Dojindo Newsletter Vol.2

Review

Oxidative Stress, DNA Damage and Human Diseases

Table of Contents

Oxidative Stress, DNA Damage and Human Diseases
Spectrum of DNA damage resulting from oxidative stress
Enzymatic repair of oxidative DNA damage
Oxidative stress and human diseases
Detection and quantification of oxidative DNA damage by the ARP assay
References
Product Information

Yoke W. Kow, Ph.D.
Division of Cancer Biology, Department of Radiation Oncology,
Emory University School of Medicine, 145 Edgewood
Avenue, Atlanta, GA 30335

Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide and hydroxyl radical are products of oxygen metabolism in all aerobic organisms. ROS are generated as a result of energy production from mitochondria (from the electron transport chain), as part of an antimicrobial (1) or antiviral (2) response, as well as detoxification reactions carried out by the cytochrome P-450 system (3, 4). Environmental agents such as ultraviolet light, ionizing radiation, redox chemicals and cigarette smoke also readily generate ROS. The antioxidant defense system in most cells is composed of two components, the antioxidant enzymes component which includes enzymes such as superoxide dismutase, catalase and glutathione peroxidase, and the low molecular weight antioxidants component that includes vitamins A and E, ascorbate, glutathione and thioredoxin. These substances are the body's natural defense against endogenous generated ROS and other free radicals, as well as ROS generated by external environmental factors. Oxidative stress occurs when the production of ROS exceeds the body's natural antioxidant defense mechanisms, causing damage to biomolecules such as lipids, proteins and DNA.

Spectrum of DNA damage resulting from oxidative stress

Oxidative damage to DNA is a result of interaction of DNA with reactive oxygen species (ROS), in particular the hydroxyl radical. Superoxide and hydrogen peroxide are normally not reactive towards DNA. However, in the presence of ferrous or cuprous ion (the Fenton reaction), both superoxide and hydrogen peroxide are converted to the highly reactive hydroxyl radical. Hydroxyl radical produces a multiplicity of modifications in DNA. Oxidative attack by OH radical on the deoxyribose moiety will lead to the release of free bases from DNA, generating strand breaks with various sugar modifications and simple abasic (AP) sites. In fact, one of the major types of damage generated by ROS is AP site, a site where a DNA base is lost.

AP sites are also formed at an appreciable rate from spontaneous depurination. It is estimated that at least 10,000 depurination events occur per cell per day under physiological conditions. A similar amount of AP site is thought to be generated by normal aerobic respiration. In addition to AP site, a wide spectrum of oxidative base modification occurs with ROS (Figure 1). The C₄-C₅ double bond of pyrimidine is particularly sensitive to attack by OH radical, generating a spectrum of oxidative pyrimidine damage including thymine glycol, uracil glycol, urea residue, 5-OHdU, 5-OHdC, hydantoin and others. Similarly, interaction of OH radical with purines will generate 8-OHdG, 8-OHdA, formamidopyrimidines and other less characterized purine oxidative products. It has been estimated that endogenous ROS can result in about 200,000 base lesions per cell per day. The biological consequences of many of the oxidative products are known. For example, unrepaired thymine glycol is a block to DNA replication and is thus potentially lethal to cells. On the contrary, 8-oxoG, an abundant oxidative damage to dG, is readily bypassed by the DNA polymerase and is highly mutagenic. Unrepaired 8-oxoG will mispair with dA, leading to an increase in G to T transition mutations.

Enzymatic repair of oxidative DNA damage

In order to maintain the fidelity of genetic material, all organ isms have evolved many different repair pathways to remove various types of DNA damage, resulting from either endogenous or external DNA reactive agents. Oxidative damage is repaired by a ubiquitous base excision repair pathway (5, 6). Base damage is recognized by DNA N-glycosylase. There are two major N-glycosylases for oxidative base damage (a deamination product such as uracil is recognized by uracil N-glycosylase). Endonuclease III from E. coli is the prototype repair enzyme that recognizes many types of oxidative pyrimidine damage (7, 8). Homologues of endonuclease III are found in all cells examined, and its gene has been cloned from bacteria, yeast, mouse, and human cells. The substrate specificity of various endonuclease III homologues appear to be similar (9, 10). The enzyme has an associated b-lyase activity. After the release of the damaged base by endonuclease III, the enzyme cleaves to the phosphodiester bond 3' to the AP site, leaving behind a 3'modified sugar moiety, 4-hydroxypentenal (11). On the other hand, oxidative purine damage is recognized by formamidopyrimidine N-glycosylase (fpg; (12, 13)). Functional homologues of the bacterial fpg protein are present in yeast and human cells. The eukaryotic enzyme that recognizes 8-oxoG is called 8-xooG glycosylase (OGG1 gene product) and shares no amino acid sequence homology with the bacterial fpg protein (14). The substrate specificity of bacterial fpg and eukaryotic OGG1 protein is similar, recognizing 8-oxoG and formamidopyrimidines. However, the bacterial fpg protein has an associated b,d-lyase activity, leaving behind a 3' phosphate terminus (15, 16). The eukaryotic OGG1 protein is a b-lyase, leaving behind a 3' 4-hydroxypentenal residue. The 3' residue (either the 4-hydroxypentenal or phosphoryl group) left behind by these N-glycosylases are further processed by AP endonucleases, generating the 3' OH that is required for repair synthesis catalyzed by DNA polymerase. There are two major types of AP endonuclease, endonuclease III and the exonuclease IV. In E. coli, both AP endonucleases are present, with exonuclease III being the major AP endonuclease. In human cells, the major AP endonuclease is exonuclease III and in yeast, the major activity is endonuclease IV. After the 3' end of the DNA is processed by AP endonucleases, the repair process is completed following repair synthesis and ligation by DNA polymerase and ligase, respectively.

Oxidative stress and human diseasesOxidative stress has been thought to contribute to the general decline in cellular functions that are associated with many human diseases including Alzheimer disease (17, 18), amyotrophic lateral sclerosis (ALS; (19, 20)), Parkinson disease (21, 22), atherosclerosis (23, 24), ischemia/reperfusion neuronal injuries, degenerative disease of the human temporomandibular-joint (25), cataract formation (26, 27), macular degeneration (20, 28), degenerative retinal damage (29), rheumatoid arthritis (30), multiple sclerosis (31), muscular dystrophy (32, 33), human cancers (34, 35) as well as the aging (36, 37) process itself. Increased cellular level of ROS due to oxidative stress can result in an increased steady state level of oxidative DNA damage. There is increasing evidence that an increased level of oxidative damage such as AP site is detected in cells obtained from ALS and Alzheimer patients or after ischemia/reperfusion. Due to the fact that many human diseases might be resulting from chronic oxidative stress and the magnitude of oxygen damage to DNA that is associated with oxidative stress, it this important to have a simple and accurate procedure for estimating the level of oxidative DNA damage in oxidative stressed cells.

Detection and quantification of oxidative DNA damage by the ARP assay

ARP reagent (N'-aminooxymethyl-carbonylhydrazino-D-biotin, Figure 2) is a biotinylated hydroxylamine derivative. The chemical reacts specifically with an aldehyde group, thus allowing the detection of DNA modifications that resulted in the formation of an aldehyde group. AP site in DNA exists in equilibrium between the ring closed and the ring opened form (Figure 3). Approximately 5% of the AP site is in the ring opened form, which has an active aldehyde group. ARP, a biotinylated alkoxyamine

Figure 2. Chemical structure of ARP reagent

Figure 3. Chemical structure of an AP site

(Figure 2) reacts specifically with the aldehyde group in the ring opened AP site. After treating DNA containing AP sites with ARPreagent, AP sites are thus tagged with a biotin residue. By using an excess amount of ARP reagent, essentially all AP sites can be converted to biotin-tagged AP sites. The amount of biotinylated AP sites can then be easily quantified with an ELISA-like assay, using avidin-biotin complex conjugated with either horseradish peroxidase or alkali phosphatase as an indicator enzyme (Figure 4). This procedure has been successfully used by laboratories for accurate measurement of AP sites in DNA (38, 39, 40, 41). A modification of the ELISA-like ARP assay was made by (38), allowing even more sensitivity in the detection of AP site. Instead of binding DNA to a microtiter plate, DNA was bound to a nitrocellulose membrane using a dot blot apparatus. A microtiter plate-based AP site assay kit is currently available from Dojindo Molecular Technologies, Inc.>

Figure 4. Principle of ARP assay

Many kind of base damage are recognized by damage specific DNA glycosylases. The substrate spectrum of DNA glycosylases varies depending on the enzymes; some have very narrow substrate specificity, such as uracil DNA N-glycosylase and T4 endonuclease V, whilesome can recognize a variety of base modifications such as endonuclease III, 8-oxoguanine N-glycosylase and alkA protein. These glycosylases remove modified bases, leaving behind either intact AP sites or modified sugar moieties (4-hydroxy-pentenal) still attached to the 3' termini of nicked DNA. Both products of N-glycosylases still retain the active aldehyde that can easily be quantified by the use of ARP assay. Therefore, treating damaged DNA with a specific repair enzyme will permit the determination of a class of base damages that is normally recognized by the repair enzyme. The advantage of the enzyme-coupled ARP assay is that it allows the investigator to assess the contribution of a whole spectrum of base damage that is normally recognized by the repair enzymes. Furthermore, if one would like to assess the amount of oxidative DNA damage due to increased oxidative stress, treatment of the damaged DNA with both endonuclease III and yeast OGG1 will provide a relatively good assessment of the total amount of oxidative base damage that has occurred on the DNA. Endonuclease III has been shown to recognize many different types of pyrimidine oxidative damages. Therefore DNA samples treated with excess endonuclease III will leave behind a 3' modified sugar moiety (4-hydroxypentenal) that can be tagged with the ARP reagent. The amount of ARP tag can then be determined and be used as a measurement for endonuclease III sensitive site or an oxidative pyrimidine lesion. In fact, the enzyme coupled-ARP assay has been used for the quantification of thymine glycols and alkylation damage in DNA (39, 41). Similarly, oxidative purine damage can be detected using either the yeast 8-oxoguanine glycosylase (yOGG1) or the human 8-oxoguanine glycosylase (hOGG1). In the latter case, the bacterial fpg protein cannot be used since the enzyme generates a phosphoryl group. OGG1 has been shown to recognize mostly 8-oxoG and formamidopyrimidines (OGG does not recognize 8-oxoA), but the amount of damage determined by the use of OGG might underestimate the total oxidative purine damage. However, it should provide a good assessment of the level of biologically important purine damage present in the cells. A kit for estimating the amount of oxidative pyrimidine and purine damage is currently under development by Dojindo Molecular Technologies, Inc., and should be available soon

References

DNA Damage Quantification Kit -AP Site Counting-

ARP (Aldehyde Reactive Probe)

Related Products

Biotin Labeling Reagents
Amine labeling
    Biotin-OSu, Biotin-AC5-OSu, Biotin-(AC5)2-OSu
    Biotin-sulfo-OSu, Biotin-AC5-sulfo-OSu
    Biotin-(AC5)2-sulfo-OSu

Thiol labeling
    Biotin-PE-maleimide, Biotin-PEAC5-maleimide

Aldehyde labeling
    Biotin-hydrazide, Biotin-AC5-hydrazide
    Biotin-(AC5)2-hydrazide

Appearance: white or slightly yellow powder
Purity: amine, aldehyde reactive biotin >95% (HPLC)
    thiol reactive biotin: >90 % (HPLC)

Topics on Chemistry
Why is the water-soluble formazan necessary?

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Table of Contents

Introduction
Application for clinical test
Application for cytotoxicity assays
            Cell proliferation assay
            Cytotoxicity assay
Conclusion
Reference
Product Information

Munetaka Ishiyama, Ph.D.
Dojindo Molecular Technologies, Inc.
211 Perry Parkway, Suite 5
Gaithersburg, MD 20877
E-mail: ishi@dojindo.com

Introduction

Tetrazolium salts are readily reduced enzymatically and generate formazan dyes. These tetrazolium salts are utilized for the determination or detection of dehydrogenase activity in tissues or body fluids for research purposes and for clinical tests. In order to obtain a clear staining result, highly lipophilic tetrazolium salts has been required, so most tetrazolium salts are barely water-soluble, and the resulting formazan dys are insoluble in water. For the determination of dehydrogenase activities in the clinical tests, these tetrazolium salts are very useful reagents. However, the low water-solubility of formazan dyes causes some troubles upon using these reagents, such as the precipitation of the dye in the detection solution, the difficulty for the preparation of the solution of the tetrazolium salt, the unwanted staining of the detection cells or tubes in autoanalyzers, and so on. Therefore, we have been developed water-soluble type tetrazolium salts over a decade. In this topics, I will introduce how water-soluble tetrazolium salts are being used an what modification or improvement will be required.

Figure 1. Structure of tetrazolium salt and electron transfer mechanism

Application for clinical test

Biochemical tests for measuring clinical important elements in body fluids are mostly done by enzymatic reactions because of their high selectivity. These enzymatic reactions are based on oxidase or dehydrogenase reactions, and these reactions are determined by colorimetrically or fluorimetrically by using several reagents, such as oxidative chromogenic dyes or reductive chromogenic dyes. Tetrazolium salts are one of the reductive chromogenic dyes, and they provide us the most sensitive detection system. The reaction cascade is shown in Fig.1. Generally, a tetrazolium salt is reduced through the reaction with a reduced form mediator or through the reaction directly with NADH (nicotineamido adenine dinucleotide reduced form) or NADPH (nicotineamido adenine dinucleotide phosphate reduced form), and NADH or NADPH are generated from NAD or NADP by the reaction of dehydrogenase and its substrate, such as lactate dehydrogenase and lactic acid. Therefore, the tetrazolium salt is utilized for the determination of the dehydrogenase activity or a substrate of the dehydrogenase. The solution of a tetrazolium salt is almost colorless and its formazan gives a strongly colored solution, like orange, red or purple. However, such formazan dyes easily aggregate and precipitate in the solution after or even during the reaction. In the clinical tests in Japan, mostly an auto-analyzer is used in the clinical test center in hospital, and the system for the detection of clinically important element should be matched to the system of the auto-analyzer. The formazan dye stains the auto-analyzer's detection cell and tubes, and gives a positive error at the detection of the next sample. Therefore, the improvement of the water-solubility of the formazan dye became one of the most interesting targets for diagnostic reagent companies.

WST-1 is a first developed compound that has high water-solubility and resulting formazan dye is also highly water-soluble. So far, five different types of WST compounds have been developed (Figure 2). The water-solubility of these WST compounds is from 10 mM to 100 mM, and no staining on plastic surface was observed. Their formazan dye's spectral properties are shown in Table 1. Since the molar extinction coefficentof INT formazan is 15,000 and Nitro-TB formazan is 36,000, these data shows that the WST compounds have similar molar absorptivity. However, it should be noted that its molar absorptivity does not always become an indicator of its sensitivity in the enzymatic reaction, the affinity between the tetrazolium and the mediator becomes the most important factor, especially in case of using diaphorase as a mediator. The maximum wavelengths of WST-4 and WST-5 formazans are around 550 nm at pH 6 or over. However, the maximum wavelengths of WST-1, 3 and 8 formazans shift from 440 nm to 600 nm by changing pH from 6 to 12.5, and these molar absorptivities increase 50 to 100 % more.

Figure 2. Structure of WST compounds

Application for cytotoxicity assays

The cytotoxicity of chemicals has been determined by animal experiments, requiring nearly 8 million animals per year in Japan. However, these assays are not only hard on animals, but also sometimes unreliable because of the ambiguous standard for the estimation of the cytotoxicity. Therefore, several alternative methods for first screening to estimate cytotoxicity have been developed. The ³H-thymidine incorporate assay and the MTT (3-(4, 5-dimethyl-2-thiazolyl)-2, 5-dihphenyl-2H-tetrazolium, bromide) assay are the most common methods among these cytotoxicity assays. The ³H-thymidine incorporate assay is based on a measurement of the amount of the tritium labeled thymidine that is taken in a DNA when DNA is synthesized. On the other hand, the MTT assay is based on a measurement of the amount of the yielded formazan dye that was reduced by dehydrogenases in living cells. The following is introduced concerning cell proliferation and the cytotoxicity assays using tetrazolium salts.

1. Cell proliferation assay

The cell proliferation assay using tetrazolium salts such as MTT is a popular method because of its simplicity (colorimetric assay) and safety (no radioisotope uses such as ³H-thymidine uptake assay). But there is only one problem in the MTT assay. The yielded formazan is not soluble in a buffer or a culture medium. So it is necessary to take an extra step, which is the solubilization of the formazan using some organic solvents before the measurement of the optical density. Dojindo Laboratories has developed and started to sell a new type of cell proliferation assay kit for a 96-well microtiter plate, "Cell Counting Kit-8 (CCK-8)," which employs WST-8 which produces a highly water-soluble formazan dye. Since CCK-8 is supplied as a one-bottled ready-to-use solution, no dilution with a buffer or a culture medium nor mixing with solutions is necessary prior to use. Furthermore, CCK-8 does not require any radioisotopes nor organic solvents, and no special skills are necessary for use. CCK-8 consists of WST-8 (5 mM) and 1-methoxy PMS (0.2 mM) as an electron mediator, and the kit solution is stable for over 1 year at -20 ºC and over 3 months at 4 ºC with protection from light. The cell proliferation assay procedure is very simple. Ten of the kit solution is added to each well of the plate on which is inoculated 100 of the cell suspension. After the plate is incubated for 1-4 hours in the incubator, the absorbance is measured using a microplate reader. The wavelength range for the measurement of the absorbance is between 450 nm and 490 nm. So the researcher is able to choose a popular filter, for example 450 nm or 490 nm. The amount of the yellow colored formazan dye generated by dehydrogenases in cells is directly proportional to the number of viable cells in a culture medium. The sensitivity using CCK-8 is higher than that using MTT or the other tetrazolium salts that produce water-soluble formazan dyes such as XTT or MTS for HeLa cells and HL60 cells. Furthermore, the cell proliferation assay data using CCK-8 correlates with that using the ³H-thymidine incorporate assay.

Figure 3. Cell proliferation assay using CCK-8 and other reagents

2. Cytotoxicity assay

The cytotoxicity assays of the several detergents and chemicals for Hela, Balb3T3, L929, RC and HL60 cells were tested by use of CCK-8. A ten of various concentration detergents or chemicals is added to 100 of the cell suspensions (5000 cells/well) that are dispensed onto each well of a 96-well microtiter plate. After the plate is incubated for 48 hours in the incubator, 10 of CCK-8 solution is added. The plate is incubated for an additional 1-4 hours, and then the absorbance is measured using the microplate reader. The IC₅₀ values (chemicals concentrations in a 50 % inhibition of growth) of detergents and chemicals are shown in Table 2. The IC₅₀ values using CCK-8 correlate with those using MTT.

Figure 4. Toxicological test of chemicals by using CCK-8

Table 2. Comparision of LD₅₀ values using CCK-8 and MTT

cell line	antitumor agent	LD50/ CCK8 (ug/ml)	LD50/ MTT (ug/ml)
HE49	bleomycin cisplatin adriamycin MMC etoposide 5-FU	154.59 1.55 0.41 0.05 13.01 0.12	157.34 1.61 0.47 0.03 13.18 0.37
MKN-28	bleomycin cisplatin adriamycin MMC etoposide 5-FU	151.18 0.57 0.11 0.02 4.11 0.05	139.51 0.60 0.13 0.02 4.63 0.05
IMR-32	bleomycin cisplatin adriamycin MMC etoposide 5-FU	314.15 5.65 2.27 0.10 9.63 0.04	207.00 5.32 0.33 0.10 9.71 0.05
H1299	bleomycin cisplatin adriamycin MMC etoposide 5-FU	406.65 0.51 0.17 0.04 14.63 0.08	1057.12 0.72 0.14 0.06 25.96 0.05
HL60	bleomycin cisplatin adriamycin MMC etoposide 5-FU	280.74 1.54 0.19 0.02 12.23 0.06	190.84 1.00 0.24 0.03 11.32 0.07
MOLT-4	bleomycin cisplatin adriamycin MMC etoposide 5-FU	218.84 0.94 0.27 0.01 3.87 0.11	187.26 1.04 0.24 0.01 4.27 0.11
KC12	bleomycin cisplatin adriamycin MMC etoposide 5-FU	739.05 3.59 0.37 0.15 52.87 0.20	880.56 2.98 0.48 0.11 53.31 0.19
HeLa	bleomycin cisplatin adriamycin MMC etoposide 5-FU	306.89 15.22 1.56 0.18 79.16 0.71	379.24 11.52 1.98 0.14 58.37 0.78

Cell lines (human)	MKN-28: gastric cancer
HE 49: normal embryo
IMR-32: neuroblastoma	H1299: lung cancer
HL60: acute promyelonic leukemia	KC12: renal cancer
MOLT-4: acute lympphoblastic leukemia	Hela: cervical cancer

Conclusion

Because of high water-solubility of formazan dyes derived from WSTs, it was found that they are preferable reagents for clinical analyses and cell proliferation. In clinical analyses, the sensitivity of the detection of analytes such as a dehydrogenase and its substrate using WSTs is equal or greater than conventional tetrazolium salts such as INT, MTT or NBT. In addition, WSTs are able to apply the cytotoxicity assay as a reductive chromogenic dye that can minimize the process of the determination of living cell number. Therefore, the cytotoxicity assay using WSTs is suitable for the first screening of the determination of cytotoxicity of chemicals, and contributes to reduce the consumption of experimental animals.Dojindo has been focused on the improvement of the water-solubility of tetrazolium salts over a decade, and we believe that the WSTs are the best reagents for the determination of dehydrogenase activity and the concentration of its substrate among water-soluble type tetrazolium salts. One of our goals of this project is to develop a WST that generates a blue or green color formazan or a fluorescent formazan in an aqueous solution.

References

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Cell Proliferation and Cytotoxicity Assay Kits

Cell Counting Kit-8 (CCK-8)

Product Code	Unit
CK04-11	1000 tests
CK04-13	3000 tests

1 bottle type (WST-8, 1-Methoxy PMS)
Colorimetric microplate assay
Storage -20 C

 

Cell Counting Kit-F (CCK-F)

Product Code	Unit
CK06-10	500 tests

DMSO solution (Calcein-AM)*
Fluorometric microplate assay
Storage: -20 ^oC
*Reagent dilution buffer is required

WST-1

Product Code	Unit
W201	100 mg
W201	500 mg

MW: 651.35
Appearance: slightly yellow powder
Purity: pass test (TLC)
Molar absorptivity (tetrazolium): >21,600 (244 nm)
Molar absorptivity (formazan): >37,000 (438 nm)
Solubility: 10 mg/ml water
Storage: 0-5 ^oC

WST-3

Product Code	Unit
W202	100 mg
W202	500 mg

MW: 696.34
Appearance: slightly yellow powder
Purity: pass test (TLC)
Molar absorptivity (tetrazolium): >36,000 (234 nm)
Molar absorptivity (formazan): >30,000 (433 nm)
Solubility: 10 mg/ml water
Storage: 0-5 ^oC

WST-4

Product Code	Unit
W203	100 mg
W203	500 mg

MW: 580.59
Appearance: slightly yellow powder
Purity: pass test (TLC)
Molar absorptivity (tetrazolium): >28,000 (264 nm)
Molar absorptivity (formazan): >10,000 (550 nm)
Storage: 0-5 ^oC

WST-5

Product Code	Unit
W204	100 mg
W204	500 mg

MW: 1331.35
Appearance: slightly yellow powder
Purity: pass test (TLC)
Molar absorptivity (tetrazolium): >50,000 (264 nm)
Molar absorptivity (formazan): >27,000 (550 nm)
Storage: 0-5 ^oC

Electron Mediator

1-Methoxy-PMS

Product Code	Unit
M003	100 mg
M003	1 g

MW: 336.36
Appearance: dark red or reddish purple powder
Purity: >95%
m.p.: >170 ^oC (dec.)
Molar absorptivity: >2,700 (505 nm)
Solubility: 34 mg/100 ml water

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