Uranium-Induced DNA Damage

Project Background

The goal of this project is to see if and how uranium (U) may

contribute to DNA damage. Radioactive forms of uranium have been used

as fuel for nuclear reactors and as fissionable material for atomic weapons,

tank armor, and ammunition shells. People who work with or live near

processed uranium have experienced higher incidence of lung cancer,

which has been attributed to radon, a radioactive daughter product (gas)

of uranium. The non-cancer effects of uranium itself (not radon), such as

DNA damage (genotoxicity), are less well studied.

What Will I Learn?

use of positive controls

use of high-pressure liquid chromatography (HPLC) for reaction mechanism and kinetics

studies

use of ChemDraw, or similar software, for drawing chemical structures and mechanisms

Who, What, Why?

Who is this project for?

This is an excellent project for students interested in

toxicology, biochemistry, or pharmacology, as well as

students interested in learning about HPLC.

What Will I Do?

In this project, you will examine the mechanism by

which a uranium complex cleaves a dinucleotide bond

(a simple model for DNA) in the presence of vitamin

C. If the dinucleotide behaves like DNA, this would

yield information on the mechanisms of DNA damage

by uranium. You will examine HPLC data to determine

which of two possible reactions are occuring between

the uranium complex and the DNA model.

To Learn More

Civitello, E. R.; Leniek, R. G.; Hossler, K. A.; Haebe, K.; Stearns, D. M. Synthesis of Peptide-Oligonucleotide Conjugates for Chromium Coordination.

Bioconjugate Chem. 2001, 12, 459 463.

Komiyama, M.; Takeda, N.; Shigekawa, H. Hydrolysis of

DNA and RNA by Lanthanide Ions: Mechanistic

Studies Leading to New Applications. Chem.

Commun. 1999, 16, 1443 1451. (available free of

charge at the RSC Publishing Web site).

Priest, N. D. Toxicity of Depleted Uranium, Lancet 2001,

357, 244 246.

Taqui Khan, M. M.; Martell, A. E. Kinetics of Metal Ion and

Metal Chelate Catalyzed Oxidation of Ascorbic Acid.

IV. Uranyl Ion Catalyzed Oxidation. J. Am. Chem. Soc.

1969, 91, 4668 4672.

Yazzie, M.; Gamble, S. L.; Civitello, E. R.; Stearns, D. M.

Uranyl Acetate Causes DNA Single Strand Breaks In

Vitro in the Presence of Ascorbate (Vitamin C).

Chem. Res. Toxicol. 2003, 16, 524 530.

Canned Research

Project 4

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Uranium-Induced DNA Damage

*Marin Robinson, Molly Costanza-Robinson, Catherina Salanga, Edgar Civitello, Diane Stearns

TABLE OF CONTENTS

PROJECT INTRODUCTION 2

The project introduction places the work in context, emphasizing why the

work is important and providing an overview of the project’s purpose and

methods. Information included in this section, together with details on the

cover sheet, will help you write the Introduction for your paper.

METHODS 3

This section describes, step-by-step, the methods used to conduct the work.

Because research projects are complex, multiple groups of individuals over

several years are often needed to finish a project. For this reason, this

section has been divided into two parts: previous work (i.e., work conducted

by others before you entered the group) and your work (i.e., work

completed by you). The Methods section of your final paper need only

include your work; however, if this were a real research project resulting in

a real manuscript, the Methods section would include all relevant work.

RESULTS 6

This section includes the HPLC results, prompts you to consider important

features of the results, and suggests ways to share the results with an expert

audience.

DISCUSSION 10

This section suggests way to interpret your findings, propose a mechanism

for the reaction, and share the mechanism in a scheme and text. The

section also prompts you to consider the broader implications of your

work.

BACKGROUND INFORMATION 11

In this section, background information is provided on the dinucleotide

and possible mechanisms for the dinucleotide cleavage reaction are

suggested.

*corresponding author

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PROJECT INTRODUCTION

Uranium exists in several forms, many of which are radioactive. Human exposure to uranium has

occurred mostly through uranium mining, such as in the Four Corners region of the U. S.

Southwest (1940 1980), and through depleted uranium use in the military. Epidemiological

studies have shown that working with, or living near, processed uranium leads to higher

incidence of lung cancer, a cancer that is attributed to radon, a radioactive gas that is a daughter

product of uranium decay. Although the cancerous effects of uranium and its decay products

have been studied, its genotoxicity (DNA-damaging effects) has only recently been addressed.

The genotoxic effects of other heavy metals have been investigated. For example, the mechanism

by which cerium(IV) cleaves DNA is known. In this project, you will compare the unknown

cleavage mechanism of uranium to the known cleavage mechanism of cerium(IV).

Purpose. The purpose of this study is to better understand the genotoxicity of uranium. Initial

studies between uranium and DNA showed no reaction, thus you will be studying the reaction

again under conditions that better mimic human physiological conditions.

Methods. You will study the reaction of uranyl acetate with thymidylyl (3'→5')-2'-

deoxyadenosine in the presence of ascorbate. Uranyl acetate was selected because it is a common

form of uranium. It is also water soluble and can enter into the cell. Thymidylyl (3'→5')-2'-

deoxyadenosine, a dinculeotide with two base pairs, was selected as a surrogate for DNA. DNA

itself is too large and complex to be detected by conventional HPLC methods. Ascorbate

(vitamin C), a common antioxidant in the body, was included to better mimic human physiology.

Representing the dinucleotide as dTpdA, two different cleavage pathways are possible:

dTpdA dT + pdA pathway 1

or

dTpdA dTp + dA pathway 2

Essentially, the dinucleotide can be cleaved in two different places (on either side of the

“p”, which represents phosphorus). You will use high-pressure liquid chromatography

(HPLC) to monitor the products of the reaction. You may observe peaks corresponding to

dTpdA (no cleavage), dT and pdA (pathway 1), or dTp and dA (pathway 2). After

observing which peaks are formed, you will propose a dinucleotide cleavage mechanism.

It is also possible, of course, that both pathways occur to different degrees or that no

dinucleotide cleavage occurs.

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METHODS

Part 1: Previous Work

Research projects are generally long-term and involve many different students over several

semesters or years. Your canned research project is no exception, and you will be joining the

project midstream. Previous members of your group have already shown that uranyl acetate in

the presence of ascorbate forms a uranyl acetate complex and causes DNA strand breaks. Their

results were published (see Excerpt 1). Additionally, these students developed the HPLC method

you will use in your work. They determined the optimal wavelength (260 nm) to use to detect the

species of interest and the average retention times for these species. These values are shown in

Excerpt 2.

Excerpt 1 (description of prior work)

The reaction of uranyl acetate and ascorbate was shown to produce plasmid

relaxation in pBluescript DNA. Reactions of uranyl ascorbate, monitored by 1H NMR

spectroscopy, showed formation of a uranyl-ascorbate complex (Yazzie et al., 2003).

Excerpt 2 (lab notebook summary of prior work)

April 10, 2008

Average HPLC retention times for absorbing species (260 nm) (n = 5). All concentrations

were 10 mM in 100 mM NaCl.

species (10 mM) ave retention time (min)

Asc 2.9

dT 8.2

pdA 8.9

dTp 9.6

dA 7.4

dTpdA 11.9

Before continuing . . .

Be sure that you

understand the terms dTpdA, dT, pdA, dTp, and dA

understand the purpose of ascorbate in the reaction

can explain why uranyl acetate is not listed in Excerpt 2

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Part 2: Your Work

The methods for your canned research project are described below. Three tasks are described

including (1) preparing buffer solutions, (2) preparing solutions for the positive control and

experimental reactions, and (3) monitoring reaction progress with HPLC. Note the change in

format. The information is written as it might appear in a lab manual or notebook. Your goal will

be to rewrite this information for an expert audience as it might appear in a journal article. (See

also the Background Information section for more information on these reactions.)

June 13, 2008

Goal: Prepare buffer solutions

Chemicals are from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.

(1) Prepare HEPES buffer (the buffer used in the positive control reaction)

A 20 mM solution of N-2-hydroxyethylpiperaxine-N'-2-ethanesulfonic acid (HEPES) buffer

will be used in the positive control reaction with cerium(IV) nitrate. To prepare the buffer,

add 2.383 g of HEPES to ~425 mL of nanopure water in a 2 L flask. Add a few scoops of

Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA) to the solution; stir for several hours.

(The Chelex will remove trace metals so they do not interfere with the desired reactions.)

Filter the solution. Adjust final pH to 7.0 (±0.1). Bring final volume to 500 mL with

nanopure water.

(2) Prepare Tris-HCl buffer (the buffer used in the experimental reaction)

It has already been shown that uranyl acetate (UA) and ascorbate (Asc) do not cleave DNA

in HEPES buffer, so the experiment will be performed in a different buffer, 20 mM

tris(hydroxymethyl) aminomethane hydrochloride (Tris-HCl). To prepare the buffer, add

1.2114 g of Tris-HCl to ~425 mL of nanopure water in a 2 L flask. Add a few scoops of

Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA) to the solution; stir for several hours.

Filter the solution. Adjust final pH to 8.0 (±0.1). Bring final volume to 500 mL with

nanopure water.

June 14, 2008

Goal: Prepare solutions for Ce(IV) and UA-Asc reactions

Chemicals

HEPES buffer (prepared on June 13, 2008)

Tris-HCl buffer (prepared on June 13, 2008)

Ce(IV) = ammonium cerium(IV) nitrate (Sigma-Aldrich, MW 548.22)

dTpdA = thymidylyl(3' 5')-2'-deoxyadenosine ammonium salt (Sigma-Aldrich, MW

570.45)

NaCl = sodium chloride (Sigma-Aldrich, MW 58.44)

UA = uranyl acetate dihydrate (Spectrum Chemical Mfg. Corp., Garden, CA, MW 424.15)

Asc = L-ascorbic acid (Sigma-Aldrich, MW 176.12)

Chelex 100 resin (Bio-Rad Laboratories, Hercules, CA)

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(1) Prepare solutions for 10 mM Ce(IV) positive control reaction

Prepare each solution in 1 mL HEPES. The final volume will be 5 mL, so masses are shown

for a 5 mL solution. Store solutions separately in centrifuge tubes until ready to start reaction.

To start a reaction, combine the three individual solutions and dilute to 5 mL with HEPES.

Ce(IV) @ 10 mM = 27.41 mg (for 5 mL)

dTpdA @ 1 mM = 2.85 mg (for 5 mL)

NaCl @ 100 mM = 29.22 mg (for 5 mL)

(2) Prepare solutions for 10 mM UA-Asc experimental reaction

Prepare each solution in 1 mL Tris-HCl. The final volume will be 5 mL, so masses are shown

for a 5 mL solution. Store solutions separately in centrifuge tubes until ready to start reaction.

To start a reaction, combine the four individual solutions and dilute to 5 mL with Tris-HCl.

dTpdA @ 1 mM = 2.85 mg (for 5 mL)

NaCl@100 mM = 29.22 mg (for 5 mL)

UA@10 mM = 21.21 mg (for 5 mL)

Asc@10 mM = 8.81 mg (add Chelex to soln; shake for 10 min; microfilter into new

tube) (for 5 mL)

June 15, 2008

Goal: Monitor reactions using HPLC

HPLC conditions

The HPLC is located in CHM 403. It is a Varian Dynamax HPLC equipped with a C-18

column (41.4 × 250 mm, 8 µm, 100 Å) and UV detector (260 nm). Column eluents will be

solvent A: 50 mM triethylamine and 46.5 mM glacial acetic acid in water and solvent B: 50

mM triethylamine and 46.5 mM glacial acetic acid in 90/10 acetonitrile/water. The linear

gradient will be 0 30% solvent B for 20 min at 1 mL/min.

Procedure

Each reaction will start when the reagents are mixed (t = 0 h). At specific times, a sample

of the reaction mixture will be injected onto the HPLC column (e.g., 0, 12, 24 h), to

monitor the progress of the reaction. Injections will be continued until the reaction is

complete (i.e., the concentrations of reactants and products are no longer changing over

time). The products will be identified by comparing the chromatographs with known

retention times (see Excerpt 2).

Product identity will be confirmed by spike additions. The UA + Asc reaction will be

prepared as before, but the reactants will be spiked with the known standards, one at a

time (e.g., first dT, then pdA). The reaction can be run immediately (t = 0 h). The spikes

should have the same retention times as the observed products.

To be sure that it is the UA-Asc complex that reacts with dTpdA and not UA or Asc,

repeat the reaction with UA and Asc separately. In each case, let the reaction run for a

long time (>72 h), to be sure that no reaction occurs.

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RESULTS

The results of the various runs are shown below in six chromatographs. Identify the products and

starting materials in each reaction by comparing the chromatographs against the retention times

in Excerpt 2. As you examine these chromatographs, consider the following questions:

Is dTpdA cleaved in the presence of Ce(IV)? If so, what products are formed?

Is dTpdA cleaved in the presence of UA and Asc? If so, what products form? Are the

products the same or different from the products in the Ce(IV) reaction?

Did the spike additions help you confirm your assignments?

How long did it take for the positive control reaction to reach completion? Compare this

time to the time needed for the UA-Asc reaction. Which reaction appears to be slower?

Does the evidence suggest that it was UA, Asc, or a UA-Asc complex that was involved

in the cleavage reaction?

These are not real chromatographs!

Have fun with them,

but do not take them too seriously.

Before continuing . . .

Think about how you will describe these methods in your paper.

Using keywords (e.g, Varian Dynamax, HPLC, HEPES, Tris-HCl) find

journal articles that describe procedures similar to those above. Read

the Methods sections of these articles to help you better understand

these procedures and to examine ways in which they are described by

experts (e.g., consider what details are included or omitted, what

subheadings are used, how concentrations are reports).

Notice that three different solutions were treated with Chelex. Can you

state that in a single sentence rather than repeating it three times?

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.

1. Positive control reaction: 10 mM Ce(IV) + 1 mM dTpdA (in HEPES and NaCl)

2. Experimental reaction: 10 mM Asc + 10 mM UA + 1 mM dTpdA (in Tris-HCl/NaCl)

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3. Spike addition of dT: 10 mM Asc + 10 mM UA + 1 mM dTpdA + 0.5 mM dT (t = 0 h)

4. Spike addition of pdA: 10 mM Asc + 10 mM UA + 1 mM dTpdA + 0.5 mM pdA (t = 0 h)

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5. Reaction with UA alone: 10 mM UA + 1 mM dTpdA (in NaCl and Tris-HCl) (t = 96 h)

6. Reaction with Asc alone: 10 mM Asc + 1 mM dTpdA (in NaCl and Tris-HCl) (t = 96 h)

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DISCUSSION

How will you discuss your results? Answer the following questions to guide you in writing the

Discussion section of your paper.

What is the mechanism for the UA-Asc reaction. (Hint: see Background Information below).

Include the mechanism in a properly formatted scheme, and “walk” the readers through the

mechanism in the text.

What did you learn about the rates of the Ce(IV) reaction vs. the UA-Asc reaction? Can you

offer an explanation for this finding?

How do your findings compare to the literature? What other mechanisms have been proposed

to explain how UA-Asc cleaves DNA? Suggest a reason for the similarity or difference

between your findings and the literature.

How can you summarize your findings? What implications can you suggest?

Before continuing . . .

Think about how you will present these results in your paper. What story do

the data tell about the UA-Asc cleavage reaction of the dinucleotide? Use

your answers to the guiding questions stated earlier to address this question.

Typically, authors do not include chromatographs in their papers; more

often, they describe the results in the text. We recommend this approach

here. Try to describe your results in the text alone, without any figures.

Also, if you follow this approach, you will not need to waste time trying to

copy & paste the chromatographs in your paper.

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BACKGROUND INFORMATION

Thymidylyl (3'→5')-2'-deoxyadenosine (dTpdA) was chosen for these experiments. It comprises

two nucleotides, thymidine and adenosine, shown in Figures 1 and 2, respectively. (The

hydrolysis literature indicates that there is not a specific base (nucleotide) that undergoes

hydrolysis preferentially; hence, it doesn’t matter very much which pair of nucleotides is

examined.)

Figure 1. Structure of the thymidine nucleotide (dT) where thymidine = thymine base pair +

ribose ring.

Figure 2. Structure of the adenosine nucleotide (dA) where adenosine = adenine base pair +

ribose ring.

thymine

ribose

ribose

adenine

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Figure 3 depicts the dinucleotide, dTpdA, where the two nucleotides dT and dA are linked by a

phosphate backbone (p). Phosphate binds together additional bases, ultimately forming DNA.

Figure 3. Structure of the dinucleotide, dTpdA.

A cleavage mechanism of a generic dinucleotide by a UA-Asc complex is shown in Scheme 1

(Yazzie et al., 2003). In your paper, you will need to adapt this mechanism for dTpdA. Based on

the data you collect, you will determine which base pair goes in which location in Scheme 1.

You will use software, such as ChemDraw, to tailor Scheme 1 to your specifics results.

Scheme 1

O

O

HO

P OHO

OO

O

H

HN

NO

O

N

N N

N

NH2

dT

p

dA