Name: formation of covalent dna adducts by enzymatically activated carcinogens and drugs in vitro and their determination by 32p-postlabeling
Uploaded: 2018-03-20T14:00:00.000+00:00
Duration: 9 min 33 s
Description: Watch this Scientific Journal Video about Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling at JoVE.com

This study was supported by Czech Science Foundation (GACR, grant 17-12816S).

The author has nothing to disclose.

In this paper, it is demonstrated a widely accessible methodology to study the potency of chemicals to be bioactivated to metabolic intermediates, resulting in generation of covalent DNA adducts. This is a crucial issue, because evaluation of the potency of environmental chemicals or drugs of their enzymatic activation to metabolites generating covalent DNA adducts is an important field in the development of cancer and its treatment. The modification of DNA by carcinogens considered the cause of tumor development is now ...

The metabolism of xenobiotics (environmental chemicals or drugs) occurs in two phases1. Phases I and II aim to render the originally hydrophobic (not water-soluble) compounds more hydrophilic (water-soluble), thus making them readily excretable via urine, feces or sweat. Phase I (functionalization) reactions include oxidation, reduction, and hydroxylation catalyzed by enzymes such as cytochrome P450s (P450s, CYPs), peroxidases (i.e., cyclooxygenase, COX), aldo-keto reductases (AKRs), and microsomal flavin-containing monooxygenases (FMOs). Phase I also includes reduction reactions, mediated by a variety of reductases i.e., microsomal NADPH:cytochrome P450 reductase (POR) and cytosolic NAD(P)H:quinone oxidoreductase (NQO1), xanthine oxidase (XO), and aldehyde oxidase (AO)1. In the second phase (conjugation), the functional groups that were attached in phase I are used to conjugate small polar molecules to further increase the polarity. Examples of enzymes considered to participate in the reaction of phase II include sulfotransferases (SULTs), N,O-acetyltransferases (NATs), methyltransferases such as catechol-O-methyltransferase (COMT), glutathione S-transferases (GSTs), and uridine diphosphate glucuronosyltransferases (UGTs)1. The classification of enzymes in phase I or II is, however, not rigid, and some enzymes can arguably be grouped into either category.
P450 enzymes (EC 1.14.14.1) are the heme containing proteins present in various organisms, which participate in the biotransformation of many chemicals, catalyzing their conversion3,4. The P450 enzymes catalyze hydroxylation of many substrates, with a reaction where one atom of dioxygen is introduced into the molecule of xenobiotics, while the second atom of oxygen is reduced to form water by the reaction that requires two electrons [the equation (1)]3,4:
RH + O2 + NADPH + H+ &#8594; ROH + H2O + NADP+ (1)
P450 enzymes localized in the endoplasmic reticulum membrane of mammalian cells (microsomal P450 systems) are members of the multienzyme monooxygenase system, which further contains NADPH:cytochrome P450 reductase (POR) and cytochrome b5, the substrate of the enzyme termed as NADH:cytochrome b5 reductase. A generally accepted theory hypothesizes that the donor of the two electrons needed for P450 is the NADPH/POR system. Nevertheless, cytochrome b5 might also act as a donor of electrons for P450, namely as a donor of the electron reducing P450 during second reduction of its reaction cycle, where it acts together with NADH:cytochrome b5 reductase2,3,4.
Mammals utilize various P450 enzymes (e.g., the enzymes of families 5, 8, 11, 17, 19, 21, 24, 26, and 27) for the synthesis of valuable endogenous compounds, such as steroids, and use them for catabolism of natural products2,3. The other CYP mammalian enzymes, such as human CYP1A2, 2C9, 2C19, 2D6, and 3A4, metabolize exogenous chemicals that are used as drugs.5,6 The most important enzymes catalyzing metabolism of drugs are CYPs of the 3A subfamily, especially CYP3A4. The conversions of xenobiotics, such as pro-carcinogens and pro-toxicants, are mediated by human CYP1A1, 1A2, 1B1, 2A6, 2E1, and 3A42,5. Most of these CYPs are present in the liver (except CYP1A1 and 1B1). Nevertheless, the CYPs are also expressed in several extrahepatic organs. Such P450s might be of great significance, predominantly when participate they in bioactivation metabolism of chemicals (drugs) to reactive intermediates in these organs7. Various P450s are induced by several compounds that are their substrates, though this is not necessarily the case.
Many P450 enzymes play a role in chemical (drug) toxicity. They can convert the xenobiotics not only into their detoxification metabolites, but also activate them to reactive species, which modify endogenous macromolecules that additionally exhibit different biological properties, usually causing their toxicity. DNA, lipids, and proteins might be the targets for their modification by reactive electrophiles and radicals generated from activated chemicals. In the case of DNA, resolving several important gene responses and their mechanisms are already known2,3,4,5.
The changes in DNA can result in a decrease in cell growth control, and this phenomenon is considered to be the predominant factor leading to development of carcinogenic processes. The generation of covalent DNA adducts with chemicals having carcinogenic potency is judged as one of the most important steps in the initiation phase of carcinogenic processes8,9,10,11. It was demonstrated that relationships between the formation of DNA adducts and tumorigenesis occur, whereas a decrease in the amount of DNA adducts is responsible for chemoprevention8,9,10,11,12,13,14. The formation of carcinogen/drug-derived DNA adducts depends on individual bases of DNA, and is affected by the sequences of these bases in DNA. The repairs of DNA adducts are dependent on their location (on the transcribed or non-transcribed DNA strand) and types of modified nucleotide sequences8,11,12,15,16.
In this article, we describe procedures utilizing the enzyme-catalyzed conversion of chemicals (drugs) to investigate their potency to be activated into metabolites which modified DNA (generating DNA adducts). For covalent DNA binding, the test compound should usually be activated either by oxidative or reductive reactions, depending on individual drugs. Oxidative or reductive activation of tested chemicals is mediated by a P450-dependent enzymatic system present in the microsomal subcellular fraction or by reduction with reductases present both in microsomes (POR, NADH:cytochrome b5 reductase, P450 enzymes) and in cellular cytosolic subcellular fractions (NQO1, XO, AO, peroxidase). Reactive metabolites thereafter bind to DNA forming DNA adducts. Because both oxidative and reductive reactions are important to activate several drugs to these reactive species, the experimental procedures employing the oxidation/reduction enzymatic system are described. Furthermore, the appropriate methods capable of detecting and quantifying these DNA adducts are described in detail.
Two independent procedures to determine whether the test chemical, activated by enzymatic systems, is bound to DNA are recommended: the 32P-postlabeling technique and utilizing radioactive-labeled compound (e.g., 3H or 14C). For the first pilot, screening the 32P-postlabeling assay is recommended. The determination of the DNA content in solutions, precisely evaluated, must precede both methods.
The 32P-postlabeling technique utilizes the enzymatic hydrolysis of DNA modified by non-radioactive chemicals (carcinogen/drug) to 3&#180;-phosphodeoxynucleosides, additional phosphorylation with radioactive phosphorus (32P) at the 5&#180;-OH position, and the separation of chemical-deoxynucleotide adducts from normal (unmodified) deoxynucleotides by chromatography17 (Figure 1). DNA modified by the chemical compound is hydrolyzed by a mixture of endonuclease, micrococcal nuclease, and exonuclease, known as spleen phosphodiesterase. The mixture of hydrolyzed DNA containing both normal (unmodified) and modified deoxyribonucleoside 3&#180;-monophosphates is reacted with [&#947;-32P]ATP in the presence of carrier (non-radioactive) ATP and T4-polynucleotide kinase at pH 9.5 to form 5&#180;-32P-labeled 3&#180;,5&#180;-bisphosphates (&#34;standard&#34; procedure in Figure 1). The used alkaline pH is capable of minimizing the enzyme activity of T4-polynucleotide kinase to dephosphorylate deoxyribonucleoside 3&#180;-monophosphates at position 3&#180;. Separation and resolution of 32P-labeled adducts from labeled deoxynucleotides that are not modified by chemicals is carried out by multidirectional anion-exchange thin layer chromatography (TLC) on polyethyleneimine (PEI) cellulose (Figure 2). In the first and second elution steps (in D1 and D2 direction), labeled normal (unmodified) deoxynucleotides as well as [32P]phosphate are eluted from the start of the TLC-PEI-cellulose plate using water solutions of electrolyte onto a short piece of chromatographic paper applied on the top of the TLC plate, whereas the deoxynucleotides containing bound chemicals exhibiting hydrophobic properties (carcinogen/drug-adducts) are maintained at the start of PEI-cellulose plate to be additionally resolved with several different solvent systems in D3 and D4 directions (Figure 2). Localization of adducts is performed using screen enhanced autoradiography; the separated adducts are detected as dark recognizable spots on X-ray films. The areas of spots are excised from the plate and used to quantify radioactivity by liquid scintillation or Cerenkov counting. A storage phosphor imaging method that has been adapted to map and quantify DNA adducts on chromatograms detected by the 32P-postlabeling assay is now also used.18 The Instant Imager machine is frequently utilized for such detection and quantification of DNA adducts. This method provides more than 10-times higher sensitivity for detecting 32P than the technique of screen enhanced autoradiography19.
Amounts of DNA adducts are determined as values of relative adduct labeling (RAL), calculated using the equation (2) as follows:
&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;cpm. in adduct deoxynucleotides 
RAL = ----------------------------------------------------------------------------------------- (2) 
&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; specific activity of 32P-ATP (in cpm./pmol) x pmol deoxynucleotides
The values of RALs are the ratio of count rates of adducted deoxynucleotides over count rates of total [adducted and normal (unmodified) deoxynucleotides] deoxynucleotides20,21. However, this calculation is based on equal labeling efficiencies of adducts and normal deoxynucleotides22. The classical (&#34;standard&#34;) procedure of the 32P-postlabeling technique is appropriate for various DNA adducts (bulky and/or non-bulky adducts), however, its sensitivity is not satisfactory to detect adducts found in low amounts in DNA. Using this procedure, the amount of an adduct in 107 unmodified deoxynucleotides in DNA (0.3 fmol adduct/&#181;g DNA) is detectable.
A variety of modifications of this classical 32P-postlabeling procedure have been utilized to elevate the sensitivity of the technique. Up to 10- to 100-times higher sensitivity of determination of adducts by 32P-labeling has been achieved using limiting levels of [&#947;-32P]ATP (the intensification procedure).23,24 A further procedure providing an increase in sensitivity of the 32P-postlabeling method utilizes an incubation of digested DNA containing adducts with nuclease P1 (from Penicillium citrinum)21 (Figure 1). This enzyme prefers to dephosphorylate unmodified deoxyribonucleoside 3&#180;-monophosphates, whereas the deoxynucleotides with bound chemicals (adducted nucleotides) are essentially not the substrates of this enzyme. Therefore, dephosphorylated deoxyribonucleoside 3&#180;-monophosphates (i.e., deoxyribonucleosides) are not phosphorylated by T4-polynucleotide kinase by [32P]phosphate from &#947;-32P]ATP. However, some of nucleotides where chemicals are bound (adducted deoxynucleotides), such as arylamine adducts substituted at C8 of deoxyguanosine, can bedephosphorylated by this enzyme. In contrast, most other adducts (e.g., adducts substituted at N2 of deoxyguanosine) are not dephosphorylated by nuclease P1. This modification of 32P-postlabeling makes this method considerably more sensitive, increasing its sensitivity by more than three orders of magnitude. Moreover, this version of 32P-postlabeling provides a method where higher amounts of DNA (5-10 &#181;g) and an excess of carrier-free [&#947;- 32P] ATP can be utilized.
Another method to enrich the adducts, described by Gupta25, utilizes the physicochemical properties of bulky deoxynucleotide adducts, which can be extracted into n-butanol in the presence of a phase transfer agent tetrabutylammonium chloride (TBA) (Figure 1) prior to [32P]phosphate labeling, whereas unmodified deoxynucleotides are poorly extracted by this organic solvent. However, less hydrophobic adducts, consisting for example of deoxynucleotides modified with non-aromatic bulky moieties or small alkyl residues, are not effectively extracted with n-butanol. Hence, they are essentially undetectable when are analyzed by this modification of the 32P-postlabeling method.
Both the previously mentioned versions of 32P-postlabeling increase the sensitivity and quantification of DNA adducts enormously (up to three orders of magnitude), being able to detect one adduct per 109,10 normal nucleotides (0.3 - 3 amol/&#181;g DNA). These two methods are recommended for testing the chemicals for their efficiency to covalently bind to DNA and, therefore, they are described in this work in details.

<table><tbody><tr><td>Tris</td><td>Sigma-Aldrich</td><td>252859</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9541</td><td></td></tr><tr><td>Sodium acetate</td><td>Sigma-Aldrich</td><td>S2889</td><td></td></tr><tr><td>Phenol</td><td>Roth</td><td>0032.8</td><td></td></tr><tr><td>Phenol/Chloroform/Isoamylalcohol</td><td>Roth</td><td>A156.1</td><td></td></tr><tr><td>Ethanol</td><td>Penta</td><td>70390-11000</td><td></td></tr><tr><td>Calf thymus DNA</td><td>Sigma-Aldrich</td><td>D4522</td><td></td></tr><tr><td>NADH</td><td>Sigma-Aldrich</td><td>N7004</td><td></td></tr><tr><td>NADP&lt;sup&gt;+&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>N5755</td><td></td></tr><tr><td>NADPH</td><td>Sigma-Aldrich</td><td>N7505</td><td></td></tr><tr><td>D-glucose 6-phosphate</td><td>Sigma-Aldrich</td><td>7647001</td><td></td></tr><tr><td>D-glucose 6-phosphate dehydrogenase</td><td>Sigma-Aldrich</td><td>G6378</td><td></td></tr><tr><td>Supersomes</td><td>Corning Gentest</td><td>456211, 456203, 456220, 456204, 456210, 456222, 456219, 456212, 456206, 456207, 456202</td><td></td></tr><tr><td>Human liver microsomes</td><td>Corning Gentest</td><td>452172</td><td></td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>P1379</td><td></td></tr><tr><td>Hypoxanthine</td><td>Sigma-Aldrich</td><td>77662</td><td></td></tr><tr><td>2-Hydroxypyrimidine</td><td>Sigma-Aldrich</td><td>H56800</td><td></td></tr><tr><td>Ethyl acetate</td><td>Sigma-Aldrich</td><td>437549</td><td></td></tr><tr><td>Diethyl ether</td><td>Sigma-Aldrich</td><td>179272</td><td></td></tr><tr><td>Micrococcal nuclease from &lt;em&gt;Staphylococcus aureus&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>N3755</td><td></td></tr><tr><td>Spleen phosphodiesterase from calf spleen, Type II</td><td>Calbiochem</td><td>524711</td><td></td></tr><tr><td>Nuclease P1 from &lt;em&gt;Penicillium citrinum&lt;/em&gt;</td><td>Sigma-Aldrich</td><td>N8630</td><td></td></tr><tr><td>Bicine</td><td>Sigma-Aldrich</td><td>163791</td><td></td></tr><tr><td>DL-Dithiotreitol</td><td>Sigma-Aldrich</td><td>D0632</td><td></td></tr><tr><td>Spermidine</td><td>Sigma-Aldrich</td><td>S2626</td><td></td></tr><tr><td>Tetrabutylammonium chloride</td><td>Sigma-Aldrich</td><td>86870</td><td></td></tr><tr><td>n-Butanol</td><td>Sigma-Aldrich</td><td>437603</td><td></td></tr><tr><td>T4-polynucleotide kinase</td><td>USB Corp</td><td>70031Y</td><td></td></tr><tr><td>[&amp;gamma;-&lt;sup&gt;32&lt;/sup&gt;P]ATP</td><td>Hartman Analytic GmbH</td><td>FP-201</td><td></td></tr><tr><td>PEI-impregnated cellulose TLC plates</td><td>Macherey-Nagel</td><td>801053</td><td></td></tr><tr><td>Packard Instant Imager A202400</td><td>Packard</td><td>G120337</td><td></td></tr><tr><td>Ellipticine</td><td>Sigma-Aldrich</td><td>285730</td><td></td></tr><tr><td>3-Nitrobenzanthrone</td><td>prepared (synthesized) as shown in ref. 40</td><td></td><td></td></tr></tbody></table>

formation of covalent dna adducts by enzymatically activated carcinogens and drugs in vitro and their determination by 32p-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of carcinogenic processes. This covalent binding, which is considered the cause of tumorigenesis, is now evaluated as a central dogma of chemical carcinogenesis. Here, methods are described employing the reactions catalyzed by cytochrome P450 and additional biotransformation enzymes to investigate the potency of chemicals or drugs for their activation to metabolites forming these DNA adducts. Procedures are presented describing the isolation of cellular fractions possessing biotransformation enzymes (microsomal and cytosolic samples with cytochromes P450 or other biotransformation enzymes, i.e., peroxidases, NADPH:cytochrome P450 oxidoreductase, NAD(P)H:quinone oxidoreductase, or xanthine oxidase). Furthermore, methods are described that can be used for the metabolic activation of analyzed chemicals by these enzymes as well as those for isolation of DNA. Further, the appropriate methods capable of detecting and quantifying chemical/drug-derived DNA adducts, i.e., different modifications of the 32P-postlabeling technique and employment of radioactive-labeled analyzed chemicals, are shown in detail.

Using the protocols described here for the utilization of enzyme-catalyzed activation (i.e., P450, peroxidase, reductase) to investigate potency of chemicals (carcinogens/drugs) to be metabolized to intermediates resulting in their covalent binding to DNA (generation of DNA adducts), we were able to resolve (i) a novel mechanism of the pharmacological action of the anticancer agent ellipticine (for a review see,29,30...

Watch this Scientific Journal Video about Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling at JoVE.com

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Covalent DNA adducts formed by chemicals or drugs with carcinogenic potency are judged as one of the most important factors in the initiation phase of ...

formation-covalent-dna-adducts-enzymatically-activated-carcinogens

Research

JoVE Journal

Biochemistry

13.8K Views.  Charles University. Evaluating the potency of environmental chemicals and drugs, to be enzymatically bioactivated to intermediates generating covalent DNA adducts, is an important field in the development of cancer and its treatment. Methods are described for compound activation to form DNA adducts, as well as techniques for their detection and quantification.

Video: Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling

Free Radicals in Chemical Biology: from Chemical Behavior to Biomarker Development

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological processes. This subject embraces diverse scientific areas, spanning from physical, biological and bioorganic chemistry to biology and medicine, with applications to the amelioration of quality of life, health and aging. Multidisciplinary skills are required for the full investigation of the many facets of radical processes in the biological environment and chemical knowledge plays a crucial role in unveiling basic processes and mechanisms. We developed a chemical biology approach able to connect free radical chemical reactivity with biological processes, providing information on the mechanistic pathways and products. The core of this approach is the design of biomimetic models to study biomolecule behavior (lipids, nucleic acids and proteins) in aqueous systems, obtaining insights of the reaction pathways as well as building up molecular libraries of the free radical reaction products. This context can be successfully used for biomarker discovery and examples are provided with two classes of compounds: mono-trans isomers of cholesteryl esters, which are synthesized and used as references for detection in human plasma, and purine 5',8-cyclo-2'-deoxyribonucleosides, prepared and used as reference in the protocol for detection of such lesions in DNA samples, after ionizing radiations or obtained from different health conditions.

Radical-based biomimetic chemistry has been applied to building-up libraries necessary for biomarker development.

The involvement of free radicals in life sciences has constantly increased with time and has been connected to several physiological and pathological ...

Chemistry

Single Molecule Analysis of Laser Localized Psoralen Adducts

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.

Lasers are frequently used in studies of the cellular response to DNA damage. However, they generate lesions whose spacing, frequency, and collisions with replication forks are rarely characterized. Here, we describe an approach that enables the determination of these parameters with laser localized interstrand crosslinks.

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live ...

Genetics

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

Cancer Research

Evaluating In Vitro DNA Damage Using Comet Assay

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer therapies, such as radio- and chemotherapy, introduce enormous amount of additional DNA damage, leading to cell cycle arrest and apoptosis to limit cancer progression. Quantitative assessment of DNA damage during experimental cancer therapy is a key step to justify the effectiveness of a genotoxic agent. In this study, we focus on a single cell electrophoresis assay, also known as the comet assay, which can quantify single and double-strand DNA breaks in vitro. The comet assay is a DNA damage quantification method that is efficient and easy to perform, and has low time/budget demands and high reproducibility. Here, we highlight the utility of the comet assay for a preclinical study by evaluating the genotoxic effect of olaparib/temozolomide combination therapy to U251 glioma cells.

Evaluating In Vitro DNA Damage Using Comet Assay

The comet assay is an efficient method to detect DNA damage including single and double-stranded DNA breaks. We describe alkaline and neutral comet assays to measure DNA damage in cancer cells to evaluate the therapeutic effect of chemotherapy.

DNA damage is a common phenomenon for each cell during its lifespan, and is defined as an alteration of the chemical structure of genomic DNA. Cancer ...

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Bioengineering

A Simple, Rapid, and Quantitative Assay to Measure Repair of DNA-protein Crosslinks on Plasmids Transfected into Mammalian Cells

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in mammalian cell lines. Rather than globally assaying removal of xenobiotic-induced or spontaneous chromosomal DPC removal, this assay examines the repair of a homogeneous, chemically defined lesion specifically introduced at one site within a plasmid DNA substrate. Importantly, this approach avoids the use of radioactive materials and is not dependent on expensive or highly-specialized technology. Instead, it relies on standard recombinant DNA procedures and widely available real-time, quantitative polymerase chain reaction (qPCR) instrumentation. Given the inherent flexibility of the strategy utilized, the size of the crosslinked protein, as well as the nature of the chemical linkage and the precise DNA sequence context of the attachment site can be varied to address the respective contributions of these parameters to the overall efficiency of DPC repair. Using this method, plasmids containing a site-specific DPC were transfected into cells and low molecular weight DNA recovered at various times post-transfection. Recovered DNA is then subjected to strand-specific primer extension (SSPE) using a primer complementary to the damaged strand of the plasmid. Since the DPC lesion blocks Taq DNA polymerase, the ratio of repaired to un-repaired DNA can be quantitatively assessed using qPCR. Cycle threshold (CT) values are used to calculate percent repair at various time points in the respective cell lines. This SSPE-qPCR method can also be used to quantitatively assess the repair kinetics of any DNA adduct that blocks Taq polymerase.

The goal of this protocol is to quantify the repair of defined DNA-protein crosslinks on plasmid DNA. Lesioned plasmids are transfected into recipient mammalian cell lines and low-molecular weight harvested at multiple time points post-transfection. DNA repair kinetics are quantified using strand-specific primer extension followed by qPCR.

The purpose of this method is to provide a flexible, rapid, and quantitative technique to examine the kinetics of DNA-protein crosslink (DPC) repair in ...

Measuring Ubiquitylated, SUMOylated, and ADP-Ribosylated DNA-Protein Crosslinks

Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, methyltransferases, etc.) malfunctioning, or exogenous agents such as chemotherapeutics and crosslinking agents. Once DPCs are induced, several types of post-translational modifications (PTMs) are promptly conjugated to them as early response mechanisms. It has been shown that DPCs can be modified by ubiquitin, small ubiquitin-like modifier (SUMO), and poly-ADP-ribose, which prime the substrates to signal their respective designated repair enzymes and, in some cases, coordinate the repair in sequential manners. As PTMs transpire quickly and are highly reversible, it has been challenging to isolate and detect PTM-conjugated DPCs that usually remain at low levels. Presented here is an immunoassay to purify and quantitatively detect ubiquitylated, SUMOylated, and ADP-ribosylated DPCs (drug-induced topoisomerase DPCs and aldehyde-induced non-specific DPCs) in vivo. This assay is derived from the RADAR (rapid approach to DNA adduct recovery) assay that is used for the isolation of genomic DNA containing DPCs by ethanol precipitation. Following normalization and nuclease digestion, PTMs of DPCs, including ubiquitylation, SUMOylation, and ADP-ribosylation, are detected by immunoblotting using their corresponding antibodies. This robust assay can be utilized to identify and characterize novel molecular mechanisms that repair enzymatic and non-enzymatic DPCs&#160;and has the potential to discover small molecule inhibitors targeting specific factors that regulate PTMs to repair DPCs.

Author Spotlight: Quantitative Detection of DNA Protein Crosslinks and Their Post-Translational Modifications  

The present protocol highlights a modified method to detect and quantify DNA-protein crosslinks (DPCs) and their post-translational modifications (PTMs), including ubiquitylation, SUMOylation, and ADP-ribosylation induced by topoisomerase inhibitors and by formaldehyde, thereby allowing the study of the formation and repair of DPCs and their PTMs.

DNA-protein crosslinks (DPCs) are frequent, ubiquitous, and deleterious DNA lesions, which arise from endogenous DNA damage, enzyme (topoisomerases, ...

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

Immunology and Infection

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

Biology

Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles

Nanomaterial exposure can cause replication stress and genomic instability in cells. The degree of instability depends on the chemistry, size, and concentration of the nanomaterials, the time of exposure, and the exposed cell type. Several established methods have been used to elucidate how endogenous/exogenous agents impact global replication. However, replicon-level assays, such as the DNA fiber assay, are imperative to understand how these agents influence replication initiation, terminations, and replication fork progression. Knowing this allows one to understand better how nanomaterials increase the chances of mutation fixation and genomic instability. We used RAW 264.7 macrophages as model cells to study the replication dynamics under graphene oxide nanoparticle exposure. Here, we demonstrate the basic protocol for the DNA fiber assay, which includes pulse labeling with nucleotide analogs, cell lysis, spreading the pulse-labeled DNA fibers onto slides, fluorescent immunostaining of the nucleotide analogs within the DNA fibers, imaging of the replication intermediates within the DNA fibers using confocal microscopy, and replication intermediate analysis utilizing a computer-assisted scoring and analysis (CASA) software.

The methodology assists in the analysis of variation in DNA replication dynamics in the presence of nanoparticles. Different methodologies can be adopted based on the cytotoxicity level of the material of interest. In addition, a description of the image analysis is provided to help in DNA fiber analysis.

Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by ³²P-postlabeling

Summary

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Quantitative Detection of DNA-Protein Crosslinks and Their Post-Translational Modifications

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Demonstration of the DNA Fiber Assay for Investigating DNA Damage and Repair Dynamics Induced by Nanoparticles