Absolute configuration of a ribonucleic acid adduct formed in vivo by metabolism of benzo[a]pyrene

126

Covalent binding of the benzo[alpyrene metabolite (+) 7#,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene to calf thymus DNA was investigated. Enzymatic hydrolysis of the carcinogen-modified DNA and subsequent separation via reversed-phase high-pressure liquid chromatography resulted in the detection and isolation of seven distinct products. High-resolution mass spectrometry indicates that these products are covalent adducts of deoxyguanosine, deoxyadenosine, and deoxycytidine. The deoxyguanosine and deoxyadenosine adducts involve binding between the activated hydrocarbon (benzo[alpyrene diol epoxide) and exocyclic amino groups of the respective purines.The potent carcinogen benzo[a]pyrene (BzaP) undergoes metabolism in vivo to a chemically reactive intermediate, which can then react with cellular RNA, DNA, and protein (1, 2). It is widely accepted that these covalent interactions with cellular macromolecules, particularly DNA, are an essential initial step in the process of carcinogenesis (3,4). Recent work has implicated 7/3,8a-dihydroxy-9a,lOa-epoxy-7,8,9,10-tetrahydrobenzo[alpyrene (BzaP diol epoxide, Structure I) as the 12 I molecular species responsible for binding to nucleic acids in vduo (5-7). Weinstein and coworkers have shown that the major RNA adduct found in cell culture is identical to the product obtained by reacting BzaP diol epoxide with poly(G), and involves a covalent bond between C-10 of the hydrocarbon and the N2 exocyclic amino group of guanine (8, 9). A similar structure has been reported for reaction of the isomeric diol epoxide, 703,8a-dihydroxy-90,10,B-epoxy-7,8,9, 10-tetrahydrobenzo[a]-pyrene, with poly(G) (10). The structures of the products obtained in the reaction between BzaP diol epoxide and DNA have not been reported, primarily because only microgram quantities of such adducts can be readily isolated from the analogous reaction with DNA.We have isolated seven distinct products from the reaction of BzaP diol epoxide with calf thymus DNA, and by the use of high-sensitivity, high-resolution mass spectrometry the structures of these products were determined. The results show that adducts are formed between BzaP diol epoxide and the bases guanine, adenine, and cytosine, and involve reactions with the exocyclic amino group of guanine and adenine.MATERIALS AND METHODS Adduct Formation and Isolation. Formation of microsomal enzyme-activated BzaP-DNA adducts has been described (11). Racemic BzaP diol epoxide was synthesized as described (12).Diol epoxide (12 nmol/mg of DNA) in 100 Al of dimethylsulfoxide was added to a solution of 100 mg of calf thymus DNA (Sigma Chemical Co.) in 100 ml of 10 mM phosphate buffer (pH 7.2) at 37'. After 24 hr, unbound BzaP diol epoxide and its hydrolysis products were removed by ethyl acetate extraction. The DNA was then precipitated with ethanol and the precipitate was heated to remove intercalated material. The DNA was enzymatically hydrolyzed with deoxyribonuclease II, spleen phosphodiesterase, and alkaline phosphatase (Sigma Chemical ...

Section: Csh4on5tmsmentioning

confidence: 99%

Identification of the major adducts formed by reaction of benzo(a)pyrene diol epoxide with DNA in vitro.

Straub¹,

Meehan²,

Burlingame³

et al. 1977

126

“…The major adduct formed in vitro (2,3), and in vivo (3), results from nucleophilic attack of the N-2 of guanine upon the C-10 position of BPDE to yield a trans opening of the 9,10-epoxide ring. This guanine derivative constitutes 80-90% of all stable adducts (2,3) and has been proposed to be responsible for the mutagenic (4) and carcinogenic (5,6) effects of BP. The adduct appears to distort DNA at its binding site; however, the secondary structure of the adduct is still disputed (7)(8)(9)(10).…”

mentioning

confidence: 99%

Site-specific carcinogen binding to DNA.

Boles¹,

Hogan²

1984

Benzo [a]pyrene diol epoxide (BPDE) is a wellstudied environmental carcinogen that binds covalently to DNA. Here we describe a photochemical technique that allows us to map BPDE-binding sites within cloned gene sequences. The technique is based upon our observation that, when irradiated with laser light at 355 nm, one single-strand DNA cut is produced at each BPDE binding site. In initial experiments we have studied the distribution of such cuts in cloned DNA from the chicken adult /8-globin gene. We rind that BPDE binding in this gene sequence is distinctly nonrandom. While several prominent BPDE-binding sites are evident, a 300-basepair sequence immediately 5' to the RNA cap site is most strongly attacked by the carcinogen. This region is believed to contain important transcriptional control sequences. We discuss the possibility that sequence-specific binding to such regulatory elements may be an important feature of the mechanism of the carcinogen. (3), results from nucleophilic attack of the N-2 of guanine upon the C-10 position of BPDE to yield a trans opening of the 9,10-epoxide ring. This guanine derivative constitutes 80-90% of all stable adducts (2, 3) and has been proposed to be responsible for the mutagenic (4) and carcinogenic (5, 6) effects of BP. The adduct appears to distort DNA at its binding site; however, the secondary structure of the adduct is still disputed (7-10).Here, we examine the photochemistry of BPDE when bound covalently to a DNA helix. We find that, when irradiated, the carcinogen cuts the DNA strand to which it is bound. We then show that the photochemical cutting process can be developed into a powerful tool for mapping carcinogen-binding sites in a eukaryotic gene.MATERIALS AND METHODS BPDE Modification. DNA fragments 146 ± 2 base pairs (bp) long were prepared from chicken erythrocyte nucleosomes (11), then modified with (± )-BPDE as described elsewhere (9), with the exception that unreacted BPDE and BP tetrol were removed by three phenol/chloroform extractions. The extent of modification was measured optically, using 6346 = 2.95 x 104 cm-' M`for the BPDE-DNA adduct and E258 = 1.3 x 104 cm-I M -I (base pairs) for DNA.Poly(dG)'poly(dC) (Boehringer) was sonicated (00C), treated with S1 nuclease (Boehringer) to remove singlestrand regions, then digested with proteinase K, extracted with phenol, and fractionated over a Sepharose 6B column as described (12). The DNA fraction used for this experiment migrated on a denaturing gel as a 330 + 50 base species.DNA was modified with [3H]BPDE and bound BPDE was determined by liquid scintillation counting.

“…The BP-7,8-diol-9,10-epoxides derived from BP-7,8-dihydrodiol exist as a pair of diastereomers in which the 7-hydroxyl group is either cis (diol epoxide 1) or trans (diol epoxide 2) to the 9,10-epoxide. The latter metabolites possess high chemical (11,12) and mutagenic (6,(13)(14)(15) activity, are potent inducers of epidermal hyperplasia (16), and interact with nucleophilic sites on cellular DNA, RNA, and protein (4,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26) (27,28). Although the diastereomeric BP-7,8-diol-9,10-epoxides are less carcinogenic than BP on mouse skin (29,30), dihydrodiol is a potent carcinogen with activity comparable to or somewhat greater than BP (29,31,32 (33).…”

mentioning

confidence: 99%

Tumorigenicity of the optical enantiomers of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides in newborn mice: exceptional activity of (+)-7beta,8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene.

Buening

Wislocki

Levin

et al. 1978

372

152

The tumorigenicities of benzo[a]pyrene and each optical enantiomer of the diastereomeric benzo[a]pyrene 7,8-diol-9,10-epoxides derived from trans-7,8-dihydroxy-7,8-dihydrobenzol[a]pyrene were tested by sequential intraperitoneal injection of mice with 1,2, and 4 nmol, or with 2, 4, and 8 nmol of each compound on the 1st, 8th, and 15th day of life, respectively. The experiment was terminated when the animals were 34--37 weeks old. (+)-7beta, 8alpha-dihydroxy-9alpha,10alpha-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene [(+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2] had exceptional tumorigenicity, whereas benzo[a]-pyrene and the other three optically pure isomers of the benzo[a]pyrene 7,8-diol,9,10-epoxides had little or no activity. These results demonstrate differences in the carcinogenic activities of optically active isomers of a polycyclic hydrocarbon diol epoxide. Eleven percent of control mice had pulmonary tumors, whereas 71% and 100% of the mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2, respectively, had pulmonary tumors. Control mice had an average of 0.12 pulmonary tumors per mouse, whereas mice treated with a total dose of 7 or 14 nmol of (+)-BP-7beta,8alpha-diol-9alpha,10alpha-epoxide 2 had 1.72 and 7.67 pulmonary tumors per mouse, respectively. Mice treated with 14 nmol of (-)-BP-7alpha,8beta-diol-9beta,10beta-epoxide 2, (-)-BP-7beta,8alpha-diol-9beta,10beta-epoxide 1, or (+)-BP-7alpha,8beta-diol-9alpha,10alpha-epoxide 1 had 0.13, 0.25, and 0.34 pulmonary tumors per animal, respectively.