Stacy Gelhaus - Academia.edu (original) (raw)
Papers by Stacy Gelhaus
Chemical Research in Toxicology
Nitric Oxide, 2014
Unsaturated fatty acids are metabolized to reactive products that can act as pro-or antiinflammat... more Unsaturated fatty acids are metabolized to reactive products that can act as pro-or antiinflammatory signaling mediators. Electrophilic fatty acid species, including nitro-and oxocontaining fatty acids, display salutary anti-inflammatory and metabolic actions. Electrophilicity can be conferred by both enzymatic and oxidative reactions, via the homolytic addition of nitrogen dioxide to a double bond or via the formation of α,β-unsaturated carbonyl and epoxide substituents. The endogenous formation of electrophilic fatty acids is significant and influenced by diet, metabolic, and inflammatory reactions. Transcriptional regulatory proteins and enzymes can sense the redox status of the surrounding environment upon electrophilic fatty acid adduction of functionally significant, nucleophilic cysteines. Through this covalent and often reversible posttranslational modification, gene expression and metabolic responses are induced. At low concentrations, the pleiotropic signaling actions that are regulated by these protein targets suggest that some classes of electrophilic lipids may be useful for treating metabolic and inflammatory diseases.
![Research paper thumbnail of 15-Hydroxyprostaglandin Dehydrogenase Generates Anti-Inflammatory [Omega]-3 Fatty Acid Derivatives](https://attachments.academia-assets.com/39185499/thumbnails/1.jpg)
](Free Radical Biology and Medicine, 2013
Background: 15-Hydroxyprostaglandin dehydrogenase (15PGDH) catalyzes the oxidation of hydroxylate... more Background: 15-Hydroxyprostaglandin dehydrogenase (15PGDH) catalyzes the oxidation of hydroxylated polyunsaturated fatty acids to ␣,-unsaturated carbonyl-containing electrophiles. Results: Hydroxylated docosahexaenoic acid (DHA) metabolites are substrates for 15PGDH, yielding electrophilic oxoDHA products that are anti-inflammatory. Conclusion: Hydroxylated ⍀-3 fatty acid species are conferred with cell signaling capabilities after oxidation by 15PGDH. Significance: Formation of biologically active metabolites by 15PGDH contributes to the salutary signaling actions of ⍀-3 fatty acids.
Journal of Visualized Experiments, 2011
The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in t... more The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers . These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer . Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs) . Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
Smart Medical and Biomedical Sensor Technology II, 2004
ABSTRACT DNA damage is caused by a variety of foreign and endogenous compounds. There are endogen... more ABSTRACT DNA damage is caused by a variety of foreign and endogenous compounds. There are endogenous photosensitizers in cells, such as porphyrins and flavins, which may create damage in the presence of UV-A light. Typically, samples are analyzed by 32P-postlabelling and electrophoretic separation or by LC-MS separation and detection. Separation by HPLC is common; however, in all instances, the DNA sample is hydrolyzed down to nucleosides prior to analysis. It will be shown here that ion-pairing reversed phase high performance liquid chromatography (IP-RPLC) has the ability to provide biophysical information concerning the sites of UV-A induced photosensitizer damage on an intact oligonucleotide concurrent with the separation. IP-RPLC is less labor intensive and faster than electrophoretic methods and it is less costly than LC-MS. IP-RPLC can also be used to purify modified oligonucleotides for further use and analysis. This technique is sensitive to the charge, conformation, and sequence characteristics of the nucleic acid sample and may be used to determine the damage or modifications made to DNA by a variety of compounds.
Analytical Instrumentation Handbook, Second Edition, 2004
![Research paper thumbnail of Multidrug resistance protein (MRP) 4 attenuates benzo[a]pyrene-mediated DNA-adduct formation in human bronchoalveolar H358 cells](https://attachments.academia-assets.com/39185494/thumbnails/1.jpg)
](Toxicology Letters, 2012
Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad subst... more 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-7,8,9,10tetrahydrobenzo[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
Rapid Communications in Mass Spectrometry, 2011
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic nitrosamine produced upon ... more 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 Oglucuronide 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).
Nucleic Acids Research, 2003
A new method for rapid puri®cation and structural analysis of oligoribonucleotides of 19 and 20 n... more A new method for rapid puri®cation and structural analysis of oligoribonucleotides of 19 and 20 nt is applied to RNA hairpins SL3 and SL2, which are stable secondary structures present on the y recognition element of HIV-1. This approach uses ion-pairing reversed-phase liquid chromatography (IP-RPLC) to achieve the separation of the stem± loop from the transcription mix. Evidence is presented that IP-RPLC is sensitive to the different conformers of these secondary structures. The purity of each stem±loop was con®rmed by mass spectrometry and PAGE. IP-RPLC puri®cation was found to be superior to PAGE in terms of time, safety and, most importantly, purity.
Journal of Chromatography B, 2005
A group of 18-mers of the same base sequence, but with differing alkyl modifications is used to i... more A group of 18-mers of the same base sequence, but with differing alkyl modifications is used to investigate effects of these modifications on retention of oligonucleotides using ion-pairing reversed-phase liquid chromatography (IP-RPLC). It is shown that IP-RPLC is able to distinguish between oligonucleotides differing only by a single alkyl group. The identity of the nucleobase and position and length of the alkyl adduct affect retention of the oligonucleotide. These separation phenomena result from changes in charge and hydrophobicity upon alkylation. As demonstrated in this paper; chromatographic selectivity, unique to IP-RPLC, greatly facilitates the purification process of modified oligonucleotides.
Journal of Allergy and Clinical Immunology, 2014
Fatty acids and consequently diet play an essential role in the formation of inflammatory mediato... more Fatty acids and consequently diet play an essential role in the formation of inflammatory mediators involved in the pathogenesis of asthma. Because intake variations of omega-6 (n-6) and omega-3 (n-3) fatty acids ultimately determine cell membrane incorporation, changes in diet have the potential to modify downstream production of inflammatory mediators derived from these compounds. It has long been hypothesized that decreasing the n-6/n-3 ratio could reduce the production of more proinflammatory mediators while increasing the formation of downstream metabolites that can serve to limit or resolve inflammation. In turn, these changes would result in improved asthma outcomes or would lower the risk for asthma incidence. This review will focus on the role of fatty acid inflammatory and resolving mediators and will summarize the clinical and epidemiologic data on how diet and obesity alter fatty acid profiles that can contribute to asthma. (J Allergy Clin Immunol 2014;133:1255-64.)
Journal of Allergy and Clinical Immunology, 2013
Background: Bronchoalveolar lavage (BAL) fluid prostaglandin D 2 (PGD 2 ) levels are increased in... more Background: Bronchoalveolar lavage (BAL) fluid prostaglandin D 2 (PGD 2 ) levels are increased in patients with severe, poorly controlled asthma in association with epithelial mast cells (MCs). PGD 2 , which is generated by hematopoietic prostaglandin D synthase (HPGDS), acts on 3 G proteincoupled receptors, including chemoattractant receptorhomologous molecule expressed on T H 2 lymphocytes (CRTH2) and PGD 2 receptor 1 (DP1). However, much remains to be understood regarding the presence and activation of these pathway elements in asthmatic patients. Objective: We sought to compare the expression and activation of PGD 2 pathway elements in bronchoscopically obtained samples from healthy control subjects and asthmatic patients across a range of disease severity and control, as well as in relation to T H 2 pathway elements. Methods: Epithelial cells and BAL fluid were evaluated for HPGDS (quantitative real-time PCR/immunohistochemistry [IHC]) and PGD 2 (ELISA/liquid chromatography mass spectrometry) in relation to levels of MC proteases. Expression of the 2 inflammatory cell receptors DP1 and CRTH2 was evaluated on luminal cells. These PGD 2 pathway markers were then compared with asthma severity, level of control, and markers of T H 2 inflammation (blood eosinophils and fraction of exhaled nitric oxide). Results: Confirming previous results, BAL fluid PGD 2 levels were highest in patients with severe asthma (overall P 5 .0001). Epithelial cell compartment HPGDS mRNA and IHC values differed among groups (P 5 .008 and P < .0001, respectively) and correlated with MC protease mRNA. CRTH2 mRNA and IHC values were highest in patients with severe asthma (P 5 .001 and P 5 .0001, respectively). Asthma exacerbations, poor asthma control, and T H 2 inflammatory markers were associated with higher PGD 2 , HPGDS, and CRTH2 levels. Conclusion: The current study identifies coordinated upregulation of the PGD 2 pathway in patients with severe, poorly controlled, T H 2-high asthma despite corticosteroid use. (J Allergy Clin Immunol 2013;131:1504-12.)
Free Radical Biology and Medicine, 2013
Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. T... more Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. The discovery of peroxynitrite and peroxidase-induced nitration of biomolecules led to the initial reports of endogenous nitrated fatty acids. These species increase during ischemia/reperfusion, but concentrations are often at or near the limits of detection. Here, we describe multiple methods for nitrated fatty acid synthesis and sample extraction from complex biological matrices and a rigorous method of qualitative and quantitative detection of nitrated fatty acids by liquid chromatography-mass spectrometry. In addition, optimized instrument conditions and caveats regarding data interpretation are discussed.
Chemical Research in Toxicology, 2008
Figure 6 was published in error without panels A, B, and C being labeled. The corrected legend wo... more Figure 6 was published in error without panels A, B, and C being labeled. The corrected legend would read: Figure 6. Mutations mapped onto the structure of p53. (Top) The locations of the top 10 hotspots in lung cancer are shown in red. (Middle) The 11 unique PAH o-quinone derived dominant mutations from are shown in red (213, 239, 244, 246, 251, 256, 273, 275, 276, 279, 281). The PAH o-quinone derived recessive mutations are shown in green The 8 unique BPDE derived dominant mutations are shown in red (156, 158, 176, 178, 196, 213, 279, 283). Note that the dominant mutations cluster in DNA contact regions. Structures were plotted using the online software of the IARC TP53 database (3).
Chemical Research in Toxicology, 2003
The effects of N 2 -ethylGua, O 6 -ethylGua, and O 6 -methylGua adducts in template DNA on polyme... more The effects of N 2 -ethylGua, O 6 -ethylGua, and O 6 -methylGua adducts in template DNA on polymerization by mammalian DNA polymerases R and η have been investigated. The N 2 -ethylGua adduct blocks polymerization by the replicative DNA polymerase R to a much greater extent than does the O 6 -ethyl-or the O 6 -methylGua adducts. The DNA polymerase η efficiently and accurately bypasses the N 2 -ethylGua lesion but like DNA polymerase R is similarly blocked by the O 6 -ethyl-or the O 6 -methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N 2 -ethylGua and the O 6 -ethylGua adducts by the DNA polymerases R and η and extension from 3′-termini positioned opposite these adducts was performed to measure the efficiency and the accuracy of DNA synthesis past these lesions. This analysis showed that insertion of Cyt opposite the N 2 -ethylGua adduct by DNA polymerase R is approximately 10 4 -fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N 2 -ethylGua:Cyt 3′-terminus by DNA polymerase R is approximately 10 3 -fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N 2 -ethylGua lesion by the DNA polymerase η is about 370fold more efficient than by the DNA polymerase R, and extension from the N 2 -ethylGua:Cyt 3′-terminus by the DNA polymerase η is about 3-fold more efficient than by the DNA polymerase R. Furthermore, the DNA polymerase η preferably inserts the correct nucleotide Cyt opposite the N 2 -ethylGua lesion with nearly the same level of accuracy as opposite an unadducted Gua, thus minimizing the mutagentic potential of this lesion. This result contrasts with the relatively high misincorporation efficiency of Thy opposite the O 6 -ethylGua adduct by the DNA polymerases R and η. In reactions containing both DNA polymerases R and η, synthesis past the N 2 -ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N 2 -ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase R and lesion bypass catalyzed by DNA polymerase η.
![Research paper thumbnail of Aldo−Keto Reductase- and Cytochrome P450-Dependent Formation of Benzo[ a ]pyrene-Derived DNA Adducts in Human Bronchoalveolar Cells](https://attachments.academia-assets.com/42397869/thumbnails/1.jpg)
](Chemical Research in Toxicology, 2007
There is substantial evidence to suggest that polycyclic aromatic hydrocarbons (PAHs) such as ben... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-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.
![Research paper thumbnail of Regulation of Benzo[ a ]pyrene-Mediated DNA- and Glutathione-Adduct Formation by 2,3,7,8-Tetrachlorodibenzo- p -dioxin in Human Lung Cells](https://attachments.academia-assets.com/39185486/thumbnails/1.jpg)
](Chemical Research in Toxicology, 2011
Environmental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), require metabolic act... more 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-7,8,9,10tetrahydro-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 2,3,7,8tetrachlorodibenzo-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-7,8dihydrodiol-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-7,8dihydrodiol 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.
Chemical Research in Toxicology, 2008
PAHs (polycyclic aromatic hydrocarbons) are suspect lung cancer carcinogens that must be metaboli... more 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.
Chemical Research in Toxicology
Nitric Oxide, 2014
Unsaturated fatty acids are metabolized to reactive products that can act as pro-or antiinflammat... more Unsaturated fatty acids are metabolized to reactive products that can act as pro-or antiinflammatory signaling mediators. Electrophilic fatty acid species, including nitro-and oxocontaining fatty acids, display salutary anti-inflammatory and metabolic actions. Electrophilicity can be conferred by both enzymatic and oxidative reactions, via the homolytic addition of nitrogen dioxide to a double bond or via the formation of α,β-unsaturated carbonyl and epoxide substituents. The endogenous formation of electrophilic fatty acids is significant and influenced by diet, metabolic, and inflammatory reactions. Transcriptional regulatory proteins and enzymes can sense the redox status of the surrounding environment upon electrophilic fatty acid adduction of functionally significant, nucleophilic cysteines. Through this covalent and often reversible posttranslational modification, gene expression and metabolic responses are induced. At low concentrations, the pleiotropic signaling actions that are regulated by these protein targets suggest that some classes of electrophilic lipids may be useful for treating metabolic and inflammatory diseases.
Free Radical Biology and Medicine, 2013
Background: 15-Hydroxyprostaglandin dehydrogenase (15PGDH) catalyzes the oxidation of hydroxylate... more Background: 15-Hydroxyprostaglandin dehydrogenase (15PGDH) catalyzes the oxidation of hydroxylated polyunsaturated fatty acids to ␣,-unsaturated carbonyl-containing electrophiles. Results: Hydroxylated docosahexaenoic acid (DHA) metabolites are substrates for 15PGDH, yielding electrophilic oxoDHA products that are anti-inflammatory. Conclusion: Hydroxylated ⍀-3 fatty acid species are conferred with cell signaling capabilities after oxidation by 15PGDH. Significance: Formation of biologically active metabolites by 15PGDH contributes to the salutary signaling actions of ⍀-3 fatty acids.
Journal of Visualized Experiments, 2011
The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in t... more The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers . These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer . Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs) . Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
Smart Medical and Biomedical Sensor Technology II, 2004
ABSTRACT DNA damage is caused by a variety of foreign and endogenous compounds. There are endogen... more ABSTRACT DNA damage is caused by a variety of foreign and endogenous compounds. There are endogenous photosensitizers in cells, such as porphyrins and flavins, which may create damage in the presence of UV-A light. Typically, samples are analyzed by 32P-postlabelling and electrophoretic separation or by LC-MS separation and detection. Separation by HPLC is common; however, in all instances, the DNA sample is hydrolyzed down to nucleosides prior to analysis. It will be shown here that ion-pairing reversed phase high performance liquid chromatography (IP-RPLC) has the ability to provide biophysical information concerning the sites of UV-A induced photosensitizer damage on an intact oligonucleotide concurrent with the separation. IP-RPLC is less labor intensive and faster than electrophoretic methods and it is less costly than LC-MS. IP-RPLC can also be used to purify modified oligonucleotides for further use and analysis. This technique is sensitive to the charge, conformation, and sequence characteristics of the nucleic acid sample and may be used to determine the damage or modifications made to DNA by a variety of compounds.
Analytical Instrumentation Handbook, Second Edition, 2004
Toxicology Letters, 2012
Multi-drug resistance protein (MRP) 4, an ATP-binding cassette (ABC) transporter, has broad subst... more 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-7,8,9,10tetrahydrobenzo[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
Rapid Communications in Mass Spectrometry, 2011
4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic nitrosamine produced upon ... more 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 Oglucuronide 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).
Nucleic Acids Research, 2003
A new method for rapid puri®cation and structural analysis of oligoribonucleotides of 19 and 20 n... more A new method for rapid puri®cation and structural analysis of oligoribonucleotides of 19 and 20 nt is applied to RNA hairpins SL3 and SL2, which are stable secondary structures present on the y recognition element of HIV-1. This approach uses ion-pairing reversed-phase liquid chromatography (IP-RPLC) to achieve the separation of the stem± loop from the transcription mix. Evidence is presented that IP-RPLC is sensitive to the different conformers of these secondary structures. The purity of each stem±loop was con®rmed by mass spectrometry and PAGE. IP-RPLC puri®cation was found to be superior to PAGE in terms of time, safety and, most importantly, purity.
Journal of Chromatography B, 2005
A group of 18-mers of the same base sequence, but with differing alkyl modifications is used to i... more A group of 18-mers of the same base sequence, but with differing alkyl modifications is used to investigate effects of these modifications on retention of oligonucleotides using ion-pairing reversed-phase liquid chromatography (IP-RPLC). It is shown that IP-RPLC is able to distinguish between oligonucleotides differing only by a single alkyl group. The identity of the nucleobase and position and length of the alkyl adduct affect retention of the oligonucleotide. These separation phenomena result from changes in charge and hydrophobicity upon alkylation. As demonstrated in this paper; chromatographic selectivity, unique to IP-RPLC, greatly facilitates the purification process of modified oligonucleotides.
Journal of Allergy and Clinical Immunology, 2014
Fatty acids and consequently diet play an essential role in the formation of inflammatory mediato... more Fatty acids and consequently diet play an essential role in the formation of inflammatory mediators involved in the pathogenesis of asthma. Because intake variations of omega-6 (n-6) and omega-3 (n-3) fatty acids ultimately determine cell membrane incorporation, changes in diet have the potential to modify downstream production of inflammatory mediators derived from these compounds. It has long been hypothesized that decreasing the n-6/n-3 ratio could reduce the production of more proinflammatory mediators while increasing the formation of downstream metabolites that can serve to limit or resolve inflammation. In turn, these changes would result in improved asthma outcomes or would lower the risk for asthma incidence. This review will focus on the role of fatty acid inflammatory and resolving mediators and will summarize the clinical and epidemiologic data on how diet and obesity alter fatty acid profiles that can contribute to asthma. (J Allergy Clin Immunol 2014;133:1255-64.)
Journal of Allergy and Clinical Immunology, 2013
Background: Bronchoalveolar lavage (BAL) fluid prostaglandin D 2 (PGD 2 ) levels are increased in... more Background: Bronchoalveolar lavage (BAL) fluid prostaglandin D 2 (PGD 2 ) levels are increased in patients with severe, poorly controlled asthma in association with epithelial mast cells (MCs). PGD 2 , which is generated by hematopoietic prostaglandin D synthase (HPGDS), acts on 3 G proteincoupled receptors, including chemoattractant receptorhomologous molecule expressed on T H 2 lymphocytes (CRTH2) and PGD 2 receptor 1 (DP1). However, much remains to be understood regarding the presence and activation of these pathway elements in asthmatic patients. Objective: We sought to compare the expression and activation of PGD 2 pathway elements in bronchoscopically obtained samples from healthy control subjects and asthmatic patients across a range of disease severity and control, as well as in relation to T H 2 pathway elements. Methods: Epithelial cells and BAL fluid were evaluated for HPGDS (quantitative real-time PCR/immunohistochemistry [IHC]) and PGD 2 (ELISA/liquid chromatography mass spectrometry) in relation to levels of MC proteases. Expression of the 2 inflammatory cell receptors DP1 and CRTH2 was evaluated on luminal cells. These PGD 2 pathway markers were then compared with asthma severity, level of control, and markers of T H 2 inflammation (blood eosinophils and fraction of exhaled nitric oxide). Results: Confirming previous results, BAL fluid PGD 2 levels were highest in patients with severe asthma (overall P 5 .0001). Epithelial cell compartment HPGDS mRNA and IHC values differed among groups (P 5 .008 and P < .0001, respectively) and correlated with MC protease mRNA. CRTH2 mRNA and IHC values were highest in patients with severe asthma (P 5 .001 and P 5 .0001, respectively). Asthma exacerbations, poor asthma control, and T H 2 inflammatory markers were associated with higher PGD 2 , HPGDS, and CRTH2 levels. Conclusion: The current study identifies coordinated upregulation of the PGD 2 pathway in patients with severe, poorly controlled, T H 2-high asthma despite corticosteroid use. (J Allergy Clin Immunol 2013;131:1504-12.)
Free Radical Biology and Medicine, 2013
Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. T... more Nitrated fatty acids are the product of nitrogen dioxide reaction with unsaturated fatty acids. The discovery of peroxynitrite and peroxidase-induced nitration of biomolecules led to the initial reports of endogenous nitrated fatty acids. These species increase during ischemia/reperfusion, but concentrations are often at or near the limits of detection. Here, we describe multiple methods for nitrated fatty acid synthesis and sample extraction from complex biological matrices and a rigorous method of qualitative and quantitative detection of nitrated fatty acids by liquid chromatography-mass spectrometry. In addition, optimized instrument conditions and caveats regarding data interpretation are discussed.
Chemical Research in Toxicology, 2008
Figure 6 was published in error without panels A, B, and C being labeled. The corrected legend wo... more Figure 6 was published in error without panels A, B, and C being labeled. The corrected legend would read: Figure 6. Mutations mapped onto the structure of p53. (Top) The locations of the top 10 hotspots in lung cancer are shown in red. (Middle) The 11 unique PAH o-quinone derived dominant mutations from are shown in red (213, 239, 244, 246, 251, 256, 273, 275, 276, 279, 281). The PAH o-quinone derived recessive mutations are shown in green The 8 unique BPDE derived dominant mutations are shown in red (156, 158, 176, 178, 196, 213, 279, 283). Note that the dominant mutations cluster in DNA contact regions. Structures were plotted using the online software of the IARC TP53 database (3).
Chemical Research in Toxicology, 2003
The effects of N 2 -ethylGua, O 6 -ethylGua, and O 6 -methylGua adducts in template DNA on polyme... more The effects of N 2 -ethylGua, O 6 -ethylGua, and O 6 -methylGua adducts in template DNA on polymerization by mammalian DNA polymerases R and η have been investigated. The N 2 -ethylGua adduct blocks polymerization by the replicative DNA polymerase R to a much greater extent than does the O 6 -ethyl-or the O 6 -methylGua adducts. The DNA polymerase η efficiently and accurately bypasses the N 2 -ethylGua lesion but like DNA polymerase R is similarly blocked by the O 6 -ethyl-or the O 6 -methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N 2 -ethylGua and the O 6 -ethylGua adducts by the DNA polymerases R and η and extension from 3′-termini positioned opposite these adducts was performed to measure the efficiency and the accuracy of DNA synthesis past these lesions. This analysis showed that insertion of Cyt opposite the N 2 -ethylGua adduct by DNA polymerase R is approximately 10 4 -fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N 2 -ethylGua:Cyt 3′-terminus by DNA polymerase R is approximately 10 3 -fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N 2 -ethylGua lesion by the DNA polymerase η is about 370fold more efficient than by the DNA polymerase R, and extension from the N 2 -ethylGua:Cyt 3′-terminus by the DNA polymerase η is about 3-fold more efficient than by the DNA polymerase R. Furthermore, the DNA polymerase η preferably inserts the correct nucleotide Cyt opposite the N 2 -ethylGua lesion with nearly the same level of accuracy as opposite an unadducted Gua, thus minimizing the mutagentic potential of this lesion. This result contrasts with the relatively high misincorporation efficiency of Thy opposite the O 6 -ethylGua adduct by the DNA polymerases R and η. In reactions containing both DNA polymerases R and η, synthesis past the N 2 -ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N 2 -ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase R and lesion bypass catalyzed by DNA polymerase η.
Chemical Research in Toxicology, 2007
There is substantial evidence to suggest that polycyclic aromatic hydrocarbons (PAHs) such as ben... more 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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-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&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;,5&amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;#39;-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.
Chemical Research in Toxicology, 2011
Environmental carcinogens, such as polycyclic aromatic hydrocarbons (PAHs), require metabolic act... more 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-7,8,9,10tetrahydro-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 2,3,7,8tetrachlorodibenzo-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-7,8dihydrodiol-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-7,8dihydrodiol 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.
Chemical Research in Toxicology, 2008
PAHs (polycyclic aromatic hydrocarbons) are suspect lung cancer carcinogens that must be metaboli... more 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.