Mechanism of inactivation of Escherichia coli aspartate aminotransferase by (S)-4-amino-4,5-dihydro-2-furancarboxylic acid

As a potential drug to treat neurological diseases, the mechanism-based inhibitor (S)-4-amino-4,5-dihydro-2-furancarboxylic acid (S-ADFA) has been found to inhibit the γ-aminobutyric acid aminotransferase (GABA-AT) reaction. To circumvent the difficulties in structural studies of a S-ADFA-enzyme complex using GABA-AT, l-aspartate aminotransferase (l-AspAT) from Escherichia coli was used as a model PLP-dependent enzyme. Crystal structures of the E. coli aspartate aminotransferase with S-ADFA bound to the active site were obtained via cocrystallization at pH 7.5 and 8. The complex structures suggest that S-ADFA inhibits the transamination reaction by forming adducts with the catalytic lysine 246 via a covalent bond while producing 1 equiv of pyridoxamine 5'-phosphate (PMP). Based on the structures, formation of the K246-S-ADFA adducts requires a specific initial binding configuration of S-ADFA in the l-AspAT active site, as well as deprotonation of the ε-amino group of lysine 246 after the formation of the quinonoid and/or ketimine intermediate in the overall inactivation reaction.     

Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectrometric evidence for the flavin-1-phenylcyclopropylamine inactivator adduct with monoamine oxidase N

1-Phenylcyclopropylamine (1-PCPA) is shown to be an inactivator of the fungal flavoenzyme monoamine oxidase (MAO) N. Inactivation results in an increase in absorbance at 410 nm and is accompanied by the concomitant loss of the flavin absorption band at 458 nm. The spectral properties of the covalent adduct formed between the flavin cofactor of MAO N and 1-PCPA are similar to those reported for the irreversible inactivation product formed with 1-PCPA and mammalian mitochondrial monoamine oxidase B [Silverman, R. B., and Zieske, P. A. (1985) Biochemistry 24, 2128-2138]. There is a hypsochromic shift of the 410 nm band upon lowering the pH to 2, indicating that an N(5)-flavin adduct formed upon inactivation. Use of the fungal enzyme, MAO N, which lacks the covalent attachment to the flavin adenine dinucleotide (FAD) cofactor present in the mammalian forms MAO A and MAO B, has allowed for the isolation and further structural identification of the flavin-inactivator adduct. The incorporation of two (13)C labels into the inactivator, [2,3-(13)C(2)]-1-PCPA, followed by analysis using on-line liquid chromatography/electrospray ionization mass spectrometry and nuclear magnetic resonance spectroscopy, provided a means to explore the structure of the flavin-inactivator adduct of MAO N. The spectral evidence supports covalent attachment of the 1-PCPA inactivator to the cofactor as N(5)-3-oxo-3-phenylpropyl-FAD.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo. S -BMO adducts were the main product and represented 77 % (n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

Regio and stereospecific DNA adduct formation in mouse lung at N6 and N7 position of adenine and guanine after 1,3 butadiene inhalation exposure

Butadiene monoepoxide (BMO) alkylated guanine N7 and adenine N 6 adducts were prepared and enriched by solid phase extraction and HPLC. The purified adducts were analysed by a modified 32P-postlabelling assay, which utilized one dimensional TLC chromatography and a subsequent HPLC analysis with UV and radioactivity detectors. In vitro with Ct-DNA the formation of N7-dGMP and N 6-dAMP adducts were linear at a concentration range of 44 to 870 nmol of BMO per mg DNA at physiological pH. N7- dGMP and N 6-dAMP adducts were formed in a ratio of 200:1. In dGMP and in dAMP 48 % and 86 % of adducts were covalently bound to the C-2 carbon of BMO. CD-1 mice were inhalation exposed to butadiene for 5 days and 6 h per day. The N7-dGMP adduct level in lung samples of animals exposed to 200, 500 and 1300 ppm was 2.8 +/- 0.9 fmol, 11 +/- 2.0 fmol and 30 +/- 6.7 fmol in 10 mug DNA, respectively. The level of N 6-dAMP adducts in lung samples after 500 ppm and 1300 ppm exposure was 0.09 +/- 0.06 fmol and 0.11 +/- 0.05 fmol in 10 mug DNA. At 200 ppm the adduct level was below the detection limit. A sub-group of animals exposed to 1300 ppm was killed 3 weeks after the last exposure. N7-dGMP adducts were not detected but the level of N 6-dAMP adducts was not affected. N7-dGMP adducts were formed in a clear stereospecific manner in vivo . S -BMO adducts were the main product and represented 77 % ( n = 4, SD = 2%) of total BMO adducts. No clear conclusion can be drawn about the enantiospecific DNA binding at the N 6 position of dAMP, because of the poor separation of the enantiomers. However, we could separate regioisomeric adducts which indicated that C-2 adducts represented 69 +/- 3 % of the total N 6 adducts formed in mice lung DNA. This observation is supported by the data derived from in vitro DNA experiments but is different to our previously published data, which indicates the 2:1 (C-1:C-2) ratio in regioisomer formation in nucleotides or nucleosides. We suggest that the data presented in this communication indicate a different mechanism between nucleotides and DNA in BMO-derived adduct formation- Dimroth rearrangement dominates in nucleotides, but in double stranded DNA a direct alkylation is probably the major mechanism of adduct formation.     

Identification of nucleic acid adducts from trans-4-acetylaminostilbene

It has been proposed that trans-4-acetylaminostilbene (AAS) is an initiator for tumor formation in rat liver and that the metabolically formed hydroxamic acid ester ultimately reacts with nucleic acids in vivo. We have now studied the generation of a major adduct in vitro. trans-4-N-Acetoxy-N-acetylaminostilbene (N-acetoxy-AAS) was reacted with guanosine at pH 7.5 and reaction products were separated by chromatography on Sephadex LH-20 and RP18 HPLC. The major adduct isolated consists of four isomers which have been tentatively identified by mass- and 1H-NMR spectroscopy as (S,S)- and (R,R)-guanosine-N2,beta-N3,alpha-N-acetylaminobibenzyl and the respective regio isomers guanosine-N2,alpha-N3,beta-N-acetylaminobibenzyl. These adducts are formed in a ratio of 9:9:1:1. Under acidic conditions (pH 2) the ribose moiety is removed and two regio isomeric base adducts are formed in the ratio 9:1. Results to be published indicate that the adducts are also formed in vivo in rat liver RNA and DNA.     

Pyridoxal 5'-phosphate-dependent histidine decarboxylase. Mechanism of inactivation by alpha-fluoromethylhistidine

Mechanism-based inactivation of pyridoxal phosphate-dependent histidine decarboxylase by (S)-alpha-(fluoromethyl)histidine was studied. The molar ratio of inactivator to enzyme subunit required for complete inactivation increased from 1.63 at 10 degrees C to 3.00 at 37 degrees C. Two inactivation products were isolated by chromatographic fractionation of the reaction mixture and identified by NMR spectroscopy as 1-(4-imidazolyl)-3(5'-P-pyridoxylidene) acetone (I), the adduct formed between pyridoxal phosphate and inactivator, and 1-(4-imidazolyl) acetone (II), an intermediate compound formed during inactivation. Formation of these two products supports a previously proposed mechanism of inactivation (Hayashi, H., Tanase, S., and Snell, E. E. (1986) J. Biol. Chem. 261, 11003-11009), with minor modifications. A precursor of I was linked covalently to the enzyme by NaBH4 reduction if the reaction was carried out immediately after inactivation, before development of the 403 nm peak of I. A mutant histidine decarboxylase (S322A) in which Ser-322 was changed to Ala was also inactivated by alpha-fluoromethylhistidine demonstrating that Ser-322 is not essential for inactivation even though it is close to the active site and is derivatized by borohydride reduction of the inactivated wild-type enzyme. Following inactivation, both the wild-type and the S322A mutant enzyme could be partially reactivated by prolonged dialysis against buffer.     

DNA adduct formation from the mutagenic air pollutant 3-nitrobenzanthrone

The environmental contaminant 3-nitrobenzanthrone (3-nitro-7H-benz[d, e]anthracen-7-one) was recently shown to be a very strong bacterial mutagen, suggesting a new class of mutagenic compounds present in airborne particulate matter and diesel exhaust. Using the 32P-postlabeling assay, we investigated the capacity for 3-nitrobenzanthrone to form DNA adducts in vitro. Calf thymus DNA was incubated with 3-nitrobenzanthrone and either xanthine oxidase, a mammalian nitroreductase or rat liver S9 or zinc. Under these conditions 3-nitrobenzanthrone formed a total of seven adducts detectable by 32P-postlabeling. Using enrichment by butanol extraction the highest level of DNA adduct formation was found with activation by zinc (RAL: 88.4+/-32 per 108 nucleotides) followed by activation with xanthine oxidase (RAL: 75.5+/-12) and activation by rat liver S9 (RAL: 48.6+/-8). Three of the seven adduct spots were detected in all activation systems, however different amounts of individual spots were obtained in the different in vitro systems. The adduct pattern observed for the enzymatic incubations consisted of three major spots and was essentially identical. Chemical reduction of 3-nitrobenzanthrone by zinc resulted in five adduct spots whose formation was found to be concentration dependent. All adducts of 3-nitrobenzanthrone observed in this study migrated primarily along a diagonal zone, typical for DNA adducts derived from extracts of airborne particulate matter. When butanol enrichment was compared with nuclease P1 enrichment one adduct was clearly sensitive to the 3'-monophosphatase activity of nuclease P1. Our results demonstrate that 3-nitrobenzanthrone binds covalently to DNA after metabolic activation, forming multiple DNA adducts in vitro all of which are reduction products. These adducts may contribute to the known genotoxicity and carcinogenicity of extracts from airborne particulates.     

DNA adducts of the enantiomers of the Pt(II) complexes of the ahaz ligand (ahaz=3-aminohexahydroazepine) and recognition of these adducts by HMG domain proteins

The bending, unwinding, and structural changes in DNA caused by the binding of each of the enantiomers of the platinum(II) complexes of the ahaz ligand (R- and S-[PtCl(2)(ahaz)], ahaz=3-aminohexahydroazepine) have been studied using 20-23 bp oligonucleotides containing TGGT and CGGA-binding sites as has the recognition of the adducts by HMG domain proteins. The domain A of HMGB1 (HMGB1a protein) binds to the adduct formed by the R enantiomer at the CGGA sequence with a similar high affinity as it does to the adduct of antitumor cisplatin, and to the adduct formed by the S enantiomer with a slightly lower affinity. In contrast, HMGB1a binds much more weakly to the ahaz adducts than to the cisplatin adducts formed at the TGGT sequence, with the binding to the adduct formed by the R enantiomer being weakest. Each enantiomer and cisplatin cause unwinding of both sequences that is in the narrow range, 19-22 degrees. There are modest but significant differences in the degree of bending induced, with the S enantiomer causing the least bending, cisplatin intermediate, and the R enantiomer the most. Molecular modeling of the {Pt(ahaz)}/GG adducts in 8-bp models reveals significant differences in the local distortion at the GG-binding sites depending on the flanking bases and shows that interactions between the thymine methyl groups and the ahaz ligand are likely to inhibit bending of the TGGT sequence.     

Crystal structures of alpha-lytic protease complexes with irreversibly bound phosphonate esters

The structures of the complexes with alpha-lytic protease of both phosphorus stereoisomers of N-[(2S)-2-[[[(1R)-1-[N-[(tert-butyloxycarbonyl)-L-alanyl-L-alanyl- L-prolyl]amino]-2-methylpropyl]-phenoxyphosphinyl]oxy]propanoyl]- L-alanine methyl ester, an analogue of the peptide Boc-Ala-Ala-Pro-Val-Ala-Ala where Val is replaced with an analogous phosphonate phenyl ester and the subsequent Ala is replaced with lactate, have been determined to high resolution (1.9 A) by X-ray crystallography. Both stereoisomers inactivate the enzyme but differ by a factor of 2 in the second-order rate constant for inactivation [Sampson, N. S., & Bartlett, P. A. (1991) Biochemistry (preceding paper in this issue)]. One isomer (B) forms a tetrahedral adduct in which the phosphonate phenyl ester is displaced by the active site serine (S195) and interacts with the enzyme across seven substrate recognition sites that span both sides of the scissile bond. Seven hydrogen bonds are formed with the enzyme, and 510 A2 of hydrophobic surface area is buried when the inhibitor interacts with the enzyme. Although two hydrogen bonds are gained by incorporation of two residues on the C-terminal side of the scissile bond into the inhibitor, there is very little adjustment in the structure of the enzyme in this region. Surprisingly, the active site histidine (H57) does not interact with the phosphonate, apparently because the phosphonate lacks negative charge in or near the oxyanion hole, and instead, the side chain rotates out of the active site cleft and hydrogen bonds with solvent. The other isomer (A) forms a mixture of two different tetrahedral adducts in the active site, both covalently bonded to Ser 195. One adduct, at approximately 58% occupancy, is exactly the same in structure as the complex formed with isomer B, and the other adduct, at 42% occupancy, has lost the two residues C-terminal to the scissile bond by hydrolysis. In the lower occupancy structure, His 57 does not rotate out of the active site and forms a hydrogen bond with the phosphonate oxygen instead. The structures of both complexes were insensitive to pH. As very little change in structure accompanies the histidine rotation, the complex with isomer B provides an excellent mimic for the structure of the transition state (or high-energy reaction intermediate) that spans both sides of the scissile bond.     

Calf thymus DNA binding/bonding properties of CC-1065 and analogs as related to their biological activities and toxicities.

CC-1065 is a potent natural antitumor antibiotic that binds non-covalently and covalently (N-3 adenine adduct) in the minor groove of B-form DNA. Synthetic analogs of CC-1065 do not exhibit the delayed death toxicity of CC-1065 and are efficacious anticancer agents, some of them curative in murine tumor models. In an attempt to understand the different biological properties of CC-1065 and analogs, we have determined the following quantities for CC-1065, enantiomeric CC-1065, and three biologically active analogs and their enantiomers: the calf thymus DNA (CT-DNA) induced molar ellipticity of the adduct (or how rigidly the adduct is held in the right-hand conformation of the minor groove); the stability of the adduct with respect to long incubation times and to digestion by snake venom phosphodiesterase I (SVPD); the stabilizing effect on the CT-DNA helix of the covalently and non-covalently bound species with respect to thermal melting; and the CT-DNA binding/bonding (non-covalent/covalent) profiles at a low molar ratio of nucleotide to drug. The major observations from these studies are as follows: (i) molecules which show large DNA interaction parameters, stable adducts, and significant non-covalent binding exhibit delayed death toxicity; (ii) molecules which show intermediate DNA interaction parameters and stable adducts, but do not show significant non-covalent binding, do not exhibit delayed death toxicity and are biologically active; (iii) molecules which show small DNA interaction parameters and unstable DNA adducts are biologically inactive. The results suggest that a window exists in the affinity for the minor groove of DNA wherein an analog may possess the correct balance of toxicity and activity to make a useful anticancer agent. Outside of this window, the analog causes delayed deaths or has no significant biological activity.     

Characterization of a covalently linked complex involving ferredoxin and ferredoxin: NADP reductase

Ferredoxin and the flavoprotein, ferredoxin: NADP reductase, have been covalently linked by incubation in the presence of a water soluble carbodiimide. The cross-linking reaction yields an adduct having a 1:1 stoichiometry. The adduct has depressed levels of diaphorase and NADPH oxidase activity and is inactive in reduction of cytochrome c using NADPH as an electron donor. Thus, although similar to an adduct described by Zanetti and coworkers [J Biol Chem 259: 6153–6157 (1984)] in its stoichiometry, the adduct described herein has significantly different enzymatic properties. It is suggested that this may be a reflection of differences in the interaction between the two proteins resulting from differences in experimental conditions in which the two adducts were prepared.Abbreviations Fd ferredoxin - Fp ferredoxin: NADP reductase - Fd Fp covalently linked Fd-Fp adduct - Fd:Fp noncovalently linked complex between Fd and Fp - EDC 1-ethyl-3-(dimethylaminopropyl) carbodiimide - Tris tris-hydroxymethylaminomethane - MOPS 3-(N-morpholino)propane sulfonic acid - DCIP 2,6-dichloropenolindophenol     

New sensitive determination method of benzidine-hemoglobin adducts by gas chromatography-electron impact mass spectrometry

A gas chromatographic-mass spectrometric assay was developed for the determination of benzidine (BZ)-hemoglobin adducts. Adducts were released from hemoglobin by alkaline hydrolysis and extraction at pH 8 with ethyl ether. The dried extract was completely derivatized with N-methyl-N-(tert.-butyldimethylsilyl) trifluoroacetamide (MTBDMSTFA)-NH(4)I (1000:3) under catalysis of dithioerythritol. The recovery of BZ, acetylbenzidine (ABZ) and diacetylbenzidine (DABZ) in the extraction procedure was 76-98%. The detection limits of the assay were 0.1 ng/g for both BZ and ABZ, and 0.5 ng/g for DABZ based upon assayed hemoglobin of 0.1 g. The method was applied to the determination of BZ-hemoglobin adducts formed in young female Sprague-Dawley rats after treatment for 1, 2 and 3 weeks with 0.008% BZ via the drinking water. Two adducts were detected by proposed procedure. The structure of these adducts could be assigned to BZ and ABZ. After 1 week, the total mean amount of adducts determined was 2.8 ng/g hemoglobin. The adduct levels increased up to about 7.5 ng/g after a week and, thereafter, remained essentially constant. The relative contribution of BZ and ABZ to the total hemoglobin adduct level was strongly treatment time-dependent. After 1 week, the BZ and ABZ adducts were formed at similar levels, whereas after 3 weeks the ABZ adducts was predominant. Treatment of rats for 3 weeks in the dose range 12.2-36.8 mg of BZ in drinking water resulted in a dose-proportional increase in the total amount of hemoglobin adducts formed.     

Adduct formation of pyrroloquinoline quinone and amino acid

When pyrroloquinoline quinone (PQQ) is mixed with an amino acid, a corresponding Schiff base PQQ adduct is readily formed between carbonyl groups of PQQ and the primary amino group. A potent growth stimulating effect for microorganisms was observed with the PQQ adduct when it was administered in a culture medium. Although PQQ itself shows a marked growth stimulating effect, PQQ adducts appeared to be more active than authentic PQQ when compared on a molar basis. Conversely, unlike authentic PQQ, PQQ adducts were shown to be less active (greater than or equal to 100-fold) as the prosthetic group for a quinoprotein apo-glucose dehydrogenase when examined by holoenzyme formation by exogenous addition of PQQ or PQQ adducts. These observations suggested that PQQ adduct formation readily occurs during isolation procedures for PQQ from biological materials or PQQ - chromophore from quinoproteins. Therefore, the presence of such adducts gives a PQQ estimation much lower than theoretically expected. As an example, formation, isolation and characterization of PQQ - serine are described.     

Simulating structural and thermodynamic properties of carcinogen-damaged DNA

A pair of stereoisomeric covalent adducts to guanine in double-stranded DNA, derived from the reaction of mutagenic and tumorigenic metabolites of benzo[a]pyrene, have been well characterized structurally and thermodynamically. Both high-resolution NMR solution structures and an array of thermodynamic data are available for these 10S (+)- and 10R (-)-trans-anti -[BP]-N(2)-dG adducts in double-stranded deoxyoligonucleotides. The availability of experimentally well-characterized duplexes containing these two stereoisomeric guanine adducts provides an opportunity for evaluating the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method for computing thermodynamic properties from molecular dynamics ensembles. We have carried out 3-ns molecular dynamics simulations, using NMR solution structures as the starting models for the 10S (+)- and 10R (-)-trans-anti-dG adducts in a DNA duplex 11-mer using AMBER 6.0. We employed the MM-PBSA method to compute the free energies, enthalpies, and entropies of the two adducts. Our complete thermodynamic analysis agrees quite well with the full experimental thermodynamic characterization of these adducts, showing essentially equal stabilities of the two adducts. We also calculated the nuclear Overhauser effect (NOE) distances from the molecular dynamics trajectories, and compared them against the experimental NMR-derived NOE distances. Our results showed that the simulated structures are in good agreement with the NMR experimental NOE data. Furthermore, the molecular dynamics simulations provided new structural and biological insights. Specifically, the puzzling observation that the BP aromatic ring system in the 10S (+)-trans-anti-dG adduct is more exposed to the aqueous solvent than the 10R (-)-trans-anti-dG adduct, is rationalized in terms of the adduct structures. The structural and thermodynamic features of these stereoisomeric adducts are also discussed in relation to their reported low susceptibilities to nucleotide excision repair.     

Formation of dopamine quinone-DNA adducts and their potential role in the etiology of Parkinson's disease

The neurotransmitter dopamine is oxidized to its quinone (DA-Q), which at neutral pH undergoes intramolecular cyclization by 1,4-Michael addition, followed by oxidation to form leukochrome, then aminochrome, and finally neuromelanin. At lower pH, the amino group of DA is partially protonated, allowing the competitive intermolecular 1,4-Michael addition with nucleophiles in DNA to form the depurinating adducts, DA-6-N3Ade and DA-6-N7Gua. Catechol estrogen-3,4-quinones react by 1,4-Michael addition to form the depurinating 4-hydroxyestrone(estradiol)-1-N3Ade [4-OHE1(E2)-1-N3Ade] and 4-OHE1(E2)-1-N7Gua adducts, which are implicated in the initiation of breast and other human cancers. The effect of pH was studied by reacting tyrosinase-activated DA with DNA and measuring the formation of depurinating adducts. The most adducts were formed at pH 4, 5, and 6, and their level was nominal at pH 7 and 8. The N3Ade adduct depurinated instantaneously, but N7Gua had a half-life of 3 H. The slow loss of the N7Gua adduct is analogous to that observed in previous studies of natural and synthetic estrogens. The antioxidants N-acetylcysteine and resveratrol efficiently blocked formation of the DA-DNA adducts. Thus, slightly acidic conditions render competitive the reaction of DA-Q with DNA to form depurinating adducts. We hypothesize that formation of these adducts could lead to mutations that initiate Parkinson's disease. If so, use of N-acetylcysteine and resveratrol as dietary supplements may prevent initiation of this disease.     

Synthesis and spectroscopic characterization of site-specific 2-amino-1-methyl-6-phenylimidazo

The aim of the present study is to determine the chemical structure and conformation of DNA adducts formed by incubation of the bioactive form of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), N-acetoxy-PhIP, with a single-stranded 11mer oligodeoxyribonucleotide. Using conditions optimized to give the C8-dG-PhIP adduct as the major product, sufficient material was synthesized for NMR solution structure determination. The NMR data indicate that in duplex DNA this adduct exists in equilibrium between two different conformational states. In the main conformer, the covalently bound PhIP molecule intercalates in the helix, whilst in the minor conformation the PhIP ligand is probably solvent exposed. In addition to the C8-dG-PhIP adduct, at least eight polar adducts are found after reaction of N-acetoxy-PhIP with the oligonucleotide. Three of these were purified for further characterization and shown to exhibit lowest energy UV absorption bands in the range 342–347 nm, confirming the presence of PhIP or PhIP derivative. Accurate mass determination of two of the polar adducts by negative ion MALDI-TOF MS revealed ions consistent with a spirobisguanidino-PhIP derivative and a ring-opened adduct. The third adduct, which has the same mass as the C8-dG-PhIP oligonucleotide adduct, may contain PhIP bound to the N² position of guanine.     

Binding of 3-hydroxybenzo[a]pyrene to bovine hemoglobin and albumin

Previous studies examined the bioavailability and first-pass biotransformation of 3-hydroxy[(3)H]benzo[a]pyrene ([(3)H]-3-OHBaP) in an isolated perfused catfish intestinal model. This work showed that 3-OHBaP, or a metabolite formed in intestine, bound covalently to blood protein. In this study, the blood adducts were characterized in vitro by incubating bovine ferric hemoglobin or albumin with [(3)H]-3OHBaP under various conditions. Incubation of 2 microM [(3)H]-3-OHBaP with hemoglobin for 1 h resulted in 7.49 pmol bound/mg protein, while albumin binding was 1.37 pmol/mg protein. Mild acid hydrolysis released only 5% of the radioactivity from 3-OHBaP-hemoglobin adducts. After gel filtration, the 3-OHBaP-hemoglobin adducts were examined by HPLC analysis. A single peak of radioactivity was detected at the same retention time as the heme component of hemoglobin. Unbound 3-OHBaP was oxidized to BaP-3,6-dione during incubation with ferric hemoglobin. Treatment of hemoglobin with ascorbic acid decreased the formation of hemoglobin adducts by 33%, while hydrogen peroxide treatment increased adduct formation by 44%. Incubation of [(3)H]-BaP-3-beta-D-glucuronide (BaP-3G) with hemoglobin and beta-glucuronidase resulted in greater binding to hemoglobin than incubation with [(3)H]-3-OHBaP alone. The hemoglobin adduct obtained from [(3)H]-BaP-3G also co-migrated with heme. These results indicate that an oxidative process is involved in formation of the heme adduct and that 3-OHBaP or BaP-3G might be a precursor of the bound metabolite.