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 ...

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