Oxidative Stress　DOJINDO LABORATORIES

Classification

Oxidative Stress

Introduction

Oxygen is a very important molecule for the synthesis of biologically active materials such as hormones and ATP. Acquisition of the ability to utilize oxygen was a significant driving force for the evolution of life. Oxygen activates various enzymes in cells and activated oxygen species are involved in the operation of cell functions. Although oxygen itself is an essential element of life, molecules in cells, such as DNA and proteins, are sometimes damaged by reactive oxygen species (ROS) in what is called oxidative stress. ROS can be created by metabolism, ionizing radiation, and carcinogenic compounds that directly interact with DNA. During metabolism, a small portion of oxygen is converted to superoxide anion by one electron reduction; superoxide anion is then converted to oxygen and hydrogen peroxide by superoxide dismutase (SOD). Hydrogen peroxide is reduced to water by catalase or glutathione peroxidase. However, if hydrogen peroxide is not completely reduced by these enzymes, it can generate an extremely reactive hydroxy radical when oxidized by iron (Fenton reaction). Hydroxy radical is also generated by UV irradiation or directly from water by ionizing radiation. Hydroxy radical reacts with lipid to generate lipid peroxide. However, not all ROS are unwanted. Hypochlorite ion, an ROS derived from hydrogen peroxide by myeloperoxidase in neutrophils, has germicidal activity. Nitric oxide, also known as endothelialderived relaxation factor, is generated by NO synthetase. However, NO and superoxide anion may react to generate peroxynitrite, which is cytotoxic. The ROS and reactive nitrogen compounds have many different activities in biological systems. In response, aerobic organisms created defense mechanisms to avoid oxidative stress. Oxidative stress has recently become the focus of many studies seeking to understand these defense mechanisms and the relationships between oxidative damage and disease or aging processes. To this end, many assay methods have been developed for the detection of ROSrelated or ROS-derived substances such as superoxide anion, superoxide dismutase, glutathione, glutathione reductase, glutathione peroxidase, DNA lesions, 8-oxoguanine, 8-nitroguanosine, and protein carbonyl.

DNA Damage by Oxidative Stress

Oxidative damage to DNA results from the interaction of DNA with ROS, in particular, hydroxy radicals. Hydroxy radicals, which are produced from superoxide anion and hydrogen peroxide by the Fenton reaction, cause multiple modifications in DNA. Oxidative attacks by hydroxy radicals on the deoxyribose moiety lead to the release of free bases from DNA, generating strand breaks with various sugar modifications and simple abasic sites. These are one of the major types of DNA damage generated by ROS. Measurement of these modifications is important for understanding the mechanisms of oxidative DNA damage and its biological repercussions. Most of the time, the DNA repair system eliminates such damage by severing the disease process and maintaining the integrity of the form of life. However, clinical data clearly indicate that oxidative DNA damage is related to a number of disease processes such as carcinogenesis and neurodegenerative diseases. DNA Damage Quantification Kit is utilized for determining the number of simple abasic sites (AP sites) in genomic DNA isolated from cell and tissue samples. The DNA damage level can be monitored in the range of 1 to 40 AP sites per 105 base pairs. Therefore, DNA Damage Quantification Kit is useful for the detection of carcinogen toxicity and for the detection of DNA repair activities. 8-Nitroguanosine, another oxidatively modified molecule, is a nitrated base of DNA or RNA. It is known that a large amount of nitric oxide and superoxide molecules, generated by inflammation, can cause nitration of guanosine. Since chemically modified nucleotides cause mutation during DNA replication, 8-nitroguanosine is thought to be a marker of DNA damage related to mutation and cancer. 8-Nitroguanosine is involved in controlling cell functions and is related to oxidizing and reducing reactions. 8-Nitroguanosine antibodies and assay kits are available from Dojindo.

Oxidative Stress Related Components

Superoxide dismutase (SOD) is one of the most important antioxidative enzymes for scavenging toxic superoxides in vivo. SOD catalyzes the dismutation of the superoxide anion into molecular oxygen and hydrogen peroxide, which are much less oxidatively reactive. SOD is a potential therapeutic agent in diseases related to oxidative stress and as a material for moderating the aging process, because of its high antioxidant efficiency (much higher than that of glutathione or other reducing agents) and early action in the initial stages of ROS metabolism. Since glutathione also has superoxidescavenging activity, it contributes to a reductive atmosphere in cells and tissues to avoid oxidative damage. SOD Assay Kit-WST is used for the determination of superoxide quenching ability, based on an inhibition assay, and is suitable for evaluating SOD or SOD-like activities in samples. Cytochrome C is a commonly utilized agent for SOD activity detection. However, its reactivity with superoxide anion is too high to determine low SOD levels. Since the reactivity of WST-1 with superoxide anion is much lower than that of cytochrome C, low levels of SOD activity can be determined. Total Glutathione Quantification Kit is for measuring the amount of glutathione in biological samples. The combination of DTNB (Ellman’s Reagent) and glutathione reductase in this kit enables highly sensitive detection of total G. DTNB generates an intense yellow-colored dye upon reaction with the reduced form of glutathione. The assay range of this kit is from 6.25 μmol/l to 100 μmol/l glutathione. GSSG/GSH Quantification Kit contains Masking reagent for glutathione reduced form (GSH). Therefore, the amount of glutathione oxidized form (GSSG) in a sample can be measured by quenching GSH. After measuring the total amount of glutathione and GSSG, the amount of GSH can be determined by the subtraction of GSSG from the total glutathione. The ratio of GSH to GSSG has been identified as one of the biological markers for oxidative stress.

Nitric Oxides

Nitric oxide (NO) has been identified as an endothelialderived relaxation factor and antiplatelet substance. It serves as a neurotransmitter when derived from a neutrophil, and as a cytotoxic substance when derived from an activated macrophage. NO reacts with superoxide anion to generate highly toxic peroxynitrite. The reaction rate of NO with superoxide is three times that of SOD. In some cases, NO also activates cyclooxygenase. The most important role of NO is thought to be the activation of guanylate cyclase. Recently, published NO research has reported many contradictory results, which are due to NO’s unique chemical properties. Since NO is a free radical, it is very reactive and unstable. NO changes its form in a complex manner immediately after appearing in a biological environment. Each of NO’s metabolites might have different bioactivities from NO itself. For this reason, it is vital to separately investigate each function of the NO-related metabolites.

Fig. 1 O2 and NO metabolism chart

Product

Stress Maker Detection

Code	Product name	Unit size
A305	Stress Marker Detection ARP(Aldehyde Reactive Probe)	10 mg
D350	Stress Marker Detection DPPP	10 mg
D535	Stress Marker Detection 3-Deoxyglucosone	1 mg
DK02	DNA Damage Detection -Nucleostain- DNA Damage Quantification Kit -AP Site Counting-	20 samples
G257	Glutathione Quantification GSSG/GSH Quantification Kit	200 tests
L248	Lipid Peroxide Detection Liperfluo	1 set (50 ug x 5)
L267	Lipid Peroxidation Assay Lipid Peroxidation Probe -BDP 581/591 C11-	200 tests
M496	MDA(malondialdehyde) Assay Kit MDA Assay Kit	100 tests
R252	ROS Detection ROS Assay Kit -Highly Sensitive DCFH-DA-	100 tests
R253	ROS Detection ROS Assay Kit -Photo-oxidation Resistant DCFH-DA-	100 tests
T419	Glutathione Quantification Total Glutathione Quantification Kit	100 tests

NO Detection

Code	Product name	Unit size
C348	NO Detection Carboxy-PTIO	10 mg
D418	NO Detection 2,3-Diaminonaphthalene (for NO detection)	10 mg
D465	NO Detection DTCS Na	100 mg

NO Donor

Code	Product name	Unit size
N377	NO Detection NOC 7	10 mg 50 mg
N378	NO Detection NOC 12	10 mg
N379	NO Detection NOC 18	10 mg 50 mg
N380	NO Detection NOC 5	10 mg 50 mg
N390	NO Detection NOR 3	10 mg
N415	NO Detection S-Nitrosoglutathione	25 mg 100 mg
S264	NO Detection Reagent SIN-1	25 mg

NO Inhibitor

Code	Product name	Unit size

ACE Inhibition Assay

Code	Product name	Unit size
A502	ACE Inhibitory Activity Assay ACE Kit - WST	50 tests 100 tests

Reagents・Kits for Sulfur Biology

Code	Product name	Unit size
SB10	Biosulfur Analysis -SulfoBiotics- SSP4	1 mg
SB12	Biosulfur Analysis -SulfoBiotics- Protein Redox State Monitoring Kit Plus	20 samples
SB15	Biosulfur Analysis -SulfoBiotics- Sulfide dibimane	10 nmol x 5
SB17	Biosulfur Analysis -SulfoBiotics- Biotin-HPDP(WS) solution	500 ul
SB20	Biosulfur Analysis -SulfoBiotics- PEG-PCMal	1 mg
SB21	Biosulfur Analysis -SulfoBiotics- HSip-1	1 mg
SB22	Biosulfur Analysis -SulfoBiotics- HSip-1 DA	50 µg

Antioxidant Assay Kit

Code	Product name	Unit size
D678	Antioxidant Ability Assay DPPH Antioxidant Assay Kit	100 tests 500 tests
S311	SOD Inhibitory Activity Assay SOD Assay Kit - WST	500 tests

Donors for Sulfur Biology

Code	Product name	Unit size
SB02	Biosulfur Analysis -SulfoBiotics- Sodium disulfide (Na₂S₂)	100 mg x 5
SB03	Biosulfur Analysis -SulfoBiotics- Sodium trisulfide (Na₂S₃)	100 mg x 5
SB04	Biosulfur Analysis -SulfoBiotics- Sodium tetrasulfide (Na2S4)	100 mg x 5
SB06	Biosulfur Analysis -SulfoBiotics- GYY4137	10 mg
SB13	Biosulfur Analysis -SulfoBiotics- Sodium Polysulfide Set	1 set

Classification