A Validated Liquid Chromatography/tandem Mass Spectrometry Assay for Cis-amminedichloro(2-methylpyridine)platinum(II) in Human Plasma Ultrafiltrate

Analytical Chemistry. Feb, 2002  |  Pubmed ID: 11838680

The clinical use of platinum drugs as anticancer agents has encountered problems when relating pharmacokinetic profiles with efficacy and toxicity is attempted. This has been mainly due to the lack of specific and sensitive analytical methodology to examine concentrations of the unbound drug in plasma. The presence of a carbocyclic ring on the new drug, cis-amminedichloro(2-methylpyridine)platinum(II) (ZD0473) suggested that it would be possible to develop the first stable isotope dilution LC/MS assay for a platinum drug in human plasma ultrafiltrate samples. The dichloro form of the drug exists in equilibrium with at least two aquated forms in plasma. The molecular form of the drug, therefore, depends on the length of time that the plasma sample is maintained at room temperature before freezing. Therefore, we have developed a method that quantitatively converts the aquated species back to the dichloro form of the parent drug so that a single molecular species can be analyzed. Selected reaction monitoring was performed on the transition of m/z 393 [M + NH4]+ to m/z 304 [M + NH4 -NH3 - 2 x HCl]- for ZD0473, and m/z 400 [M + NH4]+ to m/z 310 [M + NH4 - NH3 - HCl - 2HCl]+ for [2H7]ZD0473. The standard curves were fitted to a quadratic regression over the range from 10 to 5000 ng/mL in human plasma ultrafiltrate. The lower limit of quantitation for ZD0473 was 10 ng/mL for 100 microL of plasma ultrafiltrate. This simple, rapid, reliable, and sensitive method of quantitation had excellent accuracy and precision. The method provided adequate sensitivity for the analysis of plasma ultrafiltrate samples from a phase II study in which ZD0473 was administered to patients as an intravenous infusion at a dose of 150 mg/m2.

4,5-Epoxy-2(E)-decenal-induced Formation of 1,N(6)-etheno-2'-deoxyadenosine and 1,N(2)-etheno-2'-deoxyguanosine Adducts

Chemical Research in Toxicology. Mar, 2002  |  Pubmed ID: 11896675

trans-4,5-Epoxy-2(E)-decenal reacted with 2'-deoxyadenosine to give 1,N(6)-etheno-2'-deoxyadenosine as well as other 2'-deoxyadenosine adducts. It also reacted with 2'-deoxyguanosine to give 1,N(2)-etheno-2'-deoxyguanosine and other 2'-deoxyguanosine adducts. Synthetic trans-4,5-epoxy-2(E)-decenal was quite stable under the reaction conditions that were used. It was not contaminated with 2,3-epoxyoctanal, a potential precursor to the formation of unsubstituted etheno adducts. Furthermore, using a sensitive LC/MS assay, it was possible to show that no 2,3-epoxyoctanal was formed during prolonged incubations of trans-4,5-epoxy-2(E)-decenal. Therefore, trans-4,5-epoxy-2(E)-decenal, a primary product of lipid peroxidation, is a precursor to the formation of 1,N(6)-etheno-2'-deoxyadenosine and 1,N(2)-etheno-2'-deoxyguanosine. There is no need for an additional oxidation step such as would be required if trans,trans-2,4-decadienal or 4-hydroxy-2-nonenal were the lipid hydroperoxide decomposition products that initiated the formation of unsubstituted etheno adducts. These findings provide an important link between a primary product of lipid peroxidation and a mutagenic DNA lesion that has been detected in human tissues.

Activation of Polycyclic Aromatic Hydrocarbon Trans-dihydrodiol Proximate Carcinogens by Human Aldo-keto Reductase (AKR1C) Enzymes and Their Functional Overexpression in Human Lung Carcinoma (A549) Cells

The Journal of Biological Chemistry. Jul, 2002  |  Pubmed ID: 11978787

Polycyclic aromatic hydrocarbons (PAH) are environmental pollutants and suspected human lung carcinogens. In patients with non-small cell lung carcinoma, differential display shows that aldo-keto reductase (AKR1C) transcripts are dramatically overexpressed. However, whether AKR1C isoforms contribute to the carcinogenic process and oxidize potent PAH trans-dihydrodiols (proximate carcinogens) to reactive and redox active o-quinones is unknown; nor is it known whether these reactions occur in human lungs. We now show that four homogeneous human recombinant aldo-keto reductases (AKR1C1-AKR1C4) are regioselective and oxidize only the relevant non-K region trans-dihydrodiols. However, these enzymes are not stereo-selective, since they oxidized 100% of these racemic substrates. The highest utilization ratios (V(max)/K(m)) were observed for some of the most potent proximate carcinogens known (e.g. 7,12-dimethylbenz[a]anthracene-3,4-diol (DMBA-3,4-diol) and benzo[g]chrysene-11,12-diol). In vitro, DMBA-3,4-diol was oxidized by AKR1C4 to the highly reactive 7,12-dimethylbenz[a]anthracene-3,4-dione (DMBA-3,4-dione), which was trapped in situ as its mono- and bis-thioether conjugates, which arise from the sequential 1,6- and 1,4-Michael addition of thiol nucleophiles. Human multiple tissue expression array analysis showed that AKR1C isoform transcripts were highly expressed in the human lung carcinoma cell line A549. Isoform-specific reverse transcriptase-PCR showed that AKR1C1, AKR1C2, and AKR1C3 transcripts were all expressed. Western blot analysis and functional assays confirmed high expression of AKR1C protein and enzyme activity in these lung cells. A549 cell lysates were found to convert DMBA-3,4-diol to the corresponding o-quinone. In trapping experiments, LC/MS analysis identified peaks in the cell lysates that corresponded to the synthetically prepared mono- and bis-thioether conjugates of DMBA-3,4-dione. This quinone is one of the most electrophilic and redox-active o-quinones produced by AKRs. Its unique ability to form bis-thioether conjugates parallels the formation of bis- and tris-glutathionyl conjugates of hydroquinone, which display end organ toxicity. The ability to measure DMBA-3,4-dione formation in A549 cells implicates the AKR pathway in the metabolic activation of PAH in human lung.

Analysis of Anticancer Drugs and Their Metabolites by Mass Spectrometry

Current Drug Metabolism. Oct, 2002  |  Pubmed ID: 12369893

There is an increasing awareness that the metabolites of anticancer drugs can contribute to the pharmacodynamic effects that are observed, which has stimulated a much greater emphasis on metabolic and pharmacokinetic issues. This has coincided with the development of electrospray and related atmospheric pressure ionization mass spectrometry techniques such as ionspray (nebulizer assisted electrospray), turboionspray (heated nebulizer assisted electrospray) and atmospheric pressure chemical ionization (nebulization coupled with corona discharge). The combination of collision induced dissociation and tandem mass spectrometry coupled with a soft ionization process that produces abundant molecular species provides very powerful methodology for the trace analysis of drugs and their metabolites. The present review has emphasized the more rigorous quantitative applications that have appeared in the literature over the last five years. It is evident that modern techniques of liquid chromatography tandem mass spectrometry coupled with stable isotope dilution methodology have had a profound effect on our ability to analyze anticancer drugs and their metabolites. As new drugs emerge into the clinic, this methodology will clearly be the method of choice, particularly when many samples have to be analyzed over a short time. This approach was beautifully demonstrated in the study of the novel signal transduction inhibitor, Gleevec where thousands of clinical samples were analyzed for drug and metabolites over a relatively short period of time. The need to analyze anticancer drugs and their metabolites with such prompt turn around times has stimulated even more rapid approaches to analysis using robotic-based purification methodology and short LC chromatographic run times.

A Human Melanoma Xenograft in a Nude Rat Responds to Isolated Limb Perfusion with TNF Plus Melphalan

Surgery. Apr, 2003  |  Pubmed ID: 12717360

Isolated limb perfusion (ILP) with tumor necrosis factor-alpha (TNF) and melphalan for advanced extremity malignancies achieves significant complete response rates. To study molecular mechanisms underlying this response, a nude rat ILP model with a human melanoma xenograft was developed.

Characterization of 2'-deoxycytidine Adducts Derived from 4-oxo-2-nonenal, a Novel Lipid Peroxidation Product

Chemical Research in Toxicology. Jul, 2003  |  Pubmed ID: 12870892

Analysis of the reaction between 2'-deoxycytidine and 4-oxo-2-nonenal by LC/MS revealed the presence of three major products (adducts A(1), A(2), and B; [M + H](+) = 364). Adducts A(1) and A(2) were isomeric, and each dehydrated to form adduct B. The structure of adduct B was shown by LC/MS and NMR spectroscopy to be an etheno-2'-deoxycytidine adduct 1' '-[1-(2'-deoxy-beta-d-erythro-pentofuranosyl)-1H-imidazo[2,1-c]pyrimidin-2-oxo-4-yl]heptane-2' '-one. A time course experiment performed at 65 degrees C (pH 5-8) showed that the transformation of both A(1) and A(2) was pH-dependent. In acidic conditions, adducts A(1) and A(2) dehydrated primarily to adduct B. In contrast, in basic conditions, adducts A(1) and A(2) hydrolyzed primarily to dCyd. The data are consistent with adducts A(1) and A(2) being substituted ethano adducts that dehydrate to adduct B, a substituted 3,N(4)-etheno-2'-deoxycytidine adduct.

A Novel Lipid Hydroperoxide-derived Cyclic Covalent Modification to Histone H4

The Journal of Biological Chemistry. Oct, 2003  |  Pubmed ID: 12930824

Previous studies have established that 4-hydroxy-2-nonenal is a lipid hydroperoxide-derived aldehydic bifunctional electrophile that reacts with DNA and proteins. However, it has now been recognized that 4-oxo-2-nonenal is also a major product of lipid hydroperoxide decomposition. Furthermore, 4-oxo-2-nonenal is more reactive than 4-hydroxy-2-nonenal toward the DNA-bases 2'-deoxyguanosine, 2'-deoxyadenosine, and 2'-deoxycytidine and proteins. The formation of 4-oxo-2-nonenal can be induced through vitamin C-mediated or transition metal ion-mediated homolytic decomposition of polyunsaturated omega-3 lipid hydroperoxides such as 13(S)-hydroperoxyoctadecadienoic acid. We have discovered that synthetic 4-oxo-nonenal or 4-oxo-2-nonenal-generated from 13(S)-hydroperoxyoctadecadienoic acid recognizes the specific amino acid motifs of His75, Ala76, and Lys77 in bovine histone H4. Reaction of the histidine and lysine residues with 4-oxo-2-nonenal results in the formation of a novel cyclic structure within the protein. The cyclic structure incorporates the histidine imidazole ring and a newly formed pyrrole derived from the lysine. The cyclic imidazole-pyrrole derivative that is formed from the small Nalpha-acetyl-His-Ala-Lys peptide exists as a mixture of two atropisomers that inter-convert upon heating. Such lipid hydroperoxide-derived modifications could potentially modulate transcriptional activation in vivo. Furthermore, the ability to synthesize cyclic peptides using 4-oxo-2-nonenal will facilitate the preparation of novel structural analogs with potential biological activity.

Targeted Lipidomics Using Electron Capture Atmospheric Pressure Chemical Ionization Mass Spectrometry

Rapid Communications in Mass Spectrometry : RCM. 2003  |  Pubmed ID: 14515314

There is an increasing need to be able to conduct quantitative lipidomics analyses as a complement to proteomics studies. The highest specificity for proteomics analysis can be obtained using methodology based on electrospray ionization (ESI) or atmospheric pressure chemical ionization (APCI) coupled with liquid chromatography/tandem mass spectrometry (LC/MS/MS). For lipidomics analysis it is often necessary to be able to separate enantiomers and regioisomers. This can be very challenging when using methodology based on conventional reversed-phase chromatography. Normal-phase chromatography using chiral columns can provide dramatic improvements in the resolution of enantiomers and regioisomers. However, conventional ESI- and APCI-MS/MS has limited sensitivity, which makes it difficult to conduct studies in cell culture systems where only trace amounts of non-esterified bioactive lipids are present. The use of electron capture APCI-MS/MS overcomes this problem. Enantiomers and regioisomers of diverse bioactive lipids can be quantified using stable isotope dilution methodology coupled with normal-phase chiral chromatography and electron capture APCI-MS/MS. This methodology has allowed a lipidomics profile from rat epithelial cells maintained in culture to be delineated and allowed the effect of a non-selective lipoxygenase inhibitor to be assessed.

Reciprocal DNA Topoisomerase II Cleavage Events at 5'-TATTA-3' Sequences in MLL and AF-9 Create Homologous Single-stranded Overhangs That Anneal to Form Der(11) and Der(9) Genomic Breakpoint Junctions in Treatment-related AML Without Further Processing

Oncogene. Nov, 2003  |  Pubmed ID: 14627986

Few t(9;11) translocations in DNA topoisomerase II inhibitor-related leukemias have been studied in detail and the DNA damage mechanism remains controversial. We characterized the der(11) and der(9) genomic breakpoint junctions in a case of AML following etoposide and doxorubicin. Etoposide-, etoposide metabolite- and doxorubicin-induced DNA topoisomerase II cleavage was examined in normal homologues of the MLL and AF-9 breakpoint sequences using an in vitro assay. Induction of DNA topoisomerase II cleavage complexes in CEM and K562 cell lines was investigated using an in vivo complex of enzyme assay. The translocation occurred between identical 5'-TATTA-3' sequences in MLL intron 8 and AF-9 intron 5 without the gain or loss of bases. The 5'-TATTA-3' sequences were reciprocally cleaved by DNA topoisomerase II in the presence of etoposide, etoposide catechol or etoposide quinone, creating homologous 4-base 5' overhangs that would anneal to form both breakpoint junctions without any processing. der(11) and der(4) translocation breakpoints in a treatment-related ALL at the same site in MLL are consistent with a damage hotspot. Etoposide and both etoposide metabolites induced DNA topoisomerase II cleavage complexes in the hematopoietic cell lines. These results favor the model in which the chromosomal breakage leading to MLL translocations in DNA topoisomerase II inhibitor-related leukemias is a consequence of DNA topoisomerase II cleavage.

A Novel Lipid Hydroperoxide-derived Modification to Arginine

Chemical Research in Toxicology. Dec, 2003  |  Pubmed ID: 14680374

The guanidine group present in the amino acid arginine was found to react with the lipid hydroperoxide-derived bifunctional electrophile, 4-oxo-2-nonenal. The reaction between N(alpha)-tert-butoxycarbony-l-arginine and 4-oxo-2-nonenal resulted in the formation of an adduct (adduct A) that subsequently dehydrated on heating to adduct B. Liquid chromatography/mass spectrometry and nuclear magnetic resonance spectroscopy were used to assign the structure of adduct B as (N(delta),N(omega)(')-etheno-2'-heptanon-2' '-one)-N(alpha)-t-Boc-arginine. The reaction proceeded from initial reaction of the primary N(omega)-amino group at the C-1 aldehyde of 4-oxo-2-nonenal. Subsequently, an intramolecular Michael addition of a secondary N(delta)-amino group occurring at C-3 resulted in formation of the cyclic carbinolamine adduct A. Dehydration and rearrangement of the exocyclic imine resulted in the formation of adduct B, which contained a stable imidazole ring. The tetra peptide LRDE reacted with 4-oxo-2-nonenal primarily at arginine rather than at the amino terminus. This suggests that arginine-containing proteins can react with lipid hydroperoxide-derived 4-oxo-2-nonenal to form a novel imidazole modification.

Plasma Etoposide Catechol Increases in Pediatric Patients Undergoing Multiple-day Chemotherapy with Etoposide

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. May, 2004  |  Pubmed ID: 15131033

The purpose of this research was to determine inter- and intrapatient differences in the pharmacokinetic profiles of etoposide and its genotoxic catechol metabolite during conventional multiple-day dosing of etoposide in pediatric patients.

Kinetics and Regulation of Cytochrome P450-mediated Etoposide Metabolism

Drug Metabolism and Disposition: the Biological Fate of Chemicals. Sep, 2004  |  Pubmed ID: 15319341

Etoposide is a DNA topoisomerase II inhibitor widely used in the treatment of a variety of malignancies that is also associated with therapy-related leukemia. The cytochrome P450 (P450)-derived catechol and quinone metabolites of etoposide may be important in the damage to the MLL (mixed lineage leukemia) gene and other genes resulting in leukemia-associated chromosomal translocations. Kinetic analysis of catechol formation by recombinant P450s was determined using liquid chromatography/selected reaction monitoring/mass spectrometry. CYP3A4 was found to play a major role in etoposide metabolism (K(m) = 77.7 +/- 27.8 microM; V(max) = 314 +/- 84 pmol of catechol/min/nmol of P450). However, CYP3A5 (K(m) = 13. 9 +/- 3.1 microM; V(max) = 19.4 +/- 0.4 pmol of catechol/min/nmol of P450) may be involved in etoposide metabolism at therapeutic concentrations of free drug. Other P450s do not appear to be involved in etoposide catechol formation. Real-time polymerase chain reaction and Western blot analysis revealed significantly increased CYP3A4 mRNA and protein levels in hepatocytes treated with 10 microM rifampicin compared with untreated cells, but only modest effects of rifampicin on CYP3A5 induction. Etoposide (40, 5, 1, and 0.25 microM) caused a slight increase in CYP3A4 mRNA in three of five batches of hepatocytes but did not result in proportionately increased CYP3A4 protein levels. At high concentrations, etoposide induced only a modest increase in CYP3A5 mRNA and protein levels in four of five batches of hepatocytes. Alternatively, coadministration of other drugs with etoposide may account for the increase in etoposide catechol formation during therapy with etoposide.

Viniferin Formation by COX-1: Evidence for Radical Intermediates During Co-oxidation of Resveratrol

Journal of Natural Products. Jan, 2005  |  Pubmed ID: 15679314

Resveratrol (1) is a polyphenolic natural product, which functions as both a mechanism-based inactivator and a co-reductant of the COX-1 peroxidase. These functions are mediated through different moieties on the molecule, namely, the m-hydroquinone moiety (mechanism-based inactivator) and the phenol moiety (co-reductant). Implicit in this bifunctionality is the notion that resveratrol is oxidized at the peroxidase active site of COX-1, resulting in the formation of two hypothetical radical species. Oxidation of the m-hydroquinone moiety can generate a hypothetical m-semiquinone radical, which is unstabilized and leads to irreversible enzyme inactivation. Oxidation of the phenol moiety can generate a hypothetical phenoxy radical, which is stabilized and leads to co-reduction during peroxidase catalysis. These two radicals have been trapped as the resveratrol dimers, cis-epsilon-viniferin (4, trapped m-semiquinone radical) and trans-delta-viniferin (5, trapped phenoxy radical), and identified by liquid chromatography (LC), absorbance spectroscopy, and LC/tandem mass spectrometry (MS(n)) methods. Methoxy-resveratrol analogues, in which either the m-hydroquinone or the phenol moiety were protected as methyl ethers, were used to confirm the proposed mechanism of viniferin production by COX-1.

Liquid Chromatography/mass Spectrometry Analysis of Bifunctional Electrophiles and DNA Adducts from Vitamin C Mediated Decomposition of 15-hydroperoxyeicosatetraenoic Acid

Rapid Communications in Mass Spectrometry : RCM. 2005  |  Pubmed ID: 15723435

Reactive oxygen species convert the omega-6 polyunsaturated fatty acid arachidonic acid into 15-hydroperoxy-5,8,11,13-(Z,Z,ZE)-eicosatetraenoic acid (15-HPETE). Cyclooxygenases and lipoxygenases can also convert arachidonic acid into 15-HPETE. Vitamin C mediated decomposition of 15(S)-HPETE to protein- and DNA-reactive bifunctional electrophiles was examined by normal-phase liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry (LC/APCI-MS). The individual bifunctional electrophiles, trans-4,5-epoxy-2(E)-decenal (t-EDE), cis-4,5-epoxy-2(E)-decenal (c-EDE), 4-oxo-2(E)-nonenal (ONE), and 4-hydroxy-2(E)-nonenal (HNE), exhibited protonated molecules at m/z 169, 169, 155, and 157, respectively. The MH+ ion at m/z 173 for 4-hydroperoxy-2(E)-nonenal (HPNE) was very weak with an ion corresponding to the loss of OH at m/z 156 as the major ion in the APCI mass spectrum. The bifunctional electrophiles were all separated under normal-phase LC conditions. All five bifunctional electrophiles were formed when 15-HPETE was treated with vitamin C. The LC/MS-based methodology showed that t-EDE was the major bifunctional electrophile formed during vitamin C mediated 15(S)-HPETE decomposition. Stable isotope dilution LC/MS studies revealed that this did not result in the formation of increased levels of unsubstituted etheno-dGuo adducts in calf thymus DNA when compared with 13(S)-hydroperoxy-9,10-(Z,E)-octadecadienoic acid [13(S)-HPODE], a lipid hydroperoxide derived from linoleic acid. However, the formation of heptanone-etheno-dGuo adducts in calf thymus DNA was reduced when compared with the 13(S)-HPODE. This was attributed to the reduced formation of ONE from 15-HPETE when compared with its formation from 13-HPODE. In contrast to reactions with dGuo or DNA conducted using 13(S)-HPODE, no carboxy-containing adducts were observed with 15(S)-HPETE.

Analysis of FeII-mediated Decomposition of a Linoleic Acid-derived Lipid Hydroperoxide by Liquid Chromatography/mass Spectrometry

Journal of Mass Spectrometry : JMS. May, 2005  |  Pubmed ID: 15739161

Intracellular Fe(II), which is up-regulated during oxidative stress and during iron overload, induces the formation of a hydroxyl radical by Fenton chemistry. The hydroxyl radical can convert the prototypic omega-6 polyunsaturated fatty acid, linoleic acid, to 13-hydroperoxy-9,11-(Z,E)-octadecadienoic acid (13-HPODE). Cyclooxygenases can also convert linoleic acid to 13(S)-HPODE during oxidative stress. Subsequent Fe(II)-mediated decomposition to protein- and DNA-reactive bifunctional electrophiles was examined by normal-phase liquid chromatography (LC)/atmospheric pressure chemical ionization (APCI)/mass spectrometry. The potential individual bifunctional electrophiles trans-4,5-epoxy-2(E)-decenal (EDE), cis-EDE, 4-oxo-2(E)-nonenal (ONE) and 4-hydroxy-2(E)-nonenal (HNE) exhibited protonated molecular ions at m/z 169, 169, 155 and 157, respectively. The MH(+) ion at m/z 173 for 4-hydroperoxy-2(E)-nonenal (HPNE) was very weak with an ion corresponding to the loss of OH at m/z 156 as the major ion in the APCI mass spectrum. The bifunctional electrophiles were all separated under normal-phase LC conditions. Interestingly, ions corresponding to ONE and HNE were detected at the same retention time as HPNE, suggesting that it decomposed in the source of the mass spectrometer to ONE and HNE. All five bifunctional electrophiles were formed when 13-HPODE was treated with 50 microM Fe(II). At this concentration of Fe(II), the addition of vitamin C resulted in increased bifunctional electrophile formation. At higher concentrations of Fe(II) (500 microM to 2 mM), no HPNE was detected and there was no additive effect of vitamin C. Additional experiments with synthetic HPNE revealed that it was quantitatively converted to a mixture of ONE and HNE by Fe(II). The HNE is thought to arise from a one-electron reduction of an alkoxy radical derived from HPNE. In contrast, ONE can arise through an alpha-cleavage of the HPNE-derived alkoxy radical or by direct dehydration of HPNE.

Dioxododecenoic Acid: a Lipid Hydroperoxide-derived Bifunctional Electrophile Responsible for Etheno DNA Adduct Formation

Chemical Research in Toxicology. Mar, 2005  |  Pubmed ID: 15777096

It has been proposed that 13(S)-hydroperoxy-9Z,11E-octadecadienoic acid [13(S)-HPODE]-mediated formation of 4-oxo-2(E)-nonenal and 4-hydroxy-2(E)-nonenal arises from a Hock rearrangement. This suggested that a 4-oxo-2(E)-nonenal-related molecule, 9,12-dioxo-10(E)-dodecenoic acid (DODE), could also result from the intermediate formation of 9-hydroperoxy-12-oxo-10(E)-dodecenoic acid. A recent report has described the formation of DODE-derived etheno adducts when 13(S)-HPODE was allowed to decompose in the presence of 2'-deoxynucleosides or DNA. However, the regioselectivity of lipid hydroperoxide-derived DODE addition to 2'-deoxyguanosine (dGuo) or other 2'-deoxynucleosides was not determined. The structure of carboxynonanone-etheno-dGuo formed from vitamin C-mediated 13(S)-HPODE decomposition has now been established by a combination of 1H and 13C NMR spectroscopy studies of its bis-methylated derivative. The site of dGuo methylation was first established as being at N-5 rather than at O-9 from NMR analysis of a methyl derivative of the model compound, heptanone-etheno-dGuo. (1)H,(13)C 2D heteronuclear multiple bond correlations were then used to establish unequivocally that the bis-methyl derivative of carboxynonanone-etheno-dGuo was 3-(2'-deoxy-beta-d-erythropentafuranosyl)imidazo-7-(9' '-carboxymethylnona-2' '-one)-9-oxo-5-N-methyl[1,2-a]purine rather than its 6-(9' '-carboxymethylnona-2"-one)-9-oxo-5-N-methyl[1,2-a]purine regioisomer. Therefore, etheno adduct formation occurred by initial nucleophilic attack of the exocyclic N(2) amino group of dGuo at the C-12 aldehyde of DODE to form an unstable carbinolamine intermediate. This was followed by intramolecular Michael addition of the pyrimidine N1 of dGuo to C-11 of the resulting alpha,beta-unsaturated ketone. Subsequent dehydration gave 3-(2'-deoxy-beta-d-erythropentafuranosyl)imidazo-7-(9' '-carboxynona-2' '-one)-9-oxo-[1,2-a]purine (carboxynonanone-etheno-dGuo). An efficient synthesis of DODE was developed starting from readily available 1,8-octanediol using a furan homologation procedure. This synthetic method allowed multigram quantities of DODE to be readily prepared. Synthetic DODE when reacted with dGuo gave carboxynonanone-etheno-dGuo that was identical with that derived from vitamin C-mediated 13(S)-HPODE decomposition in the presence of dGuo.

Unexpected Formation of Etheno-2'-deoxyguanosine Adducts from 5(S)-hydroperoxyeicosatetraenoic Acid: Evidence for a Bis-hydroperoxide Intermediate

Chemical Research in Toxicology. Mar, 2005  |  Pubmed ID: 15777099

Analysis of products from the reaction between 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid and 2'-deoxyguanosine in the presence of FeII, FeIII, or vitamin C by liquid chromatography/atmospheric pressure chemical ionization/mass spectrometry revealed the presence of four DNA adducts. Surprisingly, adducts I and II had mass spectral characteristics identical to those for 1,N2-etheno-2'-deoxyguanosine and heptanone-1,N2-etheno-2'-deoxyguanosine. These adducts have previously been shown to arise from the homolytic decomposition of 13(S)-hydroperoxy-9,11-(Z,E)-octadecadienoic acid. It appears that under the reaction conditions, 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid was subjected to a previously unknown peroxidation reaction to give a bis-hydroperoxide intermediate that underwent a Hock rearrangement to produce 3(Z)-nonenal from the omega-terminus. The 3(Z)-nonenal was then converted to 4-hydroperoxy-2-nonenal, a precursor to the formation of 4-oxo-2-nonenal. 4-Oxo-2-nonenal forms heptanone-1,N2-etheno-adducts with 2'-deoxyguanosine, whereas 4-hydroperoxy-2-nonenal forms 1,N2-etheno-2'-deoxyguanosine. Two novel carboxylate adducts were also identified. The structure of the more abundant adduct (III) was characterized as its methyl ester derivative by NMR spectroscopy as 3-(2'-deoxy-beta-D-erythropentafuranosyl)imidazo-7-(5' '-carboxypenta-2' '-one)-9-oxo[1,2-alpha]purine (5-carboxy-2-pentanone-1,N2-etheno-2'-deoxyguanosine). This etheno adduct was formed by the reaction of 2'-deoxyguanosine with 5,8-dioxo-6(E)-octenoic acid. The bifunctional electrophile is proposed to arise from the alpha-terminus during the Hock rearrangement of bis-hydroperoxide derived from 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid. 5-Carboxy-2-pentanone-1,N2-etheno-2'-deoxyguanosine may serve as a biomarker of 5-lipoxygenase-mediated oxidative stress. The less abundant carboxylate adduct IV arose from a quite different pathway and was tentatively characterized as 6-carboxy-3-hydroxy-1-hexene-1,N2-etheno-2'-deoxyguanosine.

Novel Lipid Hydroperoxide-derived Hemoglobin Histidine Adducts As Biomarkers of Oxidative Stress

Journal of Mass Spectrometry : JMS. Jun, 2005  |  Pubmed ID: 15827957

Hemoglobin (Hb) adducts have long been used as dosimeters of exposure to xenobiotics and endogenously formed reactive metabolites. In this study, hemoglobin chains were separated from each other and their prosthetic heme groups and reacted with 4-oxo-2-nonenal, a major breakdown product of lipid hydroperoxides. The adducts were characterized by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-TOF/MS) analysis of the intact proteins and by a combination of liquid chromatography/electrospray ionization/tandem MS (MS/MS) and MALDI-TOF/MS/MS analysis of the tryptic peptides. Covalent modifications were found on both hemoglobin chains. The location was determined to be on H20 of the alpha-hemoglobin chain and on H(63) of the beta-hemoglobin chain. Molecular modeling revealed that these two residues were two most solvent accessible H residues present in intact Hb. The proposed reaction mechanism is based on that described for the reaction of 4-hydroxy-2-nonenal with proteins. Initial nucleophilic Michael addition is followed by hydration of the resulting aldehyde, cyclization, and two sequential dehydration reactions to give stable furan derivatives. This results in the addition of 136 Da from 4-oxo-2-nonenal to give adducts corresponding to (17)VGAH(.) AGEYGAEALER(31) from alpha-hemoglobin and (62)AH(.) GK(65) from beta-hemoglobin. These hemoglobin modifications can potentially serve as biomarkers of lipid hydroperoxide-mediated macromolecule damage and may reflect an indirect measurement of the potential for DNA damage in vivo.

DNA Topoisomerase II in Therapy-related Acute Promyelocytic Leukemia

The New England Journal of Medicine. Apr, 2005  |  Pubmed ID: 15829534

Chromosomal translocations leading to chimeric oncoproteins are important in leukemogenesis, but how they form is unclear. We studied acute promyelocytic leukemia (APL) with the t(15;17) translocation that developed after treatment of breast or laryngeal cancer with chemotherapeutic agents that poison topoisomerase II.

4-Hydroperoxy-2-nonenal-induced Formation of 1,N2-etheno-2'-deoxyguanosine Adducts

Chemical Research in Toxicology. Apr, 2005  |  Pubmed ID: 15833039

Analysis of the reaction between 4-hydroperoxy-2-nonenal (HPNE) and 2'-deoxyguanosine (dGuo) by liquid chromatography/mass spectrometry (LC/MS) revealed the formation of 1,N2-etheno-dGuo as well as heptanone-etheno-dGuo and trace amounts of dihydroxyheptane-etheno-dGuo. Identities of the dGuo adducts were confirmed by comparison with authentic standards. The minor dihydroxyheptane-etheno-dGuo adducts could be generated from 2,3-epoxy-4-hydroxynonanal (EHN), the epoxidation product of 4-hydroxy-2-nonenal (HNE). An LC/MS method was developed for the analysis of EHN. No EHN was detected by LC/MS during the decomposition of HPNE. Therefore, the dihydroxyheptane-etheno-dGuo adducts are either generated from a direct reaction between HPNE and dGuo or from another intermediate that cannot be detected by LC/MS. In addition, no HNE-derived hydroxypropano-dGuo adducts were observed. On the basis of these findings, we conclude that HPNE, a primary product of lipid peroxidation, is a major precursor to the formation of 1,N2-etheno-dGuo. We propose that it arises from the reaction of dGuo and HPNE through the intermediate formation of a cyclic hydroxy-ethano-epoxide derivative. The minor amounts of heptanone-ethano-dGuo adducts that were formed from HPNE in the absence of vitamin C suggest that heptanone-etheno-dGuo can be generated directly from HPNE without the intermediate formation of ONE. Therefore, HPNE can be considered as another lipid hydroperoxide-derived bifunctional electrophile with the potential for biological activities that are similar to HNE and ONE.

Cyclooxygenase-2-mediated DNA Damage

The Journal of Biological Chemistry. Aug, 2005  |  Pubmed ID: 15964853

Rat intestinal epithelial cells that express the cyclooxygenase-2 (COX-2) gene permanently (RIES cells) were used as a model of in vivo oxidative stress. A targeted lipidomics approach showed that 15(S)-hydroxyeicosatetraenoic acid (15(S)-HETE) was the major hydroxylated non-esterified lipid formed in unstimulated intact cells. The corresponding hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15(S)-HPETE) undergoes homolytic decomposition to the DNA-reactive bifunctional electrophile 4-oxo-2(E)-nonenal, a precursor of heptanone-etheno-2'-deoxyguanosine. This etheno adduct was identified in the DNA of RIES cells. A dose-dependent increase in adduct levels was observed in the presence of vitamin C. This suggested that vitamin C increased lipid hydroperoxide-mediated 4-oxo-2(E)-nonenal formation in the cells. The selective COX-2 inhibitor NS-398 was protective against cellular DNA damage but was less effective if vitamin C was present. Prostaglandin E(2) and 15(S)-HETE biosynthesis were completely inhibited by 110 mum NS-398 in the intact RIES cells. No inhibition of COX-1 was detected in the wild-type RIE cells at this concentration of NS-398. Arachidonic acid treatment of RIES cell lysates and ionophore stimulation of intact RIES cells produced significantly more 15(R)-HETE than the untreated intact cells. These preparations also both produced 11(R)-HETE, which was not detected in the intact cells. Aspirin treatment of the intact unstimulated RIES cells resulted in the exclusive formation of 15(R)-HETE in amounts that were slightly higher than the original 15(S)-HETE observed in the absence of aspirin, implying that significant amounts of 15(R)-HPETE had also been formed. 15(R)-HPETE should give exactly the same amount of heptanone-etheno-2'-deoxyguanosine as its 15(S)-enantiomer. However, no increase in heptanone-etheno adduct formation occurred in the aspirin-treated cells. The present study suggests a potential mechanism of tumorigenesis that involves DNA adduct formation from COX-2-mediated lipid peroxidation rather than prostaglandin formation. Therefore, inhibition of COX-2-mediated lipid hydroperoxide formation offers a potential therapeutic alternative to COX-2 inhibitors in chemoprevention strategies.

Targeted Chiral Lipidomics Analysis

Prostaglandins & Other Lipid Mediators. Sep, 2005  |  Pubmed ID: 16099399

Genomics, transcriptomics, and proteomics are proving to be very useful techniques, which have impacted significantly on our understanding mechanisms of human disease. However, this systems biology approach has several drawbacks than can be overcome by the integration of metabonomics and lipidomics. We have developed a targeted lipidomics approach that makes it possible to directly analyze chiral lipids generated in cellular systems. Bioactive lipids are usually present in trace amounts as enanatiomers and regioisomers that require separation before they can be analyzed by mass spectrometry. Normal phase chiral chromatography is generally used to resolve bioactive lipid enanatiomers. However, conventional electrospray and atmospheric pressure chemical ionization/tandem mass spectrometry have limited sensitivity when normal phase solvents are used, which makes it difficult to conduct studies when only trace amounts of the bioactive lipids are present. The use of electron capture atmospheric pressure chemical ionization/tandem mass spectrometry overcomes this problem. Enantiomers and regioisomers of targeted bioactive lipids can be quantified using stable isotope dilution methodology coupled with normal phase chiral chromatography and electron capture atmospheric chemical ionization/tandem mass spectrometry. A targeted lipidomics profile from rat epithelial cells transfected with cyclooxygenase-2 and maintained in culture was obtained. Inhibition with the non-selective cyclooxygenase inhibitor aspirin increased the formation of 15(R)-hydroxyeicosatetraenoic acid in the cells although it completely inhibited formation of the 15(S)-enantiomer and prostaglandin E2. New mass spectrometry instrumentation with an improved atmospheric pressure chemical ionization source was found to be an order of magnitude more sensitive than existing instruments for analysis of bioactive lipids using electron capture methodology. This type of mass spectrometer will permit a more detailed analysis of cellular bioactive lipid production than has been possible previously. It will also permit in vivo targeted lipidomics studies to be conducted using biological fluids derived from animal models and human subjects.

Effect of Immunoaffinity Depletion of Human Serum During Proteomic Investigations

Journal of Proteome Research. Sep-Oct, 2005  |  Pubmed ID: 16212426

Controversy exists regarding the proper mining of the human serum proteome. Because of the analytical challenges of accurately measuring samples containing a very large dynamic range of protein concentrations, current practices have employed depletion of the highly abundant housekeeping serum proteins, such as albumin and immunoglobins. There is question as to the selectivity of depletion, namely, is there loss of other non abundant serum proteins along with albumin, haptoglobin and other commonly depleted proteins. In this study, human serum was analyzed with and without immunoaffinity depletion of the six most abundant proteins by multidimensional liquid chromatography tandem mass spectrometry. Two replicates of each experiment were conducted and compared against one another. In both depleted and nondepleted replicates there was a 73% and 72% overlap of identified peptides and a 64% and 78% overlap of identified proteins, respectively. Of 262 unique proteins identified in the four experiments, 82 were found in common to all four experiments, 142 unique to the depleted serum, and 38 unique to the nondepleted serum. Although serum depletion of highly abundant proteins significantly increased the number of proteins identified, both the degree of sample complexity and this depletion method resulted in a nonselective loss of other proteins.

Characterization of Proteins in Human Pancreatic Cancer Serum Using Differential Gel Electrophoresis and Tandem Mass Spectrometry

Journal of Proteome Research. Sep-Oct, 2005  |  Pubmed ID: 16212428

The purpose of this study was to develop techniques for identifying cancer biomarkers in human serum using differential in-gel electrophoresis (DIGE), and characterizing the protein biomarkers using tandem mass spectrometry (MS/MS). A major problem in profiling protein expression by DIGE comes from the presence of high concentrations of a small number of proteins. Therefore, serum samples were first chromatographed using an immunoaffinity HPLC column (Agilent Technologies), to selectively remove albumin, immunoglobulins, transferrin, haptoglobin, and antitrypsin. Serum samples from three individuals with pancreatic cancer and three individuals without cancer were compared. Serum samples were processed using the immunoaffinity column. Differential protein analysis was performed using DIGE. A total of 56 protein spot-features were found to be significantly increased and 43 significantly decreased in cancer serum samples. These spot features were excised, trypsin digested, and analyzed by MALDI/TOF/TOF (4700 Proteomics Analyzer, Applied Biosystems). We identified 24 unique proteins that were increased and 17 unique proteins that were decreased in cancer serum samples. Western blot analysis confirmed increased levels of several of these proteins in the pancreatic cancer serum samples. In an independent series of serum samples from 20 patients with pancreatic cancer and 14 controls, increased levels of apolipoprotein E, alpha-1-antichymotrypsin, and inter-alpha-trypsin inhibitor were found to be associated with pancreatic cancer. These results suggest that affinity column enrichment and 2-D DIGE can be used to identify numerous proteins differentially expressed in serum from individuals with pancreatic cancer.

Induction of Endothelial Cell Apoptosis by Lipid Hydroperoxide-derived Bifunctional Electrophiles

Free Radical Biology & Medicine. Nov, 2005  |  Pubmed ID: 16214032

Endothelial dysfunction is considered to be the earliest event in atherogenesis. Oxidative stress, inflammation, and apoptosis play critical roles in its progression and onset. Lipid peroxidation, which occurs during oxidative stress, results in the formation of lipid hydroperoxide-derived bifunctional electrophiles such as 4-hydroxy-2(E)-nonenal that induce apoptosis. In this study, recently identified lipid hydroperoxide-derived bifunctional electrophiles 4-oxo-2(E)-nonenal (ONE; 5-30 microm) and 4,5-epoxy-2(E)-decenal (EDE; 10-20 microM) were shown to cause a dose- and time-dependent apoptosis in EA.hy 926 endothelial cells. This was manifest by morphological changes, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage. Bifunctional electrophiles caused cytochrome c release from mitochondria into the cytosol, implicating a mitochondrial pathway of apoptosis in the endothelial cells. The novel carboxylate-containing lipid hydroperoxide-derived bifunctional electrophile 9,12-dioxo-10(E)-dodecenoic acid was inactive because it could not translocate across the plasma membrane. However, its less polar methyl ester derivative (2-10 microM) was the most potent inducer of apoptosis of any bifunctional electrophile that has been tested. An acute decrease in intracellular glutathione (GSH) preceded the onset of apoptosis in bifunctional electrophile-treated cells. The ability of ONE and EDE to deplete GSH was directly correlated with their predicted reactivity toward nucleophilic amino acids. Liquid chromatography/mass spectrometry methodology was developed in order to examine the intracellular and extracellular concentrations of bifunctional electrophile-derived GSH adducts. Relative intracellular/extracellular ratios of the GSH adducts were identical with the rank order of potency for inducing caspase 3 activation. This suggests that there may be a role for the bifunctional electrophile-derived GSH adducts in the apoptotic response. N-Acetylcysteine rescued bifunctional electrophile-treated cells from apoptosis, whereas the GSH biosynthesis inhibitor d,l-buthionine-(R,S)-sulfoximine sensitized the cells to apoptosis. These data suggest that lipid hydroperoxide-derived bifunctional electrophiles may play an important role in cardiovascular pathology through their ability to induce endothelial cell apoptosis.

Analysis of Protein Expression During Oxidative Stress in Breast Epithelial Cells Using a Stable Isotope Labeled Proteome Internal Standard

Journal of Proteome Research. Nov-Dec, 2005  |  Pubmed ID: 16335945

Normal cells undergo a variety of molecular and physiological changes upon malignant transformation, including their responses to environmental factors that induce oxidative stress. Understanding the molecular pathways regulating these changes would facilitate the development of novel cancer treatments and chemoprevention strategies. Differences in the oxidative stress response were investigated between nonmalignant (S-1) and malignant (T4-2) cell lines (both derived from the HMT-3522 breast epithelial cells) using proteomic approaches. A modification of the stable isotope labeling of amino acids in cell culture (SILAC) approach was employed in which a [(13)C,(15)N]-labeled proteome was prepared from both cells. Relative quantification of the proteome derived from the S-1 cells and the T4-2 cells was then conducted using a [(13)C,(15)N]-labeled proteome as the internal standard. Differentially expressed proteins that changed in a similar manner in both cell lines were mainly stress response proteins, including heat shock proteins, peroxiredoxins, and redox proteins. Proteins that showed significant change in expression level in only one the cell lines included cytoskeleton proteins and proteins implicated in cell cycle and apoptosis regulation. Fortilin was found to be significantly up regulated in the transformed T4-2 cells after H(2)O(2) treatment but not in the parental S-1 cells. However, Ran/TC4 was up regulated by H(2)O(2) in the nonmalignant breast epithelial cells but not in the malignant cells. These results suggest that the malignant T4-2 cells have acquired more resistance to H(2)O(2)-induced apoptosis than the nonmalignant S-1 cells.

Competing Roles of Aldo-keto Reductase 1A1 and Cytochrome P4501B1 in Benzo[a]pyrene-7,8-diol Activation in Human Bronchoalveolar H358 Cells: Role of AKRs in P4501B1 Induction

Chemical Research in Toxicology. Jan, 2006  |  Pubmed ID: 16411658

Benzo[a]pyrene (BP) requires metabolic activation to electrophiles to exert its deleterious effects. We compared the respective roles of aldo-keto reductase 1A1 (AKR1A1, aldehyde reductase) and P4501B1 in the formation of BP-7,8-dione and BP-tetrols, respectively, in intact bronchoalveolar cells manipulated to express either enzyme. Metabolite formation was confirmed by HPLC/MS and quantitatively measured by HPLC/UV/beta-RAM. In TCDD-treated H358 cells (P4501B1 expression), the anti-BPDE hydrolysis product BP-tetrol-1 increased over 3-12 h to a constant level. In H358 AKR1A1 transfectants, formation of BP-7,8-dione was elevated for 3-12 h but significantly decreased after 24 h. Interestingly, BP-tetrols were also detected in AKR1A1 transfectants even though they do not constitutively express P4501A1/P4501B1 enzymes. Northern and Western blotting confirmed the induction of P4501B1 by BP-7,8-dione in parental cells and the induction of P4501B1 by BP-7,8-diol in AKR1A1-transfected cells. P4501B1 induction was blocked in AKR1A1 transfectants by the AKR1A1 inhibitor (sulfonylnitromethane), the o-quinone scavenger (N-acetyl-l-cysteine), or the cytosolic AhR antagonist (diflubenzuron). Attenuation of P4501B1 induction in these cells was verified by measuring a decrease in BP-tetrol formation. Our studies show that the formation of BP-7,8-dione by AKR1A1 in human bronchoalveolar cells leads to an induction of P4501B1 and that a functional consequence of this induction is elevated anti-BPDE production as detected by increased BP-tetrol formation. Therefore, the role of AKR1A1 in the activation of BP-7,8-diol is bifunctional; that is, it directly activates BP-7,8-diol to the reactive and redox-active PAH o-quinone (BP-7,8-dione) and it indirectly trans-activates the P4501B1 gene by generating the aryl hydrocarbon receptor (AhR) ligand BP-7,8-dione.

Endogenous Lipid Hydroperoxide-mediated DNA-adduct Formation in Min Mice

The Journal of Biological Chemistry. Apr, 2006  |  Pubmed ID: 16449227

Despite intensive research over the last two decades, there are still no specific markers of endogenous lipid hydroperoxide-mediated DNA damage. We recently demonstrated that heptanone-etheno-2'-deoxyguanosine adducts are formed in the DNA of rat intestinal epithelial cells that stably express cyclooxygenase-2. Heptanone-etheno adducts can only arise from the reaction of lipid hydroperoxide-derived 4-oxo-2(E)-nonenal with DNA. This raised the possibility that similar adducts would be formed in vivo in settings where cyclooxygenase-2 expression is increased. Therefore, DNA-adduct formation was studied in C57BL/6JAPC(min) mice, a colorectal cancer mouse model in which cyclooxygenase-2 is up-regulated. 15(S)-Hydroperoxy-5Z,8Z,11Z,13E-eicosatetraenoic acid is the major lipid hydroperoxide produced endogenously by cyclooxygenase-2. It undergoes homolytic decomposition to the DNA-reactive bifunctional electrophile 4-oxo-2(E)-nonenal, which forms heptanone-etheno adducts with DNA. A quantitative comparison was made of the heptanone-etheno-DNA adducts present in C57BL/6J and C57BL/6JAPC(min) mice. Using highly specific and sensitive methodology based on stable isotope dilution liquid chromatography/tandem mass spectrometry, we have detected the endogenous formation of heptanone-etheno adducts in mammalian tissue DNA for the first time. In addition, we found that there were statistically significant increased levels of the heptanone-etheno-2'-deoxyguanosine and heptanone-etheno-2'-deoxycytidine adducts in the C57BL/6JAPC(min) mice when compared with the control C57BL/6J mice.

Mild Folate Deficiency Induces a Proatherosclerotic Phenotype in Endothelial Cells

Atherosclerosis. Nov, 2006  |  Pubmed ID: 16469322

Low folate/high homocysteine (Hcy) is an established risk marker for cardiovascular disease (CVD). Some in vivo studies suggest low folate may independently contribute to CVD. To study the effects of mild folate deficiency on endothelial function, we adapted the EA.hy 926 endothelial cell line to growth in medium containing 23 nM folic acid (LO cells) or 9 microM folic acid (HI cells). Folate derivatives were substantially depleted in LO cells relative to HI cells. No differences were seen in intracellular homocysteine, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH), the SAM:SAH ratio, or global DNA methylation, and there was no consistent difference in secreted homocysteine. A greater percentage of LO than HI cells were in S phase of the cell cycle; supplementation of LO cells with thymidine/hypoxanthine prevented this. LO cells were more elongated than HI cells and did not form tight monolayers. Stress fibers were very prominent in LO but not HI cells. Treatment of LO cells with rho kinase inhibitors abolished stress fibers and partially normalized cell shape. LO cell monolayers were more permeable than HI cell monolayers at confluence, and MCP-1 mRNA and protein expression was higher in LO than HI cells. Our results suggest that mild folate deficiency is proatherosclerotic.

Liquid Chromatography/tandem Mass Spectrometry Characterization of Oxidized Amyloid Beta Peptides As Potential Biomarkers of Alzheimer's Disease

Rapid Communications in Mass Spectrometry : RCM. 2006  |  Pubmed ID: 16470704

Alzheimer's disease is characterized by the deposition of senile plaques that consist primarily of amyloid beta peptides. There is substantial evidence that amyloid beta is oxidized in vivo, which has led to the suggestion that oxidative stress is an important mediator of Alzheimer's disease. Metal-catalyzed oxidation can mimic in vivo oxidation of amyloid beta because the metal ion binds to the amino acid residues at the site of oxidation, which then deliver reactive oxygen species to that site. Based on electrospray mass spectrometry, it has been suggested that metal-catalyzed oxidation occurs on histidines-13 and -14. Unfortunately, the amyloid beta peptides provide complex spectra, so it is difficult to definitively characterize the sites of oxidation. Trypsin digestion of both native and oxidized amyloid beta1-16 and amyloid beta1-40 resulted in the formation of tryptic peptides corresponding to amyloid beta6-16, which could be separated by liquid chromatography (LC). Sites of oxidation were then unequivocally characterized as histidine-13 and histidine-14 by LC/tandem mass spectrometric (MS/MS) analysis of the tryptic peptides. The ability to analyze the specific amyloid beta6-16 tryptic fragments derived from full-length amyloid beta peptides will make it possible to determine whether oxidation in vivo occurs at specific histidine residues and/or at other amino acid residues such as methionine-35. Using methodology based on LC/MS/MS it will also be possible to analyze the relative amounts of oxidized peptides and native peptide in cerebrospinal fluid from patients with Alzheimer's disease as biomarkers of oxidative stress.

Quantification of Benzo[a]pyrene Diol Epoxide DNA-adducts by Stable Isotope Dilution Liquid Chromatography/tandem Mass Spectrometry

Rapid Communications in Mass Spectrometry : RCM. 2006  |  Pubmed ID: 16557497

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants found in car exhausts, charbroiled food, and tobacco smoke. Three pathways for the metabolic activation of B[a]P to ultimate carcinogens have been proposed. The most widely accepted pathway involves cytochrome-P450 (CYP) 1A1- and/or 1B1-mediated formation of B[a]P-7,8-oxide, which undergoes epoxide hydrolase-mediated metabolism to the proximate carcinogen B[a]P-7,8-dihydro-7,8-diol. Further CYP1A1- and/or CYP1B1-mediated activation of the dihydrodiol results in the formation of 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (B[a]PDE), the ultimate carcinogen. In previous studies, it was demonstrated that (+)-anti-B[a]PDE was the most potent tumorigen of the CYP-derived B[a]PDE diastereomers. We have developed a stable isotope dilution, liquid chromatography multiple reaction monitoring/mass spectrometry (LC-MRM/MS) assay for all eight (+/-)-anti-B[a]PDE-derived dGuo and dAdo DNA-adducts. The LC-MRM/MS assay was rigorously validated and used to show that (+)-anti-trans-B[a]PDE-dGuo was the major adduct formed when naked DNA and human bronchoalveolar adenocarcinoma H358 cells were treated with (+/-)-anti-B[a]PDE. The preference for DNA-adducts derived from (+)-anti-B[a]PDE was even more apparent in cellular DNA. Thus, the increased potency of (+)-anti-B[a]PDE as a tumorigen is most likely due its ability to preferentially form DNA-adducts when compared with (-)-anti-B[a]PDE. Also, the adduct profile suggests that this occurs by binding of (+)-anti-B[a]PDE to DNA in a manner that facilitates covalent binding to dGuo rather than dAdo residues.

Characterization of a Lipid Hydroperoxide-derived RNA Adduct in Rat Intestinal Epithelial Cells

Chemical Research in Toxicology. Jun, 2006  |  Pubmed ID: 16780360

Five major products (adducts A(1a), A(1b), A(2), A(3,) and B) from the reaction of guanosine (Guo) with 4-oxo-2(E)-nonenal (ONE) were detected by liquid chromatography-mass spectrometry (LC-MS). Tandem MS (MS/MS) analysis of these compounds suggested that modifications to the nucleoside had been introduced. Adducts A(1a), A(1b), A(2), and A(3) were heptanone-ethano-2'-Guo adducts that all decomposed to adduct B. Adducts A(1a) and A(1b) were isomeric hemi-ketal forms. Adducts A(2) and A(3) were diastereomers of the open chain ketone form. The structure of adduct B was shown by LC-MS/MS and NMR spectroscopy to be the heptanone-etheno-Guo (HepsilonGuo) adduct, 3-(D-erythropentafuranosyl)imidazo-7-(heptane-2' '-one)-9-hydroxyl[1,2-alpha]purine. The overall reaction of Guo with ONE was very similar to its reaction with 2'-deoxyguanosine. Reaction of ONE with yeast transfer RNA also resulted in the formation of HepsilonGuo. Finally, HepsilonGuo was detected and quantified in the RNA from rat intestinal epithelial cells that stably express cyclooxygenase-2. These data show that RNA is modified by the same bifunctional reactive electrophiles derived from lipid peroxidation that covalently modify DNA.

Translesion DNA Synthesis Across the Heptanone--etheno-2'-deoxycytidine Adduct in Cells

Chemical Research in Toxicology. Aug, 2006  |  Pubmed ID: 16918247

4-Oxo-2(E)-nonenal, a lipid peroxidation-derived product, reacts with dG, dA, and dC in DNA to form heptanone (H)-etheno (epsilon) adducts. Among the three adducts, H-epsilondC is formed in the greatest abundance in in vitro reactions, and it has been detected in the C57BL/6JAPC(min) mouse model of colorectal cancer. To establish the genotoxic properties of this adduct, a site-specifically modified oligonucleotide was synthesized and incorporated into a shuttle vector. The modified vector was replicated in Escherichia coli and human cells. Analysis of the progeny plasmid has revealed that H-epsilondC strongly blocks DNA synthesis and markedly miscodes in both hosts. The miscoding frequency was 40-50% in bacteria and more than 90% in three human cell lines (xeroderma pigmentosum A and variant cells, and DNA repair wild-type cells). There was a drastic difference in coding events in these two hosts: dG and dC were almost exclusively inserted opposite the lesion in E. coli, while dA and dT were the preferential choices in human cells. These results indicate that this endogenous DNA adduct is very genotoxic to both organisms.

Proteomics-based Strategy to Identify Biomarkers and Pharmacological Targets in Leukemias with T(4;11) Translocations

Journal of Proteome Research. Oct, 2006  |  Pubmed ID: 17022645

Translocations and other aberrations involving the MLL (mixed lineage leukemia) gene result in aggressive forms of leukemias. Heterogeneity in partner genes, in chromosomal breakpoints, in MLL itself, and in the different partner genes results in heterogeneous fusion transcripts that can be alternatively spliced, which complicates deciphering a unifying mechanism of leukemogenesis. However, recent microarray studies completed with clinical leukemia specimens have uncovered several distinct mRNA signatures within MLL leukemia that differ from other types of leukemia. A global proteomics strategy using MV4-11 and RS4:11 cells in culture was employed to investigate possible protein signatures common to different MLL leukemias and to identify disease biomarkers and protein targets for pharmacological intervention. Initial proteomics screening experiments with two-dimensional differential in-gel electrophoresis revealed heat shock protein 90 alpha (HSP90alpha) as a potential target for pharmacological inhibition and nucleoside diphosphate kinase (nm23) as a biomarker for measuring treatment efficacy. Using a modified stable isotope labeling of amino acids in cell culture (SILAC) approach, coupled with two-dimensional liquid chromatography tandem mass spectrometry (2D-LC-MS/MS), changes in abundance for over 500 proteins were measured. In addition, decreased expression of the novel biomarker nm23 was observed during HSP90 inhibition with 17-allylamino-17-demethoxygeldanamycin (17-AAG) in the MV4-11 cell line. The present study validates the use of a global proteomics strategy to uncover novel biomarkers and pharmacological targets for leukemias with MLL translocations. Additionally, several proteins were found to be expressed in concordance with microarray studies of mRNA expression in specimens from patients showing the value in comparing mRNA transcript and proteomic profiles. This work represents one of the most comprehensive proteomics screens of MLL leukemias that have been conducted to date.

Analysis of Phosphorylation Sites on Focal Adhesion Kinase Using Nanospray Liquid Chromatography/multiple Reaction Monitoring Mass Spectrometry

Rapid Communications in Mass Spectrometry : RCM. 2006  |  Pubmed ID: 17117420

An approach based on nanospray liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) was developed in order to analyze twenty-nine phosphorylated and non-phosphorylated tryptic peptides from focal adhesion kinase (FAK). All peptides monitored were resolved and showed excellent peak shape with the exception of one doubly phosphorylated peptide. Optimization of the LC method enabled the identification and subsequent monitoring of six important tyrosine phosphorylation sites on FAK, including phosphorylated Y397 (pY397), pY407, pY576, pY577, pY861, and pY925. This technique was able to identify sites of phosphorylation on FAK as well as qualitatively differentiate between autocatalytic and Src-induced phosphorylation events. FAK was shown to have autocatalytic function, which resulted in efficient phosphorylation of Y397. FAK was also capable of autophosphorylation on residues Y407 and Y576, though apparently less effectively than autophosphorylation at Y397. Src was found to phosphorylate FAK at Y407, Y576, Y577, and Y861. The presence of Src increased the abundance of pY576 at low temperature indicating Src had particularly high kinase activity toward this residue. Furthermore, Src phosphorylated FAK at Y577 to produce FAK bis-phosphorylated at Y576 and Y577. In addition, six novel sites of phosphorylation (Y148, Y347, Y441, T503, S850, and Y1007) were identified on FAK. Interestingly, Src phosphorylated FAK to form a peptide uniquely phosphorylated on Y407, together with substantial amounts of the bis-phosphorylated pY397pY407 peptide. These findings will impact significantly on future studies of FAK activity since pY397 is often used as a measure of FAK activity and Src association.

Quantitative Analysis of Amyloid Beta Peptides in Cerebrospinal Fluid of Alzheimer's Disease Patients by Immunoaffinity Purification and Stable Isotope Dilution Liquid Chromatography/negative Electrospray Ionization Tandem Mass Spectrometry

Rapid Communications in Mass Spectrometry : RCM. 2006  |  Pubmed ID: 17117458

The 40 and 42 amino-acid residue forms of amyloid beta (Abeta(1-40) and Abeta(1-42)) in cerebrospinal fluid (CSF) have been proposed as potential biomarkers of Alzheimer's disease (AD). Quantitative analyses of Abeta peptides in CSF have relied almost exclusively on the use of immunoassay-based assays such as the enzyme-linked immunosorbent assay (ELISA) procedure. However, due to the ability of the Abeta peptides to readily self-aggregate or bind to other proteins and glassware, such analyses are extremely challenging. Analyses are further complicated by the potential of the peptides to undergo post-translational modifications and the possibilities for cross-reaction in the ELISA assays with endogenous components of the CSF. An approach based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) has now been developed which overcomes these methodological issues. The key steps in implementing this new approach involved immunoaffinity purification coupled with the use of [15N]-labeled Abeta peptides as internal standards, a basic LC mobile phase, negative ion electrospray ionization, and a basic solvent for dissolving the peptides and washing the injection needle to prevent carryover of analytes during multiple injections on the LC/MS system. The validated method had limits of quantitation of 44 fmol/mL (200 pg/mL) for Abeta(1-42) and 92 fmol/mL (400 pg/mL) for Abeta(1-40). An excellent correlation was found between the LC/MS/MS assay and an ELISA assay for Abeta(1-42) in human CSF (r2 = 0.915), although less correlation was observed for Abeta(1-40) (r2 = 0.644). Mean CSF Abeta(1-42) concentrations for samples collected 2 weeks apart from a limited number of AD patients provided additional confidence in the reproducibility of the LC/MS/MS assay. Concentrations for duplicate samples from AD patients were slightly higher than most previously reported values (mean 1.06 +/- 0.25 ng/mL; n = 7). Abeta(1-40) concentrations in duplicate samples obtained from AD patients were also reproducible but were found to be slightly lower than most previously reported values (mean 6.36 +/- 3.07 ng/mL; n = 7). Consistent with literature reports, mean Abeta(1-42) concentrations were found to be lower in AD patients compared with the normal subjects (mean 1.49 +/- 0.59 ng/mL; n = 7), whereas there was no difference in Abeta(1-40) concentrations between AD patients and normal subjects (mean 5.88 +/- 3.03 ng/mL; n = 7). The accuracy and precision of the LC/MS assay mean that it will be a useful complement to existing ELISA assays for monitoring therapeutic interventions designed to modulate CSF Abeta(1-42) concentrations in individual AD patients. Moreover, the introduction of stable isotope labeled internal standards offers the potential to achieve a more rigorous account of the influence of methodological effects related to sample collection and processing.

Convenient and Efficient Syntheses of 4-hydroxy-2(E)-nonenal and 4-oxo-2(E)-nonenal

Lipids. Sep, 2006  |  Pubmed ID: 17152925

Lipid peroxidation products 4-hydroxy-2(E)-nonenal (HNE) and 4-oxo-2(E)-nonenal (ONE) were conveniently synthesized using Wittig and Horner-Wardsworth-Emmons (HWE) reaction. Wittig or HWE reaction between an easily prepared phosphorane or phosphonate with glyoxal dimethyl acetal gave a protected 4-oxo-2(E)-nonenal. Hydrolysis gave 4-oxo-2(E)-nonenal, whereas reduction followed by hydrolysis gave 4-hydroxy-2(E)-nonenal.

Interaction with Phospholipids Modulates Alpha-synuclein Nitration and Lipid-protein Adduct Formation

The Biochemical Journal. Jan, 2006  |  Pubmed ID: 16146428

Intracellular aggregates of alpha-syn (alpha-synuclein) represent pathoanatomical hallmarks of neurodegenerative disorders (synucleinopathies). The molecular mechanisms underlying alpha-syn aggregation into filamentous inclusions may involve oxidation and nitration of the protein. Whereas the effects of oxidants and nitrating species on soluble alpha-syn have been studied in detail, the effect of these reactive species on alpha-syn associated with lipids is still unknown. In the present paper, we report that alpha-syn bound to small unilamellar liposomes composed of phosphatidylcholine/phosphatidic acid is resistant to oxidation and nitration when compared with soluble alpha-syn. Additionally, increasing concentrations of unsaturated fatty acids diminished the oxidation and nitration of alpha-syn upon exposure to fluxes of peroxynitrite (8-20 microM x min(-1)). To investigate the effect of oxidized lipids on alpha-syn, the protein was incubated with the bifunctional electrophile 4-HNE [4-hydroxy-2(E)-nonenal]. MS analysis showed the formation of three major products corresponding to the native protein and alpha-syn plus one or two 4-HNE molecules. Trypsin digestion of the modified protein followed by peptide 'finger-printing' revealed that 4-HNE modified the peptide E46GVVHGVATVAEK58. Further analysis of the peptides with liquid chromatography-tandem MS identified the modified residue as His50. The data indicate that the association of alpha-syn with biological membranes protects the protein from oxidation and nitration and thus diminishes the formation of protein molecules capable of forming aggregates. However, products of lipid peroxidation can also modify alpha-syn, generating novel protein adducts that could serve as biomarkers for documenting oxidative processes in human as well as animal and cellular models of alpha-syn aggregation and pathology.

Endogenous Glutathione Adducts

Current Drug Metabolism. Dec, 2006  |  Pubmed ID: 17168687

This review provides an overview of the formation, pharmacology, and toxicology of endogenous glutathione (GSH)-adducts with particular emphasis on GSH-adducts that arise from lipid peroxidation. GSH is the major low-molecular-weight thiol in mammalian cells. It is involved in the formation of endogenous bioactive eicosanoids and is a source of reducing equivalents in a number of biosynthetic reactions. GSH has long been recognized to act as a co-factor in the reduction of reactive oxygen species and lipid hydroperoxides by glutathione peroxidases and glutathione-S-transferases (GSTs). It also plays an important role in the reduction of reactive intermediates derived from arylamines and in the conjugation of reactive intermediates to form S-substituted endogenous GSH-adducts through its nucleophilic cysteine sulfhydryl group. Although some reactive intermediates can form adducts directly, GST-mediated reactions generally predominate. This results in the formation of bioactive endogenous GSH-adducts derived from eicosanoids, isoprostanes, estrogens, catecholamines, and 4-hydroxy-2(E)-nonenal (HNE). Cellular oxidative stress causes increased lipid peroxidation with the concomitant formation of DNA- and protein-reactive bifunctional electrophiles. It has generally been considered that HNE is the most abundant bifunctional electrophile that is formed. Several years ago we discovered that 4-oxo-2(E)-nonenal (ONE) was also a major lipid hydroperoxide-derived bifunctional electrophile. From in vitro studies, we showed that ONE and HNE arose from the common intermediate, 4-hydroperoxy-2(E)-nonenal and also showed that ONE was formed in greater amounts than HNE. We have recently made the unexpected discovery that GSH addition to ONE leads to the formation of an unusual thiadiazabicyclo-ONE-GSH-adduct (TOG), which was characterized as (2S,7R) - 7 - [N - (carboxymethyl)carbamoyl] - 5 - oxo - 12 - pentyl - 9 - thia - 1,6 - diazabicyclo[8.2.1]trideca - 10(13), 11-diene-2-carboxylic acid. TOG is one of the most abundant GSH-adducts formed during peroxide/Fe(II)- or Fe(II)-mediated oxidative stress in EA.hy 926 endothelial cells. As TOG is formed from ONE, these experiments have confirmed that ONE is a major lipid hydroperoxide-derived bifunctional electrophile formed during intracellular oxidative stress. TOG represents the first member of a new class of endogenous GSH-adduct biomarkers that can be used to quantify intracellular oxidative stress. Two other members of the TOG family arise from GST-mediated GSH-adduct formation with dioxododecenoic acid and dioxooctenoic acid, bifunctional electrophiles derived from the carboxy terminus of lipid hydroperoxides. The formation of TOG and TOG-related endogenous GSH-adducts can result from free radical- as well as cyclooxygenase- and lipoxygenase-mediated pathways. Analysis of the GSH-adducts by stable isotope dilution mass spectrometry-based methodology will provide a quantitative measure of enzymatic and non-enzymatic cellular oxidative stress to complement isoprostane measurements. In future studies, it will also be important to establish the biological activity of TOG and its analogs in view of the potent activity of many other endogenous GSH-adducts such as the leukotrienes.

Characterization of an Etoposide-glutathione Conjugate Derived from Metabolic Activation by Human Cytochrome P450

Current Drug Metabolism. Dec, 2006  |  Pubmed ID: 17168690

Etoposide (VP-16), a DNA topoisomerase II poison widely used as an antineoplastic agent is also known to cause leukemia. One of its major metabolic pathways involves O-demethylation to etoposide catechol (etoposide-OH) by cytochrome P450 3A4 (CYP3A4). The catechol metabolite can undergo sequential one- and two-electron oxidations to form etoposide semi-quinone (etoposide-SQ) and etoposide quinone (etoposide-Q), respectively, which have both been implicated as cytotoxic metabolites. However, etoposide-Q is known to react with glutathione (GSH), which can protect DNA from oxidative damage by this reactive metabolite. In this study, etoposide-Q was reacted with GSH and the two etoposide-GSH conjugates were characterized. The major conjugate was etoposide-OH-6'-SG and the minor product was etoposide-OH-2'-SG. Etoposide-OH-6'-SG, which arose from Michael addition of GSH to etoposide-Q, was characterized by mass spectrometry and 2-D NMR. It was identified as the sole product from in vitro metabolism experiments using recombinant human CYP3A4 or liver microsomes incubated with etoposide in the presence of GSH. Etoposide-OH-6'-SG was also detected from incubations of etoposide-OH and GSH alone. Therefore, the presence of etoposide-OH, which can be formed from etoposide metabolism by CYP3A4, is essential for formation of the GSH conjugate. The oxidation of etoposide-OH to a quinone intermediate is likely the precursor in the formation of etoposide-OH-6'-SG.

LC/ESI/MS Analysis of Saturated and Unsaturated Fatty Acids in Rat Intestinal Epithelial Cells

Current Drug Metabolism. Dec, 2006  |  Pubmed ID: 17168692

Reactive oxygen species (ROS) can mediate damage to cellular macromolecules and lipids. Lipid peroxidation is considered to be a major pathway by which ROS can cause tissue damage and alterations in cell membranes. Other factors affecting oxidative damage include the target molecules such as fatty acids, which are readily oxidized by ROS. Thus, lipid peroxidation may depend upon the cellular fatty acid composition. Analysis of saturated fatty acids that are present by liquid chromatography/mass spectrometry (LC/MS) is difficult because they are poorly ionized under electrospray ionization (ESI) conditions. The separation of short to very long chain saturated and unsaturated fatty acids is also very challenging when LC is employed instead of gas chromatography. The use of trimethylaminoethyl (TMAE) ester iodide derivatization has been shown previously to improve the sensitivities of saturated fatty acids in the ESI mode. A reversed-phase LC method using a diphenyl column was employed to separate 14 fatty acids as their TMAE derivatives. Stable isotope dilution LC/ESI/multiple reaction monitoring/MS methodology was then developed for the quantitative analysis of seven saturated and seven unsaturated forms of short (C14) to very long (C26) chain fatty acids as their TMAE ester iodide derivatives. This methodology has allowed the analysis of fatty acid composition from parental rat intestinal epithelial cell and rat intestinal epithelial cells transfected with cyclooxygenase-2, a model system of oxidative stress.

Aldo-keto Reductase- and Cytochrome P450-dependent Formation of Benzo[a]pyrene-derived DNA Adducts in Human Bronchoalveolar Cells

Chemical Research in Toxicology. Mar, 2007  |  Pubmed ID: 17295519

There is substantial evidence to suggest that polycyclic aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (B[a]P) induce lung cancer through metabolic activation. As part of a program to delineate the routes of PAH activation, we have examined DNA adducts that are formed in human lung cells. A stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry method was used to quantify eight anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE)-derived DNA adducts in four H358 human bronchoalveolar cell lines with different phenotypes. In P450 1A1/P450 1B1-induced H358 cells exposed to (+/-)-B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), (+)-anti-trans-B[a]PDE-N2-2'-deoxyguanosine [(+)-anti-trans-B[a]PDE-N2-dGuo] was the major DNA adduct, and it formed with no lag phase. In AKR1A1-transfected H358 cells, (+)-anti-trans-B[a]PDE-N2-dGuo was also the major adduct with a 3 h lag phase before significant adduct formation was detected. In AKR1A1-transfected H358 cells with induced P450 1A1/P450 1B1, (+)-anti-trans-B[a]PDE-N2-dGuo was formed with no lag phase in amounts similar to those in the H358 cells with up-regulated P450 1A1/P450 1B1. Surprisingly, the greatest amount of (+)-anti-trans-B[a]PDE-N2-dGuo was formed in the control H358 cells. Furthermore, (+)-anti-trans-B[a]PDE-N2-dGuo formation was 2-fold higher in (-)-B[a]P-7,8-dihydrodiol-exposed H358 cells when compared with (+/-)-B[a]P-7,8-dihydrodiol-exposed cells. The P450 1A1/1B1 inhibitor 2,4,3',5'-tetramethoxystilbene did not attenuate DNA adduct formation in the control H358 cells, suggesting that another P450 was responsible. These data raise the intriguing possibility that P450 1A1/P450 1B1 and AKR1A1 may be protective against (+)-B[a]PDE-mediated DNA damage.

A Novel 4-oxo-2(E)-nonenal-derived Endogenous Thiadiazabicyclo Glutathione Adduct Formed During Cellular Oxidative Stress

Chemical Research in Toxicology. Jul, 2007  |  Pubmed ID: 17550273

Cellular oxidative stress causes increased lipid peroxidation with the concomitant formation of DNA and protein reactive bifunctional electrophiles. Glutathione (GSH) detoxifies these bifunctional electrophiles by forming GSH adducts. Several years ago we discovered 4-oxo-2(E)-nonenal (ONE) as a major bifunctional electrophile derived from lipid hydroperoxides. We have now made the unexpected discovery that glutathione-S-transferase (GST)-mediated GSH addition to ONE occurs primarily to C-1 of the alpha,beta-unsaturated ketone rather than to C-3 of the alpha,beta-unsaturated aldehyde. The resulting intermediate rapidly undergoes two intramolecular cyclizations followed by two separate dehydration reactions to provide an unusual thiadiazabicyclo-ONE-GSH adduct (TOG). Quantification of intracellular TOG was performed using stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry after the addition of ONE to cells or as an endogenously derived adduct during peroxide-induced oxidative stress. TOG represents the first member of a new class of thiadiazabicyclo GSH adducts that are formed through GST-mediated addition of GSH to reactive intermediates containing the ONE motif during intracellular oxidative stress. ONE formation can potentially result from free radical pathways as well as cyclooxygenase- and lipoxygenase-mediated pathways. Its aldo-keto reductase-mediated reduction product, 4-oxo-2(E)-nonenol (ONO), was also formed and converted to GSH adducts similar to those formed by 4-hydroxy-2(E)-nonenal (HNE). ONO is isomeric with HNE; therefore, protein and peptide adducts ascribed to arise solely from reactions with endogenous HNE will need to be re-appraised.

Metabolism of Benzo[a]pyrene in Human Bronchoalveolar H358 Cells Using Liquid Chromatography-mass Spectrometry

Chemical Research in Toxicology. Sep, 2007  |  Pubmed ID: 17702526

Benzo[ a]pyrene (B[ a]P), a representative polycyclic aromatic hydrocarbon (PAH), is metabolically activated by three enzymatic pathways: by peroxidases (e.g., cytochrome P450 peroxidase) to yield radical cations, by P4501A1/1B1 monooxygenation and epoxide hydrolase to yield diol epoxides, and by P4501A1/1B1 monooxygenation, epoxide hydrolase, and aldo-keto reductases (AKRs) to yield o-quinones. In humans, a major exposure site for environmental and tobacco smoke PAH is the lung; however, the profile of B[ a]P metabolites formed at this site has not been well characterized. In this study, human bronchoalveolar H358 cells were exposed to B[ a]P, and metabolites generated by peroxidase (B[ a]P-1,6- and B[ a]P-3,6-diones), from cytochrome P4501A1/1B1 monooxygenation [3-hydroxy-B[ a]P, B[ a]P-7,8- and 9,10- trans-dihydrodiols, and B[ a]P- r-7, t-8, t-9, c-10-tetrahydrotetrol (B[ a]P-tetraol-1)], and from AKRs (B[ a]P-7,8-dione) were detected and quantified by RP-HPLC, with in-line photo-diode array and radiometric detection, and identified by liquid chromatography-mass spectrometry (LC-MS). Progress curves showed a lag phase in the formation of 3-hydroxy-B[ a]P, B[ a]P-7,8- trans-dihydrodiol, B[ a]P-tetraol-1, and B[ a]P-7,8-dione over 24 h. Northern blot analysis showed that B[ a]P induced P4501B1 and AKR1C isoforms in H358 cells in a time-dependent manner, providing an explanation for the lag phase. Pretreatment of H358 cells with 10 nM 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) eliminated this lag phase but did not alter the levels of the individual metabolites observed, suggesting that both B[ a]P and TCDD induction ultimately yield the same B[ a]P metabolic profile. The one exception was B[ a]P-3,6-dione which was formed without a lag phase in the absence and presence of TCDD, suggesting that the peroxidase responsible for its formation was neither P4501A1 nor 1B1. Candidate peroxidases that remain include PGH synthases and uninduced P450 isoforms. This study shows that the P4501A1/1B1 and AKR pathways are inducible in human lung cells and that the peroxidase pathway was not. It also provides evidence that each of the pathways of PAH activation yields their distinctive metabolites in H358 human lung cells and that each pathway may contribute to the carcinogenic process.

Cyclooxygenase-2-mediated Metabolism of Arachidonic Acid to 15-oxo-eicosatetraenoic Acid by Rat Intestinal Epithelial Cells

Chemical Research in Toxicology. Nov, 2007  |  Pubmed ID: 17910482

Rat intestinal epithelial cells that permanently express the cyclooxygenase-2 (COX-2) gene (RIES cells) were used to investigate COX-2-mediated arachidonic acid (AA) metabolism. A targeted chiral lipidomics approach was employed to quantify AA metabolites that were secreted by the cells into the culture media. When intact RIES cells were treated with calcium ionophore A-23187 (1 microM) for 1 h, 11-(R)-hydroxyeicosatetraenoic acid (HETE) was the most abundant metabolite, followed by prostaglandin (PG) E 2, 15-(S)-HETE, 15-oxo-eicosatetraenoic acid (ETE), and 15-(R)-HETE. Incubation for a further 23 h after the calcium ionophore was removed resulted in a substantial increase in PGE 2 concentrations while HETE and 15-oxo-ETE concentrations decreased to almost undetectable levels. A similar metabolic profile was observed when RIES cells were treated with increasing concentrations of AA for 24 h. Incubation of the RIES cells with 10 microM AA revealed that maximal concentrations of 11-(R)-HETE, 15-(S)-HETE, and 15-oxo-ETE occurred after 10 min of incubation when the 15-( S)-HETE concentrations were approximately twice that of PGE 2. There was a gradual decrease in the concentrations of HETE and 15-oxo-ETE over time, whereas PGE 2 concentrations increased steadily until they reached a maximum after 24 h of incubation. The ratio of PGE 2 to 15-(S)-HETE was then approximately 20:1. 15-(S)-HETE and 15-oxo-ETE concentrations declined in the cell media during prolonged incubations with pseudo-first-order rate constants of 0.0121 and 0.0073 min(-1), respectively. 15-(S)-HETE was shown to undergo metabolism primarily to 15-oxo-ETE, which was further metabolized to a glutathione (GSH) adduct. The GSH adduct of 15-oxo-ETE was further metabolized in the extracellular milieu to a cysteinylglycine adduct. Thus, we have established for the first time that 15-oxo-ETE can be formed biosynthetically from AA, that 15-(S)-HETE is its immediate precursor, and that 15-oxo-ETE forms a GSH adduct. For ionophore-A-23187-stimulated cells and at early time points for AA-stimulated cells, 11-(R)-HETE was the major eicosanoid to be secreted into the media. Adding increasing concentrations of AA to cells in culture made it possible to estimate with surprising accuracy endogenous eicosanoid production using regression analyses. Thus, after 24 h in the absence of added AA, 11-(R)-HETE and 15-(R)-HETE were estimated to be present at concentrations close to the detection limit of our very sensitive assay. These data further highlight the importance of endogenous COX-2-mediated lipid peroxidation and illustrate the necessity to monitor eicosanoid formation from endogenous stores of AA in cell culture experiments.

Targeted Chiral Lipidomics Analysis by Liquid Chromatography Electron Capture Atmospheric Pressure Chemical Ionization Mass Spectrometry (LC-ECAPCI/MS)

Methods in Enzymology. 2007  |  Pubmed ID: 17954234

The corona discharge used to generate positive and negative ions under conventional atmospheric pressure chemical ionization (APCI) conditions also provides a source of low-energy gas-phase electrons. This is thought to occur by displacement of electrons from the nitrogen sheath gas. Therefore, suitable analytes can undergo electron capture in the gas phase in a manner similar to that observed for gas chromatography/electron capture negative chemical ionization/mass spectrometry (MS). This technique, which has been named electron-capture APCI (ECAPCI)/MS, mass spectrometry provides an increase in sensitivity of two orders of magnitude when compared with conventional APCI methodology. It is a simple procedure to tag arachidonic acid- and linoleic acid-derived oxidized lipids with an electron-capturing group such as the pentafluorobenzyl (PFB) moiety before analysis. PFB derivatives have previously been used as electron-capturing derivatives because they undergo dissociative electron capture in the gas phase to generate negative ions through the loss of a PFB radical. A similar process occurs under ECAPCI conditions. By monitoring the negative ions that are formed, it is possible to obtain extremely high sensitivity for PFB derivatives of oxidized lipids derived from arachidonic and linoleic acid. A combination of stable isotope dilution methodology and chiral liquid chromatography-ECAPCI/MS makes it possible to resolve and quantify complex mixtures of regioisomeric and enantiomeric oxidized lipids.

EBP, a Program for Protein Identification Using Multiple Tandem Mass Spectrometry Datasets

Molecular & Cellular Proteomics : MCP. Mar, 2007  |  Pubmed ID: 17164401

MS/MS combined with database search methods can identify the proteins present in complex mixtures. High throughput methods that infer probable peptide sequences from enzymatically digested protein samples create a challenge in how best to aggregate the evidence for candidate proteins. Typically the results of multiple technical and/or biological replicate experiments must be combined to maximize sensitivity. We present a statistical method for estimating probabilities of protein expression that integrates peptide sequence identifications from multiple search algorithms and replicate experimental runs. The method was applied to create a repository of 797 non-homologous zebrafish (Danio rerio) proteins, at an empirically validated false identification rate under 1%, as a resource for the development of targeted quantitative proteomics assays. We have implemented this statistical method as an analytic module that can be integrated with an existing suite of open-source proteomics software.

Quantification of Intracellular Homocysteine by Stable Isotope Dilution Liquid Chromatography/tandem Mass Spectrometry

Biomedical Chromatography : BMC. Jan, 2007  |  Pubmed ID: 17121413

A precise and accurate stable isotope dilution liquid chromatography/tandem mass spectrometry method for the analysis of intracellular homocysteine has been developed. An internal standard, [(2)H(8)]-homocystine, was added to cell pellets from EA.hy 926 cells grown in culture under low and high folate concentrations. D,L-dithiothreitol was used to reduce cellular homocystine to homocysteine. Cellular proteins were precipitated by the addition of formic acid in acetonitrile. After centrifugation, a portion of the supernatant was analyzed by liquid chromatography/tandem mass spectrometry. Using a Supelcosil cyano column with an Applied Biosystems API 4000 triple quadrupole mass spectrometer, the SRM transitions for homocysteine (m/z 136 to m/z 90) and [(2)H(4)]-homocysteine (m/z 140 to m/z 94) were monitored. The method was validated by conducting five replicate analyses on three different days at four different concentrations (concentrations at the lower limit of quantitation and expected lower quartile, mid-range and upper quartile). The limit of detection was 2 ng/10(6) EA.hy 926 cells. Using this method, the intracellular homocysteine concentration in EA.hy 926 cells ranged from 10 to 36 ng/10(6) cells.

Strategies for Synthesis of Adducts of Omicron-quinone Metabolites of Carcinogenic Polycyclic Aromatic Hydrocarbons with 2'-deoxyribonucleosides

The Journal of Organic Chemistry. Feb, 2008  |  Pubmed ID: 18181642

Polycyclic aromatic hydrocarbons (PAHs) are major environmental carcinogens produced in the combustion of fossil fuels, tobacco, and other organic matter. Current evidence indicates that PAHs are transformed enzymatically to active metabolites that react with DNA to form adducts that result in mutations. Three activation pathways have been proposed: the diol epoxide path, the radical-cation path, and the quinone path. The latter involves aldo-keto reductase mediated oxidation of PAH dihydrodiol metabolites to catechols that enter into redox cycles with quinones. This results in generation of reactive oxygen species (ROS) that attack DNA, and the PAH quinones also react with DNA to form adducts. Several strategies for synthesis of the stable adducts formed by the o-quinone metabolites of carcinogenic PAHs with 2'-deoxyribonucleosides were investigated and compared. The PAH quinones studied were benz[a]anthracene-3,4-dione and its 7-methyl- and 7,12-dimethyl- derivatives. The parent PAHs represent a range of carcinogenicity from inactive to highly potent. Two synthetic methods were devised that differ in the catalyst employed, Pd(OAc)(2) or CuI. The Pd-mediated method involved coupling a protected amino-catechol PAH derivative with a halo-2'-deoxyribonucleoside. The copper-mediated method entailed reaction of a halo-PAH catechol derivative with a 2'-deoxyribonucleoside. Adducts of benz[a]anthracene-3,4-dione (and its 7-methyl- and 7,12-dimethyl- derivatives) with 2'-deoxyadenosine and 2'-deoxyguanosine were prepared by these methods. Availability of adducts of these types through synthesis makes possible for the first time biological studies to determine the role of these adducts in tumorigenesis. The copper-mediated method offers advantages of economy, adaptability to large-scale preparation, utility for synthesis of (13)C- or (15)N-labeled analogues, and nonformation of bis-adducts as secondary products.

Analysis of the Zebrafish Proteome During Embryonic Development

Molecular & Cellular Proteomics : MCP. May, 2008  |  Pubmed ID: 18212345

The model organism zebrafish (Danio rerio) is particularly amenable to studies deciphering regulatory genetic networks in vertebrate development, biology, and pharmacology. Unraveling the functional dynamics of such networks requires precise quantitation of protein expression during organismal growth, which is incrementally challenging with progressive complexity of the systems. In an approach toward such quantitative studies of dynamic network behavior, we applied mass spectrometric methodology and rigorous statistical analysis to create comprehensive, high quality profiles of proteins expressed at two stages of zebrafish development. Proteins of embryos 72 and 120 h postfertilization (hpf) were isolated and analyzed both by two-dimensional (2D) LC followed by ESI-MS/MS and by 2D PAGE followed by MALDI-TOF/TOF protein identification. We detected 1384 proteins from 327,906 peptide sequence identifications at 72 and 120 hpf with false identification rates of less than 1% using 2D LC-ESI-MS/MS. These included only approximately 30% of proteins that were identified by 2D PAGE-MALDI-TOF/TOF. Roughly 10% of all detected proteins were derived from hypothetical or predicted gene models or were entirely unannotated. Comparison of proteins expression by 2D DIGE revealed that proteins involved in energy production and transcription/translation were relatively more abundant at 72 hpf consistent with faster synthesis of cellular proteins during organismal growth at this time compared with 120 hpf. The data are accessible in a database that links protein identifications to existing resources including the Zebrafish Information Network database. This new resource should facilitate the selection of candidate proteins for targeted quantitation and refine systematic genetic network analysis in vertebrate development and biology.

Determination of Cellular Redox Status by Stable Isotope Dilution Liquid Chromatography/mass Spectrometry Analysis of Glutathione and Glutathione Disulfide

Rapid Communications in Mass Spectrometry : RCM. 2008  |  Pubmed ID: 18215009

Oxidation of glutathione (GSH) to glutathione disulfide (GSSG) occurs during cellular oxidative stress. The redox potential of the 2GSH/GSSG couple, which is determined by the Nernst equation, provides a means to assess cellular redox status. It is difficult to accurately quantify GSH and GSSG due to the ease with which GSH is oxidized to GSSG during sample preparation. To overcome this problem, a stable isotope dilution liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) method has been developed using 4-fluoro-7-sulfamoylbenzofurazan (ABD-F) derivatization. ABD-F derivatization of the GSH thiol group was rapid, quantitative, and occurred at room temperature. The LC/MRM-MS method, which requires no sample clean-up, was validated within the calibration ranges of 5 to 400 nmol/mL in cell lysates for GSH and 0.5 to 40 nmol/mL in cell lysates for GSSG. Calibration curves prepared by adding known concentrations of GSH and GSSG to cell lysates were parallel to the standard curve prepared in buffers. GSH and GSSG concentrations were determined in two monocyte/macrophage RAW 267.4 cell lines with or without 15-LOX-1 expression (R15LO and RMock cells, respectively) after treatment with the bifunctional electrophile 4-oxo-2(E)-nonenal (ONE). R15LO cells synthesized much higher concentrations of the lipid hydroperoxide, 15(S)-hydroperoxyeicosatetraenoic acid (15-HPETE), which undergoes homolytic decomposition to ONE. GSH was depleted by ONE treatment in both RMock and R15LO cells, leading to significant increases in their redox potentials. However, R15LO cells had higher GSH concentrations (most likely through increased GSH biosynthesis) and had increased resistance to ONE-mediated GSH depletion than RMock cells. Consequently, R15LO cells had lower reduction potentials at all concentrations of ONE. GSSG concentrations were higher in R15LO cells after ONE treatment when compared with the ONE-treated RMock cells. This suggests that increased expression of 15(S)-HPETE modulates the activity of cellular GSH reductases or the transporters involved in removal of GSSG.

Fjord-region Benzo[g]chrysene-11,12-dihydrodiol and Benzo[c]phenanthrene-3,4-dihydrodiol As Substrates for Rat Liver Dihydrodiol Dehydrogenase (AKR1C9): Structural Basis for Stereochemical Preference

Chemical Research in Toxicology. Mar, 2008  |  Pubmed ID: 18251511

This study demonstrates that benzo[g]chrysene-11,12-dihydrodiol (B[g]C-11,12-dihydrodiol) derived from the fjord-region parent hydrocarbon B[g]C is oxidized by rat AKR1C9 with a k c a t/ K m 100 times greater than that observed with the commonly studied bay-region benzo[ a]pyrene-7,8-dihydrodiol (B[a]P-7,8-dihydrodiol). Conversely, despite its strikingly similar structure to B[ g]C-11,12-dihydrodiol, benzo[ c]phenanthrene-3,4-dihydrodiol (B[ c]Ph-3,4-dihydrodiol) is consumed by AKR1C9 at sluggish rates comparable to those observed with B[ a]P-7,8-dihydrodiol. CD spectroscopy revealed that only the (+)-B[ g]C-11,12-dihydrodiol stereoisomer was oxidized, while AKR1C9 oxidized both stereoisomers of B[a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol. The (+)- S, S- and (-)- R, R-stereoisomers of B[g]C-11,12-dihydrodiol were purified by chiral RP-HPLC. The 11 S,12 S-stereoisomer was oxidized at the same rate as the racemate. The 11 R,12 R-stereoisomer did not act as an inhibitor to AKR1C9, indicating that the (-)- R, R-stereoisomer was excluded from the active site. To understand the basis of stereochemical preference, we screened alanine-scanning mutants of active site residues of AKR1C9. These studies revealed that in comparison to the wild type, F129A, W227A, and Y310A enabled the oxidation of both the B[g]C-11 S,12 S-dihydrodiol and the B[g]C-11 R,12 R-dihydrodiol. Molecular modeling revealed that unlike B[a]P-7,8-dihydrodiol and B[ c]Ph-3,4-dihydrodiol, B[g]C-11,12-dihydrodiol enantiomers are significantly bent out of plane. As a consequence, the (-)- R, R-stereoisomer was prevented from binding to the active site because of unfavorable interactions with F129, W227, or Y310. Additionally, LC/MS validated that the product of the reaction of B[g]C-11,12-dihydrodiol oxidation catalyzed by AKR1C9 was B[g]C-11,12-dione, which was trapped in vitro with the nucleophile 2-mercaptoethanol. The similarity between rates of trans-dihydrodiol oxidation by the rat and human liver specific AKRs (AKR1C9 and AKR1C4) implicate these enzymes in hepatocarcinogenesis in rats observed with the fjord-region PAH.

Synthesis of Dibenzo[def,p]chrysene, Its Active Metabolites, and Their 13C-labeled Analogues

Organic Letters. Mar, 2008  |  Pubmed ID: 18284245

Dibenzo[def,p]chrysene (DBC) is a highly carcinogenic polycyclic aromatic hydrocarbon suspected to be involved in initiation of lung cancer in smokers. Efficient new syntheses of DBC, its active metabolites [DBC diol (1), DBC dione (2), DBC diol epoxide (3)], and their previously unknown 13C2-labeled analogues are reported. The 13C2-labeled analogues are required as standards for sensitive methods of analysis of their DNA adducts in human cells using stable isotope dilution liquid chromatography/tandem mass spectrometry.

DNA Adducts with Lipid Peroxidation Products

The Journal of Biological Chemistry. Jun, 2008  |  Pubmed ID: 18285329

Evidence for the Aldo-keto Reductase Pathway of Polycyclic Aromatic Trans-dihydrodiol Activation in Human Lung A549 Cells

Proceedings of the National Academy of Sciences of the United States of America. May, 2008  |  Pubmed ID: 18474869

Polycyclic aromatic hydrocarbons (PAHs) are tobacco carcinogens implicated in the causation of human lung cancer. Metabolic activation is a key prerequisite for PAHs to cause their deleterious effects. Using human lung adenocarcinoma (A549) cells, we provide evidence for the metabolic activation of (+/-)-trans-7,8dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-trans-dihydrodiol) by aldo-keto reductases (AKRs) to yield benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione), a redox-active o-quinone. We show that B[a]P-7,8-trans-dihydrodiol (AKR substrate) and B[a]P-7,8-dione (AKR product) lead to the production of intracellular reactive oxygen species (ROS) (measured as an increase in dichlorofluorescin diacetate fluores-cence) and that similar changes were not observed with the regioisomer (+/-)-trans-4,5-dihydroxy-4,5-dihydrobenzo[a]pyrene or the diol-epoxide, (+/-)-anti-7,8-dihydroxy-9alpha,10beta-epoxy-7,8,9,10-tetrahydro-B[a]P. B[a]P-7,8-trans-dihydrodiol and B[a]P-7,8-dione also caused a decrease in glutathione levels and an increase in NADP(+)/NADPH ratios, with a concomitant increase in single-strand breaks (as measured by the comet assay) and 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo). The specificity of the comet assay was validated by coupling it to human 8-oxo-guanine glycosylase (hOGG1), which excises 8-oxo-Gua to yield single-strand breaks. The levels of 8-oxo-dGuo observed were confirmed by an immunoaffinity purification stable isotope dilution ([(15)N(5)]-8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry (LC-ESI/MRM/MS) assay. B[a]P-7,8-trans-dihydrodiol produced DNA strand breaks in the hOGG1-coupled comet assay as well as 8-oxo-dGuo (as measured by LC-ESI/MRM/MS) and was enhanced by a catechol O-methyl transferase (COMT) inhibitor, suggesting that COMT protects against o-quinone-mediated redox cycling. We conclude that activation of PAH-trans-dihydrodiols by AKRs in lung cells leads to ROS-mediated genotoxicity and contributes to lung carcinogenesis.

The Pattern of P53 Mutations Caused by PAH O-quinones is Driven by 8-oxo-dGuo Formation While the Spectrum of Mutations is Determined by Biological Selection for Dominance

Chemical Research in Toxicology. May, 2008  |  Pubmed ID: 18489080

PAHs (polycyclic aromatic hydrocarbons) are suspect lung cancer carcinogens that must be metabolically converted into DNA-reactive metabolites. P4501A1/P4501B1 plus epoxide hydrolase activate PAH to (+/-)- anti-benzo[ a]pyrene diol epoxide ((+/-)- anti-BPDE), which causes bulky DNA adducts. Alternatively, aldo-keto reductases (AKRs) convert intermediate PAH trans-dihydrodiols to o-quinones, which cause DNA damage by generating reactive oxygen species (ROS). In lung cancer, the types or pattern of mutations in p53 are predominantly G to T transversions. The locations of these mutations form a distinct spectrum characterized by single point mutations in a number of hotspots located in the DNA binding domain. One route to the G to T transversions is via oxidative DNA damage. An RP-HPLC-ECD assay was used to detect the formation of 8-oxo-dGuo in p53 cDNA exposed to representative quinones, BP-7,8-dione, BA-3,4-dione, and DMBA-3,4-dione under redox cycling conditions. Concurrently, a yeast reporter system was used to detect mutations in the same cDNA samples. Nanomolar concentrations of PAH o-quinones generated 8-oxo-dGuo (detected by HPLC-ECD) in a concentration dependent manner that correlated in a linear fashion with mutagenic frequency. By contrast, micromolar concentrations of (+/-)- anti-BPDE generated (+)- trans- anti-BPDE-N (2)-dGuo adducts (detected by stable-isotope dilution LC/MS methodology) in p53 cDNA that correlated in a linear fashion with mutagenic frequency, but no 8-oxo-dGuo was detected. Previous studies found that mutations observed with PAH o-quinones were predominately G to T transversions and those observed with (+/-)- anti-BPDE were predominately G to C transversions. However, mutations at guanine bases observed with either PAH-treatment occurred randomly throughout the DNA-binding domain of p53. Here, we find that when the mutants were screened for dominance, the dominant mutations clustered at or near hotspots primarily at the protein-DNA interface, whereas the recessive mutations are scattered throughout the DNA binding domain without resembling the spectra observed in cancer. These observations, if extended to mammalian cells, suggest that mutagenesis can drive the pattern of mutations but that biological selection for dominant mutations drives the spectrum of mutations observed in p53 in lung cancer.

Quantification of Key Red Blood Cell Folates from Subjects with Defined MTHFR 677C>T Genotypes Using Stable Isotope Dilution Liquid Chromatography/mass Spectrometry

Rapid Communications in Mass Spectrometry : RCM. Aug, 2008  |  Pubmed ID: 18634122

Red blood cell (RBC) folate levels are established at the time of erythropoiesis and therefore provide a surrogate biomarker for the average folate status of an individual over the preceding four months. Folates are present as folylpolyglutamates, highly polar molecules that cannot be secreted from the RBCs, and must be converted into their monoglutamate forms prior to analysis. This was accomplished using an individual's plasma pteroylpolyglutamate hydrolase by lysing the RBCs in whole blood at pH 5 in the presence of ascorbic acid. Quantitative conversion of formylated tetrahydrofolate derivatives into the stable 5,10-methenyltetrahydrofolate (5,10-MTHF) form was conducted at pH 1.5 in the presence of [(13)C(5)]-5-formyltetrahydrofolate. The resulting [(13)C(5)]-5,10-MTHF was then used as an internal standard for the formylated forms of tetrahydrofolate that had been converted into 5,10-MTHF as well any 5,10-MTHF that had been present in the original sample. A stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry method was validated and then used for the accurate and precise quantification of RBC folic acid, 5-methyltetrahydrofolate (5-MTHF), tetrahydrofolate (THF), and 5,10-MTHF. The method was sensitive and robust and was used to assess the relationship between different methylenetetrahydrofolate reductase (MTHFR) 677C>T genotypes and RBC folate phenotypes. Four distinct RBC folate phenotypes could be identified. These were classified according to the relative amounts of individual RBC folates as type I (5-MTHF >95%; THF <5%; 5,10-MTHF <5%), type II (5-MTHF <95%; THF 5% to 20%; 5,10-MTHF <5%), type III (5-MTHF >55%; THF >20%; 5,10-MTHF >5%), and type IV (5-MTHF <55%; THF >20%; 5,10-MTHF >5%).

Genetic and Biochemical Determinants of Serum Concentrations of Monocyte Chemoattractant Protein-1, a Potential Neural Tube Defect Risk Factor

Birth Defects Research. Part A, Clinical and Molecular Teratology. Oct, 2008  |  Pubmed ID: 18937353

Women with the AA genotype at the (-2518)A>G promoter polymorphism of CCL-2, which encodes the potent pro-inflammatory chemokine monocyte chemoattractant protein 1 (MCP-1), may be at increased risk for having offspring affected by spina bifida. As the A allele at this locus has been associated with decreased transcription of MCP-1 mRNA relative to the G allele, the observed genetic association suggests that the risk of spina bifida may be increased in the offspring of women with low MCP-1 levels. The present study was undertaken to identify potential determinants of MCP-1 levels in women of reproductive age.

Two Distinct Translesion Synthesis Pathways Across a Lipid Peroxidation-derived DNA Adduct in Mammalian Cells

The Journal of Biological Chemistry. Jan, 2009  |  Pubmed ID: 18981176

Translesion DNA synthesis (TLS) of damaged DNA templates is catalyzed by specialized DNA polymerases. To probe the cellular TLS mechanism, a host-vector system consisting of mouse fibroblasts and a replicating plasmid bearing a single DNA adduct was developed. This system was used to explore the TLS mechanism of a heptanone-etheno-dC (H-epsilondC) adduct, an endogenous lesion produced by lipid peroxidation. In wild-type cells, H-epsilondC almost exclusively directed incorporation of dT and dA. Whereas knockout of the Y family TLS polymerase genes, Polh, Polk, or Poli, did not qualitatively affect these TLS events, inactivation of the Rev3 gene coding for a subunit of polymerase zeta or of the Rev1 gene abolished TLS associated with dA, but not dT, insertion. The analysis of results of the cellular studies and in vitro TLS studies using purified polymerases has revealed that the insertion of dA and dT was catalyzed by different polymerases in cells. While insertion of dT can be catalyzed by polymerase eta, kappa, and iota, insertion of dA is catalyzed by an unidentified polymerase that cannot catalyze extension from the resulting dA terminus. Therefore, the extension from this terminus requires the activity of polymerase zeta-REV1. These results provide new insight into how cells use different TLS pathways to overcome a synthesis block.

Quantification of Focal Adhesion Kinase Activation Loop Phosphorylation As a Biomarker of Src Activity

Molecular Pharmacology. Mar, 2009  |  Pubmed ID: 19098120

A recently developed stable isotope dilution liquid chromatography-multiple reaction/mass spectrometry method to quantify focal adhesion kinase (FAK) activation loop phosphorylation was used to study endogenous Src kinase activity. This revealed that bis-phosphorylated pTyr(576)/Tyr(577)-FAK was a biomarker of Src activity and inactivation in vitro and in cell culture. Mouse embryonic fibroblasts (MEFs) expressing endogenous Src family kinases contained 65% unmodified Tyr(576)/Tyr(577), 33% mono-phosphorylated-pTyr(576)-FAK, and 6% bis-phosphorylated-pTyr(576)/pTyr(577)-FAK. In contrast, MEFs expressing oncogenic Y(529)FSrc contained 38% unmodified Tyr(576)/Tyr(577)-FAK, 29% mono-phosphorylated-pTyr(576)-FAK, and 19% bis-phosphorylated-pTyr(576)/pTyr(577)-FAK. This new method has made it possible to accurately determine the absolute amounts of FAK phosphorylation that occur after Src inhibition in cell culture and in vitro with increasing concentrations of the Src inhibitor N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530). Phosphorylation of FAK at Tyr(576)/Tyr(577) was inhibited by AZD0530 in a dose-dependent manner both in cell culture and in vitro. However, there was a substantial difference in the ability of AZD0530 to inhibit Src that was constitutively activated in a cellular context (IC(50) = 2.12 muM) compared with the isolated enzyme (IC(50) = 0.14 muM). When normal MEFs and Y(529)FSrc-expressing MEFs were treated with pervanadate (a global phosphatase inhibitor), pTyr(576)/pTyr(577)-FAK accounted for almost 60% of the total FAK present in the cells. This suggests that activation loop phosphorylation is regulated by tyrosine phosphatases. These results confirm that FAK phosphorylation is a useful biomarker of Src inhibition in vivo. The accuracy and specificity of stable isotope dilution liquid chromatography-mass spectrometry methodology offers significant advantages over current immunochemical approaches for monitoring Src activity.

Synthesis of Phenol and Quinone Metabolites of Benzo[a]pyrene, a Carcinogenic Component of Tobacco Smoke Implicated in Lung Cancer

The Journal of Organic Chemistry. Jan, 2009  |  Pubmed ID: 19132942

Polycyclic aromatic hydrocarbons (PAHs) are widespread environmental pollutants produced in the combustion of organic matter. PAHs are present in automobile exhaust and tobacco smoke, and they have recently been designated as human carcinogens. Current evidence indicates that PAHs are activated enzymatically to mutagenic metabolites that interact with DNA. There is evidence for three pathways of activation: the diol epoxide path, the radical-cation path, and the quinone path. The relative importance of these paths for human lung cancer has not been established. We now report syntheses of the principal phenol and quinone isomers of the prototype PAH carcinogen benzo[a]pyrene (BP) that are known or are suspected to be formed as metabolites of BP in human bronchoalveolar cells. The methods of synthesis were designed to be adaptable to the preparation of the (13)C-labeled analogues of the BP metabolites. These compounds are needed as standards for sensitive LC-MS/MS methods for analysis of BP metabolites formed in lung cells. Efficient novel syntheses of the 1-, 3-, 6-, 9-, and 12-BP phenols and the BP 1,6-, 3,6-, 6,12-, and 9,10-quinones are now reported. The syntheses of the BP phenols (except 6-HO-BP) involve the key steps of Pd-catalyzed Suzuki-Miyaura cross-coupling of a naphthalene boronate ester with a substituted aryl bromide or triflate ester. The BP quinones were synthesized from the corresponding BP phenols by direct oxidation with the hypervalent iodine reagents IBX or TBI. These reagents exhibited different regiospecificities. IBX oxidation of the 7- and 9-BP phenols provided the ortho-quinone isomers (BP 7,8- and 9,10-diones, respectively), whereas TBI oxidation of the 1-, 3-, and 12-BP phenols furnished BP quinone isomers with carbonyl functions in separate rings (BP 1,6-, 3,6-, and 6,12-diones, respectively).

The Up-regulation of Monocyte Chemoattractant Protein-1 (MCP-1) in Ea.hy 926 Endothelial Cells Under Long-term Low Folate Stress is Mediated by the P38 MAPK Pathway

Atherosclerosis. Jul, 2009  |  Pubmed ID: 19152916

Monocyte chemoattractant protein-1 (MCP-1), encoded by the CCL2 gene, plays an important role in the initiation and progression of atherosclerosis. Ea.hy 926 endothelial cells grown under low folate conditions (LO cells) synthesize more MCP-1 mRNA and secrete more MCP-1 protein than folate-replete control cells (HI cells). We investigated the mechanisms underlying the modulation of MCP-1 expression by long-term "folate stress".

Stable Isotope Dilution Multidimensional Liquid Chromatography-tandem Mass Spectrometry for Pancreatic Cancer Serum Biomarker Discovery

Journal of Proteome Research. Mar, 2009  |  Pubmed ID: 19199705

A novel approach to pancreatic cancer biomarker discovery has been developed, which employs a stable isotope labeled proteome (SILAP) standard coupled with extensive multidimensional separation coupled with tandem mass spectrometry (MS/MS). Secreted proteins from CAPAN-2 human pancreatic cancer derived cells were collected after conducting stable isotope labeling by amino acids in cell culture (SILAC). The resulting SILAP standard contained <0.5% of individual unlabeled proteins. Pooled sera from patients with early stage pancreatic cancer or controls were prepared, and an equal amount of the SILAP standard was added to each sample. Proteins were separated by isoelectric focusing (IEF) prior to two-dimensional liquid chromatography (2D-LC)-MS/MS analysis. A total of 1065 proteins were identified of which 121 proteins were present at 1.5-fold or greater concentrations in the sera of patients with pancreatic cancer. ELISA validation of these findings was successfully performed for two proteins, ICAM-1 and BCAM. Results of these studies have provided proof of principle that a SILAP standard derived from the CAPAN-2 secreted proteome can be used in combination with extensive multidimensional LC-MS/MS for the identification and relative quantitation of potential biomarkers of pancreatic cancer. This technique allows for the detection of low-abundance proteins, and focuses only on biologically relevant proteins derived from pancreatic cancer cells.

TNFalpha-dependent Hepatic Steatosis and Liver Degeneration Caused by Mutation of Zebrafish S-adenosylhomocysteine Hydrolase

Development (Cambridge, England). Mar, 2009  |  Pubmed ID: 19201949

Hepatic steatosis and liver degeneration are prominent features of the zebrafish ducttrip (dtp) mutant phenotype. Positional cloning identified a causative mutation in the gene encoding S-adenosylhomocysteine hydrolase (Ahcy). Reduced Ahcy activity in dtp mutants led to elevated levels of S-adenosylhomocysteine (SAH) and, to a lesser degree, of its metabolic precursor S-adenosylmethionine (SAM). Elevated SAH in dtp larvae was associated with mitochondrial defects and increased expression of tnfa and pparg, an ortholog of the mammalian lipogenic gene. Antisense knockdown of tnfa rescued hepatic steatosis and liver degeneration in dtp larvae, whereas the overexpression of tnfa and the hepatic phenotype were unchanged in dtp larvae reared under germ-free conditions. These data identify an essential role for tnfa in the mutant phenotype and suggest a direct link between SAH-induced methylation defects and TNF expression in human liver disorders associated with elevated TNFalpha. Although heterozygous dtp larvae had no discernible phenotype, hepatic steatosis was present in heterozygous adult dtp fish and in wild-type adult fish treated with an Ahcy inhibitor. These data argue that AHCY polymorphisms and AHCY inhibitors, which have shown promise in treating autoimmunity and other disorders, may be a risk factor for steatosis, particularly in patients with diabetes, obesity and liver disorders such as hepatitis C infection. Supporting this idea, hepatic injury and steatosis have been noted in patients with recently discovered AHCY mutations.

Human Cytosolic Hydroxysteroid Dehydrogenases of the Aldo-ketoreductase Superfamily Catalyze Reduction of Conjugated Steroids: Implications for Phase I and Phase II Steroid Hormone Metabolism

The Journal of Biological Chemistry. Apr, 2009  |  Pubmed ID: 19218247

Aldo-ketoreductase 1C (AKR1C) enzymes catalyze the NADPH-dependent reduction of ketosteroids to hydroxysteroids. They are Phase I metabolizing enzymes for natural and synthetic steroid hormones. They convert 5alpha-dihydrotestosterone (Dht, potent androgen) to 3alpha/beta-androstanediols (inactive androgens) and the prodrug tibolone (Tib) to estrogenic 3alpha/beta-hydroxytibolones. Herein we demonstrate for the first time that human AKR1C enzymes (AKR1C1-4) are able to reduce conjugated steroids such as Dht-17beta-glucuronide (DhtG), Dht-17beta-sulfate (DhtS), and Tib-17beta-sulfate (TibS). Product identities were characterized by liquid chromatography-mass spectrometry, and kinetic parameters of the reactions were determined. The product profile of the reduction of each steroid conjugate by the individual AKR1C isoform was similar to that of the corresponding free steroid except for the reduction of DhtG catalyzed by AKR1C2, where a complete inversion in stereochemical preference to 3beta-reduction (with DhtG) from 3alpha-reduction (with Dht and DhtS) was observed. The catalytic efficiency of 3-keto reduction was modestly affected by the presence of a 17-sulfate group but severely impaired by the presence of a 17-glucuronide group for AKR1C1-3 isoforms. AKR1C4, however, showed superior catalytic efficiencies versus the other isoforms, and those were unaffected by steroid conjugation. Our findings provide evidence for alternative pathways of steroid metabolism where the phase I reaction (reduction) occurs after the phase II reaction (conjugation). Specifically, it is indicated that Dht is metabolized to its metabolite 3alpha-androstanediol-17-glucuronide via the previously unrecognized "conjugation pathway" involving the sequential reactions of UGT2B17 and AKR1C4 in liver but via the conventional "reduction pathway" involving the sequential reactions of AKR1C2 and UGT2B15/17 in prostate.

Identification and Quantification of Preterm Birth Biomarkers in Human Cervicovaginal Fluid by Liquid Chromatography/tandem Mass Spectrometry

Journal of Proteome Research. May, 2009  |  Pubmed ID: 19271751

Spontaneous preterm birth (PTB) before 37 completed weeks of gestation resulting from preterm labor (PTL) is a leading contributor of perinatal morbidity and mortality. Early identification of at-risk women by reliable screening tests could alleviate this health issue; however, conventional methods such as obstetric history and clinical risk factors, uterine activity monitoring, biochemical markers, and cervical sonography for screening women at risk for PTB have proven unsuccessful in lowering the rate of PTB. Cervicovaginal fluid (CVF) might prove to be a useful, readily available biological fluid for identifying diagnostic PTB biomarkers. Human columnar epithelial endocervical-1 (End1) and vaginal (Vk2) cell secretomes were employed to generate a stable isotope labeled proteome (SILAP) standard to facilitate characterization and relative quantification of proteins present in CVF. The SILAP standard was prepared using stable isotope labeling by amino acids in cell culture (SILAC) of End1 and Vk2 through seven passages. The labeled secreted proteins from both cell lines were combined and characterized by liquid-chromatography-tandem mass spectrometry (LC-MS/MS). In total, 1211 proteins were identified in the End1-Vk2 SILAP standard, with 236 proteins being consistently identified in each of the replicates analyzed. Individual proteins were found to contain <0.5% of the endogenous unlabeled forms. Identified proteins were screened to provide a set of 15 candidates that have either previously been identified as potential PTB biomarkers or could be linked mechanistically to PTB. Stable isotope dilution LC-multiple reaction monitoring (MRM/MS) assays were then developed for conducting relative quantification of the 15 candidate biomarkers in human CVF samples from term and PTB cases. Three proteins were significantly elevated in PTB cases (desmoplakin isoform 1, stratifin, and thrombospondin 1 precursor), providing a foundation for further validation in larger patient cohorts.

Analysis of 7,8-dihydro-8-oxo-2'-deoxyguanosine in Cellular DNA During Oxidative Stress

Chemical Research in Toxicology. May, 2009  |  Pubmed ID: 19309085

Analysis of cellular 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dGuo) as a biomarker of oxidative DNA damage has been fraught with numerous methodological problems. This is primarily due to artifactual oxidation of dGuo that occurs during DNA isolation and hydrolysis. Therefore, it has become necessary to rely on using the comet assay, which is not necessarily specific for 8-oxo-dGuo. A highly specific and sensitive method based on immunoaffinity purification and stable isotope dilution liquid chromatography (LC)-multiple reaction monitoring (MRM)/mass spectrometry (MS) that avoids artifact formation has now been developed. Cellular DNA was isolated using cold DNAzol (a proprietary product that contains guanidine thiocyanate) instead of chaotropic- or phenol-based methodology. Chelex-treated buffers were used to prevent Fenton chemistry-mediated generation of reactive oxygen species (ROS) and artifactual oxidation of DNA bases. Deferoxamine was also added to all buffers in order to complex any residual transition metal ions remaining after Chelex treatment. The LC-MRM/MS method was used to determine that the basal 8-oxo-dGuo level in DNA from human bronchoalveolar H358 cells was 2.2 +/- 0.4 8-oxo-dGuo/10(7) dGuo (mean +/- standard deviation) or 5.5 +/- 1.0 8-oxo-dGuo/10(8) nucleotides. Similar levels were observed in human lung adenocarcinoma A549 cells, mouse hepatoma Hepa-1c1c7 cells, and human HeLa cervical epithelial adenocarcinoma cells. These values are an order of magnitude lower than is typically reported for basal 8-oxo-dGuo levels in DNA as determined by other MS- or chromatography-based assays. H358 cells were treated with increasing concentrations of potassium bromate (KBrO3) as a positive control or with the methylating agent methyl methanesulfonate (MMS) as a negative control. A linear dose-response for 8-oxo-dGuo formation (r(2) = 0.962) was obtained with increasing concentrations of KBrO3 in the range of 0.05 mM to 2.50 mM. In contrast, no 8-oxo-dGuo was observed in H358 cell DNA after treatment with MMS. At low levels of oxidative DNA damage, there was an excellent correlation between a comet assay that measured DNA single strand breaks (SSBs) after treatment with human 8-oxo-guanine glycosylase-1 (hOGG1) when compared with 8-oxo-dGuo in the DNA as measured by the stable isotope dilution LC-MRM/MS method. Availability of the new LC-MRM/MS assay made it possible to show that the benzo[a]pyrene (B[a]P)-derived quinone, B[a]P-7,8-dione, could induce 8-oxo-dGuo formation in H358 cells. This most likely occurred through redox cycling between B[a]P-7,8-dione and B[a]P-7,8-catechol with concomitant generation of DNA damaging ROS. In keeping with this concept, inhibition of catechol-O-methyl transferase (COMT)-mediated detoxification of B[a]P-7,8-catechol with Ro 410961 caused increased 8-oxo-dGuo formation in the H358 cell DNA.

Overexpression of Human 15(S)-lipoxygenase-1 in RAW Macrophages Leads to Increased Cholesterol Mobilization and Reverse Cholesterol Transport

Arteriosclerosis, Thrombosis, and Vascular Biology. Jun, 2009  |  Pubmed ID: 19325142

The purpose of this study was to determine the effect of 15-lipoxygenase-1 (15-LO-1) on cholesterol mobilization from macrophages.

Absolute Quantification of Phosphorylation on the Kinase Activation Loop of Cellular Focal Adhesion Kinase by Stable Isotope Dilution Liquid Chromatography/mass Spectrometry

Analytical Chemistry. May, 2009  |  Pubmed ID: 19354260

A vital point of convergence for many signaling pathways at cellular focal adhesions is the interaction of two nonreceptor tyrosine kinases, focal adhesion kinase (FAK) and Src. The binding of Src to FAK leads to the phosphorylation of Y(576) and Y(577), located within the activation loop domain of FAK. However, it has not been possible previously to determine the absolute quantitative relationship between phosphorylated and nonphosphorylated forms of this activation loop domain in cells undergoing normal metabolism. We have developed a stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) technique that allows such determinations to be made. Isotopically labeled and phosphorylated FAK protein standards were synthesized and used to control for loss during immunoprecipitation of FAK. A control tryptic peptide, representing an unmodified region of FAK, was employed to monitor the mass balance of post-translational modifications (PTMs) on the activation loop domain. Absolute quantification was conducted using stable isotope labeled peptide standards with four endogenous amino acid overhangs at the trypsin digestion sites of both the amino and carboxy terminus. The LC-MRM/MS method was rigorously validated using in vitro kinase assays and employed to conduct absolute quantification of FAK phosphorylation in normal mouse embryonic fibroblasts (MEFs). This methodology will have particular utility for biomarker studies of kinase-inhibiting anticancer drugs and for quantitative proteomic investigations that examine kinase- and phosphatase-mediated cellular signal transduction pathways.

The Post-synaptic Density of Human Postmortem Brain Tissues: an Experimental Study Paradigm for Neuropsychiatric Illnesses

PloS One. 2009  |  Pubmed ID: 19370153

Recent molecular genetics studies have suggested various trans-synaptic processes for pathophysiologic mechanisms of neuropsychiatric illnesses. Examination of pre- and post-synaptic scaffolds in the brains of patients would greatly aid further investigation, yet such an approach in human postmortem tissue has yet to be tested. We have examined three methods using density gradient based purification of synaptosomes followed by detergent extraction (Method 1) and the pH based differential extraction of synaptic membranes (Methods 2 and 3). All three methods separated fractions from human postmortem brains that were highly enriched in typical PSD proteins, almost to the exclusion of pre-synaptic proteins. We examined these fractions using electron microscopy (EM) and verified the integrity of the synaptic membrane and PSD fractions derived from human postmortem brain tissues. We analyzed protein composition of the PSD fractions using two dimensional liquid chromatography tandem mass spectrometry (2D LC-MS/MS) and observed known PSD proteins by mass spectrometry. Immunoprecipitation and immunoblot studies revealed that expected protein-protein interactions and certain posttranscriptional modulations were maintained in PSD fractions. Our results demonstrate that PSD fractions can be isolated from human postmortem brain tissues with a reasonable degree of integrity. This approach may foster novel postmortem brain research paradigms in which the stoichiometry and protein composition of specific microdomains are examined.

5-Lipoxygenase-mediated Endogenous DNA Damage

The Journal of Biological Chemistry. Jun, 2009  |  Pubmed ID: 19390118

Lipoxygenases (LOs) convert polyunsaturated fatty acids into lipid hydroperoxides. Homolytic decomposition of lipid hydroperoxides gives rise to endogenous genotoxins such as 4-oxo-2(E)-nonenal, which cause the formation of mutagenic DNA adducts. Chiral lipidomics analysis was employed to show that a 5-LO-derived lipid hydroperoxide was responsible for endogenous DNA-adduct formation. The study employed human lymphoblastoid CESS cells, which expressed both 5-LO and the required 5-LO-activating protein (FLAP). The major lipid peroxidation product was 5(S)-hydroperoxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid, which was analyzed as its reduction product, 5(S)-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5(S)-HETE)). Concentrations of 5(S)-HETE increased from 0.07 +/- 0.01 to 45.50 +/- 4.05 pmol/10(7) cells upon stimulation of the CESS cells with calcium ionophore A23187. There was a concomitant increase in the 4-oxo-2(E)-nonenal-derived DNA-adduct, heptanone-etheno-2'-deoxyguanosine (HepsilondGuo) from 2.41 +/- 0.35 to 6.31 +/- 0.73 adducts/10(7) normal bases. Biosynthesis of prostaglandins, 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid, and 15(R,S)-hydroxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid revealed that there was cyclooxygenase (COX) activity in the CESS cells. Western blot analysis revealed that COX-1 was expressed by the cells, but there was no COX-2 or 15-LO-1. FLAP inhibitor reduced HepsilondGuo-adducts and 5(S)-HETE to basal levels. In contrast, aspirin, which had no effect on 5(S)-HETE, blocked the formation of prostaglandins, 15-HETE, and 11-HETE but did not inhibit HepsilondGuo-adduct formation. These data showed that 5-LO was the enzyme responsible for the generation of the HepsilondGuo DNA-adduct in CESS cells.

Folate and Homocysteine Phenotypes: Comparative Findings Using Research and Clinical Laboratory Data

Clinical Biochemistry. Aug, 2009  |  Pubmed ID: 19427846

A low folate/high homocysteine phenotype is associated with several pathologies, including spina bifida and cardiovascular disease. Folate and total homocysteine (tHcy) measurements are used clinically to assess risk and the need for folic acid supplementation and in research to investigate the metabolic basis of disease. Red blood cell (RBC) folate, the best indicator of long-term folate status, is usually measured as "total" folate. However, different folate derivatives support distinct biochemical functions, suggesting a need to develop more precise methods. This study was designed to evaluate a method based on stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS).

15(S)-Lipoxygenase-1 Associates with Neutral Lipid Droplets in Macrophage Foam Cells: Evidence of Lipid Droplet Metabolism

Journal of Lipid Research. Dec, 2009  |  Pubmed ID: 19528634

15(S)-lipoxygenase-1 (15-LO-1) was present in the whole-cell homogenate of an acute human monocytic leukemia cell line (THP-1). Additionally, 15-LO-1 was detected on neutral lipid droplets isolated from THP-1 foam cells. To investigate if 15-LO-1 is active on lipid droplets, we used the mouse leukemic monocytic macrophage cell line (RAW 264.7), which are stably transfected with human 15-LO-1. The RAW 15-LO-1 cells were incubated with acetylated low density lipoprotein to generate foam cells. 15(S)-hydroxyeicosatetraenoic acid [15(S)-HETE], the major 15-LO-1 metabolite of arachidonic acid, was produced in the 15-LO-1 RAW but not in the mock transfected cells when incubated with arachidonic acid. Lipid droplets were isolated from the cells and incubated with arachidonic acid, and production of 15(S)-HETE was measured over 2 h. 15(S)-HETE was produced in the incubations with the lipid droplets, and this production was attenuated when the lipid droplet fraction was subjected to enzyme inactivation through heating. Efflux of 15(S)-HETE from cholesteryl ester-enriched 15-LO RAW cells, when lipid droplets are present, was significantly reduced compared with that from cells enriched with free cholesterol (lipid droplets are absent). We propose that 15-LO-1 is present and functional on cytoplasmic neutral lipid droplets in macrophage foam cells, and these droplets may act to accumulate the anti-inflammatory lipid mediator 15(S)-HETE.

15-oxo-Eicosatetraenoic Acid, a Metabolite of Macrophage 15-hydroxyprostaglandin Dehydrogenase That Inhibits Endothelial Cell Proliferation

Molecular Pharmacology. Sep, 2009  |  Pubmed ID: 19535459

The formation of 15-oxo-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid (15-oxo-ETE) as a product from rabbit lung 15-hydroxyprostaglandin dehydrogenase (PGDH)-mediated oxidation of 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid was first reported more than 30 years ago. However, the pharmacological significance of 15-oxo-ETE formation has never been established. We have now evaluated 15-lipoxygenase (LO)-1-mediated arachidonic acid (AA) metabolism to 15-oxo-ETE in human monocytes and mouse RAW macrophages that stably express human 15-LO-1 (R15L cells). A targeted lipidomics approach was used to identify and quantify the oxidized lipids that were formed. 15-oxo-ETE was found to be a major AA-derived LO metabolite when AA was given exogenously or released from endogenous esterified lipid stores by calcium ionophore (CI) calcimycin (A-23187). This established the R15L cells as a useful in vitro model system. Pretreatment of the R15L cells with cinnamyl-3,4-dihydroxycyanocinnamate significantly inhibited AA- or CI-mediated production of 15(S)-hydroperoxy-5,8,11,13-(Z,Z,Z,E)-eicosatetraenoic acid [15(S)-HETE] and 15-oxo-ETE, confirming the role of 15-LO-1 in mediating AA metabolite formation. Furthermore, 15(S)-HETE was metabolized primarily to 15-oxo-ETE. Pretreatment of the R15L cells with the 15-hydroxyprostaglandin dehydrogenase (PGDH) inhibitor 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) reduced AA- and 15(S)-HETE-mediated formation of 15-oxo-ETE in a dose-dependent manner. This confirmed that macrophage-derived 15-PGDH was responsible for catalyzing the conversion of 15(S)-HETE to 15-oxo-ETE. Finally, 15-oxo-ETE was shown to inhibit the proliferation of human vascular vein endothelial cells by suppressing DNA synthesis, implicating a potential antiangiogenic role. This is the first report describing the biosynthesis of 15-oxo-ETE by macrophage/monocytes and its ability to inhibit endothelial cell proliferation.

A 4-oxo-2(E)-nonenal-derived Glutathione Adduct from 15-lipoxygenase-1-mediated Oxidation of Cytosolic and Esterified Arachidonic Acid

Free Radical Biology & Medicine. Oct, 2009  |  Pubmed ID: 19576981

15(S)-Hydroperoxy-[5Z,8Z,11Z,13E]-eicosatetraenoic acid (15(S)-HpETE) undergoes homolytic decomposition to bifunctional electrophiles such as 4-oxo-2(E)-nonenal. 4-Oxo-2(E)-nonenal reacts with glutathione to form a thiadiazabicyclo-4-oxo-2(E)-nonenal-glutathione adduct (TOG). Therefore, this endogenous glutathione adduct can serve as a specific biomarker of lipid hydroperoxide-mediated 4-oxo-2(E)-nonenal formation. A monocyte/macrophage cell line was generated to constitutively express human 15-lipoxygenase-1. In these cells, TOG was formed from 15(S)-HpETE-derived 4-oxo-2(E)-nonenal in a nonlinear dose-dependent manner upon arachidonic acid treatment. The lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate abolished arachidonic acid-mediated TOG formation. The calcium ionophore A23187 was also used to induce the formation of 15(S)-HpETE from esterified arachidonic acid present in the membrane lipids. In the 15-lipoxygenase-1-expressing cells, the calcium ionophore A23187 significantly increased TOG levels compared with mock-transfected cells. This was due to the 15-lipoxygenase-mediated formation of 15(S)-HpETE in the forms of free fatty acid and esterified lipids, which was subsequently converted to 4-oxo-2(E)-nonenal. The increase in TOG formation was again abrogated by pretreatment with cinnamyl-3,4-dihydroxy-alpha-cyanocinnamate. Only 8.7% 15(S)-HETE (both the free fatty acid and its esterified form in the cell membrane) was formed after ionophore A23187 stimulation compared with that formed after the addition of arachidonic acid. In contrast, the TOG levels after treatment with ionophore A23187 or arachidonic acid were comparable. Thus, it is likely that esterified 15(S)-HpETE underwent homolytic decomposition to 4-oxo-2(E)-nonenal more efficiently than the free 15(S)-HpETE that was formed in the cytosol.

Targeted Chiral Lipidomics Analysis of Bioactive Eicosanoid Lipids in Cellular Systems

BMB Reports. Jul, 2009  |  Pubmed ID: 19643036

We have developed a targeted lipidomics approach that makes it possible to directly analyze chiral eicosanoid lipids generated in cellular systems. The eicosanoids, including prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and alcohols (HETEs), have been implicated as potent lipid mediators of various biological processes. Enzymatic formations of eicosanoids are regioselective and enantioselective, whereas reactive oxygen species (ROS)-mediated formation proceeds with no stereoselectivity. To distinguish between enzymatic and non-enzymatic pathways of eicosanoid formation, it is necessary to resolve enantiomeric forms as well as regioisomers. High sensitivity is also required to analyze the eicosanoid lipids that are usually present as trace amounts (pM level) in biological fluids. A discovery of liquid chromatography-electron capture atmospheric pressure chemical ionization/mass spectrometry (LCECAPCI/MS) allows us to couple normal phase chiral chromatography without loss of sensitivity. Analytical specificity was obtained by the use of collision-induced dissociation (CID) and tandem MS (MS/MS). With combination of stable isotope dilution methodology, complex mixtures of regioisomeric and enantiomeric eicosanoids have been resolved and quantified in biological samples with high sensitivity and specificity. Targeted chiral lipidomics profiles of bioactive eicosanoid lipids obtained from various cell systems and their biological implications have been discussed.

Aryl Hydrocarbon Receptor Facilitates DNA Strand Breaks and 8-oxo-2'-deoxyguanosine Formation by the Aldo-keto Reductase Product Benzo[a]pyrene-7,8-dione

The Journal of Biological Chemistry. Oct, 2009  |  Pubmed ID: 19726680

Polycyclic aromatic hydrocarbon (PAH) o-quinones produced by aldo-keto reductases are ligands for the aryl hydrocarbon receptor (AhR) (Burczynski, M. E., and Penning, T. M. (2000) Cancer Res. 60, 908-915). They induce oxidative DNA lesions (reactive oxygen species-mediated DNA strand breaks and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dGuo) formation) in human lung cells. We tested whether the AhR enhances PAH o-quinone-mediated oxidative DNA damage by translocating these ligands to the nucleus. Using the single cell gel electrophoresis (comet) assay to detect DNA strand breaks in murine hepatoma Hepa1c1c7 cells and its AhR- and aryl hydrocarbon receptor nuclear translocator-deficient variants, benzo[a]pyrene-7,8-dione (B[a]P-7,8-dione) produced fewer DNA strand breaks in AhR-deficient cells compared with aryl hydrocarbon receptor nuclear translocator-deficient and wild type Hepa1c1c7 cells. Decreased DNA strand breaks were also observed in human bronchoalveolar H358 cells in which the AhR was silenced by siRNA. The antioxidant alpha-tocopherol and the iron chelator/antioxidant desferal decreased the formation of B[a]P-7,8-dione-mediated DNA strand breaks indicating that they were reactive oxygen species-dependent. By coupling the comet assay to 8-oxoguanine glycosylase (hOGG1), which excises 8-oxo-Gua, strand breaks dependent upon this lesion were measured. hOGG1 treatment produced more DNA single strand breaks in B[a]P-7,8-dione-treated Hepa cells and H358 cells than in its absence. The levels of hOGG1-dependent DNA strand breaks mediated by B[a]P-7,8-dione were lower in AhR-deficient Hepa and AhR knockdown H358 cells. The AhR antagonist alpha-naphthoflavone also attenuated B[a]P-7,8-dione-mediated DNA strand breaks. The decrease in 8-oxo-dGuo levels in AhR-deficient Hepa cells and AhR knockdown H358 cells was validated by immunoaffinity capture stable isotope dilution ([(15)N(5)]8-oxo-dGuo) liquid chromatography-electrospray ionization/multiple reaction monitoring/mass spectrometry. We conclude that the AhR shuttles PAH o-quinone genotoxins to the nucleus and enhances oxidative DNA damage.

Differential Secreted Proteome Approach in Murine Model for Candidate Biomarker Discovery in Colon Cancer

Journal of Proteome Research. Nov, 2009  |  Pubmed ID: 19769411

The complexity and heterogeneity of the plasma proteome have presented significant challenges in the identification of protein changes associated with tumor development. We used cell culture as a model system and identified differentially expressed, secreted proteins which may constitute serological biomarkers. A stable isotope labeling by amino acids in cell culture (SILAC) approach was used to label the entire secreted proteomes of the CT26 murine colon cancer cell line and normal young adult mouse colon (YAMC) cell line, thereby creating a stable isotope labeled proteome (SILAP) standard. This SILAP standard was added to unlabeled murine CT26 colon cancer cell or normal murine YAMC colon epithelial cell secreted proteome samples. A multidimensional approach combining isoelectric focusing (IEF), strong cation exchange (SCX) followed by reversed phase liquid chromatography was used for extensive protein and peptide separation. A total of 614 and 929 proteins were identified from the YAMC and CT26 cell lines, with 418 proteins common to both cell lines. Twenty highly abundant differentially expressed proteins from these groups were selected for liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) analysis in sera. Differential secretion into the serum was observed for several proteins when Apc(min) mice were compared with control mice. These findings were then confirmed by Western blot analysis.

12/15-lipoxygenase-dependent Myeloid Production of Interleukin-12 is Essential for Resistance to Chronic Toxoplasmosis

Infection and Immunity. Dec, 2009  |  Pubmed ID: 19822654

Interleukin-12 (IL-12) is critical for resistance to Toxoplasma gondii during both the acute and chronic stages of infection. However, the cellular and molecular pathways that regulate IL-12 production during chronic toxoplasmosis are incompletely defined. We recently discovered that 12/15-lipoxygenase (12/15-LOX), which oxidizes unsaturated lipids in macrophages, is a novel and selective regulator of IL-12 production. We now demonstrate the essential role of this enzyme in the chronic phase of toxoplasmosis. Although 12/15-LOX-deficient mice were resistant to acute T. gondii infection, 80% of 12/15-LOX-deficient mice died during chronic toxoplasmosis, compared to no deaths in wild-type controls. The morbidity of chronically infected 12/15-LOX mice was associated with an increase in brain inflammation and parasite burden. These data suggest that the evolution of the immune response to T. gondii is accompanied by an increasing requirement for 12/15-LOX-mediated signaling. Consistent with this conclusion, 12/15-LOX activity was enhanced during chronic, but not acute, toxoplasmosis. Furthermore, the enhanced susceptibility of 12/15-LOX-deficient mice to chronic toxoplasmosis was associated with reduced production of IL-12 and gamma interferon (IFN-gamma) that was not evident during acute infection. Importantly, ex vivo IFN-gamma production by 12/15-LOX-deficient splenocytes could be rescued by the addition of recombinant IL-12. These data establish that 12/15-LOX is a critical mediator of the chronic type 1 inflammatory response and that immune mediators can be subject to distinct cellular and/or molecular mechanisms of regulation at different stages of inflammation.

Secondhand Smoke Inhibits Both Cl- and K+ Conductances in Normal Human Bronchial Epithelial Cells

Respiratory Research. 2009  |  Pubmed ID: 19943936

Secondhand smoke (SHS) exposure is an independent risk factor for asthma, rhinosinusitis, and more severe respiratory tract infections in children and adults. Impaired mucociliary clearance with subsequent mucus retention contributes to the pathophysiology of each of these diseases, suggesting that altered epithelial salt and water transport may play an etiological role. To test the hypothesis that SHS would alter epithelial ion transport, we designed a system for in vitro exposure of mature, well-differentiated human bronchial epithelial cells to SHS. We show that SHS exposure inhibits cAMP-stimulated, bumetanide-sensitive anion secretion by 25 to 40% in a time-dependent fashion in these cells. Increasing the amount of carbon monoxide to 100 ppm from 5 ppm did not increase the amount of inhibition, and filtering SHS reduced inhibition significantly. It was determined that SHS inhibited cAMP-dependent apical membrane chloride conductance by 25% and Ba2+-sensitive basolateral membrane potassium conductance by 50%. These data confirm previous findings that cigarette smoke inhibits chloride secretion in a novel model of smoke exposure designed to mimic SHS exposure. They also extend previous findings to demonstrate an effect on basolateral K+ conductance. Therefore, pharmacological agents that increase either apical membrane chloride conductance or basolateral membrane potassium conductance might be of therapeutic benefit in patients with diseases related to SHS exposure.

Analysis of Endogenous Glutathione-adducts and Their Metabolites

Biomedical Chromatography : BMC. Jan, 2010  |  Pubmed ID: 20017120

The ability to conduct validated analyses of glutathione (GSH)-adducts and their metabolites is critically important in order to establish whether they play a role in cellular biochemical or pathophysiological processes. The use of stable isotope dilution (SID) methodology in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) provides the highest bioanalytical specificity possible for such analyses. Quantitative studies normally require the high sensitivity that can be obtained by the use of multiple reaction monitoring (MRM)/MS rather than the much less sensitive but more specific full scanning methodology. The method employs a parent ion corresponding to the intact molecule together with a prominent product ion that obtained by collision induced dissociation. Using SID LC-MRM/MS, analytes must have the same relative LC retention time to the heavy isotope internal standard established during the validation procedure, the correct parent ion and the correct product ion. This level of specificity cannot be attained with any other bioanalytical technique employed for biomarker analysis. This review will describe the application of SID LC-MR/MS methodology for the analysis of GSH-adducts and their metabolites. It will also discuss potential future directions for the use of this methodology for rigorous determination of their utility as disease and exposure biomarkers.

Aldo-keto Reductase 1C3 Expression in MCF-7 Cells Reveals Roles in Steroid Hormone and Prostaglandin Metabolism That May Explain Its Over-expression in Breast Cancer

The Journal of Steroid Biochemistry and Molecular Biology. Feb, 2010  |  Pubmed ID: 20036328

Aldo-keto reductase (AKR) 1C3 (type 5 17beta-hydroxysteroid dehydrogenase and prostaglandin F synthase), may stimulate proliferation via steroid hormone and prostaglandin (PG) metabolism in the breast. Purified recombinant AKR1C3 reduces PGD(2) to 9alpha,11beta-PGF(2), Delta(4)-androstenedione to testosterone, progesterone to 20alpha-hydroxyprogesterone, and to a lesser extent, estrone to 17beta-estradiol. We established MCF-7 cells that stably express AKR1C3 (MCF-7-AKR1C3 cells) to model its over-expression in breast cancer. AKR1C3 expression increased steroid conversion by MCF-7 cells, leading to a pro-estrogenic state. Unexpectedly, estrone was reduced fastest by MCF-7-AKR1C3 cells when compared to other substrates at 0.1muM. MCF-7-AKR1C3 cells proliferated three times faster than parental cells in response to estrone and 17beta-estradiol. AKR1C3 therefore represents a potential target for attenuating estrogen receptor alpha induced proliferation. MCF-7-AKR1C3 cells also reduced PGD(2), limiting its dehydration to form PGJ(2) products. The AKR1C3 product was confirmed as 9alpha,11beta-PGF(2) and quantified with a stereospecific stable isotope dilution liquid chromatography-mass spectrometry method. This method will allow the examination of the role of AKR1C3 in endogenous prostaglandin formation in response to inflammatory stimuli. Expression of AKR1C3 reduced the anti-proliferative effects of PGD(2) on MCF-7 cells, suggesting that AKR1C3 limits peroxisome proliferator activated receptor gamma (PPARgamma) signaling by reducing formation of 15-deoxy-Delta(12,14)-PGJ(2) (15dPGJ(2)).

Liquid Chromatography-mass Spectrometry (LC-MS) of Steroid Hormone Metabolites and Its Applications

The Journal of Steroid Biochemistry and Molecular Biology. Aug, 2010  |  Pubmed ID: 20083198

Advances in liquid chromatography-mass spectrometry (LC-MS) can be used to measure steroid hormone metabolites in vitro and in vivo. We find that LC-electrospray ionization (ESI)-MS using a LCQ ion trap mass spectrometer in the negative ion mode can be used to monitor the product profile that results from 5alpha-dihydrotestosterone (DHT)-17beta-glucuronide, DHT-17beta-sulfate, and tibolone-17beta-sulfate reduction catalyzed by human members of the aldo-keto reductase (AKR) 1C subfamily and assign kinetic constants to these reactions. We also developed a stable isotope dilution LC-electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the quantitative analysis of estrone (E1) and its metabolites as pentafluorobenzyl (PFB) derivatives in human plasma in the attomole range. The limit of detection for E1-PFB was 740attomole on column. Separations can be performed using normal-phase LC because ionization takes place in the gas phase rather than in solution. This permits efficient separation of the regioisomeric 2- and 4-methoxy-E1. The method was validated for the simultaneous analysis of plasma E2 and its metabolites: 2-methoxy-E2, 4-methoxy-E2, 16alpha-hydroxy-E2, estrone (E1), 2-methoxy-E1, 4-methoxy-EI, and 16alpha-hydroxy-E1 from 5pg/mL to 2000pg/mL. Our LC-MS methods have sufficient sensitivity to detect steroid hormone levels in prostate and breast tumors and should aid their molecular diagnosis and treatment.

Analysis of Estrogens in Serum and Plasma from Postmenopausal Women: Past Present, and Future

Steroids. Apr, 2010  |  Pubmed ID: 20109478

Previous studies have shown that the selection of women who are at high breast cancer risk for treatment with chemoprevention agents leads to an enhanced benefit/risk ratio. However, further efforts to implement this strategy will require the development of new models to predict the breast cancer risk of particular individuals. Postmenopausal women with elevated plasma or serum estrogens are at increased risk for breast cancer. Therefore, the roles of various enzymes involved in the biosynthesis of estrogens in postmenopausal women have been reviewed in detail. In addition, the potential genotoxic and/or proliferative effects of the different estrogen metabolites as risk factors in the etiology of breast cancer have been examined. Unfortunately, much of the current bioanalytical methodology employed for the analysis of plasma and serum estrogens has proved to be problematic. Major advances in risk assessment would be possible if reliable methodology were available to quantify estradiol and its major metabolites in the plasma or serum of postmenopausal women. High performance liquid chromatography (HPLC) coupled with radioimmunoassay (RIA) currently provides the most sensitive and best validated immunoassay method for the analysis of estrone and estradiol in serum samples from postmenopausal women. However, inter-individual differences in specificity observed with many other immunoassays have caused significant problems when interpreting epidemiologic studies of breast cancer. It is almost impossible to overcome the inherent assay problems involved in using RIA-based methodology, particularly for multiple estrogens. For reliable measurements of multiple estrogens in plasma or serum, it will be necessary to employ stable isotope dilution methodology in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS). Extremely high sensitivity can be obtained with pre-ionized estrogen derivatives when employed in combination with a modern triple quadrupole mass spectrometer and nanoflow LC. Using [(13)C(6)]-estrone as the internal standard it has proved possible to analyze estrone as its pre-ionized Girard T (GT) derivative in sub-fg (low amol) amounts on column. This suggests that in the future it will be possible to routinely conduct LC-MS assays of multiple estrogen metabolites in serum and plasma at even lower concentrations than the current lower limit of quantitation of 0.4pg/mL (1.6pmol/L). The ease with which the pre-ionization derivatization strategy can be implemented will make it possible to readily introduce high sensitivity stable isotope dilution methodology in laboratories that are currently employing LC-MS/MS methodology. This will help conserve important plasma and serum samples as it will be possible to conduct high sensitivity analyses using low sample volumes.

A 13-Oxo-9,10-epoxytridecenoate Phospholipid Analogue of the Genotoxic 4,5-epoxy-2E-decenal: Detection in Vivo, Chemical Synthesis, and Adduction with DNA

Chemical Research in Toxicology. Mar, 2010  |  Pubmed ID: 20131875

Often guided by analogy with nonphospholipid products from oxidative cleavage of polyunsaturated fatty acids, we previously identified a variety of biologically active oxidatively truncated phospholipids. Previously, 4,5-epoxy-2(E)-decenal (4,5-EDE) was found to be produced by oxidative cleavage of 13-(S)-hydroperoxy-9,11-(Z,E)-octadeca-dienoic acid (13-HPODE). 4,5-EDE reacts with deoxy-adenosine (dAdo) and deoxy-guanosine (dGuo) to form mutagenic etheno derivatives. We hypothesized that a functionally similar and potentially mutagenic compound, that is, 13-oxo-9,10-epoxytridecenoic acid (OETA), would be generated from 9-HPODE through an analogous fragmentation. We expected that an ester of 2-lysophosphatidylcoline (PC), OETA-PC, would be produced by oxidative cleavage of 9-HPODE-PC in biological membranes. An efficient, unambiguous total synthesis of trans-OETA-PC was first executed to provide a standard that could facilitate the identification of this phospholipid epoxyalkenal that was shown to be produced during oxidation of the linoleic acid ester of 2-lysoPC. Finally, trans-OETA-PC was detected in a lipid extract from rat retina. The identity of the naturally occurring oxidatively truncated phospholipid was further confirmed by derivatization with methoxylamine that produced characteristic mono and bis adducts. The average amount of trans-OETA-PC in rat retina, 0.33 pmol, is relatively low as compared to other oxidatively truncated PCs, for example, the 4-hydroxy-7-oxohept-5-enoic acid PC ester (2.5 pmol) or the 4-keto-7-oxohept-5-enoic acid PC ester (1.7 pmol), derived from the docosahexaenoic acid ester of 2-lysoPC. This, most likely, is because docosahexaenoate PCs are particularly abundant in the retina as compared to the linoleate PC ester precursor of OETA-PC. As predicted by analogy with 4,5-EDE, OETA-PC reacts with dAdo and dGuo, as well as with DNA, to form mutagenic etheno adducts.

Stable-isotope Dilution LC–MS for Quantitative Biomarker Analysis

Bioanalysis. Feb, 2010  |  Pubmed ID: 20352077

The ability to conduct validated analyses of biomarkers is critically important in order to establish the sensitivity and selectivity of the biomarker in identifying a particular disease. The use of stable-isotope dilution (SID) methodology in combination with LC–MS/MS provides the highest possible analytical specificity for quantitative determinations. This methodology is now widely used in the discovery and validation of putative exposure and disease biomarkers. This review will describe the application of SID LC–MS methodology for the analysis of small-molecule and protein biomarkers. It will also discuss potential future directions for the use of this methodology for rigorous biomarker analysis.

Hematopoietic Stem Cell Function Requires 12/15-lipoxygenase-dependent Fatty Acid Metabolism

Blood. Jun, 2010  |  Pubmed ID: 20357242

Fatty acid metabolism governs multiple intracellular signaling pathways in many cell types, but its role in hematopoietic stem cells (HSCs) is largely unknown. Herein, we establish a critical role for 12/15-lipoxygenase (12/15-LOX)-mediated unsaturated fatty acid metabolism in HSC function. HSCs from 12/15-LOX-deficient mice are severely compromised in their capacity to reconstitute the hematopoietic compartment in competitive and serial reconstitution assays. Furthermore, we demonstrate that 12/15-LOX is required for the maintenance of long-term HSC quiescence and number. The defect in HSCs is cell-autonomous and associated with a selective reduction in 12/15-LOX-mediated generation of bioactive lipid mediators and reactive oxygen species and with a decrease in canonical Wnt signaling as measured by nuclear beta-catenin staining. These results have implications for development, aging, and transformation of the hematopoietic compartment.

20-HETE Mediates Ozone-induced, Neutrophil-independent Airway Hyper-responsiveness in Mice

PloS One. 2010  |  Pubmed ID: 20422032

Ozone, a pollutant known to induce airway hyper-responsiveness (AHR), increases morbidity and mortality in patients with obstructive airway diseases and asthma. We postulate oxidized lipids mediate in vivo ozone-induced AHR in murine airways.

Genetic and Lifestyle Variables Associated with Homocysteine Concentrations and the Distribution of Folate Derivatives in Healthy Premenopausal Women

Birth Defects Research. Part A, Clinical and Molecular Teratology. Aug, 2010  |  Pubmed ID: 20544798

Low folate and high homocysteine (Hcy) concentrations are associated with pregnancy-related pathologies such as spina bifida. Polymorphisms in folate/Hcy metabolic enzymes may contribute to this potentially pathogenic biochemical phenotype.

Interferon Regulatory Factor-8-driven Myeloid Differentiation is Regulated by 12/15-lipoxygenase-mediated Redox Signaling

Experimental Hematology. Nov, 2010  |  Pubmed ID: 20647030

Several transcription factors determine the cell fate decision between granulocytes and monocytes, but the upstream signal transduction pathways that govern myelopoiesis are largely unknown. Based on our observation of aberrant myeloid cell representation in hematopoietic tissues of 12/15-lipoxygenase (12/15-LOX)-deficient (Alox15) mice, we tested the hypothesis that polyunsaturated fatty acid metabolism regulates myelopoiesis.

Analysis of Epoxyeicosatrienoic Acids by Chiral Liquid Chromatography/electron Capture Atmospheric Pressure Chemical Ionization Mass Spectrometry Using [13C]-analog Internal Standards

Rapid Communications in Mass Spectrometry : RCM. Nov, 2010  |  Pubmed ID: 20972997

The metabolism of arachidonic acid (AA) to epoxyeicosatrienoic acids (EETs) is thought to be mediated primarily by the cytochromes P450 (P450s) from the 2 family (2C9, 2C19, 2D6, and 2J2). In contrast, P450s of the 4 family are primarily involved in omega oxidation of AA (4A11 and 4A22). The ability to determine enantioselective formation of the regioisomeric EETs is important in order to establish their potential biological activities and to asses which P450 isoforms are involved in their formation. It has been extremely difficult to analyze individual EET enantiomers in biological fluids because they are present in only trace amounts and they are extremely difficult to separate from each other. In addition, the deuterium-labeled internal standards that are commonly used for stable isotope dilution liquid chromatography/mass spectrometry (LC/MS) analyses have different LC retention times when compared with the corresponding protium forms. Therefore, quantification by LC/MS-based methodology can be compromised by differential suppression of ionization of the closely eluting isomers. We report the preparation of [(13)C(20)]-EET analog internal standards and the use of a validated high-sensitivity chiral LC/electron capture atmospheric pressure chemical ionization (ECAPCI)-MS method for the trace analysis of endogenous EETs as their pentafluorobenzyl (PFB) ester derivatives. The assay was then used to show the exquisite enantioselectivity of P4502C19-, P4502D6-, P4501A1-, and P4501B1-mediated conversion of AA into EETs and to quantify the enantioselective formation of EETs produced by AA metabolism in a mouse epithelial hepatoma (Hepa) cell line.

Regulation of Benzo[a]pyrene-mediated DNA- and Glutathione-adduct Formation by 2,3,7,8-tetrachlorodibenzo-p-dioxin in Human Lung Cells

Chemical Research in Toxicology. Jan, 2011  |  Pubmed ID: 21028851

Environmental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), require metabolic activation to DNA-reactive metabolites in order to exert their tumorigenic effects. Benzo[a]pyrene (B[a]P), a prototypic PAH, is metabolized by cytochrome P450 (P450) 1A1/1B1 and epoxide hydrolase to (-)-B[a]P-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol). B[a]P-7,8-dihydrodiol then undergoes further P4501A1/1B1-mediated metabolism to the ultimate carcinogen, (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydro-B[a]P (B[a]PDE), which forms DNA-adducts primarily with 2'-deoxyguanosine (dGuo) to form (+)-anti-trans-B[a]PDE-N(2)-dGuo (B[a]PDE-dGuo) in DNA. Pretreatment of cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to induce P4501A1/1B1 mRNA expression through the aryl hydrocarbon receptor (AhR) pathway. This causes increased B[a]PDE-dGuo formation in liver cells. In contrast, TCDD induction of H358 lung cells surprisingly caused a decrease in (-)-B[a]P-7,8-dihydrodiol-mediated (+)-B[a]PDE-dGuo adduct formation when compared with the non-TCDD-induced cells. Furthermore, treatment of the TCDD-induced cells with (±)-B[a]PDE also resulted in decreased (+)-B[a]PDE-dGuo adduct formation when compared with the non-TCDD-induced cells. These data suggested that it was a detoxification pathway that had been up-regulated rather than an activation pathway that had been down-regulated. LC-MS was used to analyze B[a]PDE-dGuo and B[a]PDE-GSH-adducts in H358 lung and HepG2 liver cells. There was a significant increase in the (-)-B[a]PDE-GSH-adduct with high enantiomeric excess after treatment of the TCDD-induced H358 cells with (±)-B[a]PDE when compared with the noninduced cells. This could explain why increased activation of (-)-B[a]P-7,8-dihydrodiol through TCDD up-regulation of P4501A1/1B1 did not lead to increased (+)-B[a]PDE-dGuo adducts in the H358 lung cells. In addition, TCDD did not induce B[a]PDE-GSH-adduct formation in HepG2 liver cells. (±)-B[a]PDE-GSH-adducts were formed at much lower levels in both TCDD-induced and noninduced HepG2 cells when compared with (-)-B[a]PDE-GSH-adducts in the H358 lung cells. Therefore, our study has revealed that there is a subtle balance between activation and detoxification of B[a]P in lung-derived compared with liver-derived cells and that this determines how much DNA damage occurs.

Thermal Stability of Bimatoprost, Latanoprost, and Travoprost Under Simulated Daily Use

Journal of Ocular Pharmacology and Therapeutics : the Official Journal of the Association for Ocular Pharmacology and Therapeutics. Feb, 2011  |  Pubmed ID: 21117945

To determine the stability of bimatoprost, latanoprost, and travoprost under conditions of simulated daily use and varying degrees of thermal stress.

A New Liquid Chromatography/mass Spectrometry Method for 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in Urine

Rapid Communications in Mass Spectrometry : RCM. Jan, 2011  |  Pubmed ID: 21154658

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic nitrosamine produced upon curing tobacco. It is present in tobacco smoke and undergoes metabolism to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) in the lungs. NNAL undergoes further uridine diphosphate glucuronosyltransferase (UGT)-mediated metabolism to give N- and O-glucuronide metabolites, which together with free (non-conjugated) NNAL are then excreted in the urine. The ability to conduct validated analyses of free and conjugated NNAL in human urine is important in order to assess inter-individual differences in lung cancer risk from exposure to cigarette smoke. The use of stable isotope dilution (SID) methodology in combination with liquid chromatography/multiple reaction monitoring/mass spectrometry (LC/MRM-MS) provides the highest bioanalytical specificity possible for such analyses. We describe a novel derivatization procedure, which results in the formation of a pre-ionized N-propyl-NNAL derivative. The increased LC/MS sensitivity arising from this derivative then makes it possible to analyze free NNAL in only 0.25 mL urine. This substantial reduction in urine volume when compared with other methods that have been developed will help preserve the limited amounts of stored urine samples that are available from on-going longitudinal biomarker studies. The new high sensitivity SID LC/MRM-MS assay was employed to determine free and conjugated NNAL concentrations in urine samples from 60 individual disease-free smokers. Effects of inter-individual differences in urinary creatinine clearance on NNAL concentrations were then assessed and three metabolizer phenotypes were identified in the 60 subjects from the ratio of urinary NNAL glucuronides/free NNAL. Poor metabolizers (PMs, 14 subjects) with a ratio of NNAL glucuronides/free NNAL <2 (mean = 1.3), intermediate metabolizers (IMs, 36 subjects) with a ratio between 2 and 5 (mean = 3.4), and extensive metabolizers (EMs, 10 subjects) with a ratio >5 (mean = 11.1).

Prediction of Postoperative Recurrence-free Survival in Non-small Cell Lung Cancer by Using an Internationally Validated Gene Expression Model

Clinical Cancer Research : an Official Journal of the American Association for Cancer Research. May, 2011  |  Pubmed ID: 21242119

This study was performed to discover prognostic genomic markers associated with postoperative outcome of stage I to III non-small cell lung cancer (NSCLC) that are reproducible between geographically distant and demographically distinct patient populations.

Stable Isotope Labeling by Essential Nutrients in Cell Culture for Preparation of Labeled Coenzyme A and Its Thioesters

Analytical Chemistry. Feb, 2011  |  Pubmed ID: 21268609

Stable isotope dilution mass spectrometry (MS) represents the gold standard for quantification of endogenously formed cellular metabolites. Although coenzyme A (CoA) and acyl-CoA thioester derivatives are central players in numerous metabolic pathways, the lack of a commercially available isotopically labeled CoA limits the development of rigorous MS-based methods. In this study, we adapted stable isotope labeling by amino acids in cell culture (SILAC) methodology to biosynthetically generate stable isotope labeled CoA and thioester analogues for use as internal standards in liquid chromatography/multiple reaction monitoring mass spectrometry (LC/MRM-MS) assays. This was accomplished by incubating murine hepatocytes (Hepa 1c1c7) in media in which pantothenate (a precursor of CoA) was replaced with [(13)C(3)(15)N(1)]-pantothenate. Efficient incorporation into various CoA species was optimized to >99% [(13)C(3)(15)N(1)]-pantothenate after three passages of the murine cells in culture. Charcoal-dextran-stripped fetal bovine serum (FBS) was found to be more efficient for serum supplementation than dialyzed or undialyzed FBS, due to lower contaminating unlabeled pantothenate content. Stable isotope labeled CoA species were extracted and utilized as internal standards for CoA thioester analysis in cell culture models. This methodology of stable isotope labeling by essential nutrients in cell culture (SILEC) can serve as a paradigm for using vitamins and other essential nutrients to generate stable isotope standards that cannot be readily synthesized.

CYP3A4 Mediates Growth of Estrogen Receptor-positive Breast Cancer Cells in Part by Inducing Nuclear Translocation of Phospho-Stat3 Through Biosynthesis of (±)-14,15-epoxyeicosatrienoic Acid (EET)

The Journal of Biological Chemistry. May, 2011  |  Pubmed ID: 21402692

CYP3A4 expression in breast cancer correlates with decreased overall survival, but the mechanisms are unknown. Cytochrome P450 gene profiling by RNAi silencing demonstrates that CYP3A or 2C8 gene expression is specifically required for growth of the breast cancer lines MCF7, T47D, and MDA-MB-231. CYP3A4 silencing blocks the cell cycle at the G(2)/M checkpoint and induces apoptosis in the MCF7 line, thereby inhibiting anchorage-dependent growth and survival. CYP3A4 was profiled for NADPH-dependent arachidonic acid (AA) metabolism and synthesized AA epoxygenase products (±)-8,9-, (±)-11,12-, and (±)-14,15-epoxyeicosatrienoic acid (EET) (total turnover of ∼2 pmol/pmol CYP3A4/min) but not hydroxylase products (±)-15-, (±)-19-, or 20-hydroxyeicosatetraenoic acid. Furthermore, eicosanoid profiling revealed that MCF7 cells synthesize EETs in a CYP3A4-dependent manner. The (±)-14,15-EET regioisomer selectively rescues breast cancer cells from CYP3A4 silencing in a concentration-dependent fashion and promotes mitogenesis and anchorage-dependent cloning. Stat3 (Tyr-705) phosphorylation was inhibited by CYP3A4 silencing, providing a potential mechanism for CYP3A4 involvement in breast cancer cell growth. Silencing Stat3 blocks breast cancer cell growth and abrogates (±)-14,15-EET-induced proliferation, indicating a Stat3 requirement for (±)-14,15-EET-mediated cell growth. Although silencing of CYP3A4 reduces nuclear Tyr(P)-705-Stat3, (±)-14,15-EET restores this signaling process and promotes Tyr(P)-705-Stat3 translocation to the nucleus, suggesting that (±)-14,15-EET may be involved in an autocrine/paracrine pathway driving cell growth. These studies indicate that CYP3A4 is a highly active AA epoxygenase that promotes Stat3-mediated breast cancer cell growth in part through (±)-14,15-EET biosynthesis. Furthermore, these studies indicate an essential role for Stat3 as a mediator of epoxygenase activity in breast cancer.

Nicotine Exposure and Metabolizer Phenotypes from Analysis of Urinary Nicotine and Its 15 Metabolites by LC-MS

Bioanalysis. Apr, 2011  |  Pubmed ID: 21452992

Smokers who inhale less deeply are exposed to lower amounts of the toxic substances present in tobacco smoke. In order to more rigorously assess tobacco smoke exposure, it is necessary to have an accurate method for quantifying nicotine and all of its known metabolites.

Liquid Chromatography/mass Spectrometry of Pre-ionized Girard P Derivatives for Quantifying Estrone and Its Metabolites in Serum from Postmenopausal Women

Rapid Communications in Mass Spectrometry : RCM. May, 2011  |  Pubmed ID: 21488127

An ultrasensitive stable isotope dilution liquid chromatography/selected reaction monitoring/mass spectrometry (LC/SRM/MS) assay has been developed for serum estrone, 16α-hydroxyestrone, 4-methoxyestrone, and 2- methoxyestrone. The enhanced sensitivity was obtained by the use of Girard P (GP) pre-ionized derivatives coupled with microflow LC. The limit of detection for each estrogen using 0.5 mL of serum was 0.156 pg/mL and linear standard curves were obtained up to 20 pg/mL. Serum samples from 20 postmenopausal women (10 lifetime non-smokers and 10 current smokers) were analyzed using this new assay. Mean serum concentrations of estrone and 2-methoxyestrone were 14.06 pg/mL (±1.56 pg/mL) and 3.30 pg/mL (±1.00 pg/mL), respectively, for the 20 subjects enrolled in the study. The mean estrone concentration determined by our ultrasensitive and highly specific assay was significantly lower than that reported for the control groups in most previous breast cancer studies of postmenopausal women. In addition (and contrary to many reports) serum 16α-hydroxyestrone was not detected in any of the subjects, and 4-methoxyestrone was detected in only one of the subjects. Furthermore, there were no significant differences in the mean serum concentrations of estrone and 2-methoxyestrone or the ratio of serum 2- methoxyestrone to estrone between the non-smoking and smoking groups. Interestingly, the one subject with measurable serum 4-methoxyestrone (2.3 pg/mL) had the lowest estrone and 2-methoxyestrone concentrations. Using this assay it will now be possible to obtain definitive information on the levels of serum estrone, 4-methoxyestrone, and 2-methoxyestrone in studies of cancer risk using small serum volumes available from previous epidemiology studies.

Analysis of the Human Pancreatic Stellate Cell Secreted Proteome

Pancreas. May, 2011  |  Pubmed ID: 21499210

Pancreatic stellate cells (PSCs) are important players in pancreatic fibrosis and are major contributors to the extracellular matrix proteins observed with the stromal response characteristic of pancreatic ductal adenocarcinoma (PDAC). Pancreatic stellate cells are also believed to secrete soluble factors that promote tumor progression; however, no comprehensive analysis of the PSC proteome in either the quiescent or the activated state has been reported.

Stereospecific Reduction of 5β-reduced Steroids by Human Ketosteroid Reductases of the AKR (aldo-keto Reductase) Superfamily: Role of AKR1C1-AKR1C4 in the Metabolism of Testosterone and Progesterone Via the 5β-reductase Pathway

The Biochemical Journal. Jul, 2011  |  Pubmed ID: 21521174

Active sex hormones such as testosterone and progesterone are metabolized to tetrahydrosteroids in the liver to terminate hormone action. One main metabolic pathway, the 5β-pathway, involves 5β-steroid reductase (AKR1D1, where AKR refers to the aldo-keto reductase superfamily), which catalyses the reduction of the 4-ene structure, and ketosteroid reductases (AKR1C1-AKR1C4), which catalyse the subsequent reduction of the 3-oxo group. The activities of the four human AKR1C enzymes on 5β-dihydrotestosterone, 5β-pregnane-3,20-dione and 20α-hydroxy-5β-pregnan-3-one, the intermediate 5β-dihydrosteroids on the 5β-pathway of testosterone and progesterone metabolism, were investigated. Product characterization by liquid chromatography-MS revealed that the reduction of the 3-oxo group of the three steroids predominantly favoured the formation of the corresponding 3α-hydroxy steroids. The stereochemistry was explained by molecular docking. Kinetic properties of the enzymes identified AKR1C4 as the major enzyme responsible for the hepatic formation of 5β-tetrahydrosteroid of testosterone, but indicated differential routes and roles of human AKR1C for the hepatic formation of 5β-tetrahydrosteroids of progesterone. Comparison of the kinetics of the AKR1C1-AKR1C4-catalysed reactions with those of AKR1D1 suggested that the three intermediate 5β-dihydrosteroids derived from testosterone and progesterone are unlikely to accumulate in liver, and that the identities and levels of 5β-reduced metabolites formed in peripheral tissues will be governed by the local expression of AKR1D1 and AKR1C1-AKR1C3.

Detoxication of Structurally Diverse Polycyclic Aromatic Hydrocarbon (PAH) O-quinones by Human Recombinant Catechol-O-methyltransferase (COMT) Via O-methylation of PAH Catechols

The Journal of Biological Chemistry. Jul, 2011  |  Pubmed ID: 21622560

Polycyclic aromatic hydrocarbons (PAH) are environmental and tobacco carcinogens. Metabolic activation of intermediate PAH trans-dihydrodiols by aldo-keto reductases (AKRs) leads to the formation of electrophilic and redox-active o-quinones. We investigated whether O-methylation by human recombinant soluble catechol-O-methyltransferase (S-COMT) is a feasible detoxication step for a panel of structurally diverse PAH-catechols produced during the redox-cycling process. Classes of PAH non-K-region o-quinones (bay region, methylated bay region, and fjord region o-quinones) produced by AKRs were employed in the studies. PAH o-quinones were reduced to the corresponding catechols by dithiothreitol under anaerobic conditions and then further O-methylated by human S-COMT in the presence of S-[³H]adenosyl-l-methionine as a methyl group donor. The formation of the O-methylated catechols was detected by HPLC-UV coupled with in-line radiometric detection, and unlabeled products were also characterized by LC-MS/MS. Human S-COMT was able to catalyze O-methylation of all of the PAH-catechols and generated two isomeric metabolites in different proportions. LC-MS/MS showed that each isomer was a mono-O-methylated metabolite. ¹H NMR was used to assign the predominant positional isomer of benzo[a]pyrene-7,8-catechol as the O-8-monomethylated catechol. The catalytic efficiency (k(cat)/K(m)) varied among different classes of PAH-catechols by 500-fold. The ability of S-COMT to produce two isomeric products from PAH-catechols was rationalized using the crystal structure of the enzyme. We provide evidence that O-8-monomethylated benzo[a]pyrene-7,8-catechol is formed in three different human lung cell lines. It is concluded that human S-COMT may play a critical role in the detoxication of PAH o-quinones generated by AKRs.

Oncogene-induced Nrf2 Transcription Promotes ROS Detoxification and Tumorigenesis

Nature. Jul, 2011  |  Pubmed ID: 21734707

Reactive oxygen species (ROS) are mutagenic and may thereby promote cancer. Normally, ROS levels are tightly controlled by an inducible antioxidant program that responds to cellular stressors and is predominantly regulated by the transcription factor Nrf2 (also known as Nfe2l2) and its repressor protein Keap1 (refs 2-5). In contrast to the acute physiological regulation of Nrf2, in neoplasia there is evidence for increased basal activation of Nrf2. Indeed, somatic mutations that disrupt the Nrf2-Keap1 interaction to stabilize Nrf2 and increase the constitutive transcription of Nrf2 target genes were recently identified, indicating that enhanced ROS detoxification and additional Nrf2 functions may in fact be pro-tumorigenic. Here, we investigated ROS metabolism in primary murine cells following the expression of endogenous oncogenic alleles of Kras, Braf and Myc, and found that ROS are actively suppressed by these oncogenes. K-Ras(G12D), B-Raf(V619E) and Myc(ERT2) each increased the transcription of Nrf2 to stably elevate the basal Nrf2 antioxidant program and thereby lower intracellular ROS and confer a more reduced intracellular environment. Oncogene-directed increased expression of Nrf2 is a new mechanism for the activation of the Nrf2 antioxidant program, and is evident in primary cells and tissues of mice expressing K-Ras(G12D) and B-Raf(V619E), and in human pancreatic cancer. Furthermore, genetic targeting of the Nrf2 pathway impairs K-Ras(G12D)-induced proliferation and tumorigenesis in vivo. Thus, the Nrf2 antioxidant and cellular detoxification program represents a previously unappreciated mediator of oncogenesis.

11-Oxoeicosatetraenoic Acid is a Cyclooxygenase-2/15-hydroxyprostaglandin Dehydrogenase-derived Antiproliferative Eicosanoid

Chemical Research in Toxicology. Dec, 2011  |  Pubmed ID: 21916491

Previously, we established that 11(R)-hydroxy-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (HETE) was a significant cyclooxygenase (COX)-2-derived arachidonic acid (AA) metabolite in epithelial cells. Stable isotope dilution chiral liquid chromatography (LC)-electron capture atmospheric pressure chemical ionization (ECAPCI)/mass spectrometry (MS) was used to quantify COX-2-derived eicosanoids in the human colorectal adenocarcinoma (LoVo) epithelial cell line, which expresses both COX-2 and 15-hydroxyprostaglandin dehydrogenase (15-PGDH). 11(R)-HETE secretion reached peak concentrations within minutes after AA addition before rapidly diminishing, suggesting further metabolism had occurred. Surprisingly, recombinant 15-PGDH, which is normally specific for oxidation of eicosanoid 15(S)-hydroxyl groups, was found to convert 11(R)-HETE to 11-oxo-5,8,12,14-(Z,Z,E,Z)-eicosatetraenoic acid (ETE). Furthermore, LoVo cell lysates converted 11(R)-HETE to 11-oxo-ETE and inhibition of 15-PGDH with 5-[[4-(ethoxycarbonyl)phenyl]azo]-2-hydroxy-benzeneacetic acid (CAY10397) (50 μM) significantly suppressed endogenous 11-oxo-ETE production with a corresponding increase in 11(R)-HETE. These data confirmed COX-2 and 15-PGDH as enzymes responsible for 11-oxo-ETE biosynthesis. Finally, addition of AA to the LoVo cells resulted in rapid secretion of 11-oxo-ETE into the media, reaching peak levels within 20 min of starting the incubation. This was followed by a sharp decrease in 11-oxo-ETE levels. Glutathione (GSH) S-transferase (GST) was found to metabolize 11-oxo-ETE to the 11-oxo-ETE-GSH (OEG)-adduct in LoVo cells, as confirmed by LC-MS/MS analysis. Bromodeoxyuridine (BrdU)-based cell proliferation assays in human umbilical vein endothelial cells (HUVECs) revealed that the half-maximal inhibitory concentration (IC(50)) of 11-oxo-ETE for inhibition of HUVEC proliferation was 2.1 μM. These results show that 11-oxo-ETE is a novel COX-2/15-PGDH-derived eicosanoid, which inhibits endothelial cell proliferation with a potency that is similar to that observed for 15d-PGJ(2).

Rotenone-mediated Changes in Intracellular Coenzyme A Thioester Levels: Implications for Mitochondrial Dysfunction

Chemical Research in Toxicology. Oct, 2011  |  Pubmed ID: 21950265

Rotenone, an organic pesticide and potent mitochondrial complex I inhibitor, causes Parkinson-like neurodegeneration in rodents and is implicated in human Parkinson's disease. In this rapid report, rotenone induced a dose-dependent decrease in succinyl-coenzyme A (CoA) and increase in β-hydroxybutyryl-CoA in multiple human cell lines (IC(50) < 100 nM). Rotenone also inhibited [U-(13)C(6)]-glucose-derived [(13)C]-acetyl-CoA and [(13)C]-succinyl-CoA biosynthesis in SH-SY5Y neuroblastoma cells. These changes are compatible with a compensatory metabolic rearrangement. Stable isotope dilution liquid chromatography-mass spectrometry and CoA thioester isotopomer analysis provided insight into mechanisms of rotenone toxicity, which will facilitate the development of new biomarkers of mitochondrial dysfunction.

Quantitation of Benzo[a]pyrene Metabolic Profiles in Human Bronchoalveolar (H358) Cells by Stable Isotope Dilution Liquid Chromatography-atmospheric Pressure Chemical Ionization Mass Spectrometry

Chemical Research in Toxicology. Nov, 2011  |  Pubmed ID: 21962213

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental pollutants and are carcinogenic in multiple organs and species. Benzo[a]pyrene (B[a]P) is a representative PAH and has been studied extensively for its carcinogenicity and toxicity. B[a]P itself is chemically inert and requires metabolic activation to exhibit its toxicity and carcinogenicity. Three major metabolic pathways have been well documented. The signature metabolites generated from the radical cation (peroxidase or monooxygenase mediated) pathway are B[a]P-1,6-dione and B[a]P-3,6-dione, the signature metabolite generated from the diol-epoxide (P450 mediated) pathway is B[a]P-r-7,t-8,t-9,c-10-tetrahydrotetrol (B[a]P-tetrol-1), and the signature metabolite generated from the o-quinone (aldo-keto reductase mediated) pathway is B[a]P-7,8-dione. The contributions of these different metabolic pathways to cancer initiation and the exploitation of this information for cancer prevention are still under debate. With the availability of a library of [(13)C(4)]-labeled B[a]P metabolite internal standards, we developed a sensitive stable isotope dilution atmospheric pressure chemical ionization tandem mass spectrometry method to address this issue by quantitating B[a]P metabolites from each metabolic pathway in human lung cells. This analytical method represents a 500-fold increased sensitivity compared with that of a method using HPLC-radiometric detection. The limit of quantitation (LOQ) was determined to be 6 fmol on column for 3-hydroxybenzo[a]pyrene (3-OH-B[a]P), the generally accepted biomarker for B[a]P exposure. This high level of sensitivity and robustness of the method was demonstrated in a study of B[a]P metabolic profiles in human bronchoalveolar H358 cells induced or uninduced with the AhR ligand, 2,3,7,8-tetrachlorodibenzodioxin (TCDD). All the signature metabolites were detected and successfully quantitated. Our results suggest that all three metabolic pathways contribute equally in the overall metabolism of B[a]P in H358 cells with or without TCDD induction. The sensitivity of the method should permit the identification of cell-type differences in B[a]P activation and detoxication and could also be used for biomonitoring human exposure to PAH.

Deciphering the Human Platelet Sheddome

Blood. Jan, 2011  |  Pubmed ID: 20962327

Activated platelets shed surface proteins, potentially modifying platelet function as well as providing a source of bioactive fragments. Previous studies have identified several constituents of the platelet sheddome, but the full extent of shedding is unknown. Here we have taken a global approach, analyzing protein fragments in the supernate of activated platelets using mass spectroscopy and looking for proteins originating from platelet membranes. After removing plasma proteins and microparticles, 1048 proteins were identified, including 69 membrane proteins. Nearly all of the membrane proteins had been detected previously, but only 10 had been shown to be shed in platelets. The remaining 59 are candidates subject to confirmation. Based on spectral counts, protein representation in the sheddome varies considerably. As proof of principle, we validated one of the less frequently detected proteins, semaphorin 7A, which had not previously been identified in platelets. Surface expression, cleavage, and shedding of semaphorin 7A were demonstrated, as was its association with α-granules. Finally, cleavage of semaphorin 7A and 12 other proteins was substantially reduced by an inhibitor of ADAM17, a known sheddase. These results define a subset of membrane proteins as sheddome candidates, forming the basis for further studies examining the impact of ectodomain shedding on platelet function.

Multidrug Resistance Protein (MRP) 4 Attenuates Benzo[a]pyrene-mediated DNA-adduct Formation in Human Bronchoalveolar H358 Cells

Toxicology Letters. Feb, 2012  |  Pubmed ID: 22155354

Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad substrate specificity. It facilitates the transport of bile salt conjugates, conjugated steroids, nucleoside analogs, eicosanoids, and cardiovascular drugs. Recent studies in liver carcinoma cells and hepatocytes showed that MRP4 expression is regulated by the aryl hydrocarbon receptor (AhR) and nuclear factor E2-related factor 2 (Nrf2). The AhR has particular importance in the lung and is most commonly associated with the up-regulation of cytochrome P-450 (CYP)-mediated metabolism of benzo[a]pyrene (B[a]P) to reactive intermediates. Treatment of H358, human bronchoalveolar, cells with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or (-)-benzo[a]pyrene-7,8-dihydro-7,8-diol (B[a]P-7,8-dihydrodiol), the proximate carcinogen of B[a]P, revealed that MRP4 expression was increased compared to control. This suggested that MRP4 expression might contribute to the paradoxical decrease in (+)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene-2'-deoxyguanosine ((+)-anti-trans-B[a]PDE-dGuo) DNA-adducts observed in TCDD-treated H358 cells. We have now found that decreased MRP4 expression induced by a short hairpin RNA (shRNA), or chemical inhibition with probenecid, increased (+)-anti-trans-B[a]PDE-dGuo formation in cells treated with (-)-B[a]P-7,8-dihydrodiol, but not the ultimate carcinogen (+)-anti-trans-B[a]PDE. Thus, up-regulation of MRP4 increased cellular efflux of (-)-B[a]P-7,8-dihydrodiol, which attenuated DNA-adduct formation. This is the first report identifying a specific MRP efflux transporter that decreases DNA damage arising from an environmental carcinogen.

SILEC: a Protocol for Generating and Using Isotopically Labeled Coenzyme A Mass Spectrometry Standards

Nature Protocols. Jan, 2012  |  Pubmed ID: 22157971

Stable isotope labeling by essential nutrients in cell culture (SILEC) was recently developed to generate isotopically labeled coenzyme A (CoA) and short-chain acyl-CoA thioesters. This was accomplished by modifying the widely used technique of stable isotope labeling by amino acids in cell culture to include [(13)C(3)(15)N]-pantothenate (vitamin B(5)), a CoA precursor, instead of the isotopically labeled amino acids. The lack of a de novo pantothenate synthesis pathway allowed for efficient and near-complete labeling of the measured CoA species. This protocol provides a step-by-step approach for generating stable isotope-labeled short-chain acyl-CoA internal standards in mammalian and insect cells as well as instructions on how to use them in stable isotope dilution mass spectrometric-based analyses. Troubleshooting guidelines, as well as a list of unlabeled and labeled CoA species, are also included. This protocol represents a prototype for generating stable isotope internal standards from labeled essential nutrients such as pantothenate. The generation and use of SILEC standards takes approximately 2-3 weeks.

Relative Quantification of Serum Proteins from Pancreatic Ductal Adenocarcinoma Patients by Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry

Journal of Proteome Research. Feb, 2012  |  Pubmed ID: 22264027

We report an innovative multiplexed liquidchromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS)-based assay for rapidly measuring a large number of disease specific protein biomarkers in human serum. Furthermore, this approach uses stable isotope dilution methodology to reliably quantify candidate protein biomarkers. Human serum was diluted using a stable isotope labeled proteome (SILAP) standard prepared from the secretome of pancreatic cell lines, subjected to immunoaffinity removal of the most highly abundant proteins, trypsin digested, and analyzed by LC-MRM/MS. The method was found to be precise, linear, and specific for the relative quantification of 72 proteins when analyte response was normalized to the relevant internal standard (IS) from the SILAP. The method made it possible to determine statistically different concentrations for three proteins (cystatin M, IGF binding protein 7, and villin 2) in control and pancreatic cancer patient samples. This method proves the feasibility of using a SILAP standard in combination with stable isotope dilution LC-MRM/MS analysis of tryptic peptides to compare changes in the concentration of candidate protein biomarkers in human serum.