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Effect of anabolic steroids on behavior and physiological characteristics of female mice
Physiology & Behavior, Vol. 59, No. 1, pp. 49-55, 1996 Copyright © 1995 Elsevier Science Inc. Printed in the USA. All rights reserved 0031-9384/96 $15.00 + .00
ELSEVIER
0031-9384(95)02027-S
Effect of Anabolic Steroids on Behavior and Physiological Characteristics of Female Mice F. H. BRONSON, 1 K. Q. N G U Y E N AND J. DE LA ROSA
Institute of Reproductive Biology, Department of Zoology, University of Texas, Austin, TX 78712 USA Received 14 October 1994 BRONSON, F. H., K. Q. NGUYEN AND J. DE LA ROSA. Effect of anabolic steroids on behavior and physiological characteristics of female mice. PHYSIOL BEHAV 59(1) 49-55, 1996.--Adult female mice were exposed to a combination of four anabolic-androgenic steroids for 9 weeks at doses that were either one or five times the androgenic maintenance level for male mice. Relative to control females, steroid treatment depressed gonadotropin secretion and increased both dry body weight and fat content but without an increase in food intake. Steroid treatment depressed spontaneous use of a running wheel and open-field activity, and it increased aggressiveness. It also eliminated a behavior related to encounters between the sexes--the rejection of genital inspection. There was no effect of steroid treatment on the time required to recover from 10 h of enforced running on a treadmill. Overall, regardless of the test or measure, there was little or no difference in the effect of the high and low dose of steroids. This indicates a threshold of response below the low dose used in these studies, which itself is probably well below that used by many female athletes and body builders. Anabolic steroids Locomotor activity
Body composition Mice
Open field behavior
Aggressiveness
to have broad effects on behavior, much of the interest has been limited to potential effects on aggressiveness where most, but not all, of the available data suggest a marked increase due to steroid use, at least in males [e.g., 11,21,24,30); cf., (3,28)]. Unfortunately, in most cases the available evidence here is correlational and quite a bit is anecdotal and self-reported. Almost nothing is known about the behavioral side effects of steroid abuse in females, but certainly the potential for an increase in aggressiveness due to steroids exists in both sexes (1,25). The objective of the present study was to assess the effect of anabolic steroids on a variety of physical and behavioral characteristics in female laboratory mice using the numbers, kinds, and relative doses of steroids commonly used by female athletes and body builders. Specifically, adult female mice were exposed to a combination of four commonly abused anabolic steroids for 9 weeks at doses that were either one or five times the maintenance level for male mice. During the last 3 weeks of this period, the females were given a series of tests to assess their level of spontaneous activity, recovery time after forced running on a treadmill, response to an open field, aggressiveness, and food intake. Afterwards, the females were autopsied, body composition was assessed, and a variety of reproductive-related measures were obtained. The results of this experiment suggest broad
ANABOLIC-ANDROGENIC steroids such as testosterone and its many synthetic analogs can promote muscle-building processes while reducing muscle catabolism (16). Thus, in spite of a ban imposed by many administrative entities, these hormones are widely used by male athletes and body builders in an effort to improve their performance or appearance (17,20,31). Of concern here is the fact that many female body builders and athletes apparently also use anabolic-androgenic steroids (23,31), occasionally even during adolescence (8,10,26). Males often take as many as five different steroids simultaneously at doses that can total 10 to 40 times normal maintenance levels (17,29). They usually do this in cycles, starting weeks or months before a competition, after which they initiate a wash-out period of weeks or months. Little is known about the doses or schedules used by females except that, like males, women have been known to take as many as five steroids simultaneously at levels that are several times those recommended by manufacturers, and a degree of masculinization of secondary sexual characteristics is not uncommon in this situation (23). Supraphysiological doses of anabolic steroids can have a variety of secondary effects, some of which are decidedly pathologic (17). Of particular relevance are the potential effects of these steroids on behavior. While the anabolic steroids are thought
1 To whom requests for reprints should be addressed.
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BRONSON, NGUYEN AND DE LA ROSA
effects of anabolic steroids on the physiological and behavioral characteristics of female mice. METHOD
Animals and Experimental Design CF-1 female mice were purchased from Charles River at 7 weeks of age. Upon arrival they were housed one per 29 x 18 x 12 cm polyethylene cage and fed PMI Formulab 5008 chow. Throughout this experiment these animals were maintained on a 14 L:10 D cycle with lights off at 1800 h and on at 0400 h. At 8 weeks of age the females were organized into a 1 × 3 experimental design with 20 females per treatment. One treatment group was given a low dose of a combination of four anabolic steroids. A second group was given a high dose of the same combination of four steroids, and a control group received no exogenous hormones. The hormones administered were testosterone, testosterone cypionate, methyltestosterone, and norethandrolone. The latter three hormones span the classes of testosterone analogues proposed by Wilson (29). The doses used were defined on the basis of their androgenic potency rather than their anabolic potency, which may not be identical when pharmacological levels are given (15). The low dose was a combination of the four hormones that would maintain normal-sized seminal vesicles in a castrated male CF-1 mouse and the high dose was five times the low dose.
Steroid Administration Each hormone was administered in its own Silastic capsule, and the 20 females in the control group were given a set of four empty capsules. Capsule sizes were chosen with two objectives in mind: to ensure that each of the four steroids of concern contributed one-fourth of the total androgenicity at both dose levels and, second, to keep the length of the capsule within the range of comfort for a mouse. To obtain those objectives it was necessary to vary the diameter of the capsules as well as their length. A series of pilot studies, all based on the effect of a steroid on the weight of the seminal vesicles of castrated CF-1 males, yielded the following choices. The low dose combined: 2.5 mm of a 0.062" X0.125" capsule containing testosterone; 5 mm of a 0.062" × 0.125" capsule of testosterone cypionate; 2.5 mm of a 0.025" X 0.047" capsule of methyltestosterone and 7.5 mm of a 0.062" X 0.125" capsule of norethandrolone. The fivefold higher dose combined: 12.5 mm of a 0.062" x 0.125" capsule containing testosterone; 5 mm of a 0.078" X 0.125" capsule of testosterone cypionate; 12.5 mm of a 0.025"X 0.047" capsule of methyltestosterone; and 7.5 mm of a 0.078"X 0.125" capsule of norethandrolone.
Testing Four tests were administered over a 20-day period. The 20 females in each of the three treatment groups were divided into two subgroups of 10 each. The individuals in one subgroup of each treatment were given the spontaneous activity/treadmill recovery test. The individuals in the other subgroup of each treatment were subjected to two tests: first, their food intake was measured, and this was followed in a few days by an assessment of their reactions to an open field. The subgroups were then recombined and all 20 females in each treatment group participated in the aggression tests. All testing was accomplished without knowledge of the experimental group. Stage of the estrous cycle was deemed a potentially important variable in most of these tests. Thus, vaginal smears were
obtained daily throughout the 20-day test period. As will be detailed later, the control females cycled normally but cyclicity was completely suppressed in all females given steroids. Because the cycles of the control females were asynchronous, and because we routinely tested females only when in metestrus or diestrus, the administration of a test to a group of individuals extended over a period of several days. To keep timing constant for the three experimental groups, the testing of the androgenized females extended over the same amount of time as the control females, with approximately equal numbers of each treatment being tested each day.
Spontaneous Activity / Treadmill Recovery Test The object of this test was twofold: first, to assess the effect of anabolic steroids on the amount and daily pattern of spontaneous activity on a running wheel and, second, to determine the degree to which the expected pattern of this activity was altered by 10 h of enforced running on a treadmill. Ten females of each group were housed in 26 × 46 x 20 cm polyethylene cages, each containing a running wheel with a diameter of 16 cm and a tread width of 8 cm. The number of revolutions of the wheel in relation to time of day was stored on a computer in 10-min bins using a Dataquest III Data Acquisition System (Mini-Mitter, Sun River, OR). After a minimum of 8 days in the running wheel cages, each female was placed on a treadmill at 0900 h and left for 10 h, after which it was returned to its running wheel cage. The effective length of the treadmill was 17 cm, its angle of incline was 15°, and it was divided into eight lanes, each measuring 5 cm in width. An electric grid at the base of the treadmill ensured that the animals would remain on the treadmill and, thus, be forced to run continuously for 10 h. During the first hour of exposure to the treadmill, its speed was increased every 15 min from about 2 c m / s to a final speed of 8 cm/s. Two sets of data were collected. First, the 5 days preceding exposure to the treadmill were pooled to obtain an average amount and average daily pattern of running wheel activity for each female. Second, the running wheel data for the 48 h immediately following exposure to the treadmill were examined to determine the time required by each female to return to her normal amount and pattern of use of the running wheel. To avoid proestrus and estrns throughout the time spent on the treadmill and the subsequent 48 h, females were exposed to the treadmill only when in metestrns.
Food Intake Test Ten females of each experimental treatment were switched from their normal pelleted chow to ground chow, which was given to them in food cups designed to avoid spillage (9). After 5 days of habituation to the ground chow, food intake and was assessed over a 3-day period. Stage of the estrous cycle of the control females was ignored for this test.
Open-Field Test This test assessed a mouse's reaction to an unfamiliar environment, namely an open field enclosed by a barrier measuring 72 X 54 x 35 cm placed on a large sheet of white filter paper. The floor was marked off in a 3 × 4 grid. A female was transported to the open field in a can that was placed in a corner and left there for 2 min. Then the can was lifted and the number of line crossings made by the mouse in a 4-min period was recorded. The number of entries into the two center squares as opposed to the 10 squares bordering the walls of the barrier was recorded separately. Females were tested only when in metestrus
ANABOLIC STEROIDS AND BEHAVIOR
51
TABLE 1 REPRODUCTIVE CHARACTERISTICS,AS DETERMINEDAT THE END OF THE EXPERIMENT (MEAN 5: SE IN ALL CASES) Dose of Steroids Control Ovarian w e i g h t ( m g ) Uterine weight (mg) Pituitary w e i g h t ( m g ) Serum LH ( n g / m l ) Pituitary LH ( n g / g l a n d ) Serum F S H ( n g / m l ) Pituitary F S H ( u g / g l a n d )
32.7 175 3.4 0.34 686 14.2 6.3
Low
5:1.3 5:12 5:0.1 5:0.10 5:75 5: 3.2 5:0.4
15.2 211 3.0 0.02 488 8.1 14.8
+ 0.7 + 6 5:0.1 + 0.02 + 68 ± 2.3 5:0.6
High 14.8 276 3.1 0.05 342 4.9 14.2
Prob.
5:1.2 ± 21 5:0.1 ± 0.03 5:18 5: 1.9 5:0.5
<0.0001 < 0.0001 0.009 0.0008 0.001 0.04 < 0.0001
n = 20 in each case.
or diestrus. The paper floor was changed between individual tests.
Aggression Test Aggressiveness was assessed in 30 pairs of females using a between-groups test design that randomized the previous test experience of the females. Ten pairs of each possible combination of treatments were tested: control vs. low dose, control vs. high dose, and low vs. high dose. Each animal was tested only once, and a maximum of 10 pairs was tested on any one day. Two females of different treatments were placed one on each side of a solid wooden barrier that divided a 38 X 29 X 16 cm cage into two equal halves. Starting 4 h later, the barrier was raised and the two mice were allowed to interact for 10 min. Any fighting that occurred was recorded, as was the latency to fight. The number of attacks by each member of each pair, and whether the attacked female fought back or ran, was also recorded. Separate attacks, by operational definition, occurred a minimum of 3 s apart. Tail rattling, a common indication of impending attack, was also recorded. The definition of these behaviors are those that have been used for decades when studying aggressiveness in mice [e.g., (22)]. Control females were tested only when in metestrus or diestrus, with relatively equal numbers of steroidtreated females being tested on the same daily schedule.
Autopsy Three days after the last aggression test all females were killed and autopsied. Trunk blood was collected and frozen for later RIA of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Pituitaries were also collected and frozen for
TABLE 2 MEAN (5: SE) BODY WEIGHTAND FOOD INTAKEAS DETERMINED DURING A 3-DAY ASSESSMENT(TOP THREE ROWS OF DATA; n = 10 IN EACH CASE) AND BODY COMPOSITIONAS DETERMINEDAFTER AUTOPSY (BOTTOM THREE ROWS OF DATA; n = 8 IN EACH CASE) Dose of Steroids Control Body weight (g)* Food intake ( g / D a y ) Food intake (g/g b.wt/day) Dry weight (g) Bodyfat(g) Percent fat ( g / g BW)
31.8 5:0.6 5.9 5:0.2
Low 39.1 5:0.8 5.8 5:0.2
High 39.1 5:0.9 5.9 5:0.2
0.19 5:0.01
0.15 + 0.01
0.15 + 0.01
8.6 5:0.3 2.3 5:0.3 0.27 5:0.03
12.4 5:0.5 5.1 5:0.6 0.40 + 0.03
12.6 5:1.0 5.2 5:1.0 0.39 5:0.04
Prob. < 0.0001 NS < 0.001 0.0004 0.009 0.03
* The average of two weights, one obtained before and one after the 3-day assessment of food intake.
later RIA of LH and FSH. The uterus and ovaries were removed and weighed. Then the carcass minus the GI tract was frozen for later fat extraction. This was done with ether using a soxhlet apparatus (7). Collection and storage of the blood serum and pituitaries, and the RIAs performed on these tissues all followed procedures that have been standard in this laboratory for several years [e.g., (6)]. LH was assessed in 15/zl of serum and an aliquot of a 10,000-fold dilution of homogenized pituitary, both in the same assay. The minimal detectable amount in this assay was 0.3 n g / m l , and its CV was 2%. FSH was measured in 10 /xl of serum and an aliquot of a fivefold dilution of pituitary, both in the same assay, whose minimal detectable amount was 15 n g / m l and its CV was 6%.
Statistical Assessment Unless otherwise noted, parametric data were analyzed using a 1 X 3 ANOVA, for repeated measures when appropriate, and the nonparametric data were analyzed by a three-cell contingency table, in all cases using Macintosh StatView software. RESULTS
Reproductive Characteristics While control females showed normal vaginal cycles, no cycling was seen in any of the 40 females treated with steroids. Correlatively, steroid treatment dramatically decreased the synthesis and release of LH and significantly but not dramatically decreased the release of FSH (Table 1). FSH content of the pituitary actually increased following steroid treatment. Also correlated with these changes was a decrease in both pituitary and ovarian weight. Despite the absence of estrous cycles in the steroid-treated females, their average uterine weight was significantly greater than the control females.
Food Intake Steroid treatment increased body weight by 23%, regardless of dose (Table 2). It did so without being accompanied by an increase in food intake, which meant that on a per gram basis the steroid-treated females required significantly less food than the control females to maintain their body weight (Table 2). Females treated with the high vs. the low dose of steroids did not differ in any of these regards.
Body Composition Steroid treatment resulted in an average 45% increase in dry body weight and an average twofold increase in the absolute
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BRONSON, NGUYEN AND DE LA ROSA
i
l Day1
BoO/Fat
t)~ Body w e i ~
! 8-
15-
j
~6tOrn
~' 5-
2-
Cont.'Low 'H Runnln
Cond Low~-Iigh Running
Cont.=LOWIHigh Sedentary
tw.
IL
6 prn
Cont.=Low igh Sedentar
FIG. 1. Post hoc analyses of body composition in females given access to a running wheel for 16 days (Running) vs. females held in smaller cages without running wheels (Sedentary).
amount of body fat, regardless of dose (Table 2). There was a barely significant increase in the fat to dry body weight ratio due to steroid treatment, again, regardless of dose. In analyzing the data gathered on body composition it became apparent that the effect of the steroid treatments depended on the test history of the female. As shown in Fig. 1, steroid treatment of the females in the subgroup subjected to the spontaneous activity/treadmill test yielded a smaller increase in dry body weight and body fat than was seen in the females subjected to the food intake/deprivation open field tests. Of the 23 days that transpired from the initiation of testing to autopsy, the former females were housed in running wheel cages for the first 16 days. When the data for dry body weight shown in Fig. 1 were subjected to a 2 × 3 ANOVA, both of the main effects (presence vs. absence of wheel and steroid treatment) were significant at the 0.0001 level, and the interaction was significant at the 0.002 level. The same analysis applied to the body fat data shown in Fig. 2 revealed the same level of significance for the two main effects and an interaction that was significant at the 0.0015 level.
TABLE 3 MEAN (5: SE) NUMBER OF WHEEL REVOLUTIONSRUN PER DAY AND THE EFFECTOF 10 h OF ENFORCED RUNNING ON A TREADMILLON THE AMOUNTAND PATTERN OF WHEEL RUNNINGACTIVITY Dose of Steroids Control Number revolutions run/day* Percent change after treadmill First6h'~ Second6h Next 1 2 h Next 2 4 h
13.0+
-70 -34 63 10
Low 2.3
+ 8 +15 5:30 + 12
6.4+
-66 -29 108 -8
High 0.7
+ 8 + 8 +58 :t: 5
6.05:0.7
-70 -30 51 -1
+ 6 + 13 +23 +10
Prob. 0.0025
NS NS NS NS
In the latter regard, the data presented are the percent change in expected running activity during various time periods after the end of the 10 h exposure to the treadmill. * x 1000. This is the average for the 5 days preceding the day of exposure to the treadmill. "l"The animals were removed from the treadmill just prior to lights off at 1800 h. Thus, the first 6 h after exposure to the treadmill were in the dark phase of the light cycle and extended from 1800 h until midnight; the second 6 h extended from midnight to 0600 h and included the last 4 h of the dark phase of the light cycle and the first 2 h of the light phase; the next 12 h extended from 0600 h to 1800 h and included the final 12 h of the light phase of the light cycle; the next 24 h started at 1800 h the day after exposure to the treadmill.
I
. . . . . . .
•
10 pm
Ilk
IL
2 am
,,u
"i""=I
6 am
10 am
= I
2 prn
6 pm
FIG. 2. Typical pattern of voluntary use of a running wheel by a control female before and after 10 h of enforced running on a treadmill. Day 1 is the day before enforced running, starting at lights out at 1800 h. Exposure to the treadmill took place from 0900-1800 h during day 2, which is omitted in its entirety. Day 3 consists of the 24 h after enforced running was terminated at 1800 h, and day 4 is the subsequent 24 h. The depression in activity taking place during the 12 h following enforced running is obvious, as is the rebound effect seen over the next 12 h and the close to normal pattern of running on the second day. The spurt of activity between 0700 and 1000 h each day was caused in part by disturbing the animal to obtain a vaginal smear.
There were no significant differences associated with the ratio of body fat to dry weight, so these data are not presented.
Running Wheel/Treadmill Recovery Test Steroid treatment depressed the spontaneous use of a running wheel (Table 3). Control animals ran more than twice as much as the steroid-treated females, regardless of the dose of steroid. Use of the wheel decreased by an average of 69% during the first 6 h after a 10-h exposure to the treadmill (calculated on the basis of the expected amount of running at that time of day), with no significant differences due to hormonal treatment. Use of the wheel was depressed by an average of 31% during the next 6 h; it rebounded to an average of 73% greater than expected during the next 12 h and returned to near normal the next day (Fig. 2), all again with no significant differences due to hormonal treatment.
Open-Field Test Treatment with steroids dramatically decreased the amount of activity in the open field (Table 4). The females treated with the low dose of steroids were only about half as active as the control females, and the high-dose females were even less active (although the difference between the high-and low-dose females was not significant when tested by a post hoc Scheffe test). The steroid-treated females also showed a marginally significant tendency to avoid the center of the open field, instead choosing to stay close to the barrier wall.
Aggression Test About two-thirds of the pairings resulted in fighting, and neither the frequency with which this occurred nor the mean latency for a fight to erupt varied significantly from one kind of pairing to another (Table 5). Regardless of dose, however, when steroid-treated females were paired with control females, only the steroid-treated females showed threatening (tail rattling) and attack behavior (Table 6). Furthermore, the control females almost never fought back, attempting to run away instead; and in all cases where fighting occurred, the steroid-treated animal was the clear winner of the encounter. In contrast, when high- and a low-dose steroid-treated females were paired, threats and attacks
ANABOLIC STEROIDS AND BEHAVIOR
53
TABLE 4 MEAN (± SE) NUMBER OF SQUARES ENTERED DURING A 4 MIN EXPOSURE TO AN OPEN FIELD AND THE PERCENT THAT WERE IN THE TWO CENTER SQUARES AS OPPOSED TO THE 10 SQUARES BORDERINGTHE WALL Number of squares Percent in center
Control
Low Dose
High Dose
Prob.
94.3 ± 19.1 3.8 ± 1.2
46.5 ± 9.5 1.5 ± 0.08
38.0 ± 8.8 0.9 ± 0.7
< 0.01 0.06*
Treatment of Female Control
* Kruskall-Wallis test.
could come from either type of female, and the recipient of an attack almost always fought back. In only one case was there a clear winner at the end of the 10-min test period. Overall, there was little difference between the behavior of the females treated with the high vs. the low dose of steroids (Table 6). An interesting behavior typical of a potential sexual encounter rather than an aggressive encounter was also seen in some of these tests. Normally, when untreated female mice are exposed to a male, the male routinely inspects the female's ano-genital area. If the female is not in estrus, it will reject the male's inspection by kicking vigorously and repeatedly with the hind foot, often squealing while doing so. This sequence of behaviors was seen in almost all pairings of control and steroid-treated females. The steroid-treated female would attempt to investigate the control female's ano-genital area, and in response the control female would routinely show the rejection behavior described above. This behavior was only seen once in a steroid-treated female, despite the fact that both steroid-treated females in a paired encounter usually tried to inspect each other's ano-genital area (Table 6). DISCUSSION
The results of this experiment suggest broad effects of anabolic-androgenic steroids on the physiology and behavior of female mice. Starting with the physiological effects, the almost complete suppression of the reproductive axis that was seen here was expected. The negative feedback action of the gonadal steroids on gonadotropin secretion has been established experimentally for decades now (13), and the decrease in circulating levels of the gonadotropins that accompanies steroid abuse is well documented even in human athletes [e.g., (2)]. The effect of anabolic steroids on the uterus is of more interest. The control females were killed while in various stages of the estrous cycle, and thus, their average uterine weight represents a range from the small quiescent uterus characteristic of early diestrus to the large, ballooning uterus of late proestrus. The average weight of the uterus of the steroid-treated females was significantly greater than the average for the controls, and it was three to four times greater than the minimum level seen in the controls, despite the fact that the steroid-treated females were not cycling. This probably indicates a pathologic response of the
TABLE 5 NUMBER OF PAIRINGS IN WHICH FIGHTING OCCURRED AND THE MEAN (5: SE) LATENCY TO FIGHT IN THE PAIRINGS IN WHICH FIGHTING OCCURRED
Number pairs fighting/ number pairings Latency to fight (rain)
TABLE 6 MEAN (± SE) NUMBER OF THREATS(TAIL RATTLING) AND ATTACKSINITIATEDBY FEMALES OF THE THREE TREATMENTGROUPS, THE PROPORTIONOF THESE ATTACKS IN WHICH THE A T T A C K E D MOUSE FOUGHTB A C K (AS OPPOSED TO RUNNINGAWAY),AND THE MEAN NUMBER OF NONRECEPTIVE RESPONSES SEEN
Low vs. Control
High vs. Control
Low vs. High
Prob.
6/10
7/10
7/10
NS
5.0 ± 0.6
4.1 ± 0.9
3.1 ± 0.8
NS
Instances of tail rattling 0 Number of attacks initiated 0 Proportion of attacks fought back* 0.02 ± 0.02 Number of nonreceptive rejections 12.2 ± 1.6
Low Dose
High Dose
Prob.
4.9 ± 1.3
4.4 ± 1.3
0.009
5.8 ± 1.5
5.3 ± 1.3
0.002
0.92 + 0.08 0.96 ± 0.04
< 0.0001
0.7 + 0.7
< 0.0001
0.9 ± 0.6
The first two comparisons are based only on the pairings in which fighting occurred (tail rattling was never seen in pairings in which attack behavior was absent). * The proportion of times in which the attacked female fought back rather than running away.
uterine tissues, as suggested already by Yu-Yahiro and co-workers (32) in their studies with rats. There is a degree of confusion about the effect of the anabolic steroids on muscle mass and function in the absence of a training regimen in human males, and to the best of our knowledge, nothing is known about this subject in human females [reviewed by (12,29)]. Studies with rats and mice have tended to show either no effect or a decrease in muscle mass and body weight following treatment with anabolic steroids [e.g., (4,19)]. Again, to the best of our knowledge, little research has been directed at elucidating the effect of anabolic steroids on body fat, but the perception in humans is that it decreases in response to steroid use [e.g., (23)]. In the present study, treatment with anabolic steroids yielded a marked increase in both dry body weight and body fat, more so the latter than the former. The length of time that the fat reserves of a small mammal can protect against energetic insult is dramatically shorter than it is in the large mammal, being measured in hours or a few days at best [e.g., (7)], and, thus, different control mechanisms in small vs. large mammals probably shouldn't be too surprising. Interestingly, the increase in both dry body weight and body fat seen here in steroid-treated mice occurred in the absence of an increase in food intake and, indeed, relative to their body weight, the steroid-treated animals actually consumed less food than the controls. Little energy is required to maintain fat as opposed to muscle, but an increase in dry body weight without an accompanying increase in food intake is difficult to explain. There would seem to be two possibilities here. Either exposure to anabolic steroids induces a metabolic adjustment of some unknown kind that results in a lower food requirement or, alternatively, steroidtreated females simply expend less energy throughout the day in their home cages. The latter possibility seems of likely importance because the steroid-treated females ran only half as much as the control females when given the opportunity to use a running wheel. Complicating this picture even more is the fact that post hoc analyses suggested that access to a running wheel strongly antagonized the effects of steroids on body weight and composition, while having no such effect in control animals. This is in spite of the fact that the control animals spent much more time on their running wheels than the steroid-treated animals. All in all, the
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BRONSON, N G U Y E N A N D D E L A R O S A
present results suggest both a depression of locomotor activity and a specific antagonism of the steroidal effect on body weight and composition by exercise. In final regard to physiological effects, the only test of increased muscle capacity included in this experiment was the treadmill test, and it yielded negative results. While forced running decreased voluntary use of a running wheel markedly during the 12 h following exposure to the treadmill, there were no differences in this regard due to hormonal treatment. Behaviorally, exposure to anabolic steroids decreased activity in an open field, increased aggressiveness, and largely eliminated a specific response related to encounters between the sexes. In the first regard, two studies in male rats have shown no change in activity in an open field following treatment with anabolic steroids (5,19). In contrast, the present study with female mice showed a decided 50 to 60% decrease in activity. In theory, this decrease could be conceptualized as either an increase in timidity or a depression of locomotor activity. The marginally significant increase in wall hugging seen in the present study was also noted in one of the previously cited studies with male rats (19) and could argue for an effect on timidity. As noted earlier, however, a generalized, nonspecific depression of locomotor activity is suggested by a marked depression in wheel running. In any event, because locomotor activity is enhanced by estrogen in female rodents [e.g., (14)], the depression in activity seen here might be accounted for simply by a decrease in estrogen secretion, as suggested by the gonadotropin levels and ovarian weight of the steroid treated animals (Table 1). Alternatively, it might indicate a more central effect of the administered steroids. The dependence of aggression on testicular hormones in the male rodent is well established [e.g., (3,18,19)], and there is some documentation of an increase in aggressiveness due to anabolic
steroids in female rodents (25,27). The increase in aggressiveness seen in the present study with female mice was dramatic, as was the loss of a behavior normally associated with an encounter between the sexes--the stereotypical defensive reaction against inspection of the genitals shown by females not in estrus. None of the 20 control females showed any tail rattling, and none of them ever initiated an attack. Indeed, in only one instance in one control female was aggressive defense against an attack even seen. In sharp contrast, most steroid-treated females routinely threatened, attacked, and fought back when attacked. To the best of our knowledge, nothing is known about the effect of anabolic-androgenic steroids on aggressiveness in women athletes. The degree to which the present results with female mice are applicable to humans is also unknown. Even if not directly applicable, however, the effects seen here on locomotor activity and aggressiveness argue for changes in brain chemistry that might be generalizable to humans where, indeed, they could lead to behavioral manifestations quite different than those seen in mice. As a final comment, it is worth noting that throughout this study typically there was little difference in the effect of the two doses of anabolic steroids, regardless of the measurement being taken. This suggests a threshold effect for most of these responses below the low dose, which was the androgenic maintenance level for male mice. The present results, thus, suggest that anabolic steroids can produce physiological and behavioral effects in females at levels still lower than that which is physiological for males and below those thought to be used by some female athletes and body builders. ACKNOWLEDGEMENTS This research was supported by PHS Grant No. HD 30670.
REFERENCES 1. Albert, D. J.; Jonik, R. N.; Walsh, M. L. Hormone-dependent aggression in male and female rats: Experiential, hormonal, and neural foundations. Neurosci. Biobehav. Rev. 16:177-192; 1992. 2. Al6n, M.; Rahkila, P. Anabolic-androgenic steroid effects on endocrinology and metabolism in athletes. Sports Med. 6:327-332; 1988. 3. Archer, J. The influence of testosterone on human aggression. Br. J. Psychol. 82:1-28; 1991. 4. Bauman, D. H.; Richerson, J. T.; Britt, A. L. A comparison of body and organ weights, physiological parameters, and pathologic changes in target organs of rats given combinations of exercise, anabolic hormone, and protein supplementation. Am. J. Sports Med. 16:397402; 1988. 5. Bitrain, D.; Kellogg, C. K.; Hilvers, R. J. Treatment with an anabolic-androgenic steroid affects anxiety-related behavior and alters the sensitivity of cortical GABAA receptors in the rat. Horm. Behav. 27:568-583; 1993. 6. Bronson, F. H.; Heideman, P. D. Short-term hormonal responses to food intake in the peripubertal female rat. Am. J. Physiol. 259:R25R31; 1990. 7. Bronson, F. H.; Heideman, P. D.; Kerbeshian, M. Lability of fat stores in peripubertal wild house mice. J. Comp. Physiol [B] 161:1518; 1991. 8. Buckley, W. B.; Yesalis, C. E., III; Friedle, K. E.; Anderson, W. A.; Streit, A. L.; Wright, J. E. Estimated prevalence of anabolic steroid use among male high school seniors. JAMA 260:3441-3445; 1988. 9. Desjardins, C.; Lopez, J. M. Environmental cues evoke differential responses in pituitary-tesficular function in deer mice. Endocrinology 112:1398-1406; 1983. 10. DuRant, R. H.; Rickert, V. I.; Ashworth, C. S.; Newman, C.; Slavens, G. Use of multiple drugs among adolescents who use anabolic steroids. N. Engl. J. Med. 328:922-926; 1993. 11. Ehrenkranz, J.; Bliss, E.; Sheard, M. H. Plasma testosterone: Correla-
12. 13. 14. 15.
16. 17. 18.
19. 20. 21.
tion with aggressive behavior and social dominance in man. Psychosom. Med. 36:469-475; 1974. Elashoff, J. D.; Jacknow, A. D.; Shain, S. G.; Braunstein, G. D, Effects of anabolic-androgenic steroids on muscular strength. Ann. Intern. Med. 115:387-393; 1991. Everett, J. W. Pituitary and hypothalamus: Perspectives and overview. In: Knobil, E.; Neill, J. D., eds. The physiology of reproduction, 2rid ed. New York: Raven Press; 1994:1509-1526. Gerall, A. A.; Napoli, A. M.; Cooper, U. C. Daily and hourly estrous running in intact, spayed and estrone implanted rats. Physiol. Behav. 10:225-229; 1973. J~inne, O. A. Androgen interaction through multiple steroid receptors. In: Lin, G. C.; Erinoff, L., eds. National Institute on Drug Abuse Research Monograph Series #102. Rockville, MD: NIDA; 1990:178-186. Kochakian, C. D. History of anabolic-androgenic steroids. In: Lin, G. C.; Erinoff, L., eds. National Institute on Drug Abuse Research Monograph Series #102. RockviUe, MD: NIDA; 1990:29-59. Lukas, S. E. Current perspectives on anabolic-androgenic steroid abuse. Trends Pharmacol. Sci. 14:61-68; 1993. Lumia, A. R.; Thorner, K. M.; McGinnis, M. Y. Effects of chronically high doses of the anabolic androgenic steroid, testosterone, on inter-male aggression and sexual behavior in male rats. Physiol. Behav. 55:331-335; 1994. Minkin, D. M.; Meyer, M. E.; van Haaren, F. Behavioral effects of long-term administration of an anabolic steroid in intact and castrated male Wistar rats. Biochem. Behav. 44:959-963; 1993. Nardueci, W. A.; Wagner, J. C.; Hendrickson, T. P.; Jeffrey, T. P. Anabolic steroids--A review of the clinical toxicology and diagnostic screening. Clin. Toxicol. 28:287-310; 1990. Pope, H. G.; Katz, D. L. Psychiatric and medical effects of anabolieandrogenic steroid use: A controlled study of 160 athletes. Arch. Gen. Psychiatry 51:375-382; 1994.
A N A B O L I C STEROIDS A N D B E H A V I O R
22. Scott, J. P. Incomplete adjustment caused by frustration of untrained fighting mice. J. Comp. Psychol. 39:379-390; 1946. 23. Strauss, R. H.; Liggett, M. T.; Lanese, R. R. Anabolic steroid use and perceived effects in ten weight-trained women athletes. JAMA 253:2871-2873; 1985. 24. Su, T.; Pagliaro, M.; Schmidt, P.; Pickar, D; Wolkowitz, O.; Rubinow, D. Neuropsychiatric effects of anabolic steroids in male normal volunteers. JAMA 269:2760-2764; 1993. 25. Svare, B. Anabolic steroids and behavior: A preclinical research prospectus. In: Lin, G. C.; Erinoff, L., eds. National Institute on Drug Abuse Research Monograph Series #102. Rockville, MD: N1DA; 1990:224-241. 26. Terney, R.; McLain, L. G. The use of anabolic steroids in high school students. AJDC 144:99-103; 1990. 27. van de Poll, N. E.; van Zanten, S,; de Jonge, F. H. Effects of testosterone, estrogen and dihydrotestosterone upon aggressive and sexual behavior of female rats. Horm, Behav. 20:418-431; 1986.
55
28. Williamson, D. J.; Young, A. H. Psychiatric effects of androgenic and anabolic-androgenic steroid abuse in men: A brief review of the literature. J. Psychopharmacol. 6:20-26; 1992. 29. Wilson, J. D. Androgen abuse by athletes. Endocr. Rev. 9:181-199; 1988. 30. Yates, W. R.; Perry, P.; Murray, S. Aggression and hostility in anabolic steroid users. Biol. Psychol. 31:1232-1234; 1992. 31. Yesalis, C. E.; Kennedy, N. J.; Kopstein, A. N.; Bahrke, M. S. Anabolic-androgenic steroid use in the United States. JAMA 270:1217-1221; 1993. 32. Yu-Yahiro, J. A.; Michael, R. H.; Nasrallah, D. V.; Schofield, B. Morphologic and histologic abnormalities in female and male rats treated with anabolic steroids. Am. J. Sports Med. 17:686-689; 1989.
ELSEVIER
0031-9384(95)02027-S
Effect of Anabolic Steroids on Behavior and Physiological Characteristics of Female Mice F. H. BRONSON, 1 K. Q. N G U Y E N AND J. DE LA ROSA
Institute of Reproductive Biology, Department of Zoology, University of Texas, Austin, TX 78712 USA Received 14 October 1994 BRONSON, F. H., K. Q. NGUYEN AND J. DE LA ROSA. Effect of anabolic steroids on behavior and physiological characteristics of female mice. PHYSIOL BEHAV 59(1) 49-55, 1996.--Adult female mice were exposed to a combination of four anabolic-androgenic steroids for 9 weeks at doses that were either one or five times the androgenic maintenance level for male mice. Relative to control females, steroid treatment depressed gonadotropin secretion and increased both dry body weight and fat content but without an increase in food intake. Steroid treatment depressed spontaneous use of a running wheel and open-field activity, and it increased aggressiveness. It also eliminated a behavior related to encounters between the sexes--the rejection of genital inspection. There was no effect of steroid treatment on the time required to recover from 10 h of enforced running on a treadmill. Overall, regardless of the test or measure, there was little or no difference in the effect of the high and low dose of steroids. This indicates a threshold of response below the low dose used in these studies, which itself is probably well below that used by many female athletes and body builders. Anabolic steroids Locomotor activity
Body composition Mice
Open field behavior
Aggressiveness
to have broad effects on behavior, much of the interest has been limited to potential effects on aggressiveness where most, but not all, of the available data suggest a marked increase due to steroid use, at least in males [e.g., 11,21,24,30); cf., (3,28)]. Unfortunately, in most cases the available evidence here is correlational and quite a bit is anecdotal and self-reported. Almost nothing is known about the behavioral side effects of steroid abuse in females, but certainly the potential for an increase in aggressiveness due to steroids exists in both sexes (1,25). The objective of the present study was to assess the effect of anabolic steroids on a variety of physical and behavioral characteristics in female laboratory mice using the numbers, kinds, and relative doses of steroids commonly used by female athletes and body builders. Specifically, adult female mice were exposed to a combination of four commonly abused anabolic steroids for 9 weeks at doses that were either one or five times the maintenance level for male mice. During the last 3 weeks of this period, the females were given a series of tests to assess their level of spontaneous activity, recovery time after forced running on a treadmill, response to an open field, aggressiveness, and food intake. Afterwards, the females were autopsied, body composition was assessed, and a variety of reproductive-related measures were obtained. The results of this experiment suggest broad
ANABOLIC-ANDROGENIC steroids such as testosterone and its many synthetic analogs can promote muscle-building processes while reducing muscle catabolism (16). Thus, in spite of a ban imposed by many administrative entities, these hormones are widely used by male athletes and body builders in an effort to improve their performance or appearance (17,20,31). Of concern here is the fact that many female body builders and athletes apparently also use anabolic-androgenic steroids (23,31), occasionally even during adolescence (8,10,26). Males often take as many as five different steroids simultaneously at doses that can total 10 to 40 times normal maintenance levels (17,29). They usually do this in cycles, starting weeks or months before a competition, after which they initiate a wash-out period of weeks or months. Little is known about the doses or schedules used by females except that, like males, women have been known to take as many as five steroids simultaneously at levels that are several times those recommended by manufacturers, and a degree of masculinization of secondary sexual characteristics is not uncommon in this situation (23). Supraphysiological doses of anabolic steroids can have a variety of secondary effects, some of which are decidedly pathologic (17). Of particular relevance are the potential effects of these steroids on behavior. While the anabolic steroids are thought
1 To whom requests for reprints should be addressed.
49
50
BRONSON, NGUYEN AND DE LA ROSA
effects of anabolic steroids on the physiological and behavioral characteristics of female mice. METHOD
Animals and Experimental Design CF-1 female mice were purchased from Charles River at 7 weeks of age. Upon arrival they were housed one per 29 x 18 x 12 cm polyethylene cage and fed PMI Formulab 5008 chow. Throughout this experiment these animals were maintained on a 14 L:10 D cycle with lights off at 1800 h and on at 0400 h. At 8 weeks of age the females were organized into a 1 × 3 experimental design with 20 females per treatment. One treatment group was given a low dose of a combination of four anabolic steroids. A second group was given a high dose of the same combination of four steroids, and a control group received no exogenous hormones. The hormones administered were testosterone, testosterone cypionate, methyltestosterone, and norethandrolone. The latter three hormones span the classes of testosterone analogues proposed by Wilson (29). The doses used were defined on the basis of their androgenic potency rather than their anabolic potency, which may not be identical when pharmacological levels are given (15). The low dose was a combination of the four hormones that would maintain normal-sized seminal vesicles in a castrated male CF-1 mouse and the high dose was five times the low dose.
Steroid Administration Each hormone was administered in its own Silastic capsule, and the 20 females in the control group were given a set of four empty capsules. Capsule sizes were chosen with two objectives in mind: to ensure that each of the four steroids of concern contributed one-fourth of the total androgenicity at both dose levels and, second, to keep the length of the capsule within the range of comfort for a mouse. To obtain those objectives it was necessary to vary the diameter of the capsules as well as their length. A series of pilot studies, all based on the effect of a steroid on the weight of the seminal vesicles of castrated CF-1 males, yielded the following choices. The low dose combined: 2.5 mm of a 0.062" X0.125" capsule containing testosterone; 5 mm of a 0.062" × 0.125" capsule of testosterone cypionate; 2.5 mm of a 0.025" X 0.047" capsule of methyltestosterone and 7.5 mm of a 0.062" X 0.125" capsule of norethandrolone. The fivefold higher dose combined: 12.5 mm of a 0.062" x 0.125" capsule containing testosterone; 5 mm of a 0.078" X 0.125" capsule of testosterone cypionate; 12.5 mm of a 0.025"X 0.047" capsule of methyltestosterone; and 7.5 mm of a 0.078"X 0.125" capsule of norethandrolone.
Testing Four tests were administered over a 20-day period. The 20 females in each of the three treatment groups were divided into two subgroups of 10 each. The individuals in one subgroup of each treatment were given the spontaneous activity/treadmill recovery test. The individuals in the other subgroup of each treatment were subjected to two tests: first, their food intake was measured, and this was followed in a few days by an assessment of their reactions to an open field. The subgroups were then recombined and all 20 females in each treatment group participated in the aggression tests. All testing was accomplished without knowledge of the experimental group. Stage of the estrous cycle was deemed a potentially important variable in most of these tests. Thus, vaginal smears were
obtained daily throughout the 20-day test period. As will be detailed later, the control females cycled normally but cyclicity was completely suppressed in all females given steroids. Because the cycles of the control females were asynchronous, and because we routinely tested females only when in metestrus or diestrus, the administration of a test to a group of individuals extended over a period of several days. To keep timing constant for the three experimental groups, the testing of the androgenized females extended over the same amount of time as the control females, with approximately equal numbers of each treatment being tested each day.
Spontaneous Activity / Treadmill Recovery Test The object of this test was twofold: first, to assess the effect of anabolic steroids on the amount and daily pattern of spontaneous activity on a running wheel and, second, to determine the degree to which the expected pattern of this activity was altered by 10 h of enforced running on a treadmill. Ten females of each group were housed in 26 × 46 x 20 cm polyethylene cages, each containing a running wheel with a diameter of 16 cm and a tread width of 8 cm. The number of revolutions of the wheel in relation to time of day was stored on a computer in 10-min bins using a Dataquest III Data Acquisition System (Mini-Mitter, Sun River, OR). After a minimum of 8 days in the running wheel cages, each female was placed on a treadmill at 0900 h and left for 10 h, after which it was returned to its running wheel cage. The effective length of the treadmill was 17 cm, its angle of incline was 15°, and it was divided into eight lanes, each measuring 5 cm in width. An electric grid at the base of the treadmill ensured that the animals would remain on the treadmill and, thus, be forced to run continuously for 10 h. During the first hour of exposure to the treadmill, its speed was increased every 15 min from about 2 c m / s to a final speed of 8 cm/s. Two sets of data were collected. First, the 5 days preceding exposure to the treadmill were pooled to obtain an average amount and average daily pattern of running wheel activity for each female. Second, the running wheel data for the 48 h immediately following exposure to the treadmill were examined to determine the time required by each female to return to her normal amount and pattern of use of the running wheel. To avoid proestrus and estrns throughout the time spent on the treadmill and the subsequent 48 h, females were exposed to the treadmill only when in metestrns.
Food Intake Test Ten females of each experimental treatment were switched from their normal pelleted chow to ground chow, which was given to them in food cups designed to avoid spillage (9). After 5 days of habituation to the ground chow, food intake and was assessed over a 3-day period. Stage of the estrous cycle of the control females was ignored for this test.
Open-Field Test This test assessed a mouse's reaction to an unfamiliar environment, namely an open field enclosed by a barrier measuring 72 X 54 x 35 cm placed on a large sheet of white filter paper. The floor was marked off in a 3 × 4 grid. A female was transported to the open field in a can that was placed in a corner and left there for 2 min. Then the can was lifted and the number of line crossings made by the mouse in a 4-min period was recorded. The number of entries into the two center squares as opposed to the 10 squares bordering the walls of the barrier was recorded separately. Females were tested only when in metestrus
ANABOLIC STEROIDS AND BEHAVIOR
51
TABLE 1 REPRODUCTIVE CHARACTERISTICS,AS DETERMINEDAT THE END OF THE EXPERIMENT (MEAN 5: SE IN ALL CASES) Dose of Steroids Control Ovarian w e i g h t ( m g ) Uterine weight (mg) Pituitary w e i g h t ( m g ) Serum LH ( n g / m l ) Pituitary LH ( n g / g l a n d ) Serum F S H ( n g / m l ) Pituitary F S H ( u g / g l a n d )
32.7 175 3.4 0.34 686 14.2 6.3
Low
5:1.3 5:12 5:0.1 5:0.10 5:75 5: 3.2 5:0.4
15.2 211 3.0 0.02 488 8.1 14.8
+ 0.7 + 6 5:0.1 + 0.02 + 68 ± 2.3 5:0.6
High 14.8 276 3.1 0.05 342 4.9 14.2
Prob.
5:1.2 ± 21 5:0.1 ± 0.03 5:18 5: 1.9 5:0.5
<0.0001 < 0.0001 0.009 0.0008 0.001 0.04 < 0.0001
n = 20 in each case.
or diestrus. The paper floor was changed between individual tests.
Aggression Test Aggressiveness was assessed in 30 pairs of females using a between-groups test design that randomized the previous test experience of the females. Ten pairs of each possible combination of treatments were tested: control vs. low dose, control vs. high dose, and low vs. high dose. Each animal was tested only once, and a maximum of 10 pairs was tested on any one day. Two females of different treatments were placed one on each side of a solid wooden barrier that divided a 38 X 29 X 16 cm cage into two equal halves. Starting 4 h later, the barrier was raised and the two mice were allowed to interact for 10 min. Any fighting that occurred was recorded, as was the latency to fight. The number of attacks by each member of each pair, and whether the attacked female fought back or ran, was also recorded. Separate attacks, by operational definition, occurred a minimum of 3 s apart. Tail rattling, a common indication of impending attack, was also recorded. The definition of these behaviors are those that have been used for decades when studying aggressiveness in mice [e.g., (22)]. Control females were tested only when in metestrus or diestrus, with relatively equal numbers of steroidtreated females being tested on the same daily schedule.
Autopsy Three days after the last aggression test all females were killed and autopsied. Trunk blood was collected and frozen for later RIA of luteinizing hormone (LH) and follicle stimulating hormone (FSH). Pituitaries were also collected and frozen for
TABLE 2 MEAN (5: SE) BODY WEIGHTAND FOOD INTAKEAS DETERMINED DURING A 3-DAY ASSESSMENT(TOP THREE ROWS OF DATA; n = 10 IN EACH CASE) AND BODY COMPOSITIONAS DETERMINEDAFTER AUTOPSY (BOTTOM THREE ROWS OF DATA; n = 8 IN EACH CASE) Dose of Steroids Control Body weight (g)* Food intake ( g / D a y ) Food intake (g/g b.wt/day) Dry weight (g) Bodyfat(g) Percent fat ( g / g BW)
31.8 5:0.6 5.9 5:0.2
Low 39.1 5:0.8 5.8 5:0.2
High 39.1 5:0.9 5.9 5:0.2
0.19 5:0.01
0.15 + 0.01
0.15 + 0.01
8.6 5:0.3 2.3 5:0.3 0.27 5:0.03
12.4 5:0.5 5.1 5:0.6 0.40 + 0.03
12.6 5:1.0 5.2 5:1.0 0.39 5:0.04
Prob. < 0.0001 NS < 0.001 0.0004 0.009 0.03
* The average of two weights, one obtained before and one after the 3-day assessment of food intake.
later RIA of LH and FSH. The uterus and ovaries were removed and weighed. Then the carcass minus the GI tract was frozen for later fat extraction. This was done with ether using a soxhlet apparatus (7). Collection and storage of the blood serum and pituitaries, and the RIAs performed on these tissues all followed procedures that have been standard in this laboratory for several years [e.g., (6)]. LH was assessed in 15/zl of serum and an aliquot of a 10,000-fold dilution of homogenized pituitary, both in the same assay. The minimal detectable amount in this assay was 0.3 n g / m l , and its CV was 2%. FSH was measured in 10 /xl of serum and an aliquot of a fivefold dilution of pituitary, both in the same assay, whose minimal detectable amount was 15 n g / m l and its CV was 6%.
Statistical Assessment Unless otherwise noted, parametric data were analyzed using a 1 X 3 ANOVA, for repeated measures when appropriate, and the nonparametric data were analyzed by a three-cell contingency table, in all cases using Macintosh StatView software. RESULTS
Reproductive Characteristics While control females showed normal vaginal cycles, no cycling was seen in any of the 40 females treated with steroids. Correlatively, steroid treatment dramatically decreased the synthesis and release of LH and significantly but not dramatically decreased the release of FSH (Table 1). FSH content of the pituitary actually increased following steroid treatment. Also correlated with these changes was a decrease in both pituitary and ovarian weight. Despite the absence of estrous cycles in the steroid-treated females, their average uterine weight was significantly greater than the control females.
Food Intake Steroid treatment increased body weight by 23%, regardless of dose (Table 2). It did so without being accompanied by an increase in food intake, which meant that on a per gram basis the steroid-treated females required significantly less food than the control females to maintain their body weight (Table 2). Females treated with the high vs. the low dose of steroids did not differ in any of these regards.
Body Composition Steroid treatment resulted in an average 45% increase in dry body weight and an average twofold increase in the absolute
52
BRONSON, NGUYEN AND DE LA ROSA
i
l Day1
BoO/Fat
t)~ Body w e i ~
! 8-
15-
j
~6tOrn
~' 5-
2-
Cont.'Low 'H Runnln
Cond Low~-Iigh Running
Cont.=LOWIHigh Sedentary
tw.
IL
6 prn
Cont.=Low igh Sedentar
FIG. 1. Post hoc analyses of body composition in females given access to a running wheel for 16 days (Running) vs. females held in smaller cages without running wheels (Sedentary).
amount of body fat, regardless of dose (Table 2). There was a barely significant increase in the fat to dry body weight ratio due to steroid treatment, again, regardless of dose. In analyzing the data gathered on body composition it became apparent that the effect of the steroid treatments depended on the test history of the female. As shown in Fig. 1, steroid treatment of the females in the subgroup subjected to the spontaneous activity/treadmill test yielded a smaller increase in dry body weight and body fat than was seen in the females subjected to the food intake/deprivation open field tests. Of the 23 days that transpired from the initiation of testing to autopsy, the former females were housed in running wheel cages for the first 16 days. When the data for dry body weight shown in Fig. 1 were subjected to a 2 × 3 ANOVA, both of the main effects (presence vs. absence of wheel and steroid treatment) were significant at the 0.0001 level, and the interaction was significant at the 0.002 level. The same analysis applied to the body fat data shown in Fig. 2 revealed the same level of significance for the two main effects and an interaction that was significant at the 0.0015 level.
TABLE 3 MEAN (5: SE) NUMBER OF WHEEL REVOLUTIONSRUN PER DAY AND THE EFFECTOF 10 h OF ENFORCED RUNNING ON A TREADMILLON THE AMOUNTAND PATTERN OF WHEEL RUNNINGACTIVITY Dose of Steroids Control Number revolutions run/day* Percent change after treadmill First6h'~ Second6h Next 1 2 h Next 2 4 h
13.0+
-70 -34 63 10
Low 2.3
+ 8 +15 5:30 + 12
6.4+
-66 -29 108 -8
High 0.7
+ 8 + 8 +58 :t: 5
6.05:0.7
-70 -30 51 -1
+ 6 + 13 +23 +10
Prob. 0.0025
NS NS NS NS
In the latter regard, the data presented are the percent change in expected running activity during various time periods after the end of the 10 h exposure to the treadmill. * x 1000. This is the average for the 5 days preceding the day of exposure to the treadmill. "l"The animals were removed from the treadmill just prior to lights off at 1800 h. Thus, the first 6 h after exposure to the treadmill were in the dark phase of the light cycle and extended from 1800 h until midnight; the second 6 h extended from midnight to 0600 h and included the last 4 h of the dark phase of the light cycle and the first 2 h of the light phase; the next 12 h extended from 0600 h to 1800 h and included the final 12 h of the light phase of the light cycle; the next 24 h started at 1800 h the day after exposure to the treadmill.
I
. . . . . . .
•
10 pm
Ilk
IL
2 am
,,u
"i""=I
6 am
10 am
= I
2 prn
6 pm
FIG. 2. Typical pattern of voluntary use of a running wheel by a control female before and after 10 h of enforced running on a treadmill. Day 1 is the day before enforced running, starting at lights out at 1800 h. Exposure to the treadmill took place from 0900-1800 h during day 2, which is omitted in its entirety. Day 3 consists of the 24 h after enforced running was terminated at 1800 h, and day 4 is the subsequent 24 h. The depression in activity taking place during the 12 h following enforced running is obvious, as is the rebound effect seen over the next 12 h and the close to normal pattern of running on the second day. The spurt of activity between 0700 and 1000 h each day was caused in part by disturbing the animal to obtain a vaginal smear.
There were no significant differences associated with the ratio of body fat to dry weight, so these data are not presented.
Running Wheel/Treadmill Recovery Test Steroid treatment depressed the spontaneous use of a running wheel (Table 3). Control animals ran more than twice as much as the steroid-treated females, regardless of the dose of steroid. Use of the wheel decreased by an average of 69% during the first 6 h after a 10-h exposure to the treadmill (calculated on the basis of the expected amount of running at that time of day), with no significant differences due to hormonal treatment. Use of the wheel was depressed by an average of 31% during the next 6 h; it rebounded to an average of 73% greater than expected during the next 12 h and returned to near normal the next day (Fig. 2), all again with no significant differences due to hormonal treatment.
Open-Field Test Treatment with steroids dramatically decreased the amount of activity in the open field (Table 4). The females treated with the low dose of steroids were only about half as active as the control females, and the high-dose females were even less active (although the difference between the high-and low-dose females was not significant when tested by a post hoc Scheffe test). The steroid-treated females also showed a marginally significant tendency to avoid the center of the open field, instead choosing to stay close to the barrier wall.
Aggression Test About two-thirds of the pairings resulted in fighting, and neither the frequency with which this occurred nor the mean latency for a fight to erupt varied significantly from one kind of pairing to another (Table 5). Regardless of dose, however, when steroid-treated females were paired with control females, only the steroid-treated females showed threatening (tail rattling) and attack behavior (Table 6). Furthermore, the control females almost never fought back, attempting to run away instead; and in all cases where fighting occurred, the steroid-treated animal was the clear winner of the encounter. In contrast, when high- and a low-dose steroid-treated females were paired, threats and attacks
ANABOLIC STEROIDS AND BEHAVIOR
53
TABLE 4 MEAN (± SE) NUMBER OF SQUARES ENTERED DURING A 4 MIN EXPOSURE TO AN OPEN FIELD AND THE PERCENT THAT WERE IN THE TWO CENTER SQUARES AS OPPOSED TO THE 10 SQUARES BORDERINGTHE WALL Number of squares Percent in center
Control
Low Dose
High Dose
Prob.
94.3 ± 19.1 3.8 ± 1.2
46.5 ± 9.5 1.5 ± 0.08
38.0 ± 8.8 0.9 ± 0.7
< 0.01 0.06*
Treatment of Female Control
* Kruskall-Wallis test.
could come from either type of female, and the recipient of an attack almost always fought back. In only one case was there a clear winner at the end of the 10-min test period. Overall, there was little difference between the behavior of the females treated with the high vs. the low dose of steroids (Table 6). An interesting behavior typical of a potential sexual encounter rather than an aggressive encounter was also seen in some of these tests. Normally, when untreated female mice are exposed to a male, the male routinely inspects the female's ano-genital area. If the female is not in estrus, it will reject the male's inspection by kicking vigorously and repeatedly with the hind foot, often squealing while doing so. This sequence of behaviors was seen in almost all pairings of control and steroid-treated females. The steroid-treated female would attempt to investigate the control female's ano-genital area, and in response the control female would routinely show the rejection behavior described above. This behavior was only seen once in a steroid-treated female, despite the fact that both steroid-treated females in a paired encounter usually tried to inspect each other's ano-genital area (Table 6). DISCUSSION
The results of this experiment suggest broad effects of anabolic-androgenic steroids on the physiology and behavior of female mice. Starting with the physiological effects, the almost complete suppression of the reproductive axis that was seen here was expected. The negative feedback action of the gonadal steroids on gonadotropin secretion has been established experimentally for decades now (13), and the decrease in circulating levels of the gonadotropins that accompanies steroid abuse is well documented even in human athletes [e.g., (2)]. The effect of anabolic steroids on the uterus is of more interest. The control females were killed while in various stages of the estrous cycle, and thus, their average uterine weight represents a range from the small quiescent uterus characteristic of early diestrus to the large, ballooning uterus of late proestrus. The average weight of the uterus of the steroid-treated females was significantly greater than the average for the controls, and it was three to four times greater than the minimum level seen in the controls, despite the fact that the steroid-treated females were not cycling. This probably indicates a pathologic response of the
TABLE 5 NUMBER OF PAIRINGS IN WHICH FIGHTING OCCURRED AND THE MEAN (5: SE) LATENCY TO FIGHT IN THE PAIRINGS IN WHICH FIGHTING OCCURRED
Number pairs fighting/ number pairings Latency to fight (rain)
TABLE 6 MEAN (± SE) NUMBER OF THREATS(TAIL RATTLING) AND ATTACKSINITIATEDBY FEMALES OF THE THREE TREATMENTGROUPS, THE PROPORTIONOF THESE ATTACKS IN WHICH THE A T T A C K E D MOUSE FOUGHTB A C K (AS OPPOSED TO RUNNINGAWAY),AND THE MEAN NUMBER OF NONRECEPTIVE RESPONSES SEEN
Low vs. Control
High vs. Control
Low vs. High
Prob.
6/10
7/10
7/10
NS
5.0 ± 0.6
4.1 ± 0.9
3.1 ± 0.8
NS
Instances of tail rattling 0 Number of attacks initiated 0 Proportion of attacks fought back* 0.02 ± 0.02 Number of nonreceptive rejections 12.2 ± 1.6
Low Dose
High Dose
Prob.
4.9 ± 1.3
4.4 ± 1.3
0.009
5.8 ± 1.5
5.3 ± 1.3
0.002
0.92 + 0.08 0.96 ± 0.04
< 0.0001
0.7 + 0.7
< 0.0001
0.9 ± 0.6
The first two comparisons are based only on the pairings in which fighting occurred (tail rattling was never seen in pairings in which attack behavior was absent). * The proportion of times in which the attacked female fought back rather than running away.
uterine tissues, as suggested already by Yu-Yahiro and co-workers (32) in their studies with rats. There is a degree of confusion about the effect of the anabolic steroids on muscle mass and function in the absence of a training regimen in human males, and to the best of our knowledge, nothing is known about this subject in human females [reviewed by (12,29)]. Studies with rats and mice have tended to show either no effect or a decrease in muscle mass and body weight following treatment with anabolic steroids [e.g., (4,19)]. Again, to the best of our knowledge, little research has been directed at elucidating the effect of anabolic steroids on body fat, but the perception in humans is that it decreases in response to steroid use [e.g., (23)]. In the present study, treatment with anabolic steroids yielded a marked increase in both dry body weight and body fat, more so the latter than the former. The length of time that the fat reserves of a small mammal can protect against energetic insult is dramatically shorter than it is in the large mammal, being measured in hours or a few days at best [e.g., (7)], and, thus, different control mechanisms in small vs. large mammals probably shouldn't be too surprising. Interestingly, the increase in both dry body weight and body fat seen here in steroid-treated mice occurred in the absence of an increase in food intake and, indeed, relative to their body weight, the steroid-treated animals actually consumed less food than the controls. Little energy is required to maintain fat as opposed to muscle, but an increase in dry body weight without an accompanying increase in food intake is difficult to explain. There would seem to be two possibilities here. Either exposure to anabolic steroids induces a metabolic adjustment of some unknown kind that results in a lower food requirement or, alternatively, steroidtreated females simply expend less energy throughout the day in their home cages. The latter possibility seems of likely importance because the steroid-treated females ran only half as much as the control females when given the opportunity to use a running wheel. Complicating this picture even more is the fact that post hoc analyses suggested that access to a running wheel strongly antagonized the effects of steroids on body weight and composition, while having no such effect in control animals. This is in spite of the fact that the control animals spent much more time on their running wheels than the steroid-treated animals. All in all, the
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BRONSON, N G U Y E N A N D D E L A R O S A
present results suggest both a depression of locomotor activity and a specific antagonism of the steroidal effect on body weight and composition by exercise. In final regard to physiological effects, the only test of increased muscle capacity included in this experiment was the treadmill test, and it yielded negative results. While forced running decreased voluntary use of a running wheel markedly during the 12 h following exposure to the treadmill, there were no differences in this regard due to hormonal treatment. Behaviorally, exposure to anabolic steroids decreased activity in an open field, increased aggressiveness, and largely eliminated a specific response related to encounters between the sexes. In the first regard, two studies in male rats have shown no change in activity in an open field following treatment with anabolic steroids (5,19). In contrast, the present study with female mice showed a decided 50 to 60% decrease in activity. In theory, this decrease could be conceptualized as either an increase in timidity or a depression of locomotor activity. The marginally significant increase in wall hugging seen in the present study was also noted in one of the previously cited studies with male rats (19) and could argue for an effect on timidity. As noted earlier, however, a generalized, nonspecific depression of locomotor activity is suggested by a marked depression in wheel running. In any event, because locomotor activity is enhanced by estrogen in female rodents [e.g., (14)], the depression in activity seen here might be accounted for simply by a decrease in estrogen secretion, as suggested by the gonadotropin levels and ovarian weight of the steroid treated animals (Table 1). Alternatively, it might indicate a more central effect of the administered steroids. The dependence of aggression on testicular hormones in the male rodent is well established [e.g., (3,18,19)], and there is some documentation of an increase in aggressiveness due to anabolic
steroids in female rodents (25,27). The increase in aggressiveness seen in the present study with female mice was dramatic, as was the loss of a behavior normally associated with an encounter between the sexes--the stereotypical defensive reaction against inspection of the genitals shown by females not in estrus. None of the 20 control females showed any tail rattling, and none of them ever initiated an attack. Indeed, in only one instance in one control female was aggressive defense against an attack even seen. In sharp contrast, most steroid-treated females routinely threatened, attacked, and fought back when attacked. To the best of our knowledge, nothing is known about the effect of anabolic-androgenic steroids on aggressiveness in women athletes. The degree to which the present results with female mice are applicable to humans is also unknown. Even if not directly applicable, however, the effects seen here on locomotor activity and aggressiveness argue for changes in brain chemistry that might be generalizable to humans where, indeed, they could lead to behavioral manifestations quite different than those seen in mice. As a final comment, it is worth noting that throughout this study typically there was little difference in the effect of the two doses of anabolic steroids, regardless of the measurement being taken. This suggests a threshold effect for most of these responses below the low dose, which was the androgenic maintenance level for male mice. The present results, thus, suggest that anabolic steroids can produce physiological and behavioral effects in females at levels still lower than that which is physiological for males and below those thought to be used by some female athletes and body builders. ACKNOWLEDGEMENTS This research was supported by PHS Grant No. HD 30670.
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