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The optimal dosage of (−)deprenyl for increasing superoxide dismutase activities in several brain regions decreases with age in male Fischer 344 rats
L i f e S c i e n c e s , Vol. P r i n t e d in t h e U S A
52, pp.
1925-1934
Pergamon
Press
:7~E O P T I ~ L DOSAGE OF (-)DEPRENYL FOR INCREASING SUPEROXIDE DISMUTASE ACTIVITIES IN SEVERAL BRAIN REGIONS DECREASES WITH AGE IN MALE FISCHER 344 RATS M.C.
Carrillo 1'2, S. Kanai I, Y. Sato I, M. Nokubo I, G.O.
Ivy 3 and K. Kitani 4.
i ~Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology 35-2, Sakaecho, Itabashi-ku, Tokyo-173, JAPAN; Institute De Fisiologia Experimental Suipac~a 570, 2000, Rosario, Universidad Naciona] De Rosario, Republica Argentina; ~Division of Life Sciences, University of Toronto.at Scarborogh, o i~65 Military Trail, Scarborogh, Ontario, CANADA MIC IA4; 4 Radioisotope Research Institute, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, JAPAN. (Received
in final form March 31, 1993) Summary
We previously reported that the optimal dosage of (-)deprenyl to increase superoxide dismutase (SOD) activities in striatum in rats differs i0 fold between young male and female rats (i). Furthermore, in female rats the optimal dosage increased with age (1). In the present study in order to clarify how the optima] dosage of this effect changes with age in male rats, we examined the effects of four different dosages of deprenyl on SOD enzyme activities in striatum and several other tissues in old (28-29-month-old) ma]e Fischer 344 (F-344) rats. Continuous s.c. infusion of deprenyl for 3 wks increased activities of SOD and cata!ase (CAT) in striatum, substantia nigra and cortical regions but not in hippocampus, cerebellum or the liver. The dose of 0.5 mg/kg/day was found to be most effective, while higher (].0, 2.0 mg/kg/day) or lower (0.1 mg/kg/day) dosages were less effective. This value of 0.5 mg/kE/day was 4 fold Iower than the dosage of 2.0 mg/kg/day which was most effective in increasing SOD and CAT activities in young (5-7 month old) ma]e rats of the same straiD (1,2). The decline of the optimal dosoge with age found in male rats is best explained by a possible dec]ine with age in the hepatic microsoma! monooxygcnase enzyme activities that are involved with the metabolism of deprenyl. In view of the large differences ~n the optimal dosages sho~1 among different sexes and ages of rats, future studies regarding the unique effect of this drug in prolonging the life span of rats must be carefully investigated with the caution in mind that the optimal dosage for the life prolonging effect may we]] differ depending on sex, age and possibly strain and species of animal model used. We have previously reported that the s.c. injection of (-)deprenyl at a dose of 2.0 mg/kg/day for 3 weeks increased activities of both Cu Zn-SOD and Mn-SOD activities in striatum 3 fold in young (5-7 month old) male F-344 rats (2). Furthermore CAT activity also increases significantly (60%) by this treatment (2). Subsequent studies in our laboratory have revealed that this effect is selective for certain brain regions such as substantia nigra (s. nigra) and cerebral cortices, but not for other regions such as hippocampus, cerebellum or *Address
for correspondence Copyright
0024-3205/93 $6.00 + .OO © 1993 Pergamon Press Ltd All rights reserved.
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the liver (3). Furthermore, when this effect was examined in female rats, the optimal dosage was found to be 10 fold lower (0.2 mg/kg/day) and the optimal dosage found in young male rats (2.0 mg/kg/day) actually decreased SOD activities 3 fold in young female rats (i). Interestingly when old female rats were examined, we found an increase in optimal dosage (i.0 mg/kg/day) in comparison to young female rats (i). As possible explanations for the observed differences in the optimal dosages, we suggested two different mechanisms (i). First, the 10-fold lower optimal dosage found in young female rats than in males may be due to the possibly lower enzyme activities of the hepatic microsomal monooxygenase system involved in the metabolism of deprenyl (for review, see 4,5). Second, the increase in the optimal dosage with age seen in female rats may be due to the increase with age (both sexes) of monoamine oxidase activities in several organs including the brain (6,7), since deprenyl is irreversibly bound to an ~0 B enzyme molecule. From these considerations, we predicted that the optimal dosage in male rats may decrease with age (i), since a general decline in the hepatic microsomal monooxygenase enzyme activities with age is known to occur in male (but not in female) rat livers (4,5). Here, we demonstrate that (-)deprenyl also increases SOD and CAT sctivities in certain selective brain regions in old male rats as in their young counterparts, however, the optimal dosage is at least 4 fold lower than in young male rsts, as previously predicted (1).
Materials and Methods Chemicals (--)Deprenyl is a generous gift from Fujimoto Pharmaceutical Company (Osaka). Hydrogen peroxide, xanthine, hydroxylammonium chloride, sulfanilic acid, ~naphthylamine, potassium cyanide, reduced glutathione (GSH) and sodium azide were obtained from Wako Pure Chemicals Ltd. (Tokyo). Xanthine oxidase, glutathione reductase and SOD were from Sigma. NADPH wss purchased from Oriental Yeast Co. Ltd. (Tokyo). Animals and tissue preparations Two cohorts of old male F-344 rats (sixteen 28-month-old and seven 29-monthold) were used in this study. The average body weight of this group as well as those used in our previous studies (i-3) on young male and young and old female rats are summarized in Table I for comparison. The first cohort of old males were 20% heavier than young males on the average, and old female rats were 30% heavier than their younger counterparts. The second cohort of old males were 10% lighter than the first cohort of animals but still significantly (but only by 10%) heavier than their young counterparts. These animals were originally
TABLE I Body weights of F-344 rsts Young rats Male rats
(n)
350.1 + 31.2 (30) (5- 7-mon th-o Id )
Old rats Cohort
1
Cohort 2
Female rats
199.6 + 9.6 (32) (5- 7-mon th-o id )
(n)
423.7 + 25.0 (16) (28-non th-o id ) 391.3 + 34.4 (7) (29-month-o Id) 268.8 + 23.8 (24) (28-mon th-o Id )
*The total number of rats studied in the previous studies
(i-3).
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purchased from Charles River Japan (Atsugi) at the age of 4 weeks and raised in the aging farm of the insitute in specific pathogen-free (SPF) condition until use. Rats were treated with continuous s.c. infusion of deprenyl (dissolved in saline) for 3 weeks at different rates (the first cohort at 0.5, 1.0 and 2.0 mg/kg/day and the second at 0.1 mg/kg/day) by using an osmotic minipump (Alzet, Alza, Palo Alto CA). Control animals of same ages were given s.c. infusion of isovolumetric physiological saline solution for the same time period. On the 22nd day, animals were sacrificed by decapitation and liver and various brain regions ~substantia nigra (s. nigra), striatum, hippocampus, cerebellum and three different cortical areas (frontal, parietotempora! and occipital)" were immediately removed and dissected on an ice cold plate. Tissue preparations for enzyme activity measurements are the same as were described in the previous papers (i-3). Enzyme assays Superoxide dismutase (SOD). The activity of SOD was assayed by the method of Elstner and Heupe] (8). Differentiation of the two different types of SOD (Cu4Zn-SOD and Mn-SOD) was performed by the addition of potassium cyanide (5 X I0 M) to the incubation medium. Cu Zu-SOD activities were defined as those inhibited by potassium cyanide. The difference between total and KCN inhibited enzs~e activities was defined as Mn-SOD activity. Catalase (CAT). Catalsse activity was assayed by the method described by Beers and Sizer (9). Details of enzy~e activity measurement procedures have been described in our previous papers (i-3). Statistical analysis. Values obtained from animals from the first cohort were analyzed by one-way analysis of varlance (ANOVA). When differences were significant with respect to deprenyl dosage, comparisons between values in control and drug--treated rats and between values in rats receiving different doses were analyzed by Scheffe's test. Results for the 2nd cohort of animals were analysed by Student t test for unpaired values. P values lower than 0.05 were judged to be significant. Results FIG. 1 summarizes activities of CAT, Cu Zn-SOD and Mn-SOD in different brain regions and tile liver in old control animals and old animals given s.c. infusion of (-)deprenyl for 21 days at a rate of 0.5 mg/kg/day which caused the greatest increase in enzyme activities among the four dosages tested (see FIGs. 2-6). Basal CAT enzyme levels in old male rats were fairly comparable to those found in young male rats as we previously reported (3). However, SOD levels, especially Mn-SOD activities were generally much greater in old male rats compared to corresponding values in young animals. For example, the basal ~ - S O D activities in striatum were 0.5 u/mg in young males (3), while they were 2.0 u/mg in old males (FIG. i). With deprenyl treatment at a dose of 0.5 mg/kg/day, all these enzyme activities significant]y increased in striatum, s. nigra and cerebral cortical regions, but not in hippocampus, cerebellum or the liver. The net increase for Mn-SOD activity by deprenyl infusion was generally much greater in old males than young males. For example the greatest net increase in M_n-SOD activity ~n striatum of young males which was achieved by the dose of 2.0 mg/kg (3) was only 1.5 u/mg, while in old rats the net increase was as high as 4 u/mg with the dose of 0.5 mg/kg (FIGs. 1,2). FIGs. 2 and 3 summarize activities of these three enzymes in striata (FIG. 2) and s. nigra (FIG. 3) from control rats and rats given different doses of (-)deprenyl. Basal enzyme levels for CAT as well as two different species of SOD were mostly comparable for the two cohorts of old male rats of different ages (28 months vs. 29 months). It is apparent that the the dose of 0.5 mg/kg/day was most effective. Higher (I.0, 2.0 mg/kg/day) or lower (0.i
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i 30 20
10 0
,Oo
12! 0
8t MnSOD
8 4
S.Nigra StriatumHippo- FrontalParieto- OccipitalCerebellum campus cx. temporal cx.
0 Liver
CX.
Enz~me activities of CAT, Cu Zn-, and ~[n-SOD in different brain regions and the liver in control old male rats (n=4, white columns) and in old rats infused with depreny] at 0.5 mg/kg/day for 2] days (n=4, shadowed columns). *Significantly dilferent from respective control values (P<0.05). mg/kg/dsy)
doses were also effective but to a much lesser degree.
FIG. 4 compares the effects of different doses of deprenyl on CAT activities in striatum in young and old F-344 rats of both sexes. FIGs 5 and 6 demonstrate activities of Cu Zn-SOD and Mn-SOD respectively. All values are expressed as percentages of respective control values. Figures on young male and young and old female rats were prepared based on data reported in our previous publications (1-3). It is clear that young male rats required the greatest dosage to achieve an optimal effect of the drug in terms of increasing CAT activity as was true with both Cu Zn- and Mn-SOD activities (]). Since a dese higher than 2.0 mg/kg/day was not examined, it is possible that a more accurate optimal dosage is even greater in young male rats. In young females, the optima] dosage was at least 10 fold lower. Age, however, increased the optimal dosage in female rats. In contrast, in old male rats, the optimal dosage was about 0.5 mg/kg/day which is 4 times lower than in young males. Interestingly, when old male and female rats were compared, the dose of 1.0 mg/kg/day was about equally as effective as 0.5 mg/kg/day in females, while in males, the higher dose (i.0 mg/kg/day) was obviously less effective than the lower dose (0.5 mg/kg/day). In this regard, it is interesting to note that the dose of 0.i mg/kg/day was as effective as
Vol. 52, No. 24, 1993
Deprenyl on Brains of Old Male Rats
Catalase
Catalase 30
20 10
1929
(3)
(4)
(4)
T
3O
20
(41
,o
"~ 10l CuZnSOD~j~
~"{ I0 i CuZnSOD
C1 C2 0.1 0.5 1.0 2.0
~
iV
Cl C2 0,1 0.5 1.0 2.0
Deprenyl rng/kg b.wt./day
Deprenyl mg/kg b.wt./day
'"IG. 2
FIG. 3
FIGs. 2 and 3 Activities of CAT, Cu Zn-SOD and Mn-SOD in str!ata (FIG. 2) and s. nfgra (FIG. 3) from control old male rats and rats treated with different dosages of deprenyl. The number in parenthesis indicates the number of rats studied. CI and C2 indicate values of two control groups, cohort one (C]) and cohort two (C2) respectively. *Significantly different from corresponding control values (P<0.05) the dose of 0.5 mg/kg/day in increasing CAT as well as Mn-SOD activities, while the same dose of 0.i ms/ks/day was clearly less effective for Cu Zn-SOD activities than the higher dose (0.5 ms/ks/day) in striatum. Dose responses in s. nigra were generally similar to those feund in striatum. Discussion Knoll, who developed deprenyl (10,11) as an antidepressant and anti-Parkinson's disease agent, is also the first to have demonstrated that this drug can increase SOD activities in striatum of young male and female rats treated with s.c. injection of deprenyl at a dose of 2.0 mg/kg/day for 21 successive days (12). He also reported that activities of CAT and glutathione peroxidase (GSH Px) were to some extent increased in this brain region by the same deprenyl treatment (12). However, these increases did not attain a statistical significance (12). Our group reexamined this question and confiz~ed that (-)deprenyl when s.c. injected for 2] successive days at a dose of 2.0 mg/kg/day, caused both types of SOD activities to be increased three fold in striata of young male F-344 rats (2). Furthermore, we showed a 60% increase in CAT activity with deprenyl treatment that was also statistically significant (2). On the other hand, GSH Px activity, which was mildly (but insignificantly) increased in Knoll's study (12), remained totally unaltered in our studies (2).
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¼
(5)
youngmale
200
old male
Vol. 52, No. 24, 1993
(4) • -!-
(5)
(4).
100
.......i 0.1
0.2
0.5
1.0
2.0
youngfemale
14).
0.1
0.2
old female
(s) F~
(5)
0.5
10
(4]
(4),
0.5
1.0
2.0
FIG. 4
(7)
10C
0.1
0.2
0.5
1.0
2.0
Oeprenyl % 300
young male
0.1
0.2
2.0
mg/kg b.wt./day
0M m~e
20C
100
0.1
0.2
0.5
1.0
2.0
0.1
02
0.5
1.0
2.0
FIG. 5 % / youngfemale
old female
/
200 ~-
•
•
100 . . . . . . . . . . . . . . . . . . . . . . .
o 0.1
0.2
0.5
1.0
2.0
Deprenyl
0.1 0.2 n~/kg b.w./day
0.5
1.0
2.0
FIGs. 4 and 5 Relative enzyme activities of CAT (FIG.4) and Cu Zn-SOD (FIG. 5) in striata from young and old rats of both sexes treated with different dosages of deprenyl. All values are expressed as percentages of respective control values. Figures on young male and young and old female rats were drawn from data we previously reported (1-3). White bars indicate values in rats given 21 day s.c. infusion and shadowed bars represent vs]ues in rats given 21 day s.c. injection. *Significantly different from respective control values (P<0.05). The numbers in parenthesis indicate the number of rats studied in each group. The numbers of control animals are 4 to 7 for each group.
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%, 30O
old male
young male
20O
100
% 300
0.1
0.2
0.5
1.0
2.0
0,1
0.2
0.5
1.0
2.0
0.5
1.0
2.0
old female
young fernale
2OO
100
0.1
0.2
0.5
1,0
2.0
Deprenyl
0.1
0.2
mg/kg b.wt,/day
FIG. 6 Re]ative enzyme activities of Mn-SOD in striata from young and old rats of both sexes treated with different dosages of deprenyl. All values are expressed as percentages of respective control values. Figures on young male and young and old female rats were drawn from data previously reported by the authors (1-3). White bars indicate values in rats given 21 day s.c. infusion and shadowed bars represent values in rats given 21 day s.c. injection. *Significantly different from respective control values (P<0.05). A subsequent study in our laboratory uncovered that an increase in these enzyme activities was not specific to striatum, but was selective for certain brain regions such as s. nigra and cerebral cortices but not hippocampus, cerebellum or the liver (3). The results of the present study extend our previous observations made oil young male rats (3) that the effect is selective for specific brain regions in old male rats also. When we examined young female rats with the dosage of 2.0 mg/kg/day (s.c. injection), we encountered a 3-fold decrease (rather than an increase) ip these animals, opposite to what was observed in young male rats (i). In old female rats, the activities remained unchanged with the same dose of 2.0 mg/kg/day (i). With the speculation that an optimal dosage may differ depending on the sex and age of this species, our subsequent works cemfirmed that the optimal dosage was i0 fold lower in young female rats than males and that it increased with age in female rats (i) (see FIGs. 4-6). It has been shown that deprenyl is metabolized to demethylated deprenyl (nordeprenyl) and methamphetamine, both of which lead to the formation of amphetamine (13,14). Both amphetamine and methamphetamine are further metabolized to respective P-hydroxylated metabolites (13,14). These reactions were all shown to be mediated by the hepatic microsomal P-450 monooxygenase system, although an additional contribution of FAD containing monooxynenases has been suggested (14). It is well known that in most rat strains including F-344 (15,16, for review, see Ref. 4), lower to more than i0 fold differences exist for many enzyme activities in the hepatic microsomal monooxygenase system, with males always having higher activities than females. Higher metabolic rates in males are known to decrease drastically with age, approaching levels in young (and old) female rats at the age of 24 months
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(4,5). Thus, we predicted that if the difference in the optimal dosage in young male and female rats is due to a difference in the metabolic rates of deprenyl between livers of male and female rats, the optimal dosage in male rats may decline with age (i), since all enzyme activities in males approach levels in female rats in old age (4,5,15,16). Indeed, our prediction was proven correct in the present study, since at ]east a 4-fold decrease with age in the optimal dose of depreny! was demonstrated in old male rats. Since intermediate doses between 0.1 and 0.5 mg/kg/day were not tested, ~t is possible tilat a more precise optimal dose for old male rats is even lower tban 0.5 mg/kg/day. A factor which might work to increase the optimal dosage with age, namely an increase in ~ 0 B activity with age, may also be working in male rats as we believe worked in female rats. However, any increase in MAO B activJty in male rats does not appear to be stronger than the effect of the decrease in liver metabolic rates since the optimal dosage decreases with age in male rats. In our previous study, the optimal dosage in old females was still lower than that in young male rats (i). Thus, we were fairly confident of our prediction that in old males, the optimal dosage would be lower than in young males, even if the factor for increase in the optimal dosage (due to increasing MAO B activity with age) is taken into consideration. As is shown in the present study, our prediction was generally right but the optimal dosage in old males went dow~ to a value somewhat lower than that of old females. This difference (although minor) cannot be explained on the basis of the possible difference in metabolism of deprenyl between old male srd female rat livers, because they are practically identical for both sexes in old age (4,5,15,16). One possibility is that the second factor which increases the optimal dosage with age (i.e. ~n ~ncrease in MAO B activity with age) is weaker in males than in females. To our knowledge, however, there has been no study published with regard to sex differences in an increase of MAO B activity with age in rats. Another theoretical (but unproven) possibility is the difference in the responsiveness of old male and female rats to (-)deprenyl. in our previous study, it was noted that the responsiveness to deprenyl for increasing these ant]oxidant enzyme activities is different between young male and female rats. In males, the increase of SOD was maximally 300%, while in females it was less than 200% (FiGs. 4-6), while basal levels were fairly comparable for both sexes (i). It is possible that during aging the responsiveness to (-)deprenyl is altered, leading to some difference in optimal dosage between the two sexes. Indeed, even in the female se~, the responsiveness to deprenyl increased with age, since in old female rats, the maximal increase in Mn-SOD approached the level 3 fold the control value (FIG. 6). Furthermore, it is noteworthy that the basal SOD activity levels, (especially Mn-SOD activity) were generally higher in old male rats than young male rats. This is in accordance with our recent report (17) in the same strain of rats which showed generally higher activities of this enzyme in old males than young males, while such an age effect was not clear in female rats. Despite higher basal SOD activities, the deprenyl treatment further increased SOD activities in old male rats resulting in a much greater net increment in Mn-SOD activities, when an appropriate dose (0.5 mg/kg) was used. The physiological significance of the greater stimulation of enzyme activities in old rats of both sexes remains unknown. However, the present study clearly demonstrated that these enzyme activities can be significantly increased in old male rats as well, but with a lower dose of deprenyl than needed for young male rats. A recent study by Knoll has shown that the dose of 2.0 mg/kg/day which worked so effectively in the former Logan-Wistar derived rats of both sexes in his initial study (12) did not significantly increase SOD activities in striata of rats of another strain (18). From the results of our present and previous studies (I-3), it is conceivable that the dose of 2.0 mg/kg/day was not optimal in the strain used in Knoll's more recent study (18).
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Although the underlying mechanism(s) remains speculative, clear differences in the optimal dosage for increasing antioxidant enzyme activities depending on sexes and ages of rats as shown in our present and past studies (I-3) may raise a serious caution in future studies on the pharmacological effects of deprenyl, in particular on its life prolonging effect. Knoll reported that deprenyl, when administered at a dose of 0.25 mg/kg/day (3 times a week) after 24 months of age to a Logan-Wistar cross of rat, prolonged the remaining life expectancy by 100% compared to saline treated counterparts (12). A subsequent study from Canada using the same dosage regime on male F-344 rats has revealed that the effect of the drug in terms of life prolongation is also significant but that it quantitatively incresed lifespan by only 16% of the life expectancy after 24 months when deprenyl treatment was begun (19). In contrast to these two studies, our laboratory started to give deprenyl to F-344 male rat at a dose of 0.5 mg/kg/day at the age of 18 months of age (20). The life prolonging effect after 24 months turned out to be 34% compared to saline treated controls, its statistical significance being much higher than the Canadian study (19) on the same strain and sex (20). Our study suggests that such a dramatic effect of the drug to increase the life span of rats as reported by Knoll (12) is not always obtainable. However, our result also suggests that the life prolonging effect of the drug is a real phenomenon that was demonstrated by a higher statistical significance than that in the Canadian study (19) which was only marginally significant (P 0.048 by one-tailed ttest). Results of our present and past studies (i-3) suggest that if the effect of deprenyl for incresing SOD (and CAT) activities in certain brain regions is causally related to the reported life prolonging effect of depreny], then in future studies in this direction, the dosage to be used appears to be critically important, since the optimal dosage was shown to be so variable depending on the sex and age of rat and possibly on strain and species of animal, including humans. If a much higher dosage is used, it can easily decrease the antioxidant enzyme activities in certain brain regions (!) and may also decrease (rather than increase) the life span of the animal. Accordingly, we must b~ very cautious in our conclusions on the effects of deprenyl, keeping the facts in mind that the results may differ depending on the dosage, as well as sex, age, strain and probably species of animal used. Finally, in cur present as well as past studies, we mostlv used the 3-week treatment of s.c. continuous infusion. However, for the life span study, a s.c. injection 3 times a week has been used instead of a continuous infusion (12,17,20). Furthermore, the duration of treatment lasted for more than i year. These differences in the mode of administration may also affect the optimal dosage for increasing ant[oxidant enzyme activities. Our recent work in progress also suggests this possibility (Carri]lo et al., unpublished observation). Thus, we need to determine the precise optimal dosage of deprenyl to increase these enzyme activities by using the same way of administration as that which was used in the life span study. Studies are in progress in our laboratory to clarify these points. Acknowledgements This study was in part supported Metropolitan Institute of Gerontology. T. Ohara is gratefully acknowledged.
by the Grants in Aid from the Tokyo The skillful secretarial work by Mrs.
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52, pp.
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:7~E O P T I ~ L DOSAGE OF (-)DEPRENYL FOR INCREASING SUPEROXIDE DISMUTASE ACTIVITIES IN SEVERAL BRAIN REGIONS DECREASES WITH AGE IN MALE FISCHER 344 RATS M.C.
Carrillo 1'2, S. Kanai I, Y. Sato I, M. Nokubo I, G.O.
Ivy 3 and K. Kitani 4.
i ~Department of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology 35-2, Sakaecho, Itabashi-ku, Tokyo-173, JAPAN; Institute De Fisiologia Experimental Suipac~a 570, 2000, Rosario, Universidad Naciona] De Rosario, Republica Argentina; ~Division of Life Sciences, University of Toronto.at Scarborogh, o i~65 Military Trail, Scarborogh, Ontario, CANADA MIC IA4; 4 Radioisotope Research Institute, Faculty of Medicine, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku, Tokyo 113, JAPAN. (Received
in final form March 31, 1993) Summary
We previously reported that the optimal dosage of (-)deprenyl to increase superoxide dismutase (SOD) activities in striatum in rats differs i0 fold between young male and female rats (i). Furthermore, in female rats the optimal dosage increased with age (1). In the present study in order to clarify how the optima] dosage of this effect changes with age in male rats, we examined the effects of four different dosages of deprenyl on SOD enzyme activities in striatum and several other tissues in old (28-29-month-old) ma]e Fischer 344 (F-344) rats. Continuous s.c. infusion of deprenyl for 3 wks increased activities of SOD and cata!ase (CAT) in striatum, substantia nigra and cortical regions but not in hippocampus, cerebellum or the liver. The dose of 0.5 mg/kg/day was found to be most effective, while higher (].0, 2.0 mg/kg/day) or lower (0.1 mg/kg/day) dosages were less effective. This value of 0.5 mg/kE/day was 4 fold Iower than the dosage of 2.0 mg/kg/day which was most effective in increasing SOD and CAT activities in young (5-7 month old) ma]e rats of the same straiD (1,2). The decline of the optimal dosoge with age found in male rats is best explained by a possible dec]ine with age in the hepatic microsoma! monooxygcnase enzyme activities that are involved with the metabolism of deprenyl. In view of the large differences ~n the optimal dosages sho~1 among different sexes and ages of rats, future studies regarding the unique effect of this drug in prolonging the life span of rats must be carefully investigated with the caution in mind that the optimal dosage for the life prolonging effect may we]] differ depending on sex, age and possibly strain and species of animal model used. We have previously reported that the s.c. injection of (-)deprenyl at a dose of 2.0 mg/kg/day for 3 weeks increased activities of both Cu Zn-SOD and Mn-SOD activities in striatum 3 fold in young (5-7 month old) male F-344 rats (2). Furthermore CAT activity also increases significantly (60%) by this treatment (2). Subsequent studies in our laboratory have revealed that this effect is selective for certain brain regions such as substantia nigra (s. nigra) and cerebral cortices, but not for other regions such as hippocampus, cerebellum or *Address
for correspondence Copyright
0024-3205/93 $6.00 + .OO © 1993 Pergamon Press Ltd All rights reserved.
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the liver (3). Furthermore, when this effect was examined in female rats, the optimal dosage was found to be 10 fold lower (0.2 mg/kg/day) and the optimal dosage found in young male rats (2.0 mg/kg/day) actually decreased SOD activities 3 fold in young female rats (i). Interestingly when old female rats were examined, we found an increase in optimal dosage (i.0 mg/kg/day) in comparison to young female rats (i). As possible explanations for the observed differences in the optimal dosages, we suggested two different mechanisms (i). First, the 10-fold lower optimal dosage found in young female rats than in males may be due to the possibly lower enzyme activities of the hepatic microsomal monooxygenase system involved in the metabolism of deprenyl (for review, see 4,5). Second, the increase in the optimal dosage with age seen in female rats may be due to the increase with age (both sexes) of monoamine oxidase activities in several organs including the brain (6,7), since deprenyl is irreversibly bound to an ~0 B enzyme molecule. From these considerations, we predicted that the optimal dosage in male rats may decrease with age (i), since a general decline in the hepatic microsomal monooxygenase enzyme activities with age is known to occur in male (but not in female) rat livers (4,5). Here, we demonstrate that (-)deprenyl also increases SOD and CAT sctivities in certain selective brain regions in old male rats as in their young counterparts, however, the optimal dosage is at least 4 fold lower than in young male rsts, as previously predicted (1).
Materials and Methods Chemicals (--)Deprenyl is a generous gift from Fujimoto Pharmaceutical Company (Osaka). Hydrogen peroxide, xanthine, hydroxylammonium chloride, sulfanilic acid, ~naphthylamine, potassium cyanide, reduced glutathione (GSH) and sodium azide were obtained from Wako Pure Chemicals Ltd. (Tokyo). Xanthine oxidase, glutathione reductase and SOD were from Sigma. NADPH wss purchased from Oriental Yeast Co. Ltd. (Tokyo). Animals and tissue preparations Two cohorts of old male F-344 rats (sixteen 28-month-old and seven 29-monthold) were used in this study. The average body weight of this group as well as those used in our previous studies (i-3) on young male and young and old female rats are summarized in Table I for comparison. The first cohort of old males were 20% heavier than young males on the average, and old female rats were 30% heavier than their younger counterparts. The second cohort of old males were 10% lighter than the first cohort of animals but still significantly (but only by 10%) heavier than their young counterparts. These animals were originally
TABLE I Body weights of F-344 rsts Young rats Male rats
(n)
350.1 + 31.2 (30) (5- 7-mon th-o Id )
Old rats Cohort
1
Cohort 2
Female rats
199.6 + 9.6 (32) (5- 7-mon th-o id )
(n)
423.7 + 25.0 (16) (28-non th-o id ) 391.3 + 34.4 (7) (29-month-o Id) 268.8 + 23.8 (24) (28-mon th-o Id )
*The total number of rats studied in the previous studies
(i-3).
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purchased from Charles River Japan (Atsugi) at the age of 4 weeks and raised in the aging farm of the insitute in specific pathogen-free (SPF) condition until use. Rats were treated with continuous s.c. infusion of deprenyl (dissolved in saline) for 3 weeks at different rates (the first cohort at 0.5, 1.0 and 2.0 mg/kg/day and the second at 0.1 mg/kg/day) by using an osmotic minipump (Alzet, Alza, Palo Alto CA). Control animals of same ages were given s.c. infusion of isovolumetric physiological saline solution for the same time period. On the 22nd day, animals were sacrificed by decapitation and liver and various brain regions ~substantia nigra (s. nigra), striatum, hippocampus, cerebellum and three different cortical areas (frontal, parietotempora! and occipital)" were immediately removed and dissected on an ice cold plate. Tissue preparations for enzyme activity measurements are the same as were described in the previous papers (i-3). Enzyme assays Superoxide dismutase (SOD). The activity of SOD was assayed by the method of Elstner and Heupe] (8). Differentiation of the two different types of SOD (Cu4Zn-SOD and Mn-SOD) was performed by the addition of potassium cyanide (5 X I0 M) to the incubation medium. Cu Zu-SOD activities were defined as those inhibited by potassium cyanide. The difference between total and KCN inhibited enzs~e activities was defined as Mn-SOD activity. Catalase (CAT). Catalsse activity was assayed by the method described by Beers and Sizer (9). Details of enzy~e activity measurement procedures have been described in our previous papers (i-3). Statistical analysis. Values obtained from animals from the first cohort were analyzed by one-way analysis of varlance (ANOVA). When differences were significant with respect to deprenyl dosage, comparisons between values in control and drug--treated rats and between values in rats receiving different doses were analyzed by Scheffe's test. Results for the 2nd cohort of animals were analysed by Student t test for unpaired values. P values lower than 0.05 were judged to be significant. Results FIG. 1 summarizes activities of CAT, Cu Zn-SOD and Mn-SOD in different brain regions and tile liver in old control animals and old animals given s.c. infusion of (-)deprenyl for 21 days at a rate of 0.5 mg/kg/day which caused the greatest increase in enzyme activities among the four dosages tested (see FIGs. 2-6). Basal CAT enzyme levels in old male rats were fairly comparable to those found in young male rats as we previously reported (3). However, SOD levels, especially Mn-SOD activities were generally much greater in old male rats compared to corresponding values in young animals. For example, the basal ~ - S O D activities in striatum were 0.5 u/mg in young males (3), while they were 2.0 u/mg in old males (FIG. i). With deprenyl treatment at a dose of 0.5 mg/kg/day, all these enzyme activities significant]y increased in striatum, s. nigra and cerebral cortical regions, but not in hippocampus, cerebellum or the liver. The net increase for Mn-SOD activity by deprenyl infusion was generally much greater in old males than young males. For example the greatest net increase in M_n-SOD activity ~n striatum of young males which was achieved by the dose of 2.0 mg/kg (3) was only 1.5 u/mg, while in old rats the net increase was as high as 4 u/mg with the dose of 0.5 mg/kg (FIGs. 1,2). FIGs. 2 and 3 summarize activities of these three enzymes in striata (FIG. 2) and s. nigra (FIG. 3) from control rats and rats given different doses of (-)deprenyl. Basal enzyme levels for CAT as well as two different species of SOD were mostly comparable for the two cohorts of old male rats of different ages (28 months vs. 29 months). It is apparent that the the dose of 0.5 mg/kg/day was most effective. Higher (I.0, 2.0 mg/kg/day) or lower (0.i
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i 30 20
10 0
,Oo
12! 0
8t MnSOD
8 4
S.Nigra StriatumHippo- FrontalParieto- OccipitalCerebellum campus cx. temporal cx.
0 Liver
CX.
Enz~me activities of CAT, Cu Zn-, and ~[n-SOD in different brain regions and the liver in control old male rats (n=4, white columns) and in old rats infused with depreny] at 0.5 mg/kg/day for 2] days (n=4, shadowed columns). *Significantly dilferent from respective control values (P<0.05). mg/kg/dsy)
doses were also effective but to a much lesser degree.
FIG. 4 compares the effects of different doses of deprenyl on CAT activities in striatum in young and old F-344 rats of both sexes. FIGs 5 and 6 demonstrate activities of Cu Zn-SOD and Mn-SOD respectively. All values are expressed as percentages of respective control values. Figures on young male and young and old female rats were prepared based on data reported in our previous publications (1-3). It is clear that young male rats required the greatest dosage to achieve an optimal effect of the drug in terms of increasing CAT activity as was true with both Cu Zn- and Mn-SOD activities (]). Since a dese higher than 2.0 mg/kg/day was not examined, it is possible that a more accurate optimal dosage is even greater in young male rats. In young females, the optima] dosage was at least 10 fold lower. Age, however, increased the optimal dosage in female rats. In contrast, in old male rats, the optimal dosage was about 0.5 mg/kg/day which is 4 times lower than in young males. Interestingly, when old male and female rats were compared, the dose of 1.0 mg/kg/day was about equally as effective as 0.5 mg/kg/day in females, while in males, the higher dose (i.0 mg/kg/day) was obviously less effective than the lower dose (0.5 mg/kg/day). In this regard, it is interesting to note that the dose of 0.i mg/kg/day was as effective as
Vol. 52, No. 24, 1993
Deprenyl on Brains of Old Male Rats
Catalase
Catalase 30
20 10
1929
(3)
(4)
(4)
T
3O
20
(41
,o
"~ 10l CuZnSOD~j~
~"{ I0 i CuZnSOD
C1 C2 0.1 0.5 1.0 2.0
~
iV
Cl C2 0,1 0.5 1.0 2.0
Deprenyl rng/kg b.wt./day
Deprenyl mg/kg b.wt./day
'"IG. 2
FIG. 3
FIGs. 2 and 3 Activities of CAT, Cu Zn-SOD and Mn-SOD in str!ata (FIG. 2) and s. nfgra (FIG. 3) from control old male rats and rats treated with different dosages of deprenyl. The number in parenthesis indicates the number of rats studied. CI and C2 indicate values of two control groups, cohort one (C]) and cohort two (C2) respectively. *Significantly different from corresponding control values (P<0.05) the dose of 0.5 mg/kg/day in increasing CAT as well as Mn-SOD activities, while the same dose of 0.i ms/ks/day was clearly less effective for Cu Zn-SOD activities than the higher dose (0.5 ms/ks/day) in striatum. Dose responses in s. nigra were generally similar to those feund in striatum. Discussion Knoll, who developed deprenyl (10,11) as an antidepressant and anti-Parkinson's disease agent, is also the first to have demonstrated that this drug can increase SOD activities in striatum of young male and female rats treated with s.c. injection of deprenyl at a dose of 2.0 mg/kg/day for 21 successive days (12). He also reported that activities of CAT and glutathione peroxidase (GSH Px) were to some extent increased in this brain region by the same deprenyl treatment (12). However, these increases did not attain a statistical significance (12). Our group reexamined this question and confiz~ed that (-)deprenyl when s.c. injected for 2] successive days at a dose of 2.0 mg/kg/day, caused both types of SOD activities to be increased three fold in striata of young male F-344 rats (2). Furthermore, we showed a 60% increase in CAT activity with deprenyl treatment that was also statistically significant (2). On the other hand, GSH Px activity, which was mildly (but insignificantly) increased in Knoll's study (12), remained totally unaltered in our studies (2).
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¼
(5)
youngmale
200
old male
Vol. 52, No. 24, 1993
(4) • -!-
(5)
(4).
100
.......i 0.1
0.2
0.5
1.0
2.0
youngfemale
14).
0.1
0.2
old female
(s) F~
(5)
0.5
10
(4]
(4),
0.5
1.0
2.0
FIG. 4
(7)
10C
0.1
0.2
0.5
1.0
2.0
Oeprenyl % 300
young male
0.1
0.2
2.0
mg/kg b.wt./day
0M m~e
20C
100
0.1
0.2
0.5
1.0
2.0
0.1
02
0.5
1.0
2.0
FIG. 5 % / youngfemale
old female
/
200 ~-
•
•
100 . . . . . . . . . . . . . . . . . . . . . . .
o 0.1
0.2
0.5
1.0
2.0
Deprenyl
0.1 0.2 n~/kg b.w./day
0.5
1.0
2.0
FIGs. 4 and 5 Relative enzyme activities of CAT (FIG.4) and Cu Zn-SOD (FIG. 5) in striata from young and old rats of both sexes treated with different dosages of deprenyl. All values are expressed as percentages of respective control values. Figures on young male and young and old female rats were drawn from data we previously reported (1-3). White bars indicate values in rats given 21 day s.c. infusion and shadowed bars represent vs]ues in rats given 21 day s.c. injection. *Significantly different from respective control values (P<0.05). The numbers in parenthesis indicate the number of rats studied in each group. The numbers of control animals are 4 to 7 for each group.
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%, 30O
old male
young male
20O
100
% 300
0.1
0.2
0.5
1.0
2.0
0,1
0.2
0.5
1.0
2.0
0.5
1.0
2.0
old female
young fernale
2OO
100
0.1
0.2
0.5
1,0
2.0
Deprenyl
0.1
0.2
mg/kg b.wt,/day
FIG. 6 Re]ative enzyme activities of Mn-SOD in striata from young and old rats of both sexes treated with different dosages of deprenyl. All values are expressed as percentages of respective control values. Figures on young male and young and old female rats were drawn from data previously reported by the authors (1-3). White bars indicate values in rats given 21 day s.c. infusion and shadowed bars represent values in rats given 21 day s.c. injection. *Significantly different from respective control values (P<0.05). A subsequent study in our laboratory uncovered that an increase in these enzyme activities was not specific to striatum, but was selective for certain brain regions such as s. nigra and cerebral cortices but not hippocampus, cerebellum or the liver (3). The results of the present study extend our previous observations made oil young male rats (3) that the effect is selective for specific brain regions in old male rats also. When we examined young female rats with the dosage of 2.0 mg/kg/day (s.c. injection), we encountered a 3-fold decrease (rather than an increase) ip these animals, opposite to what was observed in young male rats (i). In old female rats, the activities remained unchanged with the same dose of 2.0 mg/kg/day (i). With the speculation that an optimal dosage may differ depending on the sex and age of this species, our subsequent works cemfirmed that the optimal dosage was i0 fold lower in young female rats than males and that it increased with age in female rats (i) (see FIGs. 4-6). It has been shown that deprenyl is metabolized to demethylated deprenyl (nordeprenyl) and methamphetamine, both of which lead to the formation of amphetamine (13,14). Both amphetamine and methamphetamine are further metabolized to respective P-hydroxylated metabolites (13,14). These reactions were all shown to be mediated by the hepatic microsomal P-450 monooxygenase system, although an additional contribution of FAD containing monooxynenases has been suggested (14). It is well known that in most rat strains including F-344 (15,16, for review, see Ref. 4), lower to more than i0 fold differences exist for many enzyme activities in the hepatic microsomal monooxygenase system, with males always having higher activities than females. Higher metabolic rates in males are known to decrease drastically with age, approaching levels in young (and old) female rats at the age of 24 months
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(4,5). Thus, we predicted that if the difference in the optimal dosage in young male and female rats is due to a difference in the metabolic rates of deprenyl between livers of male and female rats, the optimal dosage in male rats may decline with age (i), since all enzyme activities in males approach levels in female rats in old age (4,5,15,16). Indeed, our prediction was proven correct in the present study, since at ]east a 4-fold decrease with age in the optimal dose of depreny! was demonstrated in old male rats. Since intermediate doses between 0.1 and 0.5 mg/kg/day were not tested, ~t is possible tilat a more precise optimal dose for old male rats is even lower tban 0.5 mg/kg/day. A factor which might work to increase the optimal dosage with age, namely an increase in ~ 0 B activity with age, may also be working in male rats as we believe worked in female rats. However, any increase in MAO B activJty in male rats does not appear to be stronger than the effect of the decrease in liver metabolic rates since the optimal dosage decreases with age in male rats. In our previous study, the optimal dosage in old females was still lower than that in young male rats (i). Thus, we were fairly confident of our prediction that in old males, the optimal dosage would be lower than in young males, even if the factor for increase in the optimal dosage (due to increasing MAO B activity with age) is taken into consideration. As is shown in the present study, our prediction was generally right but the optimal dosage in old males went dow~ to a value somewhat lower than that of old females. This difference (although minor) cannot be explained on the basis of the possible difference in metabolism of deprenyl between old male srd female rat livers, because they are practically identical for both sexes in old age (4,5,15,16). One possibility is that the second factor which increases the optimal dosage with age (i.e. ~n ~ncrease in MAO B activity with age) is weaker in males than in females. To our knowledge, however, there has been no study published with regard to sex differences in an increase of MAO B activity with age in rats. Another theoretical (but unproven) possibility is the difference in the responsiveness of old male and female rats to (-)deprenyl. in our previous study, it was noted that the responsiveness to deprenyl for increasing these ant]oxidant enzyme activities is different between young male and female rats. In males, the increase of SOD was maximally 300%, while in females it was less than 200% (FiGs. 4-6), while basal levels were fairly comparable for both sexes (i). It is possible that during aging the responsiveness to (-)deprenyl is altered, leading to some difference in optimal dosage between the two sexes. Indeed, even in the female se~, the responsiveness to deprenyl increased with age, since in old female rats, the maximal increase in Mn-SOD approached the level 3 fold the control value (FIG. 6). Furthermore, it is noteworthy that the basal SOD activity levels, (especially Mn-SOD activity) were generally higher in old male rats than young male rats. This is in accordance with our recent report (17) in the same strain of rats which showed generally higher activities of this enzyme in old males than young males, while such an age effect was not clear in female rats. Despite higher basal SOD activities, the deprenyl treatment further increased SOD activities in old male rats resulting in a much greater net increment in Mn-SOD activities, when an appropriate dose (0.5 mg/kg) was used. The physiological significance of the greater stimulation of enzyme activities in old rats of both sexes remains unknown. However, the present study clearly demonstrated that these enzyme activities can be significantly increased in old male rats as well, but with a lower dose of deprenyl than needed for young male rats. A recent study by Knoll has shown that the dose of 2.0 mg/kg/day which worked so effectively in the former Logan-Wistar derived rats of both sexes in his initial study (12) did not significantly increase SOD activities in striata of rats of another strain (18). From the results of our present and previous studies (I-3), it is conceivable that the dose of 2.0 mg/kg/day was not optimal in the strain used in Knoll's more recent study (18).
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Although the underlying mechanism(s) remains speculative, clear differences in the optimal dosage for increasing antioxidant enzyme activities depending on sexes and ages of rats as shown in our present and past studies (I-3) may raise a serious caution in future studies on the pharmacological effects of deprenyl, in particular on its life prolonging effect. Knoll reported that deprenyl, when administered at a dose of 0.25 mg/kg/day (3 times a week) after 24 months of age to a Logan-Wistar cross of rat, prolonged the remaining life expectancy by 100% compared to saline treated counterparts (12). A subsequent study from Canada using the same dosage regime on male F-344 rats has revealed that the effect of the drug in terms of life prolongation is also significant but that it quantitatively incresed lifespan by only 16% of the life expectancy after 24 months when deprenyl treatment was begun (19). In contrast to these two studies, our laboratory started to give deprenyl to F-344 male rat at a dose of 0.5 mg/kg/day at the age of 18 months of age (20). The life prolonging effect after 24 months turned out to be 34% compared to saline treated controls, its statistical significance being much higher than the Canadian study (19) on the same strain and sex (20). Our study suggests that such a dramatic effect of the drug to increase the life span of rats as reported by Knoll (12) is not always obtainable. However, our result also suggests that the life prolonging effect of the drug is a real phenomenon that was demonstrated by a higher statistical significance than that in the Canadian study (19) which was only marginally significant (P 0.048 by one-tailed ttest). Results of our present and past studies (i-3) suggest that if the effect of deprenyl for incresing SOD (and CAT) activities in certain brain regions is causally related to the reported life prolonging effect of depreny], then in future studies in this direction, the dosage to be used appears to be critically important, since the optimal dosage was shown to be so variable depending on the sex and age of rat and possibly on strain and species of animal, including humans. If a much higher dosage is used, it can easily decrease the antioxidant enzyme activities in certain brain regions (!) and may also decrease (rather than increase) the life span of the animal. Accordingly, we must b~ very cautious in our conclusions on the effects of deprenyl, keeping the facts in mind that the results may differ depending on the dosage, as well as sex, age, strain and probably species of animal used. Finally, in cur present as well as past studies, we mostlv used the 3-week treatment of s.c. continuous infusion. However, for the life span study, a s.c. injection 3 times a week has been used instead of a continuous infusion (12,17,20). Furthermore, the duration of treatment lasted for more than i year. These differences in the mode of administration may also affect the optimal dosage for increasing ant[oxidant enzyme activities. Our recent work in progress also suggests this possibility (Carri]lo et al., unpublished observation). Thus, we need to determine the precise optimal dosage of deprenyl to increase these enzyme activities by using the same way of administration as that which was used in the life span study. Studies are in progress in our laboratory to clarify these points. Acknowledgements This study was in part supported Metropolitan Institute of Gerontology. T. Ohara is gratefully acknowledged.
by the Grants in Aid from the Tokyo The skillful secretarial work by Mrs.
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