A method for the rapid quantitation of methylated hepatic DNA-purines using high pressure liquid chromatography

A Method for the Rapid Quantitation of Methylated Hepatic DNA-Purines Using High Pressure liquid Chromatography

E. M. FAUSTMAN

An isocratic

AND J. I. GOODMAN

high

pressure

for the separation monly

found

liquid

chromatography

and quantitation

in DNA

(I-methyladenine,

system

of four of the major

after exposure

that can be employed

methylated

to carcinogenic/mutagenic

3-methyladenine,

Oh-methylguanine

purines

com-

alkylating

agents

and 7-methylguanine)

is

in which rats were treated with methyl-N-nitrosourea have been used to illustrate the applicability of the method. The amounts of the methylated bases observed were consistent with values reported in the literature. presented

in this paper.

Key Words:

Data from

Alkylation;

experiments

Carcinogen;

DNA-purine;

High pressure

liquid

chro-

matography

INTRODUCTION Previous and

studies

Lawley,

cinogens form

yield

covalent

stituents

and

Miller,

Heidelberger,

1947;

Marroquin

1964) indicating

electrophiles,

either

adducts

nucleophilic

with

that

spontaneously

appear to represent

Weinstein

centers

Thus,

The following posure

methylated

to carcinogenic

methylguanine

(Oh-MeG), Swann

DNA-purines

alkylating

(Lawley,

1966;

sistence

of “promutagenic”

in DNA

and

carthat

cellular

conThese

in carcinogenesis

of the mechanism(s)

(Miller,

of specific

to occur following (7-MeG),

and I-methyladenine

Frei et al., 1978). The

in DNA has been proposed

car-

of this process.

7-methylguanine

(3-MeA)

Brookes

activation,

other

and quantitation

in vivo;

3-methyladenine

1962;

carcinogenesis.

have been shown

agents

and Magee, 1968; lesions

events

identification

cinogen adducts is crucial to our understanding

Farber,

if not all, chemical

in chemical

one of the initial

et al., 1979).

and most,

or via metabolic

led to one of the key concepts

have

reactions 1978;

(Miller

1964;

ex06-

(I-MeA)

formation

and per-

as one of the cellular

modifications that could lead to tumor initiation following exposure to alkylating agents (Loveless, 1969; Lawley, 1972; Pegg and Nicoll, 1976; Maher and McCormick, 1976). A variety ines:

of methods

paper and thin

From

the

Department

have been used to identify layer chromatographic

of Pharmacology

and quantitate

analysis,

and Toxicology,

methylated

cation and anion

Michigan

State

University,

pur-

exchange East

Lansing,

Michigan. Address

reprint

requests

to Dr. J. I. Goodman,

State University, East Lansing, Michigan Received and accepted April 1, 1980.

Department

of Pharmacology

and Toxicology,

Michigan

48824.

305 lournalof

Pharmacologscal

‘C 1980 Elsewer

North

Methods

Holland,

Inc.,

4, 305-312 52 Vanderbilt

(198OJ Avenue,

0160.5402/80108030508/$02 New

York,

NY 10017

25

306

E. M. Faustman

column

and J. I. Goodman

chromatography,

and Sephadex

ods have been reviewed pressure Webb,

liquid 1977;

we present ylated

by Brookes

chromatography

Frei

et al., 1978;

an improved

purines

technique.

from

(HPLC) Shaikh

method

the DNA

Data from

G-10 column

chromatography.

and Lawley (1971). More systems

have been devised

et al., 1978;

Thielmann,

for the isolation

experiments

using

1979.

employing

methyl-N-nitrosourea

meth-

various

high

(Bouchert

and

In this

report

of four

meth-

an isocratic

HPLC

and quantitation

of hepatic chromatin

These

recently,

(MNU)

as the meth-

ylating carcinogen are presented to illustrate the applicability of this procedure. This methodology should prove useful in those pharmacology/toxicological studies aimed at in vivo assessments

of alkylating

agent-induced

DNA damage.

METHODS Materials [3H-methyll New England

methyl-N-nitrosourea, ‘H-MNU (1.0 Ci/mmole) was obtained from Nuclear (Boston, MA). Nonradioactivity labeled methyl-N-nitrosourea

was purchased

from

pressure

chromatographic

liquid

K and K Rare and Fine Chemicals column

man (Clifton, NJ). I-Methyladenine Sigma Chemical Co. (St. Louis, MO). 3-methyladenine

were

Federal

of Germany),

tories,

Republic

generous

(Plainview,

NY).

10 SCX) was obtained

The

from

G. H.

NC) and Dr. P. D. Lawley (Pollards

Drs.

G. Bochert

Hitchings

and J. Webb

(Wellcome

Research

Wood Research Station,

high

from What-

and 7-methylguanine were purchased Authentic standards of Ob-methylguanine

gifts Dr.

(Partisil

from and

(Berlin, Labora-

Buckinghamshire,

England).

Carcinogen

Treatment

Male, Sprague-Dawley (Madison,

rats, 225 g, purchased

WI) were used for these experiments.

100 g, was administered were

killed

hours

prior

and their

by gastric livers

to carcinogen

intubation.

excised.

from King Animal

Food was withheld

administration

Laboratories,

Inc.

‘H-MNU 15 pmoles, 10 ~Cii~molei Three hours postintubation the rats

and during

from

the animals

the following

three

for 16

hours.

Isolation of Chromatin Chromatin Rodriguez

was isolated and Becker

were homogenized

from

hepatic nuclei

(1976) as reported

in 0.25 M sucrose,

by a modification

by Schwartz

50 mM Tris

of the method

and Goodman

(1979).

of

Livers

(pH 7.9), 25 mM KCI, 5 mM MgCI,

(STKM) and filtered through cheesecloth. Nuclei were isolated by washing twice in 2% Triton X-100 in STKM followed by centrifugation at 750 g for 10 minutes. The nuclear pellet was washed once in STKM. The nuclear pellet was homogenized in 10 mM Tris

(pH 7.9) and purified

chromatin

was isolated by differential sedimentation in a discontinuous sucrose-Tris gradient of 14 ml of 1.3 M sucrose and 14 ml of 1.6 M sucrose. Centrifugation for two hours at 112,000 g in a Beckman SW27 rotor resulted in a pellet of purified chromatin. The chromatin was washed once with for 10 min. The washed chromatin

10 mM Tris (pH 7.9) and centrifuged at 12,100 g was then resuspended in 25 mM sodium acetate

Quantitation of Methylated DNA-Purines (pH 6.6) and dialyzed sate was diluted

overnight

against the same sodium

to AZ6,, units/ml

(measured

acetate buffer.

in 0.9N KOH) with sodium

The dialy-

acetate buffer.

Isolation of Purified DNA DNA

was isolated

(Marmur,

1961).

in IO mM Tris-HCI dodecyl

sulfate,

from

chromatin

Chromatin

isoamyl

alcohol

by centrifugation

with constant

(3%) solution

sion at the end of the incubation. was then

This

95% ethanol,

7.9), 5.0 mM EDTA.

with

95%

ethanol

Ribonuclease

80” C for 10 minutes,

0.5 M NaCIO,;

shaking

The

DNA from

the upper aqueous

layer

in 10 mM Tris-HCI

(pH

A (200 pg enzyme/ml),

which

was heat-treated

and incubated

90 minutes

: isoamyl

of 0.1, and 3 mg protease

at 37” C. The solution

at

at 37” C for 40

at 80” C) per ml of reaction was added. The suspension

chloroform

at 37” C.

the suspen-

and resuspended

minutes. NaCl was added to make a final molarity for an additional

1% sodium

for 40 minutes

was used to deproteinize

was added to the suspension

with

procedure resuspended

was done twice and the layers were separated

at 400 g for 10 minutes.

precipitated

for 10 minutes

of the Marmur

with

(pH 7.9), 0.1 M NaCI, 5.0 mM EDTA, and then incubated

A chloroform:

by a modification

DNA was precipitated

(heat-treated was incubated

was deproteinized

by two washes

alcohol

layers were precipitated

(3%) and one wash with redistilled phenol. The Purified DNA in the aqueous layer was separated by centrifugation. with 95% ethanol. The amount of DNA recovered was determined by

the Ceriotti

method

(Ceriotti,

1952).

Isolation of Purines Purified

DNA

suspending

was washed

one time with

ice-cold 0.1 N HCI and depurinated

it in 0.1 N HCI at 60” C for 20 minutes

(Lawley and Brooks,

1963;

by

Lawley,

1976; Bouchert and Webb, 1977). The absorbance of the supernatant was monitored at 255 nm. The concentration of purines was determined spectrophotometrically, employing

an extinction

used by Montesano to dryness

under a stream

M NH,H,PO,

HPLC

at 255 nm of 11.2. This

of N, at 35” C. These

value is similar

supernatant

samples

to those

was evaporated

were then dissolved

in 0.03

(pH 4.6).

High Pressure liquid The

coefficient

et al. (1979). The purine-containing

system

Chromatographic

employed

for these

Separation studies

was assembled

from

the following

components (Goodman, 1976): A Milton Roy minipump, model MJ, equipped with an Ashcroft pressure gauge (purchased from Laboratory Data Control, Rivera Beach, FL). A Whatman

Partisil

10 SCX (strong

cation exchange

resin)

column,

250 x 6.35

mm, fitted with a high pressure sample injection valve (cat. no. 905-01, purchased from Altex Scientific Inc., Berkeley, CA), was used. The column was equipped with a Whatman guard column, 70 mm x 6.35 mm, containing HC Pellionex SCX. Temperature was maintained at 25” C. An isocratic solvent system was utilized, 0.03 M NHZH4P04, pH 4.6. The flow rate was 1.2 ml/min (925 PSI) for the first 27 minutes and then increased to 2.1 ml/min (1575 PSI). An injection volume of 500 ~1 was used. Fractions were collected every 0.5 minute using a LKB 7000 fraction collector. The

307

308

E. M. Faustman and J. I. Goodman UV absorption

of the column

effluent

was monitored

240 spectrophotometer

(purchased

The spectrophotometer

was equipped

has a l-cm

light

recorded

on a Gilford to the portions

as determined fluent)

were

dioactivity Triton internal

Model

by using combined,

standards

and monitoring

using

(Patterson

a liquid

scintillation

and Greene,

their

a stream

1965).

in absorption

fractions

in which

(those

the specific

UV absorbance

Quench

prepared

corre-

in the efof ra-

from Toluene

correction

cell were

bases elute,

of NZ, and the amount

fluor

OH).

2428 flow cell. This

Appropriate

effluent

under

Model

Model

Co., Oberlin,

of 8 ~1. Changes

recorder.

of the column evaporated

determined

X-100

volume

6040

Instrument

with a Gilford

path and an internal

sponding

at 254 nm with a Gilford

from the Gilford

was performed

and by

standardization.

RESULTS Four methylated and guanine,

purines,

7-MeG,

06-MeG,

can be reproducibly

3-MeA,

separated

and I-MeA,

as well as adenine

using the HPLC system

described

(Fig.

I). Purines ylated column.

were isolated from control rats and authentic standards of the four methbases were added to the purine fraction prior to injection onto the HPLC Adenine

The theoretical throughout

was used to monitor

plates for adenine

these

column

remained

efficiency

constant

from one run to the next.

and were equal to 2836 ? 127

experiments.

Quantitation of alkylated purines produced in hepatic DNA following exposure to ‘H-MNU is presented in Table 1. Less than 2% of the radioactivity applied to the

TABLE 1 Identification of Methylated Purines in DNA isolated from Hepatic Chromatin of Rats Treated with (3H-Methyl) Methyl-N-nitrosourea” DPM-‘H RETENTION

ASWCIATED

TIME METHYLATEDBASE

RATIO TO 7-

WITH

(min)

FRACTION

MeG

7-MeC

12.6

06-MeG

24.0

494

0.045

I-MeA

38.5

112

0.010

3-MeA

54.0

850

0.078

a Rats received by gastric from

‘H-MNU

intubation.

hepatic

4.76 mg of DNA hydrolysate DPM. eluted

were

DNA methyl

and applied

contained

1.39

An unidentified

alkylation group/mg

with

were

The purines

separated obtained

of purine applied

the

to 1.68

(3692 DPM/mg

This purine and

purines.

x IO-” DNA).

14,615

(315 DPM)

of the alkylated

radioactive

by from

to the column

peak of radioactivity standards

g

was purified

to the column.

pmoles

was equivalent DNA

10 ~Cil~mole/lOO

later DNA

purines

of radioactivity

at 29 min. Authentic

cochromatographed

hours

in methods.

The recovery

was 88.7%.

15 (*moles,

Three

chromatin,

HPLC as described

10,858

bases Total

moles

of

Quantitation

of Methylated

DNA-Purines

-1

0.6

0.2 0

h

;

;O

it5 ;O ;5

;O

;5

i0

i5

TIME AFTER INJECTION

;O

55

60

65

70

(MIN.)

FIGURE 1. Separation of purines by high pressure liquid chromatography. Column: Whatman Partisil 1OSCX (strong cation exchange resin) 250 x 6.4 mm. Solvent: 0.03 M NH2H.,P04, pH 4.6. Temperature: 25°C. Flow rate: 1.2 ml/min, 925 psi for the first 27 minutes then increased (break in the tracing) to 2.1 ml/min, 1575 psi after break. G = guanine; A = adenine; 7-MeC = 7-methylguanine. 06-MeC = 06-methylguanine; l-MeA = I-methyladenine; 3MeA = 3-methyladenine. The arrow marks the point at which injection of the sample was made. column

was eluted

in the first

few minutes.

Frei et al. (1978) using

chromatography, has observed similar rapidly eluting radioactive cribed this to pyrimidine nucleotide containing material. Analysis sented

of purine

in Table

methylation

2. The

four

in terms

alkylated

of pmoles

purines

methyl

accounted

cation exchange material

group/mg

DNA

and asis pre-

for 70% of the total DNA

alkylation. DISCUSSION The HPLC system 7-MeG, I-MeA, ocratic solvent proposed HPLC has also used a niques.

Their

described

in this paper can effect a rapid separation

of 06-MeG,

3-MeA, guanine, and adenine. This was accomplished using an issystem and a cation exchange column. Other investigators have systems for separating various methylated bases. Frei et al. (1978) Partisil IO SCX column for HPLC but required gradient elution tech-

system

utilizes

a solvent

of 0.02 M ammonium

formate

(pH 4.0) con-

309

310

E. M. Faustman and J. I. Goodman TABLE 2 Analysis of Purine Methylation in DNA of Hepatic Chromatin Isolated from Rats Treated with (3H-Methyl) Methyl-N-nitrosourea” ~MOLESMETHYL( X IO? MEWYLATEDBASE

mg DNA

7-MeG O”-MeG l-MeA 3-MeA

% OF TOTAL DNA ALKYLATION

62 2.8 0.64 4.8

10.370 0.472 0.107 0.812

a Experimental conditions were the same as those outlined in Table I.

taining

6% methanol

centration

for 2 minutes

of 0.2 M ammonium

chromatographic containing

system

and then

formate

was found

hydrolysates

were

a 25 minute

gradient

(pH 4.0) in 8% methanol.

to be most

useful

chromatographed

when

separately

to a final

In addition,

3- and 7-alkyl from

contheir

purine

06-alkyl-guanine

and I-alkyladenine containing hydrolysates. has recently been modified by Shackleton

The HPLC system of Frei et al. (1978) et al. (1979). These later investigators

were

3- and 7-alkylpurines,

able to separate

elution. x

However,

250 mm)

linked

time of 3-MeA Bouchert isocratic

mixtures

containing

required

the use of two partisil

in series.

In this

was approximately

report

(Shackleton

for I-MeA

gradient elution

elutions.

However,

Their

and 3-MeA

techniques

isocratic

eluted

elution

almost

these investigators

the pH gradient

a cation exchange

of I-MeA other

from

7-MeG,

methylated

column

employed

but this

O”-MeG,

purines:

gradient (each 9.2

et al., 1979) the retention

55 minutes. procedure

80 minutes

employed

employing

in shorter

went from 3.9 to 8.5, which

group

and 3-MeA.

8_methylguanine,

have shown

9-methylguanine,

The pH elution

is near the

et al. (1978) along

has not demonstrated

They

both

did not include

after injection.

resulted

maximum advisable pH for most cation exchange columns. An isocratic HPLC elution technique has been used by Shaikh with

using

10 SCX columns

and Webb (1977) have also used cation exchange HPLC,

and gradient

a separation times.

this

a separation

separations

of some

and N2-methylguan-

ine. Thielmann

(1979) has reported

capable of separating separated

from

various

uncharacterized

a reverse-phase

methylated

purines.

radioactive

HPLC

system

However,

products

when

that appears to be I-MeA purine

could

not be

hydrolysates

prepared from DNA reacted with 3H-MNU were analyzed. The levels of alkylation at the 7 position of guanine-observed in our studies following exposure to 3H-MNU-were in agreement with those obtained by other investigators. Frei et al. (1978) obtained 2.82 x lop4 pmoles of 7-MeC/mg DNA at three hours after treatment of mice with 25 mg/kg of MNU, intraperitoneal injection, compared to 1.037 x 10e4 pmoles 7-MeGimg DNA three hours postintubation of 15.6 mg/kg of MNU obtained in our studies. These Oh-MeG/7-MeG, 3-MeAI7-MeG, and I-MeAl7-MeG

investigators observed ratios of of 0.046, 0.052, and 0.011, re-

Quantitation

of Methylated

311

DNA-Purines

spectively. Our corresponding values were 0.045, 0.078, and 0.010. It has been shown by Bouchert and Webb (1977) and Lawley (1976) that the 06-methyl bond of guanine is acid instable. Severe acid hydrolysis conditions can result in demethylation of 06-MeG. Therefore, our acid hydrolysis conditions (20 minutes at 60” C in 0.1 N HCI) were chosen to minimize the demethylation of 06-MeG. Using these purine hydrolysis conditions 18% of the total purines in DNA were released. Lawley (1976) has reported on the acid stability of the N-glycosidic bonds of the various methylated bases. Methylations at the N-3 position of adenine and the N7 position of guanine increase the susceptibility to acid-catalyzed hydrolysis of Nglycosidic linkages. Therefore, our depurination conditions are preferentially releasing the methylated purines. This explains why radioactivity associated with the purine hydrolysate represented 81.2% of the total DNA alkylation while only 18% of the total purines in DNA were released. In our study we can account for 70% of the total alkylation of DNA as the four methylated bases, 7-MeC, 06-MeG, I-MeA, and 3-MeA. It is known from studies by Lawley (1976) that methyl phosphotriesters account for approximately 18% of the total methylation products in DNA following exposure to MNU. These phosphotriesters are acid-stable and would be expected to remain in the apurinic DNA (Lawley, 1976). Therefore, assuming Lawley’s value for phosphotriesters is applicable in our studies, we can account for 88% of the total DNA alkylation observed. We are grateful This

for the excellent

investigation

was supported

DHEW,

and, in part, by a General

College

of Osteopathic

E.M.F.

is the recipient

ufacturer’s

Association

technical

assistance

provided

by grant No. CA-13,344, Research

Support

by Ms.

Sandra

Moore.

awarded by the National

Grant administered

Cancer

by Michigan

Institute,

State University’s

Medicine. of an advanced predoctoral Foundation,

fellowship

awarded by The

Pharmaceutical

Man-

Inc.

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