Oxidative Stress and Environmental Analysis Core

Indiana University, Bloomington

Lisa Kamendulis

The Oxidative Stress Environmental Analysis Core laboratory has established a number of measurements for assessing oxidative stress and damage in a variety of biological samples with excellent accuracy and reproducibly using state of the art LC-MSMS, GC, and PCR equipment.

• Oxidized DNA and DNA Damage i. Oxidative DNA Adduct Quantitation: It is well recognized that exogenous and endogenous agents can induce oxidative modifications in DNA. Over 20 DNA oxidation products have been identified, with 8-hydroxy-deoxyguanosine (8-OH-dG) being the most widely studied biomarker of DNA damage. Using LC-MSMS-based approaches, we now have the capability of measuring most of these oxidized DNA products. Thus, this technology allows a more comprehensive characterization of oxidized DNA and its potential linkage to chronic diseases. A panel of biologically important oxidized nucleosides has been developed for analysis in biological samples: 8-OH-dG, 8-hydroxy-2

• . Oxidized Lipid (Lipid Peroxidation) Products The most common quantitative assay for the measurement of lipid peroxidation is the malondialdehyde-based thiobarbituric acid reacting substances spectrophotometric assay. Inherent in this assay is a lack of specificity and over-estimation of lipid oxidation. Prostaglandin F2-like species are formed in vivo by free radical oxidation of arachidonic acid and are named F2-isoprostanes (F2-isoP). F2-isoP levels increase in a wide variety of diseases. Thus, quantitation of F2-IsoP has emerged as one of the most reliable indexes of lipid peroxidation in vivo. While F2-isoprostanes are the most studied class of isoprostanes due to their chemical stability, the quantitation of additional key lipid oxidation products can provide a more comprehensive assessment of oxidative stress burden. 4-hydroxy-2-nonenal (4-HNE) and acrolein are reactive aldehydes resulting from the breakdown of lipid peroxidation products. 4-HNE and acrolein have shown reactivity with protein and DNA to form protein and DNA adducts. Malondialdehyde (MDA), a highly reactive three carbon dialdehyde produced as a by-product of polyunsaturated fatty acid (PUFA) peroxidation and arachidonic acid metabolism, is one of the most intensively investigated aldehydes formed during lipid peroxidation. We use LC-MSMS-based approaches to quantify MDA, F2-isoPs, 4-HNE, and acrolein in biological samples.

• . Lipid Peroxidation-DNA Adducts Several lipid oxidation products exhibit DNA reactivity and can result in mutational events. For example, the lipid perodixation product, MDA, readily combines with DNA to form hightly mutagenic MDA-DNA adducts. Also, exocyclic DNA adducts (e.g., 1,N6-etheno-2?-deoxyadenosine (etheno-dA), are formed by the reaction between DNA bases and intermediates from the lipid peroxidation of PUFAs. These etheno

• Chemical Adducts In addition to lipid-DNA adducts, many chemicals can form adducts with DNA and proteins. These lesions may be important in disease etiology and progression. The Oxidative Stress and Environmental Toxicology core can assist with the development of methods to detect specific chemical-DNA or chemical-protein (e.g. Hb) adducts as needed.

• Measurement of total antioxidant capacity – TEAC assay : Eukaryotic organisms have developed a complex antioxidant network to counteract ROS to reduce the deleterious actions of ROS. We have modified the Trolox Equivalent Antioxidant Capacity (TEAC) assay to provide information on the overall status of antioxidants within a sample. This has proven to be a useful indicator for determining the ability of an organism to mitigate the potential damage produced by ROS. This method uses spectroscopy to evaluate antioxidant capacity in a wide array of biological samples (tissues, blood, serum, and cell lysates).

• Glutathione Status : Due in part to its multiple functions, one of the most important cellular redox systems is the glutathione system. Reduced glutathione (GSH) is a major non-enzymatic antioxidant in eukaryotes. Multiple biologically significant thiols can be assessed to provide information on the overall status of this redox system. We use LC-MSMS-based methods for the simultaneous quantification of GSH, GSSG, ?-glutamyl-cysteine, cysteinyl-glycine, cysteine, and homocysteine.

• a. Focused Gene Expression Analysis Global gene expression analysis of cells or tissues is a highly informative, but also costly determination. To provide a more cost-effective alternative, the Oxidative Stress and Environmental Analysis Core have designed assays to determine the expression of genes which are specifically involved in oxidative stress. This includes determination of expression of enzymes directly involved in oxidative stress pathways, genes which are upregulated following oxidative stress, and genes characteristic of cellular response (ie proliferation, apoptosis). Quantitative RT-PCR analysis: Total RNA is first prepared from the cells or tissues utilizing the RNeasy Mini Kit, Qiagen. RNA is reverse-transcribed (RT) using the Superscript II Reverse Transcriptase (Invitrogen) using random hexamers for priming. Real-time PCR is performed using the SYBR Green PCR Master Mix kit (Applied Biosystems) and performed on an Applied Biosystems 7900HT Fast Real Time PCR System instrument. Primers are designed for specific genes using NCBI Primer Blast Tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Data are analyzed by the threshold cycle (Ct) comparative method. The 2-??Ct value is calculated, where ?Ct = Cttarget gene-Cthousekeeping gene, and ??Ct = ?Ctsample-?Ctreference. Oxidative Stress Gene List: Genes involved in ROS generation and metabolism: catalase, cytochrome P450 isoforms, epoxide hydrolase, glutathione S-transferases, glutathione peroxidases, glutaredoxin, glutathione reductase, metallothionein, NAPDH oxidase, nitric oxide synthase, peroxiredoxins, superoxide dismutase (SOD), thioredoxin reductase Genes involved in proliferation or apoptosis: Bad, Bax, Bcl-2, Bid, Cyclin D1, Fas, Faslg Fos, Gadd45a, Jun, Myc, Tnf Genes involved in DNA damage or repair: Apex1, Atm, Ercc1, Ercc2, Cdkn1a (p21), DNMT, Mgmt, Mpg, Ogg1, Tp53, Ung, Xpa, Xrcc2 Genes involved in transcriptional responses: DUSP1, heme oxygenase, HIF1?, I?B?, NADPH dehydrogenase, Nrf-2, VEGF Housekeeping genes: ?2-microglobulin, ?-actin, GAPDH, HGPRT

• a. Focused Gene Expression AnalysisrnGlobal gene expression analysis of cells or tissues is a highly informative, but also costly determination. To provide a more cost-effective alternative, the Oxidative Stress and Environmental Analysis Core have designed assays to determine the expression of genes which are specifically involved in oxidative stress. This includes determination of expression of enzymes directly involved in oxidative stress pathways, genes which are upregulated following oxidative stress, and genes characteristic of cellular response (ie proliferation, apoptosis). rnrnQuantitative RT-PCR analysis: Total RNA is first prepared from the cells or tissues utilizing the RNeasy Mini Kit, Qiagen. RNA is reverse-transcribed (RT) using the Superscript II Reverse Transcriptase (Invitrogen) using random hexamers for priming. Real-time PCR is performed using the SYBR Green PCR Master Mix kit (Applied Biosystems) and performed on an Applied Biosystems 7900HT Fast Real Time PCR System instrument. Primers are designed for specific genes using NCBI Primer Blast Tool (http://www.ncbi.nlm.nih.gov/tools/primer-blast/). Data are analyzed by the threshold cycle (Ct) comparative method. The 2-??Ct value is calculated, where ?Ct = Cttarget gene-Cthousekeeping gene, and ??Ct = ?Ctsample-?Ctreference. rnOxidative Stress Gene List:rnGenes involved in ROS generation and metabolism: catalase, cytochrome P450 isoforms, epoxide hydrolase, glutathione S-transferases, glutathione peroxidases, glutaredoxin, glutathione reductase, metallothionein, NAPDH oxidase, nitric oxide synthase, peroxiredoxins, superoxide dismutase (SOD), thioredoxin reductasernGenes involved in proliferation or apoptosis: Bad, Bax, Bcl-2, Bid, Cyclin D1, Fas, Faslg Fos, Gadd45a, Jun, Myc, TnfrnGenes involved in DNA damage or repair: Apex1, Atm, Ercc1, Ercc2, Cdkn1a (p21), DNMT, Mgmt, Mpg, Ogg1, Tp53, Ung, Xpa, Xrcc2rnGenes involved in transcriptional responses: DUSP1, heme oxygenase, HIF1?, I?B?, NADPH dehydrogenase, Nrf-2, VEGFrnHousekeeping genes: ?2-microglobulin, ?-actin, GAPDH, HGPRTrnrnrn

• Lisa Kamendulis : lkamendu@indiana.edu 812-855-3188