Biomedical subjects

Michelle V Williams

Publications and source records attributed to Michelle V Williams.

6 recordsLinked to original sources

PubMed2006

Endogenous lipid hydroperoxide-mediated DNA-adduct formation in min mice.

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.

Animals↗

PubMed2005

Identification of the major oxidative 3alpha-hydroxysteroid dehydrogenase in human prostate that converts 5alpha-androstane-3alpha,17beta-diol to 5alpha-dihydrotestosterone: a potential therapeutic target for androgen-dependent disease.

Androgen-dependent prostate diseases initially require 5alpha-dihydrotestosterone (DHT) for growth. The DHT product 5alpha-androstane-3alpha,17beta-diol (3alpha-diol), is inactive at the androgen receptor (AR), but induces prostate growth, suggesting that an oxidative 3alpha-hydroxysteroid dehydrogenase (HSD) exists. Candidate enzymes that posses 3alpha-HSD activity are type 3 3alpha-HSD (AKR1C2), 11-cis retinol dehydrogenase (RODH 5), L-3-hydroxyacyl coenzyme A dehydrogenase , RODH like 3alpha-HSD (RL-HSD), novel type of human microsomal 3alpha-HSD, and retinol dehydrogenase 4 (RODH 4). In mammalian transfection studies all enzymes except AKR1C2 oxidized 3alpha-diol back to DHT where RODH 5, RODH 4, and RL-HSD were the most efficient. AKR1C2 catalyzed the reduction of DHT to 3alpha-diol, suggesting that its role is to eliminate DHT. Steady-state kinetic parameters indicated that RODH 4 and RL-HSD were high-affinity, low-capacity enzymes whereas RODH 5 was a low-affinity, high-capacity enzyme. AR-dependent reporter gene assays showed that RL-HSD, RODH 5, and RODH 4 shifted the dose-response curve for 3alpha-diol a 100-fold, yielding EC(50) values of 2.5 x 10(-9) M, 1.5 x 10(-9) M, and 1.0 x 10(-9) M, respectively, when compared with the empty vector (EC(50) = 1.9 x 10(-7) M). Real-time RT-PCR indicated that L-3-hydroxyacyl coenzyme A dehydrogenase and RL-HSD were expressed more than 15-fold higher compared with the other candidate oxidative enzymes in human prostate and that RL-HSD and AR were colocalized in primary prostate stromal cells. The data show that the major oxidative 3alpha-HSD in normal human prostate is RL-HSD and may be a new therapeutic target for treating prostate diseases.

3-alpha-Hydroxysteroid Dehydrogenase (B-Specific)↗

PubMed2005

Cyclooxygenase-2-mediated DNA damage.

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.

Animals↗

PubMed2005

Liquid chromatography/mass spectrometry analysis of bifunctional electrophiles and DNA adducts from vitamin C mediated decomposition of 15-hydroperoxyeicosatetraenoic acid.

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.

Ascorbic Acid↗

PubMed2005

Targeted chiral lipidomics analysis.

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.

Animals↗

PubMed2003

Targeted lipidomics using electron capture atmospheric pressure chemical ionization mass spectrometry.

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.

Animals↗