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
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).

All animal experiments were performed in accordance with the Regulations for the Care and Use of Laboratory Animals (311/1997, Ministry of Agriculture, Czech Republic), which is in compliance with the Declaration of Helsinki.
1. Isolation of Hepatic Microsomal and Cytosolic Fractions
<ol>
	<li>Prepare liver subcellular fractions (microsomes rich in P450 enzymes or cytosols rich in reductases or soluble peroxidases) from rats by simple differential centrifugation (105,000 x g). ...

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+</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 Staphylococcus aureus</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 Penicillium citrinum</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>[&#947;-32P]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 colspan="2">prepared (synthesized) as shown in ref. 40</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 ...

The overall goal of this protocol is description of methods 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 covalent DNA adducts. This method can help to answer the key questions in the field of the development of cancer and its treatment. The main advantage of this technique is that it shows you a type of method to evaluate the potency of environmental chemicals or drugs to form covalent DNA adducts representing initiation of cancer development.The implications of the technique determining the carcinogenic potency of chemicals. Environmental potence, toxicants, and drugs and also determines relatively simple methods for the formation of covalent DNA adducts formed by such compounds. Although this method can provide insight into initiation of the development of cancer and its treatment, it can also be applied to other scientific fields.Such as studies of molecular mechanisms, of DNA damage, and oral studies on enzymatic activation and the toxification of chemicals. I first heard the idea for this method when I investigated processes involved in initiation phase of chemical carcinogenesis. First prepare the incubation mixture by adding all the compounds and the enzyme systems to a final volume of 0.75 milliliter at four degrees Celsius.This is to incubate the carcinogen with the DNA in the presence of cytochrome p450. Then, add microsomes or pure recombinant p450 in supersome to the mixture. Next, add 7.5 microliters of 0.1 millimolar ellipticine drug dissolved in DMSO and 42.5 microliters of water to adjust the final volume to 0.75 milliliter.Then, mix the components by vortexing the tube for five seconds. Immediately after, incubate the tube at 37 degrees celsius with the lid open for 30 to 60 minutes. Next, mix phenol or phenol chloroform in a one to one ratio with the incubation mixture in an Eppendorf Tube.This will remove the protein contamination from the DNA in the incubation mixture. Then stir the mixture to form an emulsion. Then spin the mixture at 1600 times g for three minutes in ambient temperature.Once the centrifugation is over use a pipette to transfer the upper water phase to a new polypropylene tube. Then discard the protein interface together with the organic phase. Next, mix the water phase with equivalent volume of phenol and chloroform in one to one ratio.Then repeat the centrifugation. Next add equal volumes of chloroform to the water phase collected and again centrifuge. Then precipitate the DNA from the aqueous phase by adding two volumes of cold ethanol maintained at minus 20 degrees Celsius.Then mix the solution well. To prepare for labeling the DNA adducts, mix the bicine buffer solution with radiolabeled ATP solution to label the DNA adducts. Add the labeling mixture to NP1 solution, or butanol enrichment mix.Let the mixture stand at room temperature for 30 minutes. Then apply approximately 20 microliters at the entire sample on the PEI-cellulose TLC plate. After spotting the sample on PEI-cellulose TLC plate, leave the plate to develop in D1 direction to clean up the adducts.Once the plate is dried up, leave the plate to develop in D3 and D4 directions. To develop in D3, use lithium formate buffer at pH 3.5 with urea. For D4 development, use Tris-HCl buffer with lithium chloride and urea.In order to avoid any problems for developing in D4 direction, develop the plates in D5 direction as well. First, P32-postlabeling technique is utilized to study the efficiency of microsomal p450 mediated bioactivated ellipticine in forming DNA adducts. Results show formation of covalent DNA adducts in the deoxyguanosine of the calf thymus DNA in the presence of both rat and or human hepatic microsomes;however, no adducts are formed in the control group.Next, the potential of peroxidase enzyme dependent activated ellipticine is studied. The image shows the formation of covalent DNA adducts in calf thymus DNA in the presence of horseradish peroxidase, bovine lactoperoxidase, and human myeloperoxidase. In fact, DNA adducts are also formed from the liver DNA when the rats are treated with 40 milligrams per kilogram body weight of ellipticine in vivo.Interestingly, the human cytochrome p450 enzyme CYP3A4 also shows strong potential to efficiently oxidize ellipticine to metabolites such as 12-hydroxyellipticine and 13-hydroxyellipticine. These metabolites can easily bind to the DNA to form the adducts. Next, nuclease P1 and n-butanol enrichment methods are used to study the formation of DNA adducts upon activation of 3-nitrobenzanthrone with cytosolic reductase such as NQ01 from rat and human.The results show formation of up to five DNA adducts from the calf thymus DNA obtained from the rat, human hepatic cytosol, and also mice liver. Once mastered, this technique can be done in two days if it is performed properly. By attempting this procedure it is important to remember the stability of the enzymatic systems used.The enzyme activity may diminish if not stored on ice. After its development, this technique paved the way for researchers in the field of biochemistry, the biochemistry of chemical carcinogenesis to explore nature of phenomenon from the initiation processes of cancer development in organisms. After watching this video, you should have a good understanding of how to evaluate the potency of environmental chemicals or drugs to be enzymatically activated to metabolize genetic covalent DNA adducts.This process represents high importance for development of cancer-based treatments. Don't forget that you are working with chemicals that can be extremely hazardous. Potential carcinogens, radioactive chemicals, for example phosphorus ATP.Therefore precautions such as laboratory latex gloves and plexiglass sheets should always be taken while performing this procedure.

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Research

JoVE Journal

Biochemistry

Covalent Binding of Antibodies to Cellulose Paper Discs and Their Applications in Naked-eye Colorimetric Immunoassays

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) discs and grade No. 113 (fast-flow filter paper) discs. These cellulose paper discs were grafted with amine functional groups through a silane coupling technique before the antibodies were immobilized on them. Periodate oxidation and glutaraldehyde cross-linking methods were used to graft capture antibodies on the cellulose paper discs. In order to ensure the maximum binding capacity of the capture antibodies to their targets after immobilization, the effects of various concentrations of sodium periodate, glutaraldehyde, and capture antibodies on the surface of the paper discs were investigated. The antibodies that were coated on the amine-functionalized cellulose paper discs through a glutaraldehyde cross-linking agent showed enhanced binding activity to the target when compared to the periodate oxidation method. IgG (in mouse reference serum) was used as a reference target in this study to test the application of covalently immobilized antibodies through glutaraldehyde. A new paper-based, enzyme-linked immunosorbent assay (ELISA) was successfully developed and validated for the detection of IgG. This method does not require equipment, and it can detect 100 ng/ml of IgG. The fast-flow filter paper was more sensitive than the medium-flow filter paper. The incubation period of this assay was short and required small sample volumes. This naked-eye, colorimetric immunoassay can be extended to detect other targets that are identified with conventional ELISA.

In this manuscript, periodate oxidation and glutaraldehyde cross-linking methods for covalent immobilization of antibodies on paper discs are presented. The binding activity of immobilized antibodies was evaluated. Based on these results, a glutaraldehyde cross-linking method was used to develop a novel paper-based immunoassay for immunoglobulin G (IgG) detection.

This report presents two methods for the covalent immobilization of capture antibodies on cellulose filter paper grade No. 1 (medium-flow filter paper) ...

A G-quadruplex DNA-affinity Approach for Purification of Enzymatically Active G4 Resolvase1

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly thermally stable. There are &#62;375,000 putative G4-forming sequences in the human genome, and they are enriched in promoter regions, untranslated regions (UTRs), and within the telomeric repeat. Due to the potential for these structures to affect cellular processes, such as replication and transcription, the cell has evolved enzymes to manage them. One such enzyme is G4 Resolvase 1 (G4R1), which was biochemically co-characterized by our laboratory and Nagamine et al. and found to bind extremely tightly to both G4-DNA and G4-RNA (Kd in the low-pM range). G4R1 is the source of the majority of G4-resolving activity in HeLa cell lysates and has since been implicated to play a role in telomere metabolism, lymph development, gene transcription, hematopoiesis, and immune surveillance. The ability to efficiently express and purify catalytically active G4R1 is of importance for laboratories interested in gaining further insight into the kinetic interaction of G4 structures and G4-resolving enzymes. Here, we describe a detailed method for the purification of recombinant G4R1 (rG4R1). The described procedure incorporates the traditional affinity-based purification of a C-terminal histidine-tagged enzyme expressed in human codon-optimized bacteria with the utilization of the ability of rG4R1 to bind and unwind G4-DNA to purify highly active enzyme in an ATP-dependent elution step. The protocol also includes a quality-control step where the enzymatic activity of rG4R1 is measured by examining the ability of the purified enzyme to unwind G4-DNA. A method is also described that allows for the quantification of purified rG4R1. Alternative adaptations of this protocol are discussed.

G4 Resolvase1 binds to G-quadruplex (G4) structures with the tightest reported affinity for a G4-binding protein and represents the majority of the G4-DNA unwinding activity in HeLa cells. We describe a novel protocol that harnesses the affinity and ATP-dependent unwinding activity of G4-Resolvase1 to specifically purify catalytically active recombinant G4R1.

Higher-order nucleic acid structures called G-quadruplexes (G4s, G4 structures) can form in guanine-rich regions of both DNA and RNA and are highly ...

Real-time Tracking of DNA Fragment Separation by Smartphone

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and others. However, the traditional SGE protocol is quite tedious, and the experiment takes a long time. Moreover, the chemical consumption in SGE experiments is very high. This work proposes a simple method for the separation of DNA fragments based on an SGE chip. The chip is made by an engraving machine. Two plastic sheets are used for the excitation and emission wavelengths of the optical signal. The fluorescence signal of the DNA bands is collected by smartphone. To validate this method, 50, 100, and 1,000 bp DNA ladders were separated. The results demonstrate that a DNA ladder smaller than 5,000 bp can be resolved within 12 min and with high resolution when using this method, indicating that it is an ideal substitute for the traditional SGE method.

Traditional slab gel electrophoresis (SGE) experiments require a complicated apparatus and high chemical consumption. This work presents a protocol that describes a low-cost method to separate DNA fragments within a short timeframe.

Slab gel electrophoresis (SGE) is the most common method for the separation of DNA fragments; thus, it is broadly applied to the field of biology and ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

DNA Sequence Recognition by DNA Primase Using High-Throughput Primase Profiling

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. The binding of prokaryotic DnaG-like primases to DNA occurs at a specific trinucleotide recognition sequence. It is a pivotal step in the formation of Okazaki fragments. Conventional biochemical tools that are used to determine the DNA recognition sequence of DNA primase provide only limited information. Using a high-throughput microarray-based binding assay and consecutive biochemical analyses, it has been shown that 1) the specific binding context (flanking sequences of the recognition site) influences the binding strength of the DNA primase to its template DNA, and 2) stronger binding of primase to the DNA yields longer RNA primers, indicating higher processivity of the enzyme. This method combines PBM and primase activity assay and is designated as high-throughput primase profiling (HTPP), and it allows characterization of specific sequence recognition by DNA primase in unprecedented time and scalability.&#160;

Protein binding microarray (PBM) experiments combined with biochemical assays link the binding and catalytic properties of DNA primase, an enzyme that synthesizes RNA primers on template DNA. This method, designated as high-throughput primase profiling (HTPP), can be used to reveal DNA-binding patterns of a variety of enzymes.

DNA primase synthesizes short RNA primers that initiate DNA synthesis of Okazaki fragments on the lagging strand by DNA polymerase during DNA replication. ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

Crystallization and Structural Determination of an Enzyme:Substrate Complex by Serial Crystallography in a Versatile Microfluidic Chip

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We describe a versatile microfluidic chip that enables the production of crystals by the efficient method of counter-diffusion. The convection-free environment provided by the microfluidic channels is ideal for crystal growth and useful to diffuse a substrate into the active site of the crystalline enzyme. Here we applied this approach to the CCA-adding enzyme of the psychrophilic bacterium Planococcus halocryophilus in the presented example. After crystallization and substrate diffusion/soaking, the crystal structure of the enzyme:substrate complex was determined at room temperature by serial crystallography and the analysis of multiple crystals directly inside the chip. The whole procedure preserves the genuine diffraction properties of the samples because it requires no crystal handling.

A versatile microfluidic device is described that enables the crystallization of an enzyme using the counter-diffusion method, the introduction of a substrate in the crystals by soaking, and the 3D structure determination of the enzyme:substrate complex by a serial analysis of crystals inside the chip at room temperature.

The preparation of well diffracting crystals and their handling before their X-ray analysis are two critical steps of biocrystallographic studies. We ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...