Generation of alloxan free radicals in chemical and biological systems: Implication in the diabetogenic action of alloxan

Vol. 165, No. 1, 1989 November 30, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 278-283

GENERATION OF ALLOXAN FREE RADICALS IN CHEMICAL AND BIOLOGICAL SYSTEMS: IMPLICATION

IN THE DIABETOGENIC ACTION OF ALLOXAN

Mamoru Nukatsuka, Faculty

of

Hiromu

Pharmaceutical Shomachi

Sakurai

Sciences,

and Jun Kawada

University

1, Tokushima

770,

of Tokushima,

Japan

Received October 10, 1989 SUMMARY: Electron spin resonance (ESR) studies showed that on reaction with NADPH, alloxan was reduced forming labile anion radicals giving a 'I-line signal with g=2.005. These radicals were also produced on incubation of alloxan with rat liver subcellular fractions and their production was greatly enhanced by NADPH. Alloxan effectively scavenged superoxide anion generated by a xanthine-xanthine oxidase (XOD) system in association with its reduction to these anion radicals. radicals These were also formed during incubation of alloxan with rat pancreatic p-cells. These results suggest that the cytotoxicity of alloxan is related to the formation of alloxan anion radicals . D 1989Acadrmlc Press,Inc.

Alloxan creatic

(2,4,5,6(1H,3H)-pyrimidinetetrone; b-cells

in animals alloxan

selectively

is unknown,

been proposed. acid

oxidized

rapidly

[4]

these

radicals (SOD),

and ferrous

ion

chelator

(DETAPAC)

[5]

prevent

acid

However,

as far

induction

that

oxygen, [Z,

of

possible

is

Fig.1)

formation

31.

It

with catalase

[Z,

reduced of cytotoxic

based

is

scavengers

on such

no free

radicals

as

acetic

diabetes formed

the urea

31 or N,N-dimethyl

alloxan-induced

to auto-

as diethylenetetraaminepenta

as we know,

by have

and then

with radical

diabetes diabetes

mechanisms

alloxan

acid;

(02. )

pretreatment

dismutase

is

barbituric

by molecular

that

super-oxide

of

(5-hydroxy

anion

observation

various

pan-

destroys

insulin-dependent

mechanism of

although

One

dialuric superoxide

and causes

The exact

[l].

Fig.1)

from

[Z,

31.

alloxan

or molecular oxygen have yet been detected directly in biological systems. From results in chemical and biological systems, we here propose alloxan

alternative

an is

due to

alloxan

mechanism anion

radicals.

0006-291W89$1.50 Copyright All rights

0 1989 by Academic Press, Inc. in any form reserved.

of reproduction

that

278

the

cytotoxicity

of

BIOCHEMICAL

Vol. 165, No. 1, 1989

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

A

Figure 1. Chemical structures

MATERIALS

B

of (A) alloxan

and (B) dialuric

acid.

AND METHODS

Alloxan and trypsin were purchased from Wako Pure Chemicals Oriental Yeast Co. (Tokyo). Xanthine (Osaka). NADPH was from and bovine serum albumin oxidase (XOD), collagenase (Type I) (BSA, Fraction V) were from Sigma Co. 5,5'-Dimethyl-1-pyrroline N-oxide (DMPO) was from Daiichi Chem. Co. (Tokyo). Fetal calf serum and medium-199 were obtained from Flow Lab. (Sydney) and Nissui (Tokyo), respectively. ESR spectra were measured at 22 'C in a JEOL-FElXG ESR spectrometer. As standards, TCNQ-Li salt, used. Five Fremy's salt and Mn" doped in MgO were groups of experiments were performed: (1) Alloxan (6.3 mM) in Krebs-Ringer phosphate buffer (KRPB, pH= 7.5) was mixed with NADPH (1-12 mM) and the ESR spectra were measured. (2) Alloxan (6.3 mM) in 10 mM citrate buffer (pH=4-5.5) or in 10 mM phosphate buffer (pH=6-8.5) was mixed with NADPH (1 mM) and the ESR spectra were recorded. (3) Rat liver was homogenized in 10 volumes of KRPB and subcellular fractions were obtained differential by centrifugation [6]. Then each rat subcellular fraction (8 mg protein/ml and 0.51 nmole cytochrome P-450/mg protein for microsomal fractions) was mixed with alloxan (6.3 or mM) with without NADPH (1 mM) and the ESR spectrum was measured. The protein concentration of each fraction was measured by the method of Lowry et al. [7] with BSA as a standard. Cytochrome P-450 in microsomal fractions was determined by the method of Omura and Sat.0 [8]. (4) The reaction of alloxan with rat pancreatic B-cells (6 X lo6 cells/sample), isolated from neonatal rats [9], was monitored by ESR spectra. (5) Hypoxanthine in KRPB (2 mM, 100 ul), XOD (12 mu, lo wl), various concentrations of alloxan (O-150 PM, 10 and DMPO ul) (10 ~1) were mixed and the ESR spectra were recorded within 30 set after mixing [lo]. The activity of XOD was estimated by the method of Hashimoto [ll].

RESULTS A freshly

prepared

solution

ESR signal

(data

not

shown),

mixed

NADPH or

NADH,

with

consisting of of

of

7 symmetrical

of

alloxan

in KRPB (pH 7.5),

but

when the

solution

an

intense

ESR

peaks with

a relative

gave no

of alloxan

signal

was

intensity

was seen, ratio

The g-value was 2.005 and the coupling constant 1:3:5:7:5:3:1. adjacent peaks was 0.46 G (Fig. 2 A). The optimum conditions 279

Vol. 165, No. 1, 1989

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

oL-r-774 10 20 30

40

50

GO(Min)

Figure 2. ESR spectra generated in a mixture of alloxan (6.3 mM) and NADPH (1 mM) at pH 7.4 and 22 'C. (A) Observed spectrum. Magnetic field = 3371-3381 G, modulation amplitude = 0.1 G at field modulation frequency, 100 KHz; power, 8 mW; amplitude, 1600 ; response 0.1 set at 22 'C. The arrow shows 3366 G in the magnetic field. (B) Computer-simulated spectrum. (C) ESR spectrum of alloxan anion radicals generated by alloxan (6.3 mM) and rat pancreatic D-cells (6 X 10" cells). The arrow shows 3366 G in the magnetic field. Figure 3. Time course of disappearance of alloxan anion radicals by alloxan (6.3 mM) and NADPH (7.6 mM) at pH 7.0. The ordinate indicates the signal height in arbitrary of the 4th peak from the lower magnetic field.

for

the

ratio

generation

of NADPH to

The signal

of

alloxan

of radicals

disappeared

the

radicals of

an

units

(mm)

approximate

molar

1.2 at pH 7.0.

appeared

within

with

time-dependently,

When alloxan

were

generated

30 set

a half

was added to subcellular

life

after of

fractions

mixing

13 min of

rat

and

(Fig.

3)

liver,

the same radicals were seen as in the chemical systems. The postmitochondrial supernatant (PMS, 12000 X g supernatant) showed the strongest

activity

post-nuclear

for

formation

supernatant

(PNS,

105000 X g supernatant) (CYto, microsomal fractions exhibited NADPH to the especially In the

fractions

microsomal

estimated

systems,

to be 39 min

(data

the

radicals,

700 X g supernatant)

followed and

by the cytosol

(Fig. 4). The mitochondrial and weaker Addition of activities. enhanced generation of radicals,

greatly

in the microsomal

of

fractions the

half

not

(6.6-fold life

shown). 280

of

these

increase; radicals

Fig.4) was

Vol. 165, No. 1, 1989

PiiS

BIOCHEMICAL

M’t

Pk

MiLro

CytO

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

05

KdPB

0I

30I

GO I

120 I

30

150I W-V

Figure 4. Formations of alloxan anion radicals in reactions with subcellular fractions of rat liver. Open columns reactions of alloxan and subcellular fractions containing 8 'mg protein/ml. Closed columns, the same reactions in the presence of 1 mMNADPH. The ordinate indicates the signal height in arbitrary units (mm) of the 4th peak from the lower magnetic field of alloxan anion radicals. Values are means ? SEM (n = 4). Figure 5. Effect of alloxan in scavenging superoxide anion radicals (02-I system and ESR spectrum of generated in the hypoxanthine-XOD alloxan anion radicals produced in the reactions of alloxan with 02.. The arrow shows 3366 G in the magnetic field (inset). The scavenging effect was monitored by ESR spin-trapping method with DMPO. The ordinate indicates the signal height at the lowest magnetic field of DMPO-OOH(%). Values are means ? SEM (n = 4). Addition

of alloxan

signal

height

during

the

observed.

reactions,

in the

examined cells

the

the

the

. After

less

a

of

were detected

(Fig.

the

of

alloxan (Fig.5,

1 mM alloxan

(data

(6.3

of

mM) to

exactly

the

but XOD was

radical

was

inset),

but

not

alloxan-induced with intact rat

of alloxan

weak signal

the

strong superoxidemM (Fig. 5). In

0.03

due to

than

of alloxan

addition

has

being

6.3 mM alloxan

mechanism

reaction

cells/sample), radicals

of

of

decreased

activity

alloxan

ESR signal

system

dose-dependently,

specific

EDsO value

presence

presence

To elucidate

of

showed that the

experiments, in the

DMPO-OOH adduct

no loss

activity,

detected, not

due to the

The results

scavenging these

to the hypoxanthine-XOD

diabetes,

we

pancreatic

p-cells same

shown).

X

(6

alloxan

o10' anion

2 C).

DISCUSSION A previous reduced reagents

to

study

by ESR

alloxan

anion

radicals

such as glutathione

we examined

the

subcellular

fractions.

reactions

spectrometry during

or ascorbic

of alloxan with Both NADPH and 281

showed

that

reaction acid

[12].

alloxan with

is

reducing

In this

work,

NADPH and rat liver liver subcellular

rat

Vol. 165, No. 1, 1989

fractions

showed strong

radicals anion

BIOCHEMICAL

(Fig.

2 A).

radicals,

and AH

=

simulation (Fig. electron

coupling

that

regenerate

of

mechanism of alloxan is reduced

*OH + OH- + of

alloxan

with

to

202)

like

with

reduction

OZ- , 02. of

the

by

presence

which

we

latter

and aH = 0.415G acid

is

reaction

was

2.005 computer-

has been explained

the 31,

However,

c131.

alloxan

c2,

alloxan =

and

dialuric

in

and form

(g

[12]

aN = 0.425G

free

as

ESR parameters

previously

02*OH by a Haber-Weiss

reaction

alloxan-derived

were identified

and autooxidized

alloxan

to cytotoxic

induce

both

constants

alloxan

reduction

of

reported

The cytotoxic

by supposing

to

These radicals as

with

2 B).

activities

on the basis

0.46G)

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

of

then

found

to

+

2H+

+

during

that

to anion

02

converted

(302~

scavenged

two-

the

effectively

by

radicals

(Fig.

5,

inset). From these cytotoxic anion

we propose

activity radicals

chemical for

results

of alloxan; produced

producing

NADPH (Fig.

A),

in microsomes

a-cells

(Fig.

Pancreatic

o-cells of

may be reduced

to

reduced cellular

contain alloxan

to anion suggest

cytotoxicity

that

radicals

on reaction

that

alloxan

anion

with or

with P-450

5,

pancreatic

inset).

XOD,

which

is

so

alloxan

SOD [14],

a

in o-cells.

diabetogenic

D-cells

intact

of

radicals

pathways

cytochrome

OZ- (Fig. of

in

reaction

with

level

alloxan

four

direct

NADPH and

a low level

the

intact

of alloxan

a

with

anion

of

seem to be

reaction

a high

02. , but

fractions,

results

4),

it

reduction

There

with

(Fig.

we found

that

radicals:

2 C) and reaction

source

In summary,

anion reaction

systems

typical

systems.

alloxan 2

namely

by one-electron

and biological

for mechanism the depends on alloxan

an alternative

reagent NADPH,

superoxide

radicals

alloxan

are

was

liver

subThese

anion. involved

in

the

in p-cells.

REFERENCES [l] [2]

Dunn, D.S., Sheehan, H.L., and McLetchin, N.G.B. (1943) Lancet I, 484-487. Cohen, G., and Heikkila, R.E. (1974) J. Biol. Chem. 249, 2447-2452.

[3]

[4] [5]

Grankvist, K., Marklund, S., Sehlin, J., and Taljedal, I. (1979) Biochem. J. 182, 17-25. Heikkila, R.E., and Cabbat, F.S., (1978) Europ. J. Pharmacol. 52, 57-60. Fischer, L.J., and Hamburger, S.A. (1980) Diabetes 29, 213216. 282

Vol. 165, No. 1, 1989

IS] [7] [S] [S] [lo] [ll] [12] [13] [14]

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hogeboom, G.H. (1955) in Methods in Enzymology (Colowick, S. P and Kaplan, N.O., Ed.), vol 1, pp. 16-19. Academic-Press, New York. Lowry, O.H., Pssebrough , N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2370-2378. Lambert, A.E., Blondel,B., Kanazawa, Y., Orcil, L., and Renold, A.E. (1972) Endocrinology, SO, 239-248. Ueno,I., Kohno, M., Yoshihira, K., and Hirono, I. (1984) J. Pharmaco-bio. Dyn. 7, 563-569. Hashimoto, S. (1974) Anal. Biochem. 62, 426-435. Lagercrantz, C., and Yhland, M., (1963) Act. Chem. Stand. 1677-1682. iikere, M.S., and Gebicki, J.M. (1984) Arch. Biochem. Bio258-264. PWs . 234, Nukatsuka, M., Sakurai, H., Yoshimura, Y., Nishida, M., and Kawada, J. (1988) FEBS Lett. 239, 295-298.

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