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Biomedical subjects

D C Liebler

Publications and source records attributed to D C Liebler.

At least 19 recordsLinked to original sources
PubMed2003

Induction of redox instability of bovine myoglobin by adduction with 4-hydroxy-2-nonenal.

The redox stability of myoglobin (Mb) is compromised by many factors, including lipid oxidation and its products. 4-Hydroxy-2-nonenal (HNE) is an alpha,beta-unsaturated aldehyde derived from the oxidation of omega-6 polyunsaturated fatty acids and is highly reactive and cytotoxic. Our objective was to study potential binding of HNE to Mb and determine how it affects redox stability. OxyMb (0.15 mM) was incubated with HNE (1 mM) at 4, 25, and 37 degrees C at pH 7.4 or 5.6. Samples were analyzed for MetMb formation and by Western blot analyses, LC-MS, LC-MS-MS, circular dichroism (CD), and differential scanning calorimetry (DSC). MetMb formation increased with increasing temperature and was greater at pH 5.6 than at pH 7.4 (P < 0.05). At 37 degrees C, HNE accelerated oxidation at pH 7.4 but not at pH 5.6 (P < 0.05). At both 25 and 4 degrees C, HNE accelerated oxidation at pH 7.4 and 5.6 (P < 0.05). LC-MS revealed the covalent binding of HNE to Mb at both pH values via Michael addition, while Western blot analysis indicated that HNE was bound to histidine (HIS) residues. LC-MS-MS identified six histidine residues of Mb that were readily adducted by HNE, including the proximal (HIS 93) and distal (HIS 64) histidine associated with the heme group. Secondary structure differences between control Mb and Mb incubated with HNE were not detected by CD. However, DSC revealed a decreased T(m) for Mb reacted with HNE at pH 7.4, indicating Mb tertiary structure was altered in a manner consistent with destabilization. These results suggest that HNE accelerates bovine skeletal muscle OxyMb oxidation in vitro by covalent modification at histidine residues.

Aldehydes↗
PubMed2001

SALSA: a pattern recognition algorithm to detect electrophile-adducted peptides by automated evaluation of CID spectra in LC-MS-MS analyses.

A pattern recognition algorithm called SALSA (scoring algorithm for spectral analysis) has been developed to rapidly screen large numbers of peptide MS-MS spectra for fragmentation characteristics indicative of specific peptide modifications. The algorithm facilitates sensitive and specific detection of modified peptides at low abundance in an enzymatic protein digest. SALSA can simultaneously score multiple user-specified search criteria, including product ions, neutral losses, charged losses, and ion pairs that are diagnostic of specific peptide modifications. Application of SALSA to the detection of peptide adducts of the electrophiles dehydromonocrotaline, benzoquinone, and iodoacetic acid permitted their detection in a complex tryptic peptide digest mixture. SALSA provides superior detection of adducted peptides compared to conventional tandem MS precursor ion or neutral loss scans.

Algorithms↗
PubMed2001

Quantitative analysis by liquid chromatography-tandem mass spectrometry of deuterium-labeled and unlabeled vitamin E in biological samples.

A method for quantification of unlabeled alpha-tocopherol and the deuterated tocopherols, RRR-alpha-5-(CD(3))-tocopherol (d(3)RRR) and all rac-alpha-5,7-(CD(3))(2) tocopherol (d(6)all-rac) in plasma by HPLC-tandem mass spectrometry (LC-MS/MS) has been developed. Deuterated and unlabeled alpha-tocopherols were separated by HPLC and were detected by positive ion multiple-reaction monitoring using a triple-quadrupole mass spectrometer equipped with a heated nebulizer-atmospheric pressure chemical ionization interface, following routine extraction of vitamin E from plasma. The accuracy and precision were evaluated by replicate analysis of standards and samples. Human plasma samples, which were obtained at different times after the subject had consumed a capsule containing 1:1 ratio of d(3)RRR and d(6)all rac-alpha-tocopheryl acetates, were analyzed with this method. Plasma deuterated alpha-tocopherols measured by LC-MS/MS followed the same pattern as previously demonstrated by GC-MS measurement, without requiring an extra derivitization step. The detection limit was 10 pmol for each form of alpha-tocopherol injected.

Animals↗
PubMed2001

Mechanisms of dimer and trimer formation from ultraviolet-irradiated alpha-tocopherol.

Alpha-Tocopherol (alpha-TH) undergoes ultraviolet (UV)-induced photooxidation on the surface of mouse skin to produce a dihydroxydimer, a spirodimer, and trimers as the major products. To study the photochemistry involved, we UV-irradiated alpha-TH in a thin film on a glass petri dish. Photooxidation yielded a mixture of dihydroxydimer, spirodimer, and trimers. In the time-course studies, the dihydroxydimer accumulated and then was further oxidized, whereas the spirodimer and trimers accumulated more gradually. Reaction of two tocopheroxyl radicals forms the dihydroxydimer, whereas the spirodimer may be formed either by photooxidation of alpha-TH to an orthoquinone methide (o-QM) followed by a Diels-Alder reaction or by photooxidation of alpha-TH to the dihydroxydimer, followed by two-electron oxidation. Irradiation of a mixture of d10-labeled and unlabeled (d0) dihydroxydimer produced a mixture of labeled and unlabeled spirodimers as detected by positive atmospheric pressure chemical ionization-mass spectrometry. The absence of mixed label spirodimers among products indicated that direct oxidation of the dihydroxydimer is a facile route to the spirodimer and is probably the major spirodimer-forming reaction in alpha-TH photooxidations. Trimer formation from the dihydroxydimer and the spirodimer was observed, however, and requires an o-QM intermediate. Photooxidation of dl0-labeled and unlabeled (d0) dihydroxydimers yielded mixed isotopomers of the trimer products, thus demonstrating that the dihydroxydimer and spirodimers underwent conversion to o-QM intermediates. Photochemical conversion of alpha-TH to UV-absorbing dimer and trimer products may contribute to photoprotection by topically applied alpha-TH.

Chemical Phenomena↗
PubMed2001

Human monomethylarsonic acid (MMA(V)) reductase is a member of the glutathione-S-transferase superfamily.

The drinking of water containing large amounts of inorganic arsenic is a worldwide major public health problem because of arsenic carcinogenicity. Yet an understanding of the specific mechanism(s) of inorganic arsenic toxicity has been elusive. We have now partially purified the rate-limiting enzyme of inorganic arsenic metabolism, human liver MMA(V) reductase, using ion exchange, molecular exclusion, and hydroxyapatite chromatography. When SDS-beta-mercaptoethanol-PAGE was performed on the most purified fraction, seven protein bands were obtained. Each band was excised from the gel, sequenced by LC-MS/MS and identified according to the SWISS-PROT and TrEMBL Protein Sequence databases. Human liver MMA(V) reductase is 100% identical, over 92% of sequence that we analyzed, with the recently discovered human glutathione-S-transferase Omega class hGSTO 1-1. Recombinant human GSTO1-1 had MMA(V) reductase activity with K(m) and V(max) values comparable to those of human liver MMA(V) reductase. The partially purified human liver MMA(V) reductase had glutathione S-transferase (GST) activity. MMA(V) reductase activity was competitively inhibited by the GST substrate, 1-chloro 2,4-dinitrobenzene and also by the GST inhibitor, deoxycholate. Western blot analysis of the most purified human liver MMA(V) reductase showed one band when probed with hGSTO1-1 antiserum. We propose that MMA(V) reductase and hGSTO 1-1 are identical proteins.

Amino Acid Sequence↗
PubMed2001

Interactions of beta-carotene and cigarette smoke in human bronchial epithelial cells.

Results from recent intervention trials indicated that supplemental beta-carotene enhances lung cancer incidence and mortality among smokers. It was hypothesized that beta-carotene was exerting its deleterious effects through a prooxidant effect in the smoke-exposed lung. To test this hypothesis we examined the interactions of beta-carotene and cigarette smoke in transformed human bronchial epithelial cells. We studied the effects of beta-carotene supplementation on rates of gas phase smoke-induced lipid peroxidation, membrane damage and depletion of endogenous antioxidants in BEAS-2B cells. Gas phase cigarette smoke caused cellular beta-carotene levels to decrease over time. The oxidation of beta-carotene by smoke generated various oxidation products, including 4-nitro-beta-carotene, beta-apo-carotenals and beta-carotene epoxides. Peroxidation of membrane lipids by gas phase smoke progressed at a slower rate than did oxidation of beta-carotene and incorporation of beta-carotene into the cells did not enhance the overall rate of lipid peroxidation. Additionally, lactate dehydrogenase release during smoke exposure was also unaffected by the presence or absence of beta-carotene in cells. beta-Carotene incorporation in cells was not found to accelerate the rates of alpha-tocopherol and glutathione depletion by cigarette smoke. Our results indicate that beta-carotene is more sensitive than lipids to cigarette smoke oxidation, but that this preferential oxidation of beta-carotene does not lead to a prooxidant effect in human bronchial epithelial cells.

Bronchi↗
PubMed2000

Antioxidant reactions of alpha-tocopherolhydroquinone.

Alpha-tocopherolhydroquinone (TQH2) is a product of alpha-tocopherol oxidation/reduction that exerts antioxidant effects in biological systems. TQH2 inhibited autoxidation of methyl linoleate initiated by peroxyl radicals derived from thermolysis of 2,2'-azobis(2,4-dimethylvaleronitrile) in acetonitrile. TQH2 oxidation yielded alpha-tocopherolquinone (TQ) as a major product and 2,3-epoxy-alpha-tocopherolquinone and 5,6-epoxy-alpha-tocopherolquinone as minor products. Each TQH2 consumed approximately two peroxyl radicals in the course of the oxidation. The data suggest that TQH2 scavenges peroxyl radicals primarily by electron transfer to form TQ and secondarily by addition-elimination to form the epoxyquinones.

Antioxidants↗
PubMed2000

Reactions of Genistein with alkylperoxyl radicals.

Antioxidant actions of the soy isoflavone genistein are believed to contribute to its overall chemopreventive activity. However, the mechanisms of its antioxidant reactions remain unknown. The objective of this study was to characterize the reaction products of genistein (5,7,4'-trihydroxyisoflavone) with peroxyl radicals generated by thermolysis of 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN). Genistein oxidations with AMVN-derived peroxyl radicals yielded orobol (5,7,3',4'-tetrahydroxyisoflavone), a hydroxylated derivative of genistein, and several stable adducts of 4'-oxogenistein with AMVN-derived radicals. Some of these adducts include novel structures resulting from secondary oxidations of the AMVN-derived moiety. For all the observed oxidation products, the modifications occurred on the B-ring of the molecule. Genistein oxidation product structures provide potentially useful markers of genistein antioxidant chemistry.

Acetonitriles↗
PubMed2000

Characterization of benzoquinone-peptide adducts by electrospray mass spectrometry.

Benzoquinone adducts were prepared with model peptides to identify characteristic features of adduct fragmentation in tandem mass spectrometry (MS) experiments. Model peptides contained cysteine and had a molecular mass of less than 2 kDa to facilitate peptide fragmentation in tandem MS analyses. Peptides were adducted with an excess of benzoquinone, and the adducts were analyzed by LC/MS. Adducts were identified by addition of 108 Da to the monoisotopic mass of the peptide, except in the case of oxytocin, which formed a bis adduct with addition of 216 Da. Tandem MS experiments were performed on the [M + 2H](2+) ions and/or the [M + H](+) ions. Sequence information obtained from modified peptides was comparable to that of their unmodified counterparts. A unique ion pair separated by 141 or 142 Da corresponding to beta-elimination of benzoquinol-S or benzoquinol-SH from a b(n) or y(n) series ion indicated attachment at the sulfur of the cysteine residue. An alternate ion pair of 211 Da corresponded to fragmentation at the peptide bond on either side of the adducted cysteine. Enzymatic digestion of BSA and a 2560 Da frog peptide with trypsin yielded tryptic peptides, which were treated with benzoquinone. In addition to ion pairs of 142 and 211 Da, singly and doubly charged tryptic peptide adducts showed a neutral loss of 142 Da from the precursor. Either one or both ion pairs were present in more than half of all the peptides that were examined. The neutral loss of 142 Da was present in all singly charged tryptic peptide adducts and in 11 out of 14 doubly charged tryptic peptide adducts. The data indicate that reliable detection of benzoquinone-cysteinyl peptide adducts requires monitoring of multiple spectral characteristics.

Amino Acid Sequence↗
PubMed2000

Antioxidant chemistry of green tea catechins. New oxidation products of (-)-epigallocatechin gallate and (-)-epigallocatechin from their reactions with peroxyl radicals.

The green tea catechins (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) react with peroxyl radicals generated by thermolysis of the azo initiator 2,2'-azobis(2, 4-dimethylvaleronitrile) (AMVN) to produce several oxidation products. Structure elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. We isolated and identified a previously unreported reaction product of EGCG and three reaction products of EGC. In the EGCG product, the B-ring was transformed into a ring-opened unsaturated dicarboxylic acid moiety. The EGC products include a seven-membered B-ring anhydride and a symmetrical EGC dimer, both analogues of previously described EGCG oxidation products. The third EGC product was an unsymmetrical dimer. In all identified products, changes occurred solely in the B-ring of EGCG or EGC. This confirmed our previous observation that the principal site of antioxidant reactions in EGCG and EGC is the trihydroxyphenyl B-ring, regardless of the presence of a 3-galloyl moiety. A stoichiometric factor n of 4.16 +/- 0.51 was measured for EGCG, whereas factors of 2.20 +/- 0.26 was found for EGC and 2.33 +/- 0.18 measured for methyl gallate. These values represent the net peroxyl radical trapping per catechin molecule by several competing reactions. EGCG and EGC oxidation involves addition of oxygen, which is not derived from water, but most likely from atmospheric oxygen via peroxyl radicals. Characteristic oxidation products may be useful markers for antioxidant actions in living systems.

Anticarcinogenic Agents↗
PubMed2000

Tandem MS analysis of model peptide adducts from reactive metabolites of the hepatotoxin 1,1-dichloroethylene.

Dichloroethylene (DCE) is a hepatotoxin that undergoes cytochrome P450-catalyzed bioactivation in hepatocytes to form 2-chloroacetyl chloride and 1,1-dichloroethylene oxide. 2-Chloroacetyl chloride reacts with nucleophilic residues and with N-terminal amines to produce 2-chloroacetylated residues and with glutathione to form the reactive electrophile S-(2-chloroacetyl)glutathione (ClCH(2)COSG), which, in turn, is capable of sulfhydryl alkylation. 1,1-DCE oxide can bind to cysteine sulfhydryl groups and subsequently hydrolyze to form an S-carboxymethylated cysteine residue. S-Carboxymethylated, 2-chloroacetylated, and GSCOCH(2)-S-Cys-peptide adducts of model cysteine-containing peptides were synthesized, and their fragmentation patterns were characterized by electrospray tandem mass spectrometry. Synthesis of GSCOCH(2)-S-Cys-peptide adducts was achieved via a novel tert-butoxycarbonyl (tBOC) derivative of ClCH(2)COSG. CID of GSCOCH(2)-S-Cys-peptide adducts resulted in product ions and neutral losses indicative of the GSCOCH(2)-S-Cys moiety as well fragment ion pairs in the b- and y-ion series corresponding to the modified cysteine residue. S-Carboxymethylated peptides exhibited only a characteristic b- or y-series ion pair separated by 161 Da, corresponding to cysteine + CH(2)COOH. CID of 2-chloroacetylated peptides showed neutral losses of 36 (HCl), 78 (HCOCH(2)Cl), 96 (HCOCH(2)Cl + H(2)O), and 114 Da (HCOCH(2)Cl + 2H(2)O). Combinations of characteristic fragment ions, neutral losses, and ion pairs thus are characteristic for DCE-derived adducts. These features can be used in an MS/MS data reduction algorithm for the selective identification of protein targets of DCE metabolites.

Dichloroethylenes↗
PubMed2000

Effects of UV light and tumor promoters on endogenous vitamin E status in mouse skin.

Recent reports indicate that both orally administered and topically applied alpha-tocopherol (vitamin E, TH) prevent UVB-induced skin carcinogenesis in mice. Because UVB exposure causes the formation of oxidants associated with tumor promotion, epidermal TH status may be an important determinant of susceptibility to photocarcinogenesis. To test this hypothesis, we studied the status of epidermal TH in C3H mice following exposure to single and repeated UVB exposures at doses typical of chronic photocarcinogenesis protocols. Exposure of mice to a single 13 kJ/m(2) dose over 60 min resulted in no acute depletion of epidermal TH and a modest increase in TH within 6-12 h. Daily exposure to 6.5 kJ/m(2) over 30 min resulted in a gradual increase in epidermal TH, which reached 5-fold after five daily exposures. The increase in epidermal TH was accompanied by an increase in the TH oxidation products alpha-tocopherolquinone (TQ) and alpha-tocopherolhydroquinone (THQ). We also studied the effect of the prooxidant chemical tumor promoter benzoyl peroxide and the prooxidant azo initiators azobis(amidinopropane HCl) and azobis(2, 4-dimethylvaleronitrile). Topical application of these prooxidant chemicals acutely oxidized epidermal TH to TQ and THQ. Topical treatments with the phorbol ester tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) increased epidermal TH levels without producing a significant accumulation of TH oxidation products. The results indicate that UVB and tumor promoting chemicals all exert qualitatively different effects on epidermal TH status and that UVB and TPA trigger an adaptive response involving epidermal TH accumulation.

Amidines↗
PubMed1999

Prevention of DNA photodamage by vitamin E compounds and sunscreens: roles of ultraviolet absorbance and cellular uptake.

Topical application of alpha-tocopherol (alphaTH), the most prominent naturally occurring form of vitamin E, inhibits ultraviolet (UV) B-induced photocarcinogenesis and DNA photodamage in C3H mice in vivo. In this study, we compared alphaTH with other vitamin E compounds and with three commercial sunscreen compounds for their ability to inhibit DNA photodamage in C3H mouse skin in vivo. When applied in a 5% dispersion in a neutral cream vehicle, alpha-tocopherol (alphaTH), gamma-tocopherol (gammaTH), and delta-tocopherol (deltaTH) each produced a statistically significant inhibition of thymine dimer formation, whereas alpha-tocopherol acetate (alphaTAc) and alpha-tocopherol methyl ether (alphaTOMe) did not. Application of 5% dispersions of the commercial sunscreen agent octylmethoxycinnamate also inhibited dimer formation, whereas ethylhexyl salicylate and oxybenzone did not, despite their considerably greater UVB absorbances than alphaTH. To test the hypothesis that cellular uptake and distribution are necessary for optimal photoprotection by tocopherols, photoprotection was studied in mouse 308 keratinocyte cells in vitro. Preincubation of 308 cells with 1 microM alphaTH for at least 2 h before exposure to 2.5 J/m2/s UVB for 10 min significantly (P < 0.05) attenuated thymine dimer formation. Pre-incubation with 1 microM gammaTH, deltaTH, alphaTAc, or alphaTOMe for 2 h did not inhibit thymine dimer formation significantly. Uptake of alphaTH was measured after incubation with 1 microM [2H3]alphaTH (d3-alphaTH) and resulted in a time-dependent increase in alphaTH levels. Use of d3-alphaTH allowed separate, simultaneous measurement of added d3-alphaTH and unlabeled endogenous alphaTH by gas chromatography-mass spectrometry. Accumulation of 167 +/- 62 pmol d3-alphaTH/mg protein was measured within 1 h in whole-cell fractions. d3-AlphaTH in the nuclear fraction reached levels of 15 +/- 4 pmol d3-alphaTH/mg protein at 2 h. Accumulation of alphaTH in the whole cell and nuclei corresponded temporally with significant protection against DNA photodamage. The kinetics of accumulation of the three tocopherols in whole cells and in nuclei were similar. Although only alphaTH conferred significant protection compared with irradiated controls at 2 h, the differences between individual tocopherols were not statistically significant. This work suggests that incorporation of tocopherol compounds into sunscreen products confers protection against procarcinogenic DNA photodamage and that cellular uptake and distribution of tocopherol compounds is necessary for their optimal photoprotection.

Administration, Cutaneous↗
PubMed1999

alpha-tocopherol oxidation in beef and in bovine muscle microsomes.

The oxidation of alpha-tocopherol (TH) in beef was analyzed using a stable isotope dilution capillary gas chromatography-mass spectrometry assay. TH decreased while alpha-tocopherolquinone (TQ) and 2,3-epoxy-alpha-tocopherolquinone (TQE(2)) increased in ground longissimus lumborum (LL) and psoas major (PM) muscles during storage (P < 0.10). In LL steaks, the relative concentrations of TH decreased and TQ and TQE(2) increased in surface samples; changes were less dramatic in deep samples. Deuterated alpha-tocopherolhydroquinone (THQ) standard was not recovered and endogenous THQ was not detected in meat; THQ was measurable in microsomes isolated from PM and incubated in the presence of 2, 2'-azobis(2-amidopropane)HCl (ABAP) or myoglobin. ABAP-challenged microsomes yielded a tocopherol product profile which favored 5, 6-epoxy-alpha-tocopherolquinone (TQE(1)) and TQE(2), while the use of myoglobin as prooxidant resulted in a higher proportion of TQ and THQ. Results demonstrated that concentrations of TH decreased and TQ and TQE(2) increased in meat during storage and are consistent with the peroxy-radical scavenging function of tocopherol.

Animals↗