DNA strand breaks in pancreatic islets by invivo administration of alloxan or streptozotocin

Vol. 103, No. 3,198l December

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH

COMMUNICATIONS Pages

15, 1981

1014-2020

DNA STRAND BREAKS IN PANCREATIC ISLETS BY -in vivo ADMINISTRATION OF ALLOXAN OR STREPTOZOTOCIN Hiroshi

Yamamoto',

Yasuko Uchigata

and Hiroshi

Okamoto"

Department of Biochemistry Medical and Pharmaceutical University School of Medicine Toyama 930-01, Toyama, Japan

Toyama

Received October 30.1981 SUMMARY: Administration of diabetogenic doses of alloxan or streptozotocin to rats caused extensive DNA strand breaks in pancreatic islets. DNA of pancreatic exocrine cells was not affected by either alloxan or streptozotocin. Hepatocyte DNA was fragmented by streptozotocin but not by alloxan. Intracellular NAD level was decreased in tissues whose DNA was fragThe results may raise a novel aspect concerning the mented. the diabetogenic agents mechanisms of action of as well as concerning the organotropisms of the agents. Chemical constitute

compounds

a class alloxan

pounds,

diabetes

of

diabetogenic

With islets

diabetogenic

and are

mental

elucidating

(l-3).

in vitro -rats,

streptozotocin poly(ADP-ribose) and inhibiting

To

agents.

used

for

agents

seems

under

experimental we have

cause

the

great

which

diabetes

systems

using that

DNA strand

breaks

to

proinsulin

synthesis

depleting (4,5). fulfillment

whomall correspondence should be addressed.

0006-291X/81/231014-07$01.00/0 Copyright 0 1981 by Academic Press, Inc. All righrs of reproduction in any form reserved.

1014

com-

experi-

of

action

importance

for

develops. isolated

found

thereby

of

mechanisms

recently

synthetase,

cells

the most potent

production

be of

to

islet Among such

by far

To understand

!l Submitted part of this work as partial of Medical Science at Kanazawa University. *

pancreatic

exhibit

widely

the conditions

of

damage

and streptozotocin

diabetogenicity

the

of

that

both

pancreatic alloxan

stimulate

nuclear

intracellular The finding

and

NAD may

open

of the degree of Doctor

Vol. 103, No. 3,1981

a novel

and important

diabetes, are

if

the

actually

diabetogenic

of

aspect

induced

the

events

in --

vivo

alloxan

breaks

diabetogenic

BIOPHYSICAL

on the

we describe

doses of

DNA strand

effects

AND

biochemical

Here

treatment.

islet

BIOCHEMICAL

RESEARCH COMMUNICATIONS

cause

of

initiated by

alloxan

evidence

that

or streptozotocin

and depresses agents

islet

on non-islet

insulin-dependent

by islet or

DNA breaks

streptozotocin

administration to

rats

generates

NAD level. tissues

of

The are also

examined. MATERIALS AND METHODS Chemicals : Alloxan monohydrate was purchased from Wako Pure Chemical Industries, streptozotocin from Upjohn, and nicotinamide [U-14C]adenine dinucleotide (286 mCi/uunole) from the Radiochemical Center. Treatment of animals with alloxan or streptozotocin : Experiments were carried out with male Wistar rats weighing 200-250 g, which were fed --ad libitum. Alloxan monohydrate or streptozotocin was dissolved in saline just before use, and injected via the tail vein of ether-anesthetized rats in doses of 40 mg/kg or 50 mg/kg, respectively. The doses have been shown to yield 100%diabetogenesis in rats (1). Alkaline sucrose gradient analysis of DNA : Pancreatic islets of Langerhans, pancreatic exocrine cells and hepatocytes from alloxan- or streptozotocintreated rats were submitted to alkaline sucrose gradient centrifusation. and velosity sedimentation of DNA was examined. -Islets of Langerhans -were isolated by a collagenase digestion method as described previously (6). It may be noteworthy that the number of islets isolated by this procedure was significantly reduced when the pancreas was removed later than 20 min posttreatment with 40 mg/kg alloxan. A similar result was also observed with Pancreatic exocrine cells were separated from streptozotocin treatment. pancreatic islets by the same procedure of islet isolation. A hepatocyte suspension was prepared as described by Cox --et al. (7) from liver that had been perfused with cold Hanks solution. One hundred islets and 2.5~5 x lo5 hepatocytes or pancreatic exocrine cells were applied on 0.5 ml lysis solution (1.0 N NaOH, 0.01 M EDTA, 1% (v/v) Triton X-100) that had just been layered over 14.8 ml of a 5 to 20% (w/v) linear sucrose gradient containing 0.3 N NaOH, 0.7 M NaCl and 0.01 M EDTA. On the bottom of each gradient was a 1 ml 80% (w/v) sucrose shelf. The loaded gradients were placed in the dark at Then the gradients were centrifuged at 26,000 room temperature for 30 min. rpm at 2O'C for 200 min in a Beckman SW27.1 rotor. After centrifugation, fractions of 33 drops were collected from the gradient. DNA in each fraction was precipitated by adding 2 ml of 20% cold trichloroacetic acid (TCA) with 200 ug of bovine serum albumin as carrier. The precipitate was washed 3 times with cold 5% TCA, once with cold 0.1 N potassium acetate in ethanol, twice with ethanol and then assayed for DNA content by a fluorometric method described by Kissane and Robins (8). NADdetermination : One hundred islets were disrupted by sonication in 1 ml of One g liver which had been perfused with cold 0.5 N perchloric acid (PCA). cold Hanks solution was homogenized with Polytron in 5 ml of cold 0.6 N PCA. The acid-soluble extract was brought to pH 5.0 with KOH, and the NADcontent in the extract was determined as described previously (5,9). The DNAcontent in the islet PCA-precipitate or the liver homogenate was determined by the fluorometric method (8).

1015

Vol. 103, No. 3,198l

BIOCHEMICAL

AND

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RESULTS Alloxan

(40 mg/kg)

intravenously

into

or streptozotocin

male

have

been

shown

to

Islets

of

Langerhans

isolated

after

the

S-20

min

tation

in

the

alkaline

single

peak

position

at

Considerable

which

undamaged

of the

20

at

min

after

fragmented

w/kg)

pancreas been

Liver

of that

liver

of DNA of

of untreated

DNA strand

of

the 2).

alloxan

in

1,

as a

5 and

decrease &,

or

in

the

undamaged

c,

almost

tissues

alloxan

removed

were

alloxan

< and 3). completely

sucrose

conditions 2,

rats

gradient

either

alloxan

5 and

20 min

b). after

exocrine or

rat

As

cells

was

streptozotocin rat

whereas

(50

analysis.

streptozotocin-treated rat,

where

or streptozotocin

pancreatic

alloxan-treated

--in vitro breaks in

from

(40 mg/kg)

untreated

rat

the

streptozotocin

(Fig.

DNA of by

or

under

liver

DNA of

that

Our previous that

1,

was

is

peak

DNA was

effect

2, c and a,

treatment.

profile

DNA

a broad

islet

to alkaline

unaffected

than

been

sedimen-

with

(Fig.

fragmented

with

essentially

slower

treated as

(1).

had

which

(Fig.

completely

and

treated

Fig.

rats

was recovered

treatment

non-islet

I and submitted

shown in

from

the

DNA was almost

having

in

islet

gradient,

rats

agents

1, d and 1).

on DNA of

Exocrine

the

a concomitant

after

we examined

treatment

rats

sediment

treatment,

(Fig.

Next,

islet

min

the

which

of

DNA sediments

with

gradient, S-10

of

to

of

and velosity

gradient

from

found

doses

pancreas

untreated

bottom

islets

was

from treatment,

from

the

DNA of

DNA even At

islets

The

was injected

diabetogenesis

sucrose

near

streptozotocin middle

100%

were

at

DNA of

rats.

yield

removed

examined.

Wistar

(50 mg/kg)

the

sedimented

sedimentation

was indistinguishable

(Fig.

2, e- and f). study with isolated

islets

result

1016

in

islets

a significant

has

shown deple-

Vol. 103, No. 3,198l

BIOCHEMICAL

AND

BIOPHYSICAL

B

RESEARCH

COMMUNICATIONS

c

I

I

-ILd 20

10

1

Fraction

10

20

no.

s

h

I

I

(0 Bottom

Fig. 1. Sedimentation profile in alkaline sucrose gradient of DNA of islets from alloxanor streptozotocin-treated rats. Experimental details are described in MATERIALS AND METHODS. Each point represents the percentage of total DNA recovered; recovery was between 85 and 100%. l : DNA of islets from pancreas of untreated rats (5 and 2). from 0 ; DNA of islets pancreas removed at 5 min (h), 10 min (2) or 20 min (2) after intravenous administration of 40 mg/kg alloxan. A ; DNA of islets from pancreas removed at 5 min (L), 10 min (8) or 20 min Q) after intravenous administration of 50 mg/kg streptozotocin. Sedimentation was from left to right. The arrow indicates the position of a bacteriophage X DNA (3.2 x lo7 daltons, New England BioLabs).

tion

of

dation the

through effect

liver

NAD content

poly(ADP-ribose)

of

alloxan-

NAD content

tissues

in

depletion also

of

islet

caused liver

liver

no significant in

untreated

when the

value

due (4,5).

parallel

shown

in

was

NAD Content. to

change rats

increased

We

experiments

mg/kg)

NAD

to

NAD

therefore

streptozotocin-treatment

AS

(50

decreased

islets

or

was analyzed.

streptozotocin

or

of

intracellular

Table found

which

1,

alloxan

to

lead

Streptozotocin 74% of in

the

NAD.

was about

2-fold

was calculated

1017

DNA

islet

or

of

the

(40 mg/kg) to

a marked

administration

control,

liver

examined on

in

degra-

Control larger

per 1-14 DNA.

while

alloxan

NAD content than

that

of

Vol. 103, No. 3,198l

BIOCHEMICAL

AND

BIOPHYSICAL

Fmction

RESEARCH

no.

COMMUNICATIONS

eottml

Fig. 2. Sedimentation profile in alkaline sucrose gradient of DNA of rat tissues after alloxanor streptozotocin-treatment. Experimental details are described In MATERIALS AND METHODS. Each point represents the percentage of total DNA recovered ; recovery was between 85 and 100%. a and b ; Islet DNA. c and d ; DNA of pancreatic exocrine cells. e andf ; hepatocyte DNA. +*a.4 7 untreated rats. M ; rats treated with alloxan (40 &kg). &d ; rats treated with streptozotocin (50 mg/kg). Sedimentation was from left to right. Arrow indicates the position of a bacteriphage ,% DNA.

DISCUSSION

The

present

tration

to

zotocin

causes

content.

rats

This

alloxan

and

biochemical

study of

has

demonstrated

diabetogenic

islet

through

DNA strand

inhibition

of proinsulin

addition,

we also

breaks

our

carried that

out

or

streptoislet

proposal

NAD

that

following

stimulation of

of

intracellular

(4,5). the

experiments

DNA of pancreatic

1018

adminis-

the

+

synthetasel)depletion synthesis

vivo

depletes

diabetes

islet

The finding

and

induce

NAD I)

tissues.

breaks

to

poly(ADP-ribose)

islet

of

support

streptozotocin events:

--in alloxan

a strong

nuclear

In

doses

DNA strand

may be

that

exocrine

on noncells

Vol. 103, No. 3,198l

BIOCHEMICAL.

Table 1.

Effect

AND

BIOPHYSICAC

RESEARCH

COMMUNICATIONS

of alloxan- or streptozotocin-administration on iSlet or liver NAD content

Tissue

Administration

NAD Content pmole/pg DNA

50.0

none (control) islet

(100)

40 mg/kg alloxan

5.9 ( 12)

50 mg/kg streptozotocin

4.8 ( 10)

liver

none (control)

110.9 (100)

40 mg/kg alloxan

113.6 (102)

50 mg/kg streptozotocin

82.1 ( 74)

After administration of alloxan or streptozotocin, pancreatic islets and liver were simultaneously removed from each rat, and NADcontent of the tissues was determined as described in MATERIALS AND METHODS. 14C-NAD was added to each sample before the extraction in PCA, and the recovery of 14C-radioactivity in the final preparation was referred to for correction of the NAD value for overall recovery. Recovery was between 85% and 100%. The numbers in parentheses give the percentage of the control.

not

is

affected

zotocin to

zotocin

pancreas.

the

the it

fact,

jected

in

mice

in

recently

are level

was recovered alloxan

by

in

islets

(11).

decreased not

that

reported with

liirer

liver

following

1019

strepto-

difference

by the

DNA

tissue.

streptozotocin as in

in-

islets

be accumulated Bennett

DNA is

NAD levels

streptozotocin

agents

on liver

agents

administration

that

changed

action

as well to

by The

the

streptothe

fragmented

Furthermore,

streptozotocin

been

in

or of

alloxan.

was shown

after

is

DNA was

not

alloxan

preference

in uptake of the 14 C-labelled shown that

evidence

has

for

agents

described

methylated

of

difference

has been

exclusively

It

but

14 C-labelled

whereas

NAD

Liver

two diabetogenic

reflect

liver

administration indication

administration

between

In

the

may be a definite

endocrine

may

by

almost

and Pegg have

also

significantly

(12).

in (13,14),

alloxan

(lo),

mouse

islets

while treatment

the

and liver (15).

Vol. 103, No. 3,198l

Since

liver

NAD level

considered However,

that the

alloxan

data

of

in

altered

VAD level

also

present

DNA strand

breaks

of

the

than

smaller

may be due to

pancreatic

islet

cells of

fact

alloxan

agents

understanding

of

the etiology

has been

be changed clearly

islets

between

(2,3).

shown that

as well

as NAD

decrease

NAD even

islet

liver

a hepatocyte

evidence

that

though

in the

and islets.

contains

DNA strand

preferentially

induced

streptozotocin.

the

and may

have

it

greater

Cell.

or

concerning

aspect

in

that

here are

not

rat

COMMUNICATIONS

alloxan,

may

was similar

an islet

described

administration

diabetogenic

the

by

paper of

RESEARCH

streptozotocin-induced

that

DNA fragmentation

We have

BIOPHYSICAL

the

amounts of NAD than

novel

AND

was not

The extent

NAD was

extent This

islet

causes

depletion. liver

BIOCHEMICAL

mechanism

have of

breaks

by the -in vivo This

of

important

action

raises of

implications

insulin-dependent

in

a

typical for

our

diabetes.

ACKNOWLEDGEMENTS: This work has been supported in part by Grants-in-Aid for Cancer Research and for Scientific Research from the Ministry of Education, Science and Culture, Japan. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Rerup, C.C. (1970) Pharmacol. Rev. 22, 485-518. Dulin, W.E., and Soret, M.G. (1977) The Diabetic Pancreas (Volk, B.W., and Wellmann, K.F., eds), pp. 425-465, Plenum Press, New York. Agarwal, M.K. (1980) FEBS Lett. 120, l-3. Okamoto, H. (1981) Mol. Cell. Biochem. 37, 43-61. Yamamoto, H., Uchigata, Y., and Okamoto, H. (1981) Nature (London), in press. Okamoto, H., Noto, Y., Miyamoto, S., Mabuchi, H., and Takeda, R. (1975) FEBS Lett. 54. 103-105. Cox, R., Damjanov, I., Abanobi, S.E., and Sarma, D.S.R. (1973) Cancer Res. 33, 2114-2121. Kissane, J.M., and Robins, E. (1958) J. Biol. Chem. 2.33, 184-188. Yamamoto, H., and Okamoto, H. (1980) Biochem. Biophys. Res. Commun. 95, 474-481. TjIlve, H., Wilander, E., and Johansson, E.B. (1976) J. Endocrinol. 69, 455-456. Hammarstrb'm, L., and Ullberg, S. (1966) Nature (London) 212, 708-709. Bennett, R.A., and Pegg, A.E. (1981) Cancer Res. 41, 2786-2790. Gunnarsson, R., Berne, C., and Hellerstram, C. (1974) Biochem. J. 140,

487-494. Schein, P.S., 2324-2332. Schein, P.S.,

Cooney, and Loftus,

D.A.,

and

S. (1968)

Vernon, Cancer

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(1967) 28,

Cancer

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