Exp. Geront. Vol. 10. pp. 185-188.PergamonPress 1975.Printed in Great Britain.
EFFECT PREDNISOLONE
OF B-AMINOPROPIONITRILE ON
SURVIVAL
OF MALE
OR LAF/J
MICE*
FRANK S. LABELLA,t a n d STANLEY VIVIAN Department of Pharmacology and Therapeutics, University of Manitoba, Faculty of Medicine, Winnipeg, Manitoba, Canada (Received 26 December 1974)
Abstract--Authors examined the effects of B-aminopropionitrile (BAPN) and prednisolone, added to the drinking water at 1 mg/ml and 21xg/mi, respectively, on survival of 2-month old male LAF/J mice. In one experiment BAPN, prednisolone, or both were administered throughout the entire lifespan, with no significant differences from control in either mean lifespan, 28-29 months, or maximum lifespan, 33-35 months. In a second experiment, BAPN was administered for 0, 3, 6, 9 or 12 months only. Median lifespan of 26 months for control animals was significantly increased for 3 of the 4 test groups, the animals treated with BAPN for 12 months showing the greatest increase, i.e. median 31 months. Maximum lifespan of control animals was 33 months, and was 1-3 months longer for BAPN treated groups.
INTRODUCTION IN A previous study (LaBella, 1972) a modest increase in survival of rats was achieved by chronic administration of lathyrogenic agents. The beneficial effect of B-aminopropionitrile (BAPN) or semicarbazide added to the diet was evident in animals aged 2-15 months at the start of the experimental period. Survival curves of the albino rats used in that study were quite steep throughout the lifespan, indicating the presence of morbid processes resulting in accelerated mortality. Furthermore, control animals showed maximum lifespans in several experiments of only 20--30 months, and unaltered by the lathyrogens. In this report are presented results of BAPN administration to mice which proved to be more vigorous than rats and gave survival curves approximating the ideal for a population experiencing senescence. In this same series of experiments, groups of mice were treated with prednisolone, in an attempt to confirm, in the relatively long-lived LAF/J mice, the report of Bellamy (1968) of a marked increase in survival and longevity resulting from administration of the drug to a strain of mice with a maximum lifespan of about one year, METHODS AND MATERIALS Male LAF/J mice, strain 0305, were obtained at approximately six weeks of age from Jackson Labs., Bar Harbor, Maine. Ten to twenty mice, constant for a given experiment, were placed in clear plastic cages with sliding stainless steel tops. The mice were provided with a standard pelleted diet and tap water, both supplied ad libitum. After 2-3 weeks mice were randomly assigned to control or various test groups. Prednisolone phosphate (Sigma Chemical Co.) and BAPN fumarate (Aldrich Chemical Co.) were added to the drinking water at a level of 2 ~tg/ml or 1 mg/ml, respectively. Stock solutions of the drugs were made up fresh each week, kept refrigerated, and diluted with fresh drinking water each day. In Experiment I prednisolon¢ was added to the drinking water and BAPN to the powdered diet (0.10 per cent) for the first year, and both to the drinking water thereafter. The drug was started at 2 months and administered throughout life. There were 50 mice in the control group and 51 in each of the test groups, i.¢. those receiving BAPN or prednisolone. In Experiment II, only BAPN was added to the drinking water, again starting at age 2 months, but administered only for 3, 6, 9 or 12 months. *Supported by the Manitoba Heart Foundation and the Medical Research Council of Canada. 1"Associate of the Medical Research Council. 185
186
F R A N K S. LABELLA
and
STANLEY VIVIAN
The data (number of months survived) was subjected to an analysis of variance (completely random design) (Steel and Torrie, 1960) and by multiple Kruskal-Wallis Tests (Sokal and Pohlf, 1960). A preliminary test for homogeneity of variances by Bartlett's test (Steel and Torrie, 1960) indicated some heterogeneity in Experiment I, apparently resulting from the premature death of one prednisolone treated mouse which died at age 8.6 months. This observation was considered a non-representative outlying value and was discarded. The resulting variances were stable to a retest of variance homogeneity. All mice survived the first year in Experiment II, and variances were homogeneous. Duncan's new-multiple-range test (Steel and Torrie, 1960) was used to compare means of test groups with that of the control group. Multiple Kruskal-Wallis tests were also carried out between treated and untreated groups as an appropriate test of significance relating to the median lifespan as this parameter is frequently expressed in studies on aging populations. RESULTS
AND
DISCUSSION
In Experiment I there were no significant differences from control in mean lifespan for any of the drug-treated groups (Fig. 1, Table 1). These results certainly do not approach the dramatic findings of Bellamy (1968) with prednisolone administered to his short-lived
survivors (?o) 100-
~,., ~ •
• CONTROL ~'~ ~ ~' ~,i t
80-
o
PRED
•
BAPN+PRED
60-
40 -
20-
.o:,o
2'0
2'5
3'0
3f5
4'0
AGE (months) FIG. 1. Effect of BAPN, prednisolone or both on survival of male LAF/J mice. BAPN was added to the powdered diet of 0.10 per cent by weight of the first year only and to the drinking water thereafter; BAPN: 1 mg/ml; prednisolone, 2/ag/ml. Drugs were administered to the mice at age 2 months and continued throughout life. TABLE 1. EEEECTor BAPN ANWORPREDNISOLONEON SURVIVAL NO.
of
Survival Mean
(~lonths)
Treatment
Mice
Control
50
BAPN
51
28.49
~ 0.49
ns T
28.20
ns
34
Prednisolone
5~ W
27.91
~ 0.49
ns
28.20
ns
34
BAPN + P r e d .
49
27.90
~ 0.49
ns
27.90
ns
33
28.96
Medinn
~ 0.49
Maximum
29.14
35
* One mouse failed to survive the first year and was not included in this group for the parametric (mean) test due to variance heterogeneity. "~ns: non-significant (P > 0"05) by Duncan's new-multiple-range test on the means, or by multiple Kruskal.-Wallis tests on the medians.
EFFECT OF B-AMINOPROPIONITRILE
187
OR PREDNISOLONE
mice. Perhaps, the dosage of prednisolone required to yield comparable effects with our LAF/J mice is greater than that for Bellamy's mice. No systematic measurement of body weight was carded out in our study; however, on a few occasions when mice were weighed, the steroid-treated animals were 10--15 per cent lighter than controls. The lower weights o f the prednisolone treated animals suggest that the steroid was present in pharmacologically active levels, since the adrenocortical hormones are known to be catabolic. In Experiment II, BAPN was administered to mice at 2 months of age and for periods of only 3-12 months. This design was chosen because, in our earlier work with rats, there was an indication that chronic toxicity with the drug was manifested later in life and counteracted the beneficial effects apparently afforded earlier by the agent. It can be seen in Fig. 2 and Table 2 that there is an increase both in survival and maximum lifespan for all experimental groups, irrespective of the duration of BAPN administration. The beneficial effect on survival was significant for three of the four drug-treated groups. Median survival was greatest for the mice treated for 12 months and was about 20 per cent greater than SU
(re)
V,VORS 100"
c ~
•
•
,-
~a
•
Q CONTROL ~ ~
80.
[]
•.~: ~
~
3
•
6
Months
~9 c
o~-
G
o~, •
D
12
60-
40-
.,r,
e~
o:n
oA~r: $ ll(~j
20-
•/¢~ • • u ,~
2'0
A
AGE
a'o
t
A
,'o
(months)
Fie. 2. Effect of BAPN on survival of male LAF/J mice. The drug was added to the drinking water (1 mg/ml) at age 2 months, and groups of mice were treated for 3, 6, 9 or 12 months. TABLE 2.
EFFECT OF THE DURATION
No. of months treated with BAPN
OF
BAPN
TREATMENT ON SURVIVAL
Survlval (months) No,
Mice
Of Median
M e a n . + S, E,
0
25
27,14
~ 0.66 +
3
24
29,02
~ 0.67
ns
Maximum
26.4
33
28.7*
36
6
25
28,71. ~ 0,66 ns
27,9 ns
35
9
26
29°75
~ 0.65*
30.7
*
35
12
26
30.60
~ 0,65
30.9
~
34
~
Comparison of means with that of the control group by Duncan's new-multiple-range test or of medians by multiple Kruskal-Walli8 tests. * P < 0.05. ~ P < 0-01. ns P > 0-05. +One mouse failed to survive the first year and was not included in this group for the parametric (mean) test due to variance heterogeneity.
FRANK S. LABELLA and STANLEY VIVIAN
188
that of controls. Maximum lifespan of mice in the drug-treated groups was 1-3 months greater than the 33 months of the control. Thus, animals kept on the BAPN for the longest period seemed to be protected most. The beneficial effect of the drug would not appear to be due to diminished caloric intake, since there were no marked differences among groups in body weights (Table 3). On the TABLE 3. EFFECT OF B A P N a t age 10 months
ON BODY WEIGHT a t age 15 months
% ol Group*
wt,
?; of
control
wt.
control
I
31.7
10O
33.9
i00
2
28.6
90
33.5
99
3
29,6
93
32.5
96
4
28,2
89
33.0
97
5
30,2
95
31.5
93
*Groups 1-5 represent mice which were given, starting at 2 months of age, BAPN for 0, 3, 6, 9 or 12 months, r-espectivel-y.Weights shown represent average body weights, since all mice in a group were weighed together.
basis of these encouraging results, we have initiated another study in which BAPN administration is being continued for longer than 12 months and with higher doses of the drug. In order to determine the possible confounding influence of food intake on survival, pair feeding will be carried out in future studies. Our studies on BAPN in relation to its effect on longevity of rodents were initiated at a time when the chemical nature of the lysine-derived crosslinks in elastin and collagen was unknown. The rationale for instituting lathyrogen treatment as a possible inhibitor of the aging process was based on the hypothesis that the drug might delay age-related and deleterious connective tissue crosslinking processes in the post-maturation phase. There is no evidence to suggest that the crosslinks inhibited by BAPN in the developing individual are, in fact, of the same type presumed to increase in the fibrous proteins with age (see reviews, LaBella, 1971, 1972). The basis for observed beneficial effects of lathyrogens on survival is yet to be identified. We have previously suggested that potential pharmacological control of the aging process would probably entail a variety of specifically tailored drugs administered at specific life phases (LaBella, 1972). Our current results, indeed, suggest that life-long treatment with BAPN has no salutary influence on survival of mice, in contrast to those animals receiving the drug during an early portion of the lifespan. REFERENCES BELLAMY,D. (1968) Exp. Geront. 3, 327. LABELLA,F. S. (1971) In: Biophysical Properties of the Skin (ELDEN, H., ed.), p. 243, Wiley, New York. LABELLA,F. S. (1972) In: Search for New Drugs (RuB~, A., ed.), p. 347, Dekker, New York. SOKAL, R. R. and ROI-IOLF,F. J. (1960) Biometry: The Principles and Practice of Statistics in Biological Research. Freeman, San Francisco. STEEL, R. G. D. and TORRIE,J. H. (1960) Principles and Procedures of Statistics. McGraw-Hill, New York.
EFFECT PREDNISOLONE
OF B-AMINOPROPIONITRILE ON
SURVIVAL
OF MALE
OR LAF/J
MICE*
FRANK S. LABELLA,t a n d STANLEY VIVIAN Department of Pharmacology and Therapeutics, University of Manitoba, Faculty of Medicine, Winnipeg, Manitoba, Canada (Received 26 December 1974)
Abstract--Authors examined the effects of B-aminopropionitrile (BAPN) and prednisolone, added to the drinking water at 1 mg/ml and 21xg/mi, respectively, on survival of 2-month old male LAF/J mice. In one experiment BAPN, prednisolone, or both were administered throughout the entire lifespan, with no significant differences from control in either mean lifespan, 28-29 months, or maximum lifespan, 33-35 months. In a second experiment, BAPN was administered for 0, 3, 6, 9 or 12 months only. Median lifespan of 26 months for control animals was significantly increased for 3 of the 4 test groups, the animals treated with BAPN for 12 months showing the greatest increase, i.e. median 31 months. Maximum lifespan of control animals was 33 months, and was 1-3 months longer for BAPN treated groups.
INTRODUCTION IN A previous study (LaBella, 1972) a modest increase in survival of rats was achieved by chronic administration of lathyrogenic agents. The beneficial effect of B-aminopropionitrile (BAPN) or semicarbazide added to the diet was evident in animals aged 2-15 months at the start of the experimental period. Survival curves of the albino rats used in that study were quite steep throughout the lifespan, indicating the presence of morbid processes resulting in accelerated mortality. Furthermore, control animals showed maximum lifespans in several experiments of only 20--30 months, and unaltered by the lathyrogens. In this report are presented results of BAPN administration to mice which proved to be more vigorous than rats and gave survival curves approximating the ideal for a population experiencing senescence. In this same series of experiments, groups of mice were treated with prednisolone, in an attempt to confirm, in the relatively long-lived LAF/J mice, the report of Bellamy (1968) of a marked increase in survival and longevity resulting from administration of the drug to a strain of mice with a maximum lifespan of about one year, METHODS AND MATERIALS Male LAF/J mice, strain 0305, were obtained at approximately six weeks of age from Jackson Labs., Bar Harbor, Maine. Ten to twenty mice, constant for a given experiment, were placed in clear plastic cages with sliding stainless steel tops. The mice were provided with a standard pelleted diet and tap water, both supplied ad libitum. After 2-3 weeks mice were randomly assigned to control or various test groups. Prednisolone phosphate (Sigma Chemical Co.) and BAPN fumarate (Aldrich Chemical Co.) were added to the drinking water at a level of 2 ~tg/ml or 1 mg/ml, respectively. Stock solutions of the drugs were made up fresh each week, kept refrigerated, and diluted with fresh drinking water each day. In Experiment I prednisolon¢ was added to the drinking water and BAPN to the powdered diet (0.10 per cent) for the first year, and both to the drinking water thereafter. The drug was started at 2 months and administered throughout life. There were 50 mice in the control group and 51 in each of the test groups, i.¢. those receiving BAPN or prednisolone. In Experiment II, only BAPN was added to the drinking water, again starting at age 2 months, but administered only for 3, 6, 9 or 12 months. *Supported by the Manitoba Heart Foundation and the Medical Research Council of Canada. 1"Associate of the Medical Research Council. 185
186
F R A N K S. LABELLA
and
STANLEY VIVIAN
The data (number of months survived) was subjected to an analysis of variance (completely random design) (Steel and Torrie, 1960) and by multiple Kruskal-Wallis Tests (Sokal and Pohlf, 1960). A preliminary test for homogeneity of variances by Bartlett's test (Steel and Torrie, 1960) indicated some heterogeneity in Experiment I, apparently resulting from the premature death of one prednisolone treated mouse which died at age 8.6 months. This observation was considered a non-representative outlying value and was discarded. The resulting variances were stable to a retest of variance homogeneity. All mice survived the first year in Experiment II, and variances were homogeneous. Duncan's new-multiple-range test (Steel and Torrie, 1960) was used to compare means of test groups with that of the control group. Multiple Kruskal-Wallis tests were also carried out between treated and untreated groups as an appropriate test of significance relating to the median lifespan as this parameter is frequently expressed in studies on aging populations. RESULTS
AND
DISCUSSION
In Experiment I there were no significant differences from control in mean lifespan for any of the drug-treated groups (Fig. 1, Table 1). These results certainly do not approach the dramatic findings of Bellamy (1968) with prednisolone administered to his short-lived
survivors (?o) 100-
~,., ~ •
• CONTROL ~'~ ~ ~' ~,i t
80-
o
PRED
•
BAPN+PRED
60-
40 -
20-
.o:,o
2'0
2'5
3'0
3f5
4'0
AGE (months) FIG. 1. Effect of BAPN, prednisolone or both on survival of male LAF/J mice. BAPN was added to the powdered diet of 0.10 per cent by weight of the first year only and to the drinking water thereafter; BAPN: 1 mg/ml; prednisolone, 2/ag/ml. Drugs were administered to the mice at age 2 months and continued throughout life. TABLE 1. EEEECTor BAPN ANWORPREDNISOLONEON SURVIVAL NO.
of
Survival Mean
(~lonths)
Treatment
Mice
Control
50
BAPN
51
28.49
~ 0.49
ns T
28.20
ns
34
Prednisolone
5~ W
27.91
~ 0.49
ns
28.20
ns
34
BAPN + P r e d .
49
27.90
~ 0.49
ns
27.90
ns
33
28.96
Medinn
~ 0.49
Maximum
29.14
35
* One mouse failed to survive the first year and was not included in this group for the parametric (mean) test due to variance heterogeneity. "~ns: non-significant (P > 0"05) by Duncan's new-multiple-range test on the means, or by multiple Kruskal.-Wallis tests on the medians.
EFFECT OF B-AMINOPROPIONITRILE
187
OR PREDNISOLONE
mice. Perhaps, the dosage of prednisolone required to yield comparable effects with our LAF/J mice is greater than that for Bellamy's mice. No systematic measurement of body weight was carded out in our study; however, on a few occasions when mice were weighed, the steroid-treated animals were 10--15 per cent lighter than controls. The lower weights o f the prednisolone treated animals suggest that the steroid was present in pharmacologically active levels, since the adrenocortical hormones are known to be catabolic. In Experiment II, BAPN was administered to mice at 2 months of age and for periods of only 3-12 months. This design was chosen because, in our earlier work with rats, there was an indication that chronic toxicity with the drug was manifested later in life and counteracted the beneficial effects apparently afforded earlier by the agent. It can be seen in Fig. 2 and Table 2 that there is an increase both in survival and maximum lifespan for all experimental groups, irrespective of the duration of BAPN administration. The beneficial effect on survival was significant for three of the four drug-treated groups. Median survival was greatest for the mice treated for 12 months and was about 20 per cent greater than SU
(re)
V,VORS 100"
c ~
•
•
,-
~a
•
Q CONTROL ~ ~
80.
[]
•.~: ~
~
3
•
6
Months
~9 c
o~-
G
o~, •
D
12
60-
40-
.,r,
e~
o:n
oA~r: $ ll(~j
20-
•/¢~ • • u ,~
2'0
A
AGE
a'o
t
A
,'o
(months)
Fie. 2. Effect of BAPN on survival of male LAF/J mice. The drug was added to the drinking water (1 mg/ml) at age 2 months, and groups of mice were treated for 3, 6, 9 or 12 months. TABLE 2.
EFFECT OF THE DURATION
No. of months treated with BAPN
OF
BAPN
TREATMENT ON SURVIVAL
Survlval (months) No,
Mice
Of Median
M e a n . + S, E,
0
25
27,14
~ 0.66 +
3
24
29,02
~ 0.67
ns
Maximum
26.4
33
28.7*
36
6
25
28,71. ~ 0,66 ns
27,9 ns
35
9
26
29°75
~ 0.65*
30.7
*
35
12
26
30.60
~ 0,65
30.9
~
34
~
Comparison of means with that of the control group by Duncan's new-multiple-range test or of medians by multiple Kruskal-Walli8 tests. * P < 0.05. ~ P < 0-01. ns P > 0-05. +One mouse failed to survive the first year and was not included in this group for the parametric (mean) test due to variance heterogeneity.
FRANK S. LABELLA and STANLEY VIVIAN
188
that of controls. Maximum lifespan of mice in the drug-treated groups was 1-3 months greater than the 33 months of the control. Thus, animals kept on the BAPN for the longest period seemed to be protected most. The beneficial effect of the drug would not appear to be due to diminished caloric intake, since there were no marked differences among groups in body weights (Table 3). On the TABLE 3. EFFECT OF B A P N a t age 10 months
ON BODY WEIGHT a t age 15 months
% ol Group*
wt,
?; of
control
wt.
control
I
31.7
10O
33.9
i00
2
28.6
90
33.5
99
3
29,6
93
32.5
96
4
28,2
89
33.0
97
5
30,2
95
31.5
93
*Groups 1-5 represent mice which were given, starting at 2 months of age, BAPN for 0, 3, 6, 9 or 12 months, r-espectivel-y.Weights shown represent average body weights, since all mice in a group were weighed together.
basis of these encouraging results, we have initiated another study in which BAPN administration is being continued for longer than 12 months and with higher doses of the drug. In order to determine the possible confounding influence of food intake on survival, pair feeding will be carried out in future studies. Our studies on BAPN in relation to its effect on longevity of rodents were initiated at a time when the chemical nature of the lysine-derived crosslinks in elastin and collagen was unknown. The rationale for instituting lathyrogen treatment as a possible inhibitor of the aging process was based on the hypothesis that the drug might delay age-related and deleterious connective tissue crosslinking processes in the post-maturation phase. There is no evidence to suggest that the crosslinks inhibited by BAPN in the developing individual are, in fact, of the same type presumed to increase in the fibrous proteins with age (see reviews, LaBella, 1971, 1972). The basis for observed beneficial effects of lathyrogens on survival is yet to be identified. We have previously suggested that potential pharmacological control of the aging process would probably entail a variety of specifically tailored drugs administered at specific life phases (LaBella, 1972). Our current results, indeed, suggest that life-long treatment with BAPN has no salutary influence on survival of mice, in contrast to those animals receiving the drug during an early portion of the lifespan. REFERENCES BELLAMY,D. (1968) Exp. Geront. 3, 327. LABELLA,F. S. (1971) In: Biophysical Properties of the Skin (ELDEN, H., ed.), p. 243, Wiley, New York. LABELLA,F. S. (1972) In: Search for New Drugs (RuB~, A., ed.), p. 347, Dekker, New York. SOKAL, R. R. and ROI-IOLF,F. J. (1960) Biometry: The Principles and Practice of Statistics in Biological Research. Freeman, San Francisco. STEEL, R. G. D. and TORRIE,J. H. (1960) Principles and Procedures of Statistics. McGraw-Hill, New York.
