Exp. Geront. Vol. 15, pp. 593-603. Pergamon Press Ltd. 1980. Printed in Great Britain.

A G E - D E P E N D E N T MODIFICATION OF DRUG INTERFERENCE ON THE ENZYMATIC ACTIVITIES OF THE RAT BRAIN

G. BENZI, E. ARRIGONI, F. DAGANI, F. MARZATICO, D. CURTI, S. RAIMONDO, M. DOSSENA, M. POLGATTI and R. F. VILLA

Department of Science, Institute of Pharmacology, University of Pavia, Italy

(Received4 June 1980)

INTRODUCTION A FAIRLY interesting theoretical and practical problem concerns the ability of drugs to interfere on the cerebral biochemical processes which are subject to modifications with age. In this field, the potential ability to release energy can be assayed by studying the changes occurring in the enzymatic activities related to energy transduction (anaerobic glycolysis, Krebs' cycle, electron transport chain).

Data concerning the behavior of enzymatic activities as a function of age are often in disagreement (Wilson, 1973). This fact can be accounted for in the first place by differences in evaluation techniques. Indeed, data are often recorded over short periods during the animals' life (Kuhlman and Lowry, 1956; Hamburgh and Flexner, 1957; Sims et aL, 1979), measurements being carried out at long and irregular intervals (Pitts and Quick, 1967; Hollander and Barrows, 1968; Epstein and Barrows, 1969; Adlard and Dobbing, 1971), while it would be more useful to get systematic data, obtained at short, regular intervals over prolonged periods of the life of the animal species studied (Kellog and Fridovich, 1976; Benzi, 1979). On the other hand, one should also take into account the complex relationships existing between ageing, protein synthesis and en- zymatic activities (Haining and Correl, 1969; von Hahn, 1970; Adelman, 1970; Reiss and Rothstein, 1974; Sharma etal . , 1976; Rothstein, 1977; 1979).

The effect induced in the adult rat (16-28 weeks of age) on cerebral enzymatic activities by a four-, eight- or twelve-week treatment with "vasoact ive" or "vasculometabolic" drugs can be observed (Benzi et al., 1979)--the pharmacological response tended to be stronger after the first four weeks of treatment, while after 12 weeks it became less evident. This fact migh~t be related to a reduction in drug power due to prolonged treatment (e.g. because of drug induction, changes in pharmacokinetics, etc.), or to an intrinsic inefficiency of the drugs to interfere with the age-dependent decrement of cerebral en- zymatic activities taking place from the 20th week of age in the strain of rats used (Benzi, 1979). In order to verify this latter hypothesis the influence of age on the same subchronic (4-week) treatment, performed daily with one dose level of several drugs was evaluated in this study. We investigated a long span in the life of the Sprague-Dawley strain, evaluations being carried out at regular time intervals, approximately every 9 months. The ages studied (20, 60, 100 and 140 weeks of life) roughly corresponded to "adu l t " , "mature" , "senescent" and "o ld" rats. Obviously, these attributions are totally arbitrary and imprecise; however, they are used to facilitate the singling out of given times during rats' life. The cerebral enzymatic activities studied were: lactate dehydrogenase (L-lactate: NAD ÷ oxidoreductase, EC 1.1.1.27) for the glycolytic pathway; citrate synthase (citrate oxalocetate-lyase, EC 4.1.3.7) and malate dehydrogenase (L-malate: NAD ÷ oxidoreductase: EC 1.1.1.37) for the Krebs' cycle; total NADH cytochrome c reductase (NADH-cytochrome c:

593

594 G. BENZl et al.

o x y g e n o x i d o r e d u c t a s e , E C 1 .6 .99 .3 ) a n d c y t o c h r o m e o x i d a s e ( f e r r o c y t o c h r o m e c: o x y g e n

ox ido reduc t a s e , E C 1.9.3.1) fo r the e lec t ron t r a n s p o r t cha in . S o m e o f the e n z y m a t i c act ivi t ies

we re e v a l u a t e d in b o t h t he h o m o g e n a t e in to to a n d t he c r u d e m i t o c h o n d r i a l f r a c t i o n , s ince

m a n y o f t h e m a r e v a r i o u s l y l o c a t e d in t he c y t o p l a s m .

A r a t h e r d i f f i c u l t p r o b l e m in th i s i n v e s t i g a t i o n was the c h o i c e o f t h e d r u g s to be u sed

in o r d e r to tes t t he r e s p o n s e o f t he c e r e b r a l e n z y m e s m e n t i o n e d a b o v e as a f u n c t i o n o f

age ing . A s is wel l k n o w n , t h e r e exis t d r u g s w ide ly u s e d c l in ica l ly in h u m a n s , w h i c h a re

g e n e r i c a l l y a n d c o n f u s e d l y c l a s s i f i ed as c e r e b r a l " v a s o d i l a t i n g " , " v a s o a c t i v e " o r " v a s -

c u l o m e t a b o l i c " a g e n t s . W e c h o s e the f o l l o w i n g m o l e c u l e s : (a) p a p a v e r i n e , t h e o p h y l l i n e ,

a n d t r i m e t a z i d i n e as d r u g s e x e r t i n g a m a i n l y v a s c u l a r a c t i o n ; (b) c y t i d i n e d i p h o s p h a t e

c h o l i n e , as a n a g e n t e x e r t i n g m a i n l y a m e t a b o l i c a c t i o n ; (c) n i c e r g o l i n e , d i h y d r o e r g o -

c r i s t i ne , su loc t i d i l , ( - ) e b u r n a m o n i n e a n d v i n c a m i n e , as d r u g s e x e r t i n g b o t h a v a s c u l a r

a n d a m e t a b o l i c a c t i o n . C l e a r l y th i s s u b d i v i s i o n is a v a g u e a n d s u p e r f i c i a l one . O n the

o t h e r h a n d th i s w o r k is i n t e n d e d a l so to o v e r c o m e it, b e s ide s b e i n g a i m e d a t d e t e c t i n g

t he c h a n g e s in t he a c t i o n o f t h e s e s u b s t a n c e s o n seve ra l e n z y m a t i c ac t iv i t i e s r e l a t e d to

e n e r g y t r a n s d u c t i o n , d u r i n g m a t u r i t y , s e n e s c e n c e a n d o ld age s ince n o d a t a a re a v a i l a b l e

in t he l i t e r a t u r e o n th i s s u b j e c t .

A t a n y r a t e , t h i s p a p e r d o e s n o t i n t e n d to t a c k l e t he p r o b l e m o f d e s c r i b i n g o r p r e d i c t i n g

t h e r a p e u t i c m e a s u r e s w i t h r e g a r d to a g e i n g in h u m a n s . R a t h e r , d r u g s a r e u s e d as exo-

g e n o u s f a c t o r s a b l e o r u n a b l e to a f f e c t t he c e r e b r a l e n z y m a t i c r e a c t i v i t y in a b i o l o g i c a l

m o d e l o f n a t u r a l age i ng .

M A T E R I A L S A N D M E T H O D S

The study was carried out in male rats (Sprague-Dawley strain) fed a standard diet as pellets and water ad libitum and housed three, and subsequently two, per cage under optimal environmental conditions: 22°; 55-60070 relative humidity; 12-h day cycle (light from 7.00 a.m. to 7.00 p.m.) until they reached the age of 16-140 weeks. The rats were housed at first for one month under the fixed dark-light rhythm. In fact, the circadian activities of various enzymes are stable only after about 3 weeks in the adult rat at the same time of the nycthemeron. The initial allocation of animals to the different lots was made by randomization. The time course of the examinations performed in the lots was established by means of permutation tables.

The drugs were administered between 9:10 and 10:40 a.m. Within this time interval, the treatments were alternated so as to balance the circadian rhythm effect. Treatment was carried out daily (6 days a week, by intraperitoneal administration) using one dose of drug. The dose was within the range used experimentally in order to characterize the pharmacodynamic action of the different substances tested: (a) papaverine hydro- chloride (papaverine) = 1 mg/kg as papaverine base; (b) vincamine theophyllinylpropane sulfonate (vincamine TPS) = 5 mg/kg as vincamine base; (c) sodium theophyllinylpropane sulfonate (theophylline PS) = 5 mg/kg as theophylline base; (d) cytidine diphosphate choline (CDP-eholine) = 5 mg/kg; (e) trimetazidine dihydro- chloride (trimetazidine) = 5 mg/kg as trimetazidine base; (f) nicergoline tartrate (nicergoline) = 1 mg/kg as nicergoline base; (g) dihydroergocristine methane sulfonate (dihydroergocristine MS) = 0.1 mg/kg as dihydroergocristine base; (h) (-) eburnamonine phosphate (eburnamonine) = 5 mg/kg as base; (i) suloctidil in tween 80 suspension (suloctidil) = 1 mg/kg. Control animals were given the vehicle only by the same route. Blind biochemical evaluations were performed after 4 weeks of treatment at 20, 60, 100 and 140 weeks of age. In order to point out the existence of a linear dose/action relationship in 20-week old rats, the effect of a 4-week nicergoline treatment from the 16th to the 20th week by i.p. route at the following doses: 0.0625; 0.25; 1.00; 4.00 mg/kg was assayed.

All animals were killed between 9.30 and 10.10 a.m., 48 h after the last injection. At the set time the animals were sacrificed by decapitation and their brains removed from the skull within 15 s in a precooled box at 5 °. The 0.32 M sucrose washed and weighed brains (without cerebellum) were homogenized in 0.32 M sucrose for 30 s (precooled Potter-Braun S homogenizer with a teflon pestle). The homogenate obtained was diluted with 0.32 M sucrose (10% w/v) and an aliquot of each sample was taken for the assay of enzymatic activities. The remaining homogenate was submitted to a series of centrifugations (Sorvall RC-5 Supercentrifuge and J-21 C Beckman Supercentrifuge) for the preparation of the crude mitochondrial fraction (De Robertis et al., 1962) pelleted at 14,000 g for 20 min. On both the homogenate and the mitochondrial preparation samples, protein content was evaluated (Lowry et al., 1951) and the following enzymatic activities were measured: malate dehydrogenase (Ochoa, 1955), total NADH-cytochrome c reductase (Nason and Vasington, 1963);

MODIFICATION OF DRUG INTERFERENCE ON ENZYME ACTIVITY 595

cytochrome oxidase (Smith, 1955; Wharton and Tzagsloff, 1967). The activity of lactate dehydrogenase was evaluated only in homogenate samples (Bergmeyer and Bernt, 1974) while that of citrate synthase was measured only in the mitochondrial preparation samples (Sugden and Newsholme, 1975). Enzymatic activities were recorded (Beckman 25 Spectrophotometer Recorder) and calculated using the straight portion of the reaction curves. Results were expressed as specific activities: ~,moles min z (mg protein) 1. Two statistical tests (ANOVA and Dunnett's tests) were applied to the results, after checking the homogeneity of variance by the Bartlett's test. ANOVA was employed to evaluate the "enzymatic activities by times" interactions and the "treatments by times" interactions. The Dunnett's test was used, at each individual time, to assess differences between the cerebral enzymatic activities of controls and those of treated rats.

RESULTS

In the "treatments by times" interactions, ANOVA failed to exhibit any significant difference between the values of the enzymatic activities evaluated in the brain of control rats and of those treated for one month with a single dose of the various drugs, at the different ages. Therefore, the physiological behavior of cerebral enzymatic activities during ageing prevailed over the changes which could be altogether induced by the drugs tested. The results reported below are thus described with regard to significant differences pointed out by the Dunnett's test at each tested time (20, 60, 100 or 140 weeks of age).

Each pharmacological treatment (Tables 1, 2, 3 and 4) induced different and specific changes in some of the enzymatic activities tested in the homogenate in toto and/or in the crude mitochondrial fraction. However, the range of the pharmacological interference progressively narrowed with age. Thus at 20 weeks of age, one month treatment with papaverine caused an increase of lactate dehydrogenase, of malate dehydrogenase and of cytochrome oxidase, as evaluated in the homogenate in toto. At 60 and 100 weeks, the same treatment led only to the enhancement of lactate dehydrogenase and cytochrome oxidase, no effect being observed at 140 weeks in the homogenate in toto. At 20, 60 and 100 weeks of age, one month treatment with theophylline affected only lactate dehydro- genase, no effect being detected at 140 weeks.

At 20 weeks of age, one month treatment with trimetazidine caused the inhibition of all the enzymatic activities tested in the mitochondrial fraction, and the inhibition of cytochrome oxidase in the homogenate in toto. Subsequently (60 and 100 weeks of age) only cytochrome oxidase in the homogenate in toto and citrate synthase in the mito- chondrial fraction were found to be inhibited: no action was observed at 140 weeks of age. At 20 weeks of age, after one month treatment with nicergoline, cytocbrome oxidase (homogenate in toto), malate dehydrogenase (homogenate in toto and mitochondrial fraction) and citrate synthase (mitochondrial fraction) were inhibited, while the actitivity of NADH-cytochrome c reductase (homogenate in toto and mitochondrial fraction) appeared to be increased. At 60 and 100 weeks of age, these effects concerned only the total NADH-cytochrome c reductase and citrate synthase, while at 140 weeks they were no longer detected. CDP-choline basically affected the activity of citrate synthase: this action, however limited, persisted in time. Indeed, at 20, 60, 100 and 140 weeks of age, one month treatment with CDP-choline led to the inhibition of mitochondrial citrate synthase.

At 20 weeks of age, one month treatment with vincamine was responsible for an increase of the activity of lactate dehydrogenase and cytochrome oxidase (as measured in the homogenate in toto) and of citrate synthase, malate dehydrogenase, NADH- cytochrome c reductase, cytochrome oxidase (as measured in the crude mitochondrial fraction). At 60 and 100 weeks of age, one month treatment with vincamine Led to increase of lactate dehydrogenase and cytochrome oxidase enzymatic activities, as measured in

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600 G. BENZI et al.

the homogenate in toto, as well as of citrate synthase and malate dehydrogenase in the mitochondrial fraction. At 140 weeks of age, after one month treatment with the same drug, only lactate dehydrogenase and cytochrome oxidase (as measured in the homogenate in toto) were found to be affected.

At 20 weeks of age, a one-month treatment with suloctidil caused an increase of several enzymatic activities, as evaluated in both the homogenate in toto (lactate dehydrogenase, total NADH-cytochrome c reductase, cytochrome oxidase) and in the mitochondrial fraction (citrate synthase, malate dehydrogenase). At 60 weeks, the same treatment limited its influence to the mitochondrial enzymatic activities related to the tricarboxylic acid cycle, while at 100 and 140 weeks only the maximal rate of the mitochondrial malate dehydrogenase was increased.

In 20-week-old animals, the one-month treatment with eburnamonine increased both the activity of lactate dehydrogenase and that of the mitochondrial cytochrome oxidase, citrate synthase being on the contrary decreased. This interference on the mitochondrial fraction was observed also with regard to treatment of mature rats and partially (cyto- chrome oxidase only) of senescent rats, while it was no longer found in old rats (140 weeks of age).

At 20 weeks of age, dihydroergocristine decreased the activity of lactate dehydrogenase and increased that of the total NADH-cytochrome c reductase, evaluated in the homo- genate in toto, while the activity of malate dehydrogenase was reduced in both the preparations tested. At 60 weeks, the same treatment led to the same kind of inter- ference, except for the lack of action on the total NADH-cytochrome c reductase. At 100 and 140 weeks of age, the only action still observed was the inhibition of malate dehydrogenase, as evaluated in the homogenate in toto.

DISCUSSION

The present data refer to the cerebral tissue as a whole; therefore, some important areas of the brain might undergo different biochemical adjustments, because of the functional and anatomical heterogeneity of the organ. This is no major drawback, since the purpose of this work is to establish, at the level of cerebral enzymatic activities, whether any age-dependent changes occur in the response to subchronic pharmacological treatment. This because, at least as far as the drugs tested are concerned, no data are available concerning their action on age-dependent modifications of the cerebral en- zymatic activities related to energy transduction. It should also be remembered that the values indicated by us for the enzymatic activities studied specifically refer to their maximal rate, since the methods used to determine the enzymatic activities were applied to brain preparations in which the autoregulative interactions that maintain the cell as a functionally integrated system were abolished.

A problem to be preliminarily discussed concerns the linear dose/action relationship. Table 5 concerns the subchronic treatment with nicergoline carried out between the 16th and 20th week of age with four different doses of drug (0.0625; 0.25; 1.00; 4.00 mg/kg i.p.). The measurements performed on some cerebral enzymatic activities showed the absence of any linear dose/effect relationship, a threshold dose level being present. This is in agreement with what has been observed with regard to the effect of the sub- chronic treatment on muscular enzymatic activities related to energy transduction (Benzi etal., 1977).

MODIFICATION OF DRUG INTERFERENCE ON ENZYME ACTIVITY 601

TABLE 5. CEREBRAL ENZYMATIC ACTIVITIES RELATED TO ENERGY TRANSDUCTION. EFFECT OF I.P. TREATMENT WITH NICERGOLINE A'r DIFFERENT DOSE LEVELS (0.0625; 0.25; 1.00; 4.00 mg/kg) IN RAT FOR 4 WEEKS (6 DAYS A WEEK):

FROM 16TO 20 WEEKS OF AGE

Nicergoline Total NADH dose levels Lactate Citrate Malate cytochrome c Cytocbrome

(mg/kg) n dehydrogenase synthase dehydrogenase reductase oxidase

0.00 6 0.812 0.092 1.73 0.140 0.714 ±0. 008 ±0. 003 ±0. 04 ±0.004 ±0.015

0.0625 4 0.826 0.089 1.65 0.137 0.696 +-0. 019 ±0. 006 ±0.09 ±0. 006 ±0.015

0.25 4 0.865 0.077* 1.60* 0.178"i" 0.738 ±0.033 ±0. 004 ±0. 07 +_0. 012 ±0. 023

1.00 6 0.852 0.071 "1" 1,54" 0.184']- 0.701 ±0.018 ±0.002 ±0.04 ±0.005 +_0.015

4.00 4 0.870 0.084 1.63 0.169" 0.685 +_0. 03 7 ±0. 004 +_0.06 ±0.014 +_0. 031

The enzymatic activities 0amoles min - I m g protein -1) were evaluated both in the homogenate in toto (lactate dehydrogenase) and in the crude mitochondrial fraction from the rat brain (age: 20 weeks) and are expressed as the mean values ± S.E.M. for each group of n animals.

*Differs from control: p < 0.05 I, "['Differs from control: p < 0.01 1 Dunnett ' s test

The behavior of the curves depicting the decrease of the different enzymatic activities as a function of ageing was never different in the rats given saline solution as compared to those receiving the different drugs tested. This emphasizes the preminence of the physio- logical or physiopathological phenomenon of cerebral ageing, over the pharmacological treatments carried out. Only at individual times (20, 60, 100 and 140 weeks of age) did the various pharmacological treatments cause different modifications in some cerebral enzymatic activities. This work shows that, at 20 weeks of age ( "adu l t " animals) the various drugs exert different actions on the different cerebral enzymatic activities assayed. It is therefore possible to propose the classification reported in Table 6, again with the reservation that these biological findings cannot be acritically transferred to the field of human physiopathology.

At any rate, it can be seen that these different agents, generically grouped as "vasculo- metabolic" drugs, exhibit extremely different, or even contrasting, interferences on the same cerebral enzymatic activities. Moreover, even when these changes in cerebral enzymatic activities do occur, their range is positively wider in " adu l t " rats (20 weeks of age) than during maturity, and old age (60, 100 and 140 weeks of age in rats). Therefore, as the animal gets older, the pharmacological treatment reduces its spectrum of action on enzymatic activities, without affecting those enzymatic activities which were not modified at earlier times. Each drug therefore exerts an effect on cerebral enzymes, while ageing progressively narrows this effect, which becomes almost null during old age (140 weeks of age in the rat). In order to explain this behavior, the following hypotheses can be put forward: (1) modification of enzyme conformation without covalent change, as supported by several experimental observations and related to a slowing of protein turnover in old organisms (Sharma et al., 1976; Rothstein, 1979). The resulting increase in the "dwell t ime" for the enzymes in cells would provide the opportunity for both a functional change and a subtle denaturation to occur, without replacement of the altered enzymatic molecules (Reiss and Rothstein, 1974; Rothstein, 1979); (2) changes in the cerebral bio- availability of drugs, due to age-dependent modifications of drug absorption, distribution

602 G. BENZI et al.

TABLE 6. CLASSIFICATION OF THE DRUGS ACTING ON THE MAXIMAL RATE OF THE CEREBRAL ENZYMATIC ACTIVITIES (C.E.A.) RELATED

TO ENERGY TRANSDUCTION

l--Drugs which generically interfere with the C.E.A. : 1. l - -By an activating trend of action: medibazine

vincamine 1.2--By an inhibitory trend of action: trimetazidine

2--Drugs which interfere with the C.E.A. o f the glycolytic path way:

2. l - -By an activating trend of action: papaverine theophylline

2.2--By an inhibitory trend of action: dihydroergocristine

3--Drugs which interfere with the C.E.A. o f the Krebs" cycle: 3.1--By an activating trend of action: suloctidil 3.2--By an inhibitory trend of action: ( )eburnamonine

CDP-choline nicergoline dihydroergocristine

4--Drugs which interfere with the C.E.A. o f the electron transfer chain:

4.1 --By an activating trend of action: nicergoline (-)eburnamonine

and metabolization processes; (3) changes in the function of organs and apparatus involved in the extracerebral action of the drugs, when the cerebral pharmacological effect is indirectly mediated by them. However, these are mere hypotheses since, with regard to the drugs tested, no reliable explanations can be found in the literature.

In conclusion, the trend of interference on enzymatic activities can be established for each drug even if a more detailed description of pharmacological interference on tissue enzymatic systems also requires their evaluation: (a) in more homogeneous subcellular fractions (purified mitochondrial fraction, synaptosomal fraction, etc.); (b) in the individual brain areas (cortex, forebrain, hippocampus, substantia nigra, etc.). At any rate, this first study showed that age progressively narrows the range of drug effects on the enzy- matic activities tested, and consequently a classification of drug action of cerebral enzy- matic activities must necessarily take into account the age of the animal. However, aged rats (in particular, 140-week-old ones) can not be regarded as a representative sample of the whole population, but only of the population of survivors.

SUMMARY

In "adu l t " , "ma tu re" , "senescent" and " o l d " rats (20, 60, 100 and 140 weeks of age) the age-dependent changes of some cerebral enzymatic activities (lactate dehydrogenase; citrate synthase and malate dehydrogenase; total NADH-cytochrome c reductase and cytochrome oxidase) were studied in the homogenate in toto and/or in the crude mito- chondrial fraction of the brain. With age, from "adu l t " to " o l d " rats, the activities studied exhibited a natural decrease.

The drugs tested (trimetazidine, papaverine, vincamine, theophylline, nicergoline, CDP-choline, suloctidil, eburnamonine, dihydroergocristine) were administered daily for periods of 4 weeks each (16-20, 56-60, 96-100 and 136-140 weeks of life) by intra- peritoneal route and at one dose level. The drugs tested exerted typical effects on the

MODIFICATION OF DRUG INTERFERENCE ON ENZYME ACTIVITY 603

various enzymatic activities of the brain. At any rate, the range of drug interference with these enzymatic activities narrowed remarkably during maturity and senescence and even more during old age. The possible mechanisms of such behavior are discussed.

Acknowledgements--We thank Dr. M. L. Riva for assistance in the preparation of the manuscript, Mrs. G. Garlaschi, Mr. G. Arioli and Mr. L. Maggi for technical assistance.

R E F E R E N C E S

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