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Inherent stereospecificity in the reaction of aflatoxin B(1) 8,9-epoxide with deoxyguanosine and efficiency of DNA catalysis.

ABSTRACT: Kinetic analysis of guanine alkylation by aflatoxin B(1) exo-8,9-epoxide, the reactive form of the hepatocarcinogen aflatoxin B(1), shows the reaction to be >2000 times more efficient in DNA than in aqueous solution, that is, with free 2'-deoxyguanosine. Thermodynamic analysis reveals AFB(1) exo-8,9-epoxide intercalation as the predominant source of the observed DNA catalytic effect. However, the known exo > endo epoxide stereospecificity of the DNA alkylation is observed even with free deoxyguanosine (ratio >20:1 determined by LC-MS and NMR measurements), as predicted by theoretical calculations [ Bren , U. , et al. ( 2007 ) Chem. Res. Toxciol. 20 , 1134 - 1140 ].

SUBMITTER: Brown KL

PROVIDER: S-EPMC3141577 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Inherent stereospecificity in the reaction of aflatoxin B(1) 8,9-epoxide with deoxyguanosine and efficiency of DNA catalysis.

Brown Kyle L KL Bren Urban U Stone Michael P MP Guengerich F Peter FP

Chemical research in toxicology 20090501 5

Kinetic analysis of guanine alkylation by aflatoxin B(1) exo-8,9-epoxide, the reactive form of the hepatocarcinogen aflatoxin B(1), shows the reaction to be >2000 times more efficient in DNA than in aqueous solution, that is, with free 2'-deoxyguanosine. Thermodynamic analysis reveals AFB(1) exo-8,9-epoxide intercalation as the predominant source of the observed DNA catalytic effect. However, the known exo > endo epoxide stereospecificity of the DNA alkylation is observed even with free deoxygua ...[more]

PMID: 19301826

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Project description:Resistance to the carcinogenic effects of aflatoxin B1 (AFB1) in the mouse is due to the constitutive expression of an Alpha-class glutathione S-transferase (GST), YcYc, with high detoxification activity towards AFB1-8,9-epoxide. A cDNA clone (pmusGST Yc) for a murine GST Yc polypeptide has been isolated. Sequencing has shown the cDNA insert of pmusGST Yc to be 922 bp in length, with an open reading frame of 663 bp that encodes a polypeptide of M(r) 25358. The primary structure of the murine GST Yc subunit predicted by pmusGST Yc is in complete agreement with the partial amino acid sequence of the aflatoxin-metabolizing mouse liver GST described previously [McLellan, Kerr, Cronshaw & Hayes (1991) Biochem. J. 276, 461-469]. A plasmid, termed pKK-musGST Yc, which permits the expression of the murine Yc subunit in Escherichia coli, has been constructed. The murine GST expressed in E. coli was purified and found to be catalytically active towards several GST substrates, including AFB1-8,9-epoxide. This enzyme was also found to possess electrophoretic and immunochemical properties closely similar to those of the GST Yc subunit from mouse liver. However, the GST synthesized in E. coli and the constitutive mouse liver Alpha-class GST exhibited small differences in their chromatographic behaviour during reverse-phase h.p.l.c. Automated Edman degradation revealed alanine to be the N-terminal amino acid in the GST Yc subunit expressed in E. coli, whereas the enzyme in mouse liver possesses a blocked N-terminus. Although sequencing showed that the purified Yc subunit from E. coli lacked the initiator methionine, the amino acid sequence obtained over the first eleven N-terminal residues agreed with that predicted from the cDNA clone, pmusGST Yc. Comparison of the deduced amino acid sequence of the mouse Yc polypeptide with the primary structures of the rat Alpha-class GST enzymes revealed that it is more closely related to the ethoxyquin-induced rat liver Yc2 subunit than to the constitutively expressed rat liver Yc1 subunit. The significance of the fact that both mouse Yc and rat Yc2 exhibit high catalytic activity towards AFB1-8,9-epoxide, whereas rat Yc1 possesses little activity towards this compound, is discussed in terms of structure/function.

Project description:A purification scheme has been devised for two ethoxyquin-inducible Alpha-class glutathione S-transferases (GSTs) which possess at least 25-fold greater activity towards aflatoxin B1 (AFB1)-8,9-epoxide than that exhibited by the GSTs (i.e. F, L, B and AA) that have been described previously. These two enzymes are both heterodimers and both contain a subunit of Mr 25,800. This subunit has been isolated from both of the GST isoenzymes and, after cleavage with CNBr, it has been subjected to automated amino acid sequencing. The primary structure of the Mr 25,800 subunit revealed that it forms part of a subfamily of Alpha-class GSTs which possess closest identity (about 92%) with the Yc subunit of apparent Mr 27,500, which is encoded by the recombinant cDNA clone pGTB42 [Telakowski-Hopkins, Rodkey, Bennett, Lu & Pickett (1985) J. Biol. Chem. 260, 5820-5825]. As these two GSTs possess less than 70% sequence identity with the Ya1 and Ya2 subunits, both of Mr 25,500, the constitutively expressed Yc subunit of Mr 27,500 has been renamed Yc1 and the ethoxyquin-inducible GST of Mr 25,800 has been designated Yc2. Using this nomenclature, the two GSTs with high activity for AFB1-8,9-epoxide are Ya1Yc2 and Yc1Yc2. Although evidence suggests that induction of Yc2 is responsible for the high detoxification capacity of livers from ethoxyquin-treated rats for AFB1-8,9-epoxide, resistance towards AFB1 may be multifactorial in this instance as dietary ethoxyquin also induces the Ya1, Ya2 and Yc1 subunits about 2.2-, 10.9- and 2.7-fold respectively. Besides the induction of GST by ethoxyquin, activity towards AFB1-8,9-epoxide is also elevated in the livers of neonatal rats and in livers that contain preneoplastic nodules. Western blotting experiments show that Yc2 is not present in hepatic cytosol from adult rats fed on normal diets but is expressed in neonatal rat livers and in the livers of adult rats that contain preneoplastic nodules that have arisen as a consequence of consuming diets contaminated with AFB1.

Project description:DNA interstrand cross-links (ICLs) are cytotoxic DNA lesions derived from reactions of DNA with a number of anti-cancer reagents as well as endogenous bifunctional electrophiles. Deciphering the DNA repair mechanisms of ICLs is important for understanding the toxicity of DNA cross-linking agents and for developing effective chemotherapies. Previous research has focused on ICLs cross-linked with the N7 and N2 atoms of guanine as well as those formed at the N6 atom of adenine; however, little is known about the mutagenicity of O6-dG-derived ICLs. Although less abundant, O6-alkylated guanine DNA lesions are chemically stable and highly mutagenic. Here, O6-2'-deoxyguanosine-butylene-O6-2'-deoxyguanosine (O6-dG-C4-O6-dG) is designed as a chemically stable ICL, which can be induced by the action of bifunctional alkylating agents. We investigate the DNA replication-blocking and mutagenic properties of O6-dG-C4-O6-dG ICLs during an important step in ICL repair, translesion DNA synthesis (TLS). The model replicative DNA polymerase (pol) Sulfolobus solfataricus P2 DNA polymerase B1 (Dpo1) is able to incorporate a correct nucleotide opposite the cross-linked template guanine of ICLs with low efficiency and fidelity but cannot extend beyond the ICLs. Translesion synthesis by human pol ? is completely inhibited by O6-dG-C4-O6-dG ICLs. Moderate bypass activities are observed for human pol ? and S. solfataricus P2 DNA polymerase IV (Dpo4). Among the pols tested, pol ? exhibits the highest bypass activity; however, 70% of the bypass products are mutagenic containing substitutions or deletions. The increase in the size of unhooked repair intermediates elevates the frequency of deletion mutation. Lastly, the importance of pol ? in O6-dG-derived ICL bypass is demonstrated using whole cell extracts of Xeroderma pigmentosum variant patient cells and those complemented with pol ?. Together, this study provides the first set of biochemical evidence for the mutagenicity of O6-dG-derived ICLs.

Dataset Information

Inherent stereospecificity in the reaction of aflatoxin B(1) 8,9-epoxide with deoxyguanosine and efficiency of DNA catalysis.

Publications

Inherent stereospecificity in the reaction of aflatoxin B(1) 8,9-epoxide with deoxyguanosine and efficiency of DNA catalysis.

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