Changes of mechanisms and product distributions in the hydrolysis of benzo[a]pyrene-7,8-diol 9,10,-epoxide metabolites induced by changes in pH

The heme enzyme indoleamine 2,3-dioxygenase (IDO) was found to catalyze the oxidation of indole by H 2 O 2 , with generation of 2-and 3-oxoindole as the major products. This reaction occurred in the absence of O 2 and reducing agents and was not inhibited by superoxide dismutase or hydroxyl radical scavengers, although it was strongly inhibited by L-Trp. The stoichiometry of the reaction indicated a one-to-one correspondence for the consumption of indole and H 2 O 2 . The 18 O-labeling experiments indicated that the oxygen incorporated into the monooxygenated products was derived almost exclusively from H 2 18 O 2 , suggesting that electron transfer was coupled to the transfer of oxygen from a ferryl intermediate of IDO. These results demonstrate that IDO oxidizes indole by means of a previously unrecognized peroxygenase activity. We conclude that IDO inserts oxygen into indole in a reaction that is mechanistically analogous to the "peroxide shunt" pathway of cytochrome P450.monooxygenase | oxygen isotopic labeling T he heme enzyme indoleamine 2,3-dioxygenase (IDO) catalyzes the first and rate-determining step of L-tryptophan metabolism in nonhepatic mammalian tissues by inserting both atoms of O 2 into the indole ring to form N-formylkynurenine (N-FK) (1). This dioxygenase activity of IDO requires O 2 and the reduced (Fe 2+ ) enzyme or O 2•− and the oxidized (Fe 3+ ) enzyme. In recent years, an increasing number of physiological consequences of IDO activity have been identified, including links to neural, ocular, and immunological disorders (2). Of these roles, the ability of IDO expressed by tumors to suppress the normal response of T lymphocytes and enable tumor survival and growth has attracted the most intense interest, owing to its implications for the development of new therapeutic approaches in cancer treatment (3).Aside from tryptophan (Trp), the dioxygenase activity of IDO extends to oxidation of other indoleamines, such as 5-hydroxy-LTrp, serotonin, and tryptamine, whereas indole, 3-methylindole, and indoleacetic acid are not substrates (4-6). Although indole can apparently bind to the oxygenated enzyme (IDOFe 3+ -O 2•− ) or to the IDOFe 2+ -CO complex (7), autoxidation to IDOFe 3+ is unaffected by the presence of indole, and oxidation of indole does not occur (5). Notably, IDO does not catalyze oxidation of L-Trp by H 2 O 2 (4, 8, 9). Consequently, the reactivity of IDO with H 2 O 2 received little attention for more than 30 years. During that time, IDO was noted to have peroxidase activity (6) and to catalyze the H 2 O 2 -supported N-demethylation of benzphetamine and hydroxylation of aniline (10), but these activities were not studied further.Recently, reactivity of the enzyme with H 2 O 2 has received considerable attention in light of spectroscopic detection of a compound II-like ferryl species (11, 12) and quantum mechanics/ molecular mechanics simulations implicating a role for this intermediate in the catalytic cycle (13). Subsequently, the peroxidase activity of IDO has received renewed atten...

Section: •−mentioning

confidence: 99%

Indole peroxygenase activity of indoleamine 2,3-dioxygenase

Kuo

Mauk

2012

“…AFB 1 exo-8,9-epoxide is 30-and 1,200-fold more reactive than syn-BPDE and anti-BPDE, respectively, at pH Ն 7 (18). AFB 1 exo-8,9-epoxide has similar acid-catalyzed rates of hydrolysis, 3.4-fold higher than syn-BPDE and 1.4 times that of anti-BPDE (18). Thus, AFB 1 exo-8,9-epoxide should provide a good system for testing the DNA acidic domain/DNA-mediated acid catalysis theory because of its instability in H 2 O.…”

mentioning

confidence: 97%

“…The local concentration of protons has been mathematically modeled by combining Poisson-Boltzmann, Monte Carlo, and molecular mechanics techniques (6), and the theory has been shown to fit some experimental data well (7). The hydrolysis of BPDE has a spontaneous as well as an acidcatalyzed component that elevates the rate in acidic medium (18). The presence of either ds or ssDNA has been shown to clearly enhance the rate of hydrolysis of both the syn and anti isomers and is DNA-concentration-dependent (19)(20)(21).…”

mentioning

confidence: 99%

Reaction of aflatoxin B ₁ exo -8,9-epoxide with DNA: Kinetic analysis of covalent binding and DNA-induced hydrolysis

Johnson

Guengerich

1997

114

The exo isomer of af latoxin B 1 (AFB 1 ) 8,9-epoxide appears to be the only product of AFB 1 involved in reaction with DNA and reacts with the N 7 atom of guanine via an S N 2 reaction from an intercalated state. Although the epoxide hydrolyzes rapidly in H 2 O (0.6 s ؊1 at 25°C), very high yields of DNA adduct result. Experimental binding data were fit to a model in which the epoxide forms a reversible complex with calf thymus DNA (K d ‫؍‬ 0.43 mg ml ؊1 , or 1.4 mM monomer equivalents) and reacts with guanine with a rate of 35 s ؊1 . Stopped-f low kinetic analysis revealed attenuation of f luorescence in the presence of DNA that was dependent on DNA concentration. Kinetic spectral analysis revealed that this process represents conjugation of epoxide with DNA, with an extrapolated rate maximum of 42 s ؊1 and half-maximal velocity at a DNA concentration of 1.8 mg ml ؊1 (5.8 mM monomer equivalents). The rate of hydrolysis of the epoxide was accelerated by calf thymus DNA in the range of pH 6-8, with a larger enhancement at the lower pH (increase of 0.23 s ؊1 at pH 6.2 with 0.17 mg DNA ml ؊1 ). The same rate enhancement effect was observed with poly[dA-dT]⅐poly[dAdT], in which the epoxide can intercalate but not form significant levels of N 7 purine adducts, and with singlestranded DNA. The increased rate of hydrolysis by DNA resembles that reported earlier for epoxides of polycyclic hydrocarbons and is postulated to involve a previously suggested localized proton field on the periphery of DNA. The epoxide preferentially intercalates between base pairs, and the proton field is postulated to provide acid catalysis to the conjugation reaction.Aflatoxin B 1 (AFB 1 ) exposure is a major factor in human liver cancer in some parts of the world. The compound is a natural product of the Aspergillus genus of molds, which grow on several foodstuffs stored in hot moist conditions, and is among the most potent hepatocarcinogens and genotoxins presently known. AFB 1 is enzymatically activated by cytochrome P450 3A4 to a very reactive epoxide (1, 2). AFB 1 exo-8,9-epoxide is the genotoxic isomer and reacts efficiently with DNA at the N 7 position of Gua, evidently after intercalation (Fig. 1) (3). We recently showed that AFB 1 exo-8,9-epoxide reacts spontaneously with H 2 O with a pseudo-first order rate of 0.6 s Ϫ1 at 25°C (4). Although DNA adduct yields have been determined (5), the instability of the AFB 1 exo-8,9-epoxide has precluded direct measurement of the reaction rate and the affinity for DNA.A proton-rich or low pH microenvironment at the surface of DNA molecules has recently been alleged, based on Poisson-Boltzmann distributions and Monte Carlo mathematical simulations (6, 7). We previously found the hydrolysis of AFB 1 exo-8,9-epoxide to be elevated by acid catalysis at pH Ͻ 5.4 but not base-catalyzed (4). Consequently, we considered this to be an excellent system for testing the theory of a proton-rich microenvironment around DNA, because such a field should enhance the hydrolysis rate. We show here that t...

“…Chemical and kinetic studies have demonstrated that bayregion diol epoxides are subject to general acid catalysis to form chemically unreactive and biologically inactive tetraols (7)(8)(9)(10)(11)(12) as well as to form covalent adducts with low molecular weight nucleophiles such as p-nitrothiophenol and 2-mercaptoethanol (7,8,12). Thus a chemical basis exists for a rational approach to the identification of nontoxic compounds that can block the adverse biological effects of the ultimate carcinogens of polycyclic aromatic hydrocarbons.…”

mentioning

confidence: 99%

Inhibition of the mutagenicity of bay-region diol epoxides of polycyclic aromatic hydrocarbons by naturally occurring plant phenols: Exceptional activity of ellagic acid

Wood

Huang

Chang

et al. 1982

Self Cite

203

Ferulic, caffeic, chlorogenic, and ellagic acids, four naturally occurring plant phenols, inhibit the mutagenicity and cytotoxicity of (+)-7,8,8a-dihydroxy-9a,10a-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]P 7,8-diol-9,10-epoxide-2), the only known ultimate carcinogenic metabolite of benzo [a]pyrene. The mutagenicity of 0.05 nmol of B[a]P 7,8-diol-9,10-epoxide-2 in strain TA100 of Salmonella typhimurium is inhibited 50% by incubation ofthe bacteria and the diol epoxide with 150 nmol offerulic acid, 75 nmol of caffeic acid, 50 nmol of chlorogenic acid or, most strikingly, 1 nmol of ellagic acid in the 0.5-ml incubation mixture. A 3-nmol dose of ellagic acid inhibits mutation induction by 90%. Ellagic acid is also a potent antagonist of B[a]P 7,8-diol-9,10-epoxide-2 in Chinese hamster V79 cells. Mutations to 8-azaguanine resistance induced by 0.2 ,AM diol epoxide are reduced by 50% when tissue culture media also contains 2 ,uM ellagic acid. Similar to results obtained with the bacteria, ferulic, caffeic, and chlorogenic acids are approximately two orders of magnitude less active than ellagic acid in the mammalian cell assay. The antimutagenic effects of the plant phenols result from their direct interaction with B[a]P 7,8-diol-9,10-epoxide-2, because a concentration-dependent increase in the rate ofdiol epoxide disappearance in cell-free solutions of 1:9 dioxane/water, pH 7.0, is observed with all four phenols. In parallel with the mutagenicity studies, ellagic acid is 80-300 times more effective than the other phenols in accelerating the disappearance of B[a]P 7,8-diol-9,10-epoxide-2. Ellagic acid at 10 ,M increases the disappearance of B[a]P 7,8-diol-9,10-epoxide-2 by approximately 20-fold relative to the spontaneous and hydronium ion-catalyzed hydrolysis of the diol epoxide at pH 7.0. Ellagic acid is a highly potent inhibitor of the mutagenic activity of bay-region diol epoxides of benzo[a]pyrene, dibenzo[a,h]pyrene, and dibenzo[a,ijpyrene, but higher concentrations of ellagic acid are needed to inhibit the mutagenic activity of the chemically less reactive bay-region diol epoxides of benz [a]anthracene, chrysene, and benzo [c]phenanthrene. These studies demonstrate that ellagic acid is a potent antagonist of the adverse biological effects of the ultimate carcinogenic metabolites of several polycyclic aromatic hydrocarbons and suggest that this naturally occurring plant phenol, normally ingested by humans, may inhibit the carcinogenicity ofpolycyclic aromatic hydrocarbons.Polycyclic aromatic hydrocarbons are relatively inert environmental precarcinogens that must be metabolized by mammalian enzymes to their biologically active products (ultimate carcinogens). Mutagenicity, metabolism, DNA-binding, and tumorigenicity studies during the past 8 years have shown that benzoring diol epoxides, in which the epoxide moiety forms part of the bay region (1, 2) of the hydrocarbons, are ultimate carcinogens of polycyclic hydrocarbons (3-6).Chemical and kinetic studies have demonstrated that bayregion diol epoxid...