Sites Of Adduct Formation Research Articles

Covalent adducts of melphalan with free amino acids and a model peptide studied by liquid chromatography/tandem mass spectrometry.

Melphalan is a frequently used chemotherapeutical agent for the treatment of myeloma, breast cancer, ovarian cancer and sarcoma of soft tissue. A good knowledge of the reactivity of the drug toward the different amino acids, e.g. covalent adduct formation, is crucial for the understanding of its activity and side effects during cancer treatment. The reactivity of melphalan and sites of adduct formation were studied by in vitro incubation of melphalan with free amino acids and glutathione as a model peptide. The formed covalent adducts were investigated using ultra-performance liquid chromatography tandem mass spectrometry (UPLC/MS/MS) using a triple-quadrupole instrument. Accurate mass measurements for the confirmation of characteristic product ions were performed on a quadrupole time-of-flight (QTOF) mass spectrometer. The incubation of melphalan with different classes of amino acids resulted in the formation of adducts on the amino and carboxyl termini, as well as adduct formation in the reactive side chains of Cys, Met, Tyr, His, Lys, Asp and Glu. All these melphalan adducts could be identified by their characteristic collision-induced dissociation (CID) product ion patterns. The present study demonstrates the reactivity of melphalan towards the functional groups of amino acids. The different alkylation site products show distinctive fragmentation patterns, which enable a fast identification of the different melphalan adducts. This study is a first important step towards a better understanding of the adduct formation in more complex molecules, e.g. peptides and proteins.

Inhibition of Bupropion Metabolism by Selegiline: Mechanism-Based Inactivation of Human CYP2B6 and Characterization of Glutathione and Peptide Adducts

Selegiline, the R-enantiomer of deprenyl, is used in the treatment of Parkinson's disease. Bupropion, an antidepressant, often used to treat patients in conjunction with selegiline, is metabolized primarily by CYP2B6. The effect of selegiline on the enzymatic activity of human cytochrome CYP2B6 in a reconstituted system and its effect on the metabolism of bupropion were examined. Selegiline was found to be a mechanism-based inactivator of the 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation (7-EFC) activity of CYP2B6 as well as bupropion metabolism. The inactivations were time-, concentration-, and NADPH-dependent and were characterized by K(I) values of 0.14 and 0.6 μM, k(inact) values of 0.022 and 0.029 min⁻¹, and t(½) values of 31.5 and 24 min, respectively. In standard inhibition assays, selegiline increased the K(m) of CYP2B6 for bupropion from 10 to 92 μM and decreased the k(cat) by ∼50%. The reduced carbon-monoxide difference spectrum revealed over a 50% loss in the cytochrome P450 spectrum in the inactivated sample, with no loss in heme, and there was ∼70% loss in enzyme activity. Trapping of the reactive metabolite using GSH led to the identification of a GSH-selegiline conjugate with a m/z 528 that could be explained by hydroxylation of selegiline followed by the addition of glutathione to the propargyl moiety after oxygenation to form the ketene intermediate. Liquid chromatography-tandem mass spectrometry analysis of the labeled protein following digestion with trypsin revealed the peptide ⁶⁴DVFTVHLGPR⁷³ as the peptide modified by the reactive metabolite of selegiline and the site of adduct formation is Asp64.

Covalent Modification and Time-Dependent Inhibition of Human CYP2E1 by themeta-Isomer of Acetaminophen

The hypothesis that N-acetyl-m-aminophenol (AMAP), the meta isomer of acetaminophen, will covalently bind to and inhibit human CYP2E1 in a time- and NADPH-dependent manner was investigated. Liquid chromatography/electrospray ionization-mass spectrometry analysis indicated that AMAP metabolites (i.e., AMAP*) selectively and covalently modified CYP2E1 apoprotein in a ratio of 1.4:1 (AMAP*/CYP2E1) in a reconstituted system. The deconvoluted spectra of CYP2E1 apoprotein from incubations containing NADPH and AMAP displayed mass shifts of 167.2 ± 7.1 and 334.4 ± 6.5 Da, suggesting the addition of one and two hydroxylated AMAP metabolites to CYP2E1, respectively. Mass shifts in cytochrome P450 reductase, cytochrome b(5), and heme from these samples were not observed. CYP2E1 inhibition by AMAP increased with time in the presence of NADPH; a reversible inhibition component was also observed. The results support a bioactivation process that involves formation of a hydroquinone metabolite that undergoes further oxidation to a quinone, which reacts with CYP2E1 nucleophilic residues. The data are consistent with evidence from previous studies that identified hydroxylated AMAP glutathione conjugates collected from mice and indicate that cysteine residues are the most likely sites for adduct formation. This study reports the first direct evidence of AMAP-derived hydroquinone metabolites bound to human CYP2E1.

Synthesis and structure determination of the adducts formed by electrochemical oxidation of Dibenzo[a,l]pyrene in the presence of adenine.

Because the radical cations of polycyclic aromatic hydrocarbons (PAH) are involved in tumor initiation, determination of the structures of biologically formed PAH-DNA adducts is important and relies on comparison of their properties with those of synthesized adducts. One of the possible sites of adduct formation is the N-3 position of Ade, but this depurinating adduct is not obtained by one-electron oxidation of dibenzo[a,l]pyrene (DB[a,l]P) in the presence of deoxyadenosine. Therefore, we turned to electrochemical oxidation of DB[a,l]P in the presence of Ade in dimethylformamide and produced the following adducts: DB[a,l]P-10-N1Ade (47%), DB[a, l]P-10-N3Ade (5%), DB[a,l]P-10-N7Ade (2%), and DB[a,l]P-10-N(6)Ade (6%). In Me(2)SO, this reaction afforded the same four adducts, but in slightly different yields: DB[a,l]P-10-N1Ade (44%), DB[a, l]P-10-N3Ade (9%), DB[a,l]P-10-N7Ade (1%), and DB[a,l]P-10-N(6)Ade (3%). These adducts were purified by reverse-phase HPLC, and the subtle differences between the isomers were revealed by NMR, tandem mass spectrometry, and fluorescence line-narrowing spectroscopy. The relative yields of the N1Ade, N3Ade, and N7Ade adducts reflect the nucleophilicity and steric accessibility of these three nitrogen atoms in Ade.

Influence of quinone methide reactivity on the alkylation of thiol and amino groups in proteins: studies utilizing amino acid and peptide models

Quinone methides (QMs) are electrophiles formed in several biological processes including direct oxidations of 4-alkylphenols by cytochromes P450. These species may be responsible for the adverse effects of certain phenolic compounds through protein alkylation, but little information is available concerning specific targets or the resulting mechanisms of cell injury. The present goal was to determine the most likely sites of adduct formation among competing protein nucleophiles utilizing QMs of varying electrophilicity. Reactions of poorly reactive, moderately reactive, and highly reactive QMs, 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone (BHT-QM), 6-tert-butyl-2-(2′-hydroxy-1′,1′-dimethylethyl)-4-methylene-2,5-cyclohexadienone (BHTOH-QM), and 2-tert-butyl-6-methyl-4-methylene-2,5-cyclohexadienone (BDMP-QM), respectively, were investigated in aqueous solutions with nucleophilic amino acids. Each QM rapidly formed a thioether derivative of cysteine with little or no competition from the addition of water (hydration). The α-amino groups were the primary sites of alkylation for all other amino acids examined including lysine, histidine, tyrosine, and serine, and the pseudo-first order rates were 5 to 8-fold greater than the rates of hydration. Alkylation of the side chain nitrogens of lysine and histidine occurred at about one-fourth the rate of hydration for BDMP-QM, but no reaction was detectable for BHT-QM and no reactions occurred between QMs and amino acid hydroxyl groups. The results indicate that, based on chemical reactivity, peptide alkylation should occur in the order cysteine thiol>N-terminal amino>N ε-lysine=NIm-histidine, with side chain modifications occurring only with the more electrophilic QMs. Reactions of QMs with the tripeptide Gly-His-Lys confirmed the results with amino acids as N α-glycine alkylation predominated, but side chain adducts also formed with BHTOH-QM and BDMP-QM. Human hemoglobin was treated with QMs, hydrolyzed, and assayed by HPLC-thermospray mass spectrometry. This work revealed that N ε-lysine was the main alkylation site, emphasizing the importance of factors, in addition to chemical reactivity, which influence protein modification by electrophiles.

Detection of mutagen specific adduct formation in DNA using sequencing methodology

It has been reported that single stranded viral DNA reacts with the carcinogen, chloroacetaldehyde at specific hot spots (Premaratne et al., 1993 Int. J. Biochem. 25, 1669–1672). We tested this occurrence with several other mutagens and potential carcinogens. A series of chemicals (chloroacetaldehyde, methyl, ethyl, and propyl nitro nitrosoguanidine, hydrazine, 2,4 dinitrophenyl hydrazine, hydroxylamine and methyl methanesulfonate) were each separately reacted with viral M13mp18 DNA for 2 hr at 37°C and pH 4.9. The locations of adduction were identified as points of chain termination (or polymerase fall off) when the reacted DNA was subjected to a modified sequencing procedure that had ample regular labeled and unlabeled nucleotides but lacked dideoxy chain termination mixtures. Chain termination was observed to occur at specific, non-random, sites rather than with equal probability at all bases of the DNA. Chemicals with similar structures had identical points of “fall off”. The pattern of chain termination appears to be unique to each class of compounds and is independent of temperature, pH, and salt concentration. Termination is believed to occur when the DNA polymerase encounters an adduct. Mutagens of different unrelated structures when reacted with this DNA produced different sites of adduct formation, while the alkyl nitro nitrosoguanidines, compounds with homologous structure showed identical points of chain termination.

Proteinuria in passive Heymann nephritis is associated with lipid peroxidation and formation of adducts on type IV collagen.

Passive Heymann nephritis (PHN) is a model of human membranous nephropathy that is characterized by formation of granular subepithelial immune deposits in the glomerular capillary wall which results in complement activation. This is causally related to damage of the filtration barrier and subsequent proteinuria. The local accumulation of injurious reactive oxygen species (ROS) is a major effector mechanism in PHN. ROS may induce tissue damage by initiating lipid peroxidation (LPO). In turn, this leads to adduct formation between breakdown products of LPO with structural proteins, such as formation of malondialdehyde (MDA) or 4-hydroxynonenal-lysine adducts. To examine the role of LPO in the development of proteinuria we have localized MDA and 4-hydroxynonenal-lysine adducts in glomeruli of PHN rats by immunofluorescence microscopy, using specific monoclonal antibodies. By immunogold electron microscopy, MDA adducts were localized to cytoplasmic vesicles and cell membranes of glomerular epithelial cells, to the glomerular basement membrane (GBM), and also to immune deposits. Type IV collagen was specifically identified as being modified by MDA adducts, using a variety of techniques. Collagenase pretreatment of GBM extracts indicated that the NC-1 domain of type IV collagen was a site of adduct formation. When LPO was inhibited by pretreatment of PHN rats with the antioxidant probucol, proteinuria was reduced by approximately 85%, and glomerular immunostaining for dialdehyde adducts was markedly reduced, even though the formation of immune deposits was not affected. By contrast, lowering of the serum cholesterol levels had no influence on the development of proteinuria. These findings are consistent with the premise that ROS-induced glomerular injury in PHN involves LPO and that this results not only in damage of cell membranes but in modification of type IV collagen in the GBM as well. The close temporal correlation of the occurrence of LPO with proteinuria and the ability of probucol to inhibit proteinuria support a causal role for LPO in the the alteration of the glomerular permselectivity which results in proteinuria.

Sequence specificity of DNA repair by Escherichia coli Fpg protein.

The sequence specificity of guanine methylation in DNA by N-methyl-N-nitrosourea and the subsequent repair of ring-opened N7-methylguanine was studied using oligonucleotides of defined sequence. It was found that the methylation of TAGGGGCCCCTA was less than 2-fold greater than that occurring in TAGAGATCTCTA or TATGTGCACATA and 6-fold greater than in TACGCGCGCGTA. This is consistent with the concept that guanine methylation is least when the 5' preceding base is a pyrimidine and greatest when the 5' base is another guanine. These dodecamers were used to study repair by the Escherichia coli Fpg protein (formamidopyrimidine-DNA glycosylase) after the 7-methyl-guanine present in them was converted to the ring-opened form by alkaline treatment. The repair of ring-opened 7-methylguanine was much faster in self-complementary double-stranded 12mer substrates and was twice as rapid at 37 degrees C in TAGGGGCCCCTA compared with TACGCGCGCGTA. However, at 15 degrees C, the relative rates were reversed since TACGCGCGCGTA was repaired at the same rate as at 37 degrees C, whereas the repair of TAGGGGCCCCTA was much slower at 15 degrees C. The repair of TAGGGGCCCCTA at 37 degrees C was also much faster than the repair of TAGAGATCTCTA and was slightly more rapid than repair of TATGTGCACATA. Ligation of the dodecamer substrates to form 24mers or 36mers slightly reduced the initial rates of repair but did not abolish these differences. These results indicate that under physiological conditions, the Fpg protein is more active against adducts in guanine-rich regions and such regions may be the most likely sites of adduct formation at the N7-position of guanine which can then give rise to derivatives attacked by this enzyme.

Attack at coordinated carbon monoxide in a set of dihydrido triosmium carbonyl clusters

Abstract Methoxide reacts with triosmium carbonyl clusters of formula (μ-H) 2 Os 3 (CO) 9 (μ 3 ,η 3 -O 3 ER) (E = S, R = O ( 1 ); E = P, R = OH ( 2 ), Ph ( 3 ); E = As, R = Ph ( 4 )) at a coordinated CO ligand to form methoxycarbonyl adducts. For the sulfate ( 1 ), phenylphosphonate ( 3 ), and phenylarsonate ( 4 ) compounds one equivalent of base is consumed to give anionic clusters of formula [(μ-H) 2 Os 3 (CO) 8 (C(O)OMe)(μ 3 ,η 3 -O 3 ER] − . For the hydrogen phosphate cluster ( 2 ) two equivalents of base are consumed to give the dianion [(μ-H) 2 Os 3 (CO) 8 (C(O)OMe)(μ 3 ,η 3 -O 3 PO)] 2- . The salts of two adducts, [ n Bu 4 N][(μ-H) 2 Os 3 (CO) 8 (C(O)OMe)(μ 3 ,η 3 -O 3 SO)] ( 5 ) and [PPN][(μ-H) 2 Os 3 (CO) 8 (C(O)OMe)(μ 3 ,η 3 -O 3 PPh)] ( 6 ), were isolated. A proposed site of adduct formation, based on 13 C NMR spectra, is presented.

Effect of the diaminocyclohexane carrier ligand on platinum adduct formation, repair, and lethality

Platinum compounds with the diaminocyclohexane (dach) carrier ligand are of particular interest because cell lines that have developed resistance to platinum compounds in general often retain sensitivity to dach-platinum compounds, suggesting that the dach carrier ligand affects the formation, repair, or lethality of platinum-DNA adducts. The effect of the dach ligand on platinum adduct formation was assessed by using the (HaeIII-HindIII)146 fragment of pBR322 treated to give equal amounts of dach- or ethylene-diamine-platinum adducts. The sites of adduct formation were mapped by digestion with Escherichia coli ABC excinuclease. There were no significant effects of the dach carrier ligand on the types or sites of platinum adduct formation. The effect of the dach ligand on platinum adduct repair was determined by using synthetic oligomers designed to have single, specific platinum adducts (G monoadduct; GG, AG, or GNG diadduct) with either the dach or ethylenediamine (en) carrier ligand. These adducts differed significantly in their ability to serve as substrates for ABC excinuclease with GNG greater than or equal to G greater than AG greater than GG. The dach carrier ligand had little effect on the recognition of AG and GG adducts by ABC excinuclease, but significantly improved the ability of ABC excinuclease to excise G monoadducts and GNG diadducts. These data suggest that if the carrier ligand has any effect on the repair of platinum adducts, it is more likely to exert that effect on the repair of platinum monoadducts or GNG diadducts rather than on the more abundant AG or GG diadducts. [14C]Thiourea incorporation was used to quantitate the rate of monoadduct to diadduct conversion.(ABSTRACT TRUNCATED AT 250 WORDS)