8-substituted dGuo. Little or no reaction with deoxyadenosine residues is detected. The N 7 adduct is unstable in DNA at 37 OC and is released as the modified base with a half-life of about 24 h. This adduct lability apparently leads to single-strand breaks and alkali-sensitive sites in the DNA and may account in part for some of the biological properties of BPDE-I11 adducts. This represents the first description of an N7-dGuo adduct that is formed in DNA as the major adduct by a diol epoxide derived from a carcinogenic polycyclic aromatic hydrocarbon. Polycyclic aromatic hydrocarbons, an often-studied group of chemical carcinogens, produce many of their biological effects by way of electrophilic metabolites that attack cellular macromolecules (Miller
Carcinogenesis, 1997
C3H10T1/2CL8 (C3H10T1/2) mouse embryo fibroblasts were used to study the in vitro carcinogenic activities of dibenzo[a,l]pyrene (DB[a,l]P) and benzo[a]pyrene (B[a]P). The morphological transforming activities of these rodent carcinogens were compared using replicate concentrationresponse studies. In concentration ranges where both polycyclic aromatic hydrocarbons (PAHs) were active, DB[a,l]P proved to be four to 12 times as potent as B[a]P based on concentration. At lower concentrations DB[a,l]P was active at 0.10 and 0.20 µM, concentrations where B[a]P was inactive. This makes DB[a,l]P the most potent non-methylated PAH evaluated to date in C3H10T1/2 cells. DNA adducts of DB[a,l]P in C3H10T1/2 cells were analyzed by both TLC and TLC/HPLC 32 P-postlabeling methods using mononucleotide 3Ј-phosphate adduct standards derived from the reactions of anti-DB[a,l]P-11,12-diol-13,14-epoxide (anti-DB[a,l]PDE) and syn-DB[a,l]P-11,12-diol-13,14-epoxide (syn-DB[a,l]PDE) with deoxyadenosine 3Ј-monophosphate and deoxyguanosine 3Ј-monophosphate. All of the DNA adducts observed in C3H10T1/2 cells treated with DB[a,l]P were identified as being derived from the metabolism of DB[a,l]P to its fjord region diol epoxides through DB[a,l]P-11,12-diol. The predominant adduct was identified as an anti-DB[a,l]PDE-deoxyadenosine adduct. Other major adducts were anti-DB[a,l]PDE-deoxyguanosine and syn-DB[a,l]PDE-deoxyadenosine adducts with minor amounts of syn-DB[a,l]PDE-deoxyguanosine adducts. These DNA adduct data are consistent with similar findings of DB[a,l]PDE-deoxyadenosine adducts in mouse skin studies and human mammary cells in culture.
Relationships between benzo(a)pyrene-DNA adduct levels and genotoxic effects in mammalian cells
Cancer research, 1987
The effectiveness of benzo(a)pyrene [B(a)P]-DNA binding as an internal dosimeter was evaluated. Data were obtained from concurrent studies, measuring B(a)P induced genotoxic effects and DNA adducts in several short-term bioassay systems: cytotoxicity, gene mutation, and sister chromatid exchange in Chinese hamster V79 cells; cytotoxicity, gene mutation, and chromosome aberrations in mouse lymphoma L5178Y TK+/-; cytotoxicity and enhanced virus transformation in Syrian hamster embryo cells; and cytotoxicity and morphological transformation in C3H10T1/2CL8 mouse embryo fibroblasts. Both total B(a)P-DNA binding and specific B(a)P-DNA adducts were measured. N2-(10 beta-[7 beta,8 alpha,9 alpha-trihydroxy-7,8,9,10-tetrahydrobenzo(a)pyrene]yl)deoxyguanosine [BPDE I-dGuo] was one of the major adducts identified in all bioassay systems. DNA binding and genotoxic responses varied significantly between bioassays. Each genetic end point was induced with a differing efficiency on a per adduct bas...
Analytical Biochemistry, 2004
The two major metabolic pathways of benzo[a]pyrene (BP) that lead to DNA lesions are monooxygenation that results in diolepoxides (BPDE) and one-electron oxidation that yields a BP radical cation. These pathways result in formation of stable and depurinating DNA adducts, respectively. Most in vivo animal studies with BP, however, have employed dosage/DNA adduct levels several orders of magnitude higher than the DNA damage level expected from environmentally relevant exposures. Presented are results of experiments in which A/J strain mice were intraperitoneally exposed to 50-g/g doses of BP. It is shown that non-line-narrowed Xuorescence and Xuorescence line-narrowing spectroscopies possess the selectivity and sensitivity to distinguish between helix-external, base-stacked, and intercalated conformations of DNA-BPDE adducts formed in lung tissue. Concentrations measured by 32 P postlabeling 2 and 3 days after intraperitoneal injection were 420-430 and 600-830 amol BPDE-type adducts per g DNA. The external and base-stacked conformations are attributed mainly to (+)-trans-anti-BPDE-N 2 dG and the intercalated conformations to (+)-cisanti adducts. A stable adduct derived from 9-OH-BP-4,5-epoxide was also detected at a concentration about a factor of 10 lower than the above concentrations. The DNA supernatants were analyzed for the presence of depurinating BP-derived adducts by capillary electrophoresis laser-induced Xuorescence and high-performance liquid chromatography mass spectrometry. 2004 Elsevier Inc. All rights reserved.
Biochemistry, 1995
pyrene (DB[a,l]P) is the most potent carcinogen known among aromatic hydrocarbons. DB[a,l]P-11,12-dihydrodiol, precursor to the bay-region diol epoxide, is slightly less carcinogenic than the parent compound. DB[a,l]P and its 11,12-dihydrodiol were covalently bound to DNA by cytochrome P-450 in 3-methylcholanthrene-induced rat liver microsomes, and DB [a,l]P was also bound to DNA by horseradish peroxidase. The "stable" (remaining intact in DNA under normal conditions of purification) and "depurinating" (released from DNA by cleavage of the glycosidic link between the purine base and deoxyribose) adducts were identified and quantified. Stable adducts were analyzed by the 32P-postlabeling technique. Depurinating adducts were identified by comparison of their retention times with those of standard adducts on HPLC in two solvent systems. Confirmation of their identity was obtained by means of fluorescence line-narrowing spectroscopy. When DB[a,l]P was activated by horseradish peroxidase, the depurinating adducts 3-(DB[a,l]P-10-y1)adenine (DB[a,l]P-lO-N3Ade, 33%), 7-(DB[a,llP-10-y1)adenine (DB[a,l]P-10-N7Ade, 27%), and 7-DB[a,l]P-10-y1)guanine (DB[a,l]P-10-N7Gua, 5 % ) were formed. Unidentified stable adducts comprised the remaining 35% of the detected adducts. When DB[a,l]P was activated by microsomes, the one-electron oxidation depurinating adducts DB[a,l]P-10-N3Ade (28%), DB[a,l]P-lO-N7Ade (14%), DB[a,l]P-lO-N7Gua (2%), and DB[a,l]P-lO-C8Gua (6%), as well as the diol epoxide depurinating adducts (f)-syn-DB[a,l]P-diol epoxide (DE)-14-N7Ade (3 1 %) and (k)-anti-DB[a,l]PDE-14-N7Gua (3%), were formed. Stable adducts predominantly formed via the DB[a,l]PDE pathway represented 16% of the adducts detected. When DB[a,l]P-l1,12-dihydrodiol was activated by microsomes, the same two depurinating adducts arising from DB [a,l]PDE were found, but they constituted only 19% of the adducts because the amount of stable adducts was much higher than with DB[a,l]P. Analysis of stable DNA adducts by the 32P-postlabeling method indicates that the profiles formed from DB[a,l]P and its 11,12-dihydrodiol were qualitatively similar. These results demonstrate that the major depurinating adducts formed by both DB[a,l]P and its 11,12-dihydrodiol are at the N-3 and N-7 of adenine, resulting in apurinic sites in the DNA. Dibenzo[a,l]pyrene (DB[a,l]P)' ) has been shown to be the most potent carcinogen among PAH . The carcinogenicity of DB[a,l]P in rat mammary gland and mouse skin was found to be significantly stronger than that of DMBA, previously considered to be the most
Cancer research, 1990
Male CD rats were injected with single i.p. doses of benzo(a)pyrene (B(a)P), and peripheral blood lymphocytes (PBLs), livers, and lungs were removed at various times after administration. DNA adducts were analyzed in each tissue by 32P postlabeling with nuclease P1 enhancement. Sister chromatid exchange frequencies were concomitantly measured in cultured whole blood. B(a)P-DNA adducts were observed in all three tissues from animals sacrificed between 1 and 56 days after injection. Maximal adduction levels occurred at about 4 days after administration, followed by a gradual loss of adducts over the period examined. The apparent half-lives of total DNA adducts were 15 days in liver, 17 days in PBLs, and 22 days in lung. Induced sister chromatid exchanges were linearly related to the amount of DNA adducts remaining in the PBLs at the time of harvest up to 56 days and were significantly elevated above concurrent controls up to 14 days. One of the major adducts found in each tissue was N...
Role of activated oxygen species in benzo[a]pyrene: DNA adduct formation in vitro
Free Radical Biology and Medicine, 1991
The role of several activated oxygen species in the oxidation and binding of B[a]P to calf thymus DNA in vitro was investigated. B[a]P was reacted with calf thymus DNA in the presence and absence of scavengers of active oxygen species. Reactions were performed in the dark at 37 °C for 30 rain in a buffered aqueous solution with 250 ttg of calf thymus DNA. The levels of B[a]P:DNA adducts formed were determined using the 32p-postiabeling assay. B[a]P:DNA adduct levels ranged from 1.5-2.6 and 0.25 pmol adducts/mg DNA in reactions with 120 or 12 nmol of B[a]P, respectively. The addition of scavengers of reactive oxygen species to reaction mixtures resulted in a considerable decrease in the levels of DNA adducts formed in comparison to control reactions. Reactions performed with 500 units catalase or 100 units superoxide dismutase significantly inhibited DNA adduct formation. In these reactions adduct levels were 32 and 48% of control levels, respectively. The addition of both catalase and superoxide dismutase to reactions inhibited adduct formation by 95% relative to control reactions. A decrease in adduct levels was also observed when reactions were performed with citrate-Fe 3÷ chelate, a scavenger of superoxide. In reactions with 50 mM mannitol and 50 mM sodium benzoate, both of which are hydroxyl radical scavengers, adduct formation was significantly inhibited with adduct levels being 30 and 51% of control values, respectively. Adduct levels were decreased to 26% of control values in reactions with 10 mM 2,5-dimethylfuran, a scavenger of singlet oxygen. These data demonstrate that hydrogen peroxide and reactive oxygen species such as superoxide, hydroxyl radicals, and singlet oxygen are involved in the nonenzymatic binding of B[a]P to DNA in vitro.