Exp. Brain Res. 7, 250--257 (1969)

The Effect of Polyhydroxyphenols on Brain ATP in the Mouse A. ANGEL, R.N. L~Mo~*, K . J . ROG~I~S and P. BANKS Departments of Physiology, Pharmacology and Biochemistry, University of Sheffield. S10 2TN, England Received August 14, 1968 Summary. The penetration of pyrogallol and catechol into the brains of mice after an intraperitoneal injection has been studied together with their effects on locomotor activity and on the concentration of cerebral ATP. A 60 mg/kg dose of catechol produced convulsive activity together with a decrease in concentration of brain ATP. The time courses of these effects were similar, and closely followed the time course of catechol penetration into the brain, all showing peak changes at 3 min. 60 mg/kg pyrogallo] did not produce convulsive activity although there was a fall in concentration of cerebral ATP of the same order (20--30o/0) as that produced by the same dose of catechol. There was also a marked similarity between the cerebral penetration of pyrogallol and the time course of the fall of ATP it produced, although penetration was slower and maximal changes were observed 10 rain after injection. Both catechol and pyrogallol produced similar changes of A T P in the cerebral hemispheres, thalamus, medulla, and cerebellum of mice whose brains were divided prior to estimation. I n a further a t t e m p t to determine if there was any correlation between brain levels of A T P and convulsive activity it was found that: 1. A lower dose of catechol (40 mg/kg), which still produced convulsions, had less effect on the cerebral ATP than did a non-convulsive dose of pyrogallol (60 mg/kg). 2.60 mg/kg eatechol produces typical convulsions in mice respiring a 95% 02, 5~o COs gas mixture, but there was no significant change in brain levels of A T P in these mice. I t is concluded that there is no correlation between the convulsive action of catechol and the fall in brain concentration of A T P produced by this compound. Key Words: Polyhydroxyphenols - - Mouse - - Brain - - ATP - - Convulsions Introduction BACQ (1936) first demonstrated that the intravenous administration of catechol and other po]yhydroxyphenols produced convulsive jerks in cats. At the present time we are investigating the mode of action of catechol on the central nervous system. In theory, cateehol may act in a number of ways, but principally two mechanisms have been suggested. First, catechol is an inhibitor of cerebral catechol-O-methyl transferase, both in vivo (Ross and HALIAS~A~ 196~b) and in vitro * M.R.C. Scholar

Effect of Polyhydroxyphenols on Mouse Brain ATP

251

(Ross a n d IIALJASMAA 1964a; ABBS et al. 1967) a n d it has been suggested t h a t the central effect of catechol m a y be m e d i a t e d b y a n adrenergic m e c h a n i s m (BAcQ et al. 1959). Recently, however, ANGEL a n d R o c E ~ s (1968) a n d ROGEnS et al. (1968) have provided evidence i n d i c a t i n g t h a t this m a y n o t be so. Secondly, it has been f o u n d (HocHSTErN a n d COHEN 1960) t h a t catechol inhibits glyeolysis i n b r a i n cells a n d thus reduces the supply of A T P . This m i g h t t h e n be expected to result i n w h a t are essentially anoxic convulsions. To investigate this problem, changes i n t o t a l A T P of b r a i n in mice injected with either catechol (1,2-dihydroxybenzene) or pyrogallol (1,2,3-trihydroxybenzene) have been measured. These changes have also been studied i n various b r a i n regions. The p e n e t r a t i o n of catechol a n d of pyrogallol into the mouse b r a i n a n d their effect on the spontaneous m o t o r a c t i v i t y of the a n i m a l have been i n v e s t i g a t e d i n relation to the time course of the change of A T P concentration.

Methods Estimation of Changes in Brain A T P Concentration. Female mice of an inbred strain, weighing 2 0 - 2 5 g, were injected intraperitoneally with either eateehol (60, 40 and 30 mg/kg) or pyrogallol (60 mg/kg) and killed by immersion in liquid nitrogen at zero, 30 see, 1, 3, 5, 10, 15 and 30 rain after injection. Mice injected with 0.9 ~ (w/v) saline and killed at similar times were used as controls. The concentration of cerebral ATP was also measured in mice without any injection. One group of mice was kept in an atmosphere of 95 ~ O~ and 5 ~ C02 during the experimental period. The brains (excluding the olfactory lobes) were chipped out of the frozen heads, weighed and crushed in a stainless steel anvil of a type described by STONE (1938). Liquid nitrogen was used to cool the brain tissue and the anvil throughout this procedure. The crushed brains were then homogenised in ice-cold 3 ~ (w/v) perehloric acid and centrifuged at 400 • g for 15 min at - - 5 ~ C. The supernatant was neutralised with 1 M K~C03 and the preeipitate spun off. Estimation of ATP in the supernatant fraction was carried out with the luciferin-luciferase system as described by McEL~oY and ST~E~LE~ (1949) and ST~E~LER (1963). In order to investigate the possibility that any changes in levels of ATP might be loealised to one or more areas of the brain, the topographical distribution of the cerebral ATP during treatment with eateehol and pyrogailol was examined. The frozen brain was divided into four main parts: the cerebral hemispheres, the thalamus and midbrain, the cerebellum, and the medulla. The concentration of ATP of these parts was then estimated separately. The Penetration Into the Mouse Brain of Catechol and Pyrogallol. The cerebral concentration of catechol or pyrogallol, after an intraperitoneal injection of 60 mg/kg of either compound, was measured at time intervals similar to those given above. The method used was a modification of that described by S w a n and HILLIS (1959). E#eet on Spontaneous Motor Activity. The spontaneous activity of the mice was measured by placing them in a light plastic beaker attached to a strain gauge (Type 2ST02, Devices Ltd., Welwyn Garden City, I-Ierts, England). The movements of the beaker recorded by the strain gauge were integrat@ over six-second periods with an operational amplifier (Type SD-5, Nexus-Philbrick I~esearch, 18a North Street, Chichester, Sussex, England). The integrated output was sampled and digitalised 1 msec before the end of the integration period with a Biomae 1000 computer (Data Laboratories Ltd., 28 Wates Way, Mitcham, Surrey, England). After an initial control period, mice were given an intraperitoneal injection of either eateehol or pyrogallol and recording was resumed.

Results The c o n c e n t r a t i o n of A T P i n the whole b r a i n of u n i n j e c t e d control mice was 2.15 pmoles/g wet weight of b r a i n (S.D. • 0.67/~moles/g). This relatively large value for the s t a n d a r d d e v i a t i o n has been o b t a i n e d because the A T P yield varied considerably from one e x p e r i m e n t to a n o t h e r (range 1 . 8 6 ~ 2 . 9 4 pmoles/g). The

252

A. ANGEL, R.N. LEMON,K.J. ROGERSand P. BANKS:

effect of intraperitoneal injections of saline, cateehol or pyrogallol on the whole brain concentrations of A T P is shown in Fig. 1. All mice, control or experimental, killed immediately after injection have a brain level of A T P 15% less t h a n t h a t of mice killed without injection. This fall was presumably produced b y the stress caused during handling and injecting of the mice. Since it took several seconds to inject the mouse and transfer it to the liquid nitrogen and because it m a y take 10--20 see to freeze the innermost parts of the brain (RIcHTE~ and D a w s o ~ 1948), a n y changes in A T P produced b y this stress would be evident in animals killed 'immediately' after injection. The top curve in Fig. 1 (saline-injected controls) 0-

,0-

§

E 20P .c_ 30-

......i 0

,

i

2

,

I

4

,

I

6

,

I

8

,

I

10

1

12

,

I

14

ir~

"30

minutes a f t e r injection

Fig. 1. Changes in the concentration of whole brain A T P following an intraperitoneal injectio# of either 0.9 % saline, 60 mg/kg catechol, or 60 mg/kg pyrogallol at time zero. All values represent a percentage fall from the brain ATP concentration found in uninjected control mice. The vertical lines represent the standard deviations (S.D.) of the mean for 5--8 observations. The S.D. of the results at zero time for saline, (e), catechol (.) and pyrogallol (A) treated mice are 1.7 %, 6.6 % and 1.9 % respectively

shows that the level of ATP is quickly restored within a minute, approaching, but never quite reaching, the level of brain ATP in uninjeeted controls. Both catechol and pyrogallol caused a fall in ATP of brain at a dose of 60 mg/ kg, but the time courses of these changes were completely different. Maximum depression was seen at 3 rain for mice given catechol (28.6% fall; S.D. ~ 4.8) but at l0 min for mice given pyrogallol (23.7%; S.D. + 5.8). After 15 min, the level of ATP in catechol-treated mice approached the control value (4.7%; S.D. + 1.1) but in pyrogallol-treated mice it was still appreciably depressed (16.1%; S.D. 4- ~.4) and did not return to the control level until 30 min after injection (3.2% ; S.D. d- 1.0). Table ] shows typical brain levels of ATP in three experiments for

Effect of Polyhydroxyphenols on Mouse Brain ATP

253

b o t h catechol a n d pyrogallol a t t h e t i m e s of m a x i m u m change. I t can be seen t h a t t h e falls p r o d u c e d b y b o t h catechol a n d p y r o g a l l o l were significantly different from t h e b l a n k control levels. Table 1. Three typical experiments to show the peak changes in concentration of mouse brain A T P after intraperitoneal injections of 60 mg/kg catechol or pyrogallol. The A T P concentration in mice injected with 0.9 ~ saline is also shown. The statistical significance of the di~erences between the injected and the unin]ected control mice was evaluated with the Student 't' test (P) Experi- ] ment[ Nlo.

Group Control . . . . . . . . . . . . . . .

3 rain after saline

2 3 1 2 3 1 2 3

..........

3 rain after 60 mg/kg catechol . . . . .

Control . . . . . . . . . . . . . . .

I

10 min after saline . . . . . . . . . .

10 min after 60 mg/kg Pyrogallol

. . .

3

!

ATE mean • S.D. /lmoles/g

No.

2.94 1.86 2.94 2.81 1.77 2.82 2.06 1.33 2.18

+ 0.05 + 0.08 2 0.20 + 0.02 _+ 0.08 + 0.003 + 0.23 + 0.25 + 0.002

<0.02 N.S. N.S. <0.001 <0.01 <0.02

2.12 2.94 2.94 2.04 2.80 2.81 1.65 2.28 2.4l

+ 0.05 + 0.20 +_ 0.05 + 0.08 + 0.113 • 0.07 + 0.12 _+ 0.23 + 0.09

N.S. N.S. N.S. <0.01 < 0.05
]

Abbreviations: S.D. = Standard Deviation; N.S. = Not Significant I n Fig. 2B t h e levels of A T P of t h e saline i n j e c t e d controls h a v e been s u b t r a c t e d from t h e values for A T P o b t a i n e d with r e s p e c t i v e l y eatechol a n d p y r o g a l l o l in order t h a t a n e s t i m a t e of t h e effect due d i r e c t l y to t h e i n j e c t e d m a t e r i a l m i g h t be o b t a i n e d . F o r comparison, t h e c o n c e n t r a t i o n s of cateehol a n d p y r o g a l l o l (Fig. 2A) in t h e b r a i n are shown after a 60 m g / k g i n t r a p e r i t o n e a l injection a t t i m e zero. Catechol p e n e t r a t e d t h e b r a i n r a p i d l y a n d r e a c h e d a m a x i m u m c o n c e n t r a t i o n after 3 rain (33.5/~g/g wet weight of brain). Pyrogallo] p e n e t r a t e d more slowly a n d r e a c h e d a m a x i m u m after l 0 rain (28.4 pg/g). F i g u r e 2C shows h i s t o g r a m s of t h e s p o n t a n e o u s m o t o r a c t i v i t y over a 15 rain e x p e r i m e n t a l p e r i o d after t h e injection of two mice, one given eatechol a n d t h e o t h e r p y r o g a l l o l (60 mg/kg). I n this s t u d y t h e doses of p y r o g a l l o l used h a d no convulsive effect, whereas catechol caused convulsions 20 see after injection. Violent jerks l a s t e d for 5 - - 6 min, with a p e a k a t a b o u t 3 rain (160 units m a x i m u m a c t i v i t y c o m p a r e d to 30 units m a x i m u m a c t i v i t y in t h e control period). This was followed b y a p e r i o d of depression (15 units m a x i m u m a c t i v i t y ) lasting for 6 - - 8 rain. F i g u r e 3 shows t h e n e t p e r c e n t a g e change in t h e A T P level following an injection of a lower dose of catechol, 40 mg/kg. The convulsions in this ease are less violent, last for a s h o r t e r time, a n d t h e fall in level of A T P is less m a r k e d t h a n t h a t

254

A. A~G~L, R.N. LEPtoN, K.J. ROGERSand P. BANKS:

produced b y 60 mg/kg. The m a x i m u m fall at 40 mg/kg catechol (14.6%) was less t h a n the m a x i m u m fall for a 60 mg/kg dose of pyrogallol (18.2%). I n a separate experiment mice injected with 30 mg/kg catechol did not convulse at all but 3 rain after the injection, ~he brain concentration of A T P had fallen b y 10.1% (S.D. _+ 2.2%) from the level of saline controls. 40-

A

o cn c~

2O c

~c

r

O-Q

10 0

< c

.c_ & =

g

"K > V <

160~120[-

4O 0 L-

i I III 2

iIi

I ~ Ii

4 8 10 12 minutes after injection

I i 14

Fig. 2. Penetration, e~e~t on concentration of brain A T P and locomotor activity of catechol and pyrogallol (60 mg/kg). A. The concentration of phenols in the brain is shown at various times after the injection of cutechol (.) or pyrogallol (A) at time zero. B. The net change in brain ATP concentration is shown for injections of catechol (.) or pyrogallol (A) at time zero. All values represenl~ the percentage fall from the level found in saline injected controls. C. The motor activity integrated over 30 sec periods for two mice, one given 60 mg/kg catechol, the other 60 mg/kg pyrogMlol (shaded) Changes in the level of A T P in the various regions of the brains of mice given 60 mg/kg catechol or pyrogallol are shown in Fig. 4A. I n the eontrol animals the highes~ concentration of A T P was found in the cerebral hemispheres (2.13//moles A T P / g wet weight of brain tissue). Concentrations of 1.81, 1.78 and 1.78/lmoles/g were found in the thalamus, medulla, and cerebellum respectively. Three minutes after an injection of 60 mg/kg catechol, the greatest change in A T P occurred in the medulla (39.5% fall from the control concentration). Changes of a smaller degree were found in all the other parts studied. This p a t t e r n is m u c h the same for the different brain areas of mice killed l0 rain after injection of 60 mg/kg pyrogallol.

Effect of Polyhydroxyphenols on Mouse Brain ATP

255

The average fall in concentration of ATP for the four parts was 27.6 % in catecholtreated mice and 20.4% in pyrogallol-treated mice. These figures agree well with the values found in whole brain at corresponding times after injection. A r@

0 r-

=~J

@

%

t~

E

s rt

5 ~o

x~ 15 C

20--

~120

l 0

0

I

'

2

4

,

'

6

!

8

10

12

14

minutes ofter injection

Fig. 3. Egects of gO mg/lcg catechol compared to 60 mg/lcg pyrogallol on brain A T P concentration. A. Net percentage changes in brain ATP following an injection of 40 mg/kg catechol at time zero. The net changes produced by 60 mg/kg pyrogallol are shown for comparison. B. Histogram of mouse motor activity after an injection of 40 mg/kg catechol at time zero Finally, in mice respiring a 95% 0 2 and 5% CO S gas mixture the change in cerebrM A T P 3 rain after an injection of 60 mg/kg catechol was much less than that found in animals breathing room air (Fig. 4B). A fall of only 6.5% was found compared to 28.4~o in the control (room air) animals. However, mice breathing the oxygen mixture exhibited a typical convulsive pattern, and this was identical in both duration and intensity to that of the control group. Discussion

The question therefore arises whether there is any relationship between the concentration of brain ATP and convulsive state of the animal. The concentration of ATP is reduced to about the same level by either 60 mg/kg pyrogallol or 40 rag/ kg catechol. Because no convulsions are seen following the dose of pyrogallol this would seem to suggest that the convulsive action of catechol cannot be ascribed to a general reduction of cerebral ATP. This is supported by the fact that catechol is able to induce convulsions in animals kept in oxygen, which have approximately normal concentrations of A T P at the height of convulsion. A subconvulsive dose

256

A. ANGEr~,I~.N. LEMON,K.J. ROGERSand P. BANKS:

(30 mg/kg) of catechol still produces a 10% fall from the control level and this might possibly be due to an intrinsic action of catechol on brain metabolism not directly related to the production of convulsions. This action m a y be related to t h a t of pyrogallol on the brain. On the other h a n d it m a y be the result of increased nervous activity which is below the threshold for observable convulsions. I n support of this it is well k n o w n t h a t increased cerebral activity resulting in convulsions, jerks and seizures is often associated with a fall in cerebral A T P (KLEr~ and OLS]~N 1947 ; PSCHEIDT et al. 1954; LEOXARD 1965 ; KING et al. 1967). ANGEL (unpublished) has found t h a t catechol causes an arousal p a t t e r n in the rat E E G , changing it from a high voltage, low frequency p a t t e r n to one t h a t is of low voltage and high A ~0

[ ] 60 mg/kg CATECHOL 3 minutes [ ] 60 mg/kg PYROGALLOL10minutes ~

B

~0

% o

30

30

20 n

c

Z ._c

CEREBRAL THALAMUSMEDULLA CEREBELLUM HEMISPHERES

0I

ROOM 95% 02 AIR 5% CO2

Fig. 4. A. Net percentage changes in concentration of A T P in various regions of the mouse brain after injection of 60 mg/kg catechol or pyrogallol. The ATP was estimated in each region 3 rain after a 60 mg/kg injection of catechol, and 10 rain after a 60 mg/kg injection of pyrogallol. B. Net percentage changes in concentration of brain A T P in mice respiring room air or 95 ~ 02, 50/0 CO 2 gas mixture after a 60 mg/kg injection of catechol. The change for ATP concentration

shown is that found 3 rain after a 60 mg/kg injection of catechol. Values shown are percentage changes from sMine-injected controls. Vertical lines in both figures represent the standard deviations of the mean frequency. Thus the greater fall in concentration of A T P observed with convulsive doses of catechol could be the effect rather t h a n the cause of the increased neuronal activity and overt convulsion. I f this is the case, the relatively larger percentage change in concentration of A T P in the medulla of the brain (Fig. 4A) might be explained b y the finding t h a t cateehol is a powerful reticular excitant (Yos•II et al. 1960; ANGEL, unpublished). The present study indicates t h a t the fall in the concentration of A T P of the brain observed during eatechol-induced convulsions is a consequence, rather t h a n the cause of the increased neuronal activity. This is in agreement with the conclusions reached b y other workers who have studied different eonvulsants (PscEEDT et al. 1954; KING et al. i957). Acknowledgements: One of us (A. ANGEL)would like to express his gratitude to the Medical t~esearch Council for providing apparatus used in this investigation.

Effect of Polyhydroxyphenols on Mouse Brain ATP

257

References AB~S, E.T., K . J . BROADLEYand D.J. RO]3ERTS: Inhibition of catechol-O-methyl transferase by some acid degradation products of adrenaline and noradrenaline. Biochem. Pharmacol. 16, 279--282 (1967). ANGEL, A., and K . J . ROOERS: Convulsant action of polyphenols. Nature (Lond.) 217, 84--85 (1968). BACQ, Z. M. : Recherehes sur la physiologie at la pharmacologie du systgme nerveux autonome. XX. Sensibilisation s l'adr6naline et l'excitation des nerfs adr6nergiques par les antioxyg~nes. Arch. int. Physiol. 42, 340--366 (1936). --, J. SCHLAG, J. FAIIIERBE et G. CI{AILLET : ]~tude de Faction excitatrice centrale du catSchol. C.R. Acad. Sci. (Paris) 249, 2839--2841 (1959). HOt,STEIN, P., and G. COHEn: The inhibitory effects of quinones and dihydric phenols on glucose metabolism in sub-cellular systems of brain. J. Neurochem. 5, 370--378 (1960). KING, L.J., O.H. LowRY, J.V. PASSONEAU and V. VENSON: Effects of eonvulsants on energy reserves in the cerebral cortex. J. Neurochem. 14, 599--611 (1967). KLEIN, J.R., and N.S. OLSEN: Effect of convulsive activity upon the concentration of brain glucose, glycogen, lactate and phosphates. J. biol. Chem. 167, 747--756 (1947). LEO~ARD, B.E. : The effect of audiogenic seizures on labile nitrogen and phosphorus containing compounds in the rat brain. Biochem. Pharmacol. 14, 1293--1298 (1965). McEL]~oY, W.D., and B.L. STREELER: Factors influencing the response of the biolumniescent reaction to ATP. Arch. Biochem. Biophys. 22, 4 2 0 - 4 3 3 (1949). PSCttEIDT, G.R., :D. BENITEZ, L.B. KIRSCHNER and W.E. STONE: Effects of fluoroacetate poisoning on citrate, lactate and energy-rich phosphates in the cerebrum. Amer. J. Physiol. 176, 483--487 (1954). RICHTER, D., and R.M.C. DAWSON: Brain metabolism in emotional excitment and in sleep. Amer. J. Physiol. 154, 73--79 (1948). ROGERS, K.J., A. ANGEL and L. BUTTERFIELD: The penetration of catechol and pyrogallol into mouse brain and the effect on cerebral monoamine levels. J. Pharm. Pharmacol. 20, 727--729 (1968). Ross, S.B., and 0. HAr~JAS~AA: Catechol-O-methyl transferase inhibitors. I n vitro inhibition of the enzyme in mouse brain extract. Acta pharmacol. (Kbh.) 21, 205~214 (1964a). - - - - Catechol-0-methyl transferase inhibitors. I n vivo inhibition in mice. Acta pharmacol. (Kbh.) 21, 215--225 (1964b). STONE, W . E . : The effects of anaesthetics and convulsants on the lactic acid content of the brain. Biochem. J. 32, 1908--1918 (1938). STREm~ER, B.L. : In: Methods of Enzymatic Analysis, pp. 559--572. Ed. by H.U. BERG)~EYER. New York: Academic Press, and Weinheim: Verlag Chemie 1963. SWAIN, T., and W.E. HILLIS : The phenolic constituents of Prunus domestica. I. The quantitative analysis of phenolic constituents. J. Sci. Food Agric. 10, 63--68 (1959). Y o s ~ I , N., J. M~TSUMOTOand H. OGVRA: Studies on the unit discharge of brainstem reticular formation in the cat. II. Effects of catechol, amphetamine, nembutal and megimide. Med. J. Osaka Univ. 11, 19--33 (1960). Dr. A. ANGEL Department of Physiology University of Sheffield S10 2TN, England