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The effects of anabolic steroids on collagensynthesis in rat skeletal muscle and tendonA preliminary report

JARMO A. KARPAKKA,*† MD, MAIJA K. PESOLA,‡ MD, ANDTIMO E. S. TAKALA,* MD

From the *Department of Sports Medicine, Deaconess Institute of Oulu and ‡Department ofPhysiology, University of Oulu, Finland

ABSTRACT

We measured the activities of prolyl 4-hydroxylase andgalactosylhydroxylysyl glucosyltransferase (both en-

zymes of collagen biosynthesis) and the concentrationof hydroxyproline in male rat soleus muscle and Achillestendon during anabolic steroid treatment at 1 and 3weeks. The rats were treated using a therapeutic dos-age or a dosage that was five times the therapeuticlevel. After 1 week, the activity of prolyl 4-hydroxylasedecreased significantly (P < 0.01) in both treated

groups in the soleus muscle, but the activity of galac-tosylhydroxylsyl glucosyltransferase decreased signifi-cantly (P < 0.05) only in the group given a therapeuticdose. After 3 weeks, the activities were at the controllevel. In the Achilles tendon, the activity of prolyl 4-hydroxylase and the hydroxyproline concentration de-creased significantly (P < 0.05) in the group given highdoses at 3 weeks. Anabolic steroid treatment seemsto have at least a transitory effect on collagen biosyn-thesis in male rat muscle and tendon; in tendon thiseffect is seen only with high doses.

It is generally believed that the use of anabolic steroids toenhance athletic performance is widespread among ath-letes.4° The dose is usually self-administered and can be asmuch as 40 to 100 times above the therapeutic level.&dquo; Thereis still some dispute about the effect of these drugs on musclemass and strength.’ Analyzing the world literature on thesubject, Haupt and Rovere’ concluded that anabolic steroidswill consistently result in significant strength increase if

they are given to athletes who have had intensive strengthtraining before the drug intervention and who continueintensive strength training during the steroid regimen whilemaintaining a high-protein diet. Increased body size andweight were also related to increased muscle strength.On the other hand, anabolic steroids have been associated

with many undesirable side effects. The most commonlyreported psychologic effects are changes in libido and in-creased aggressiveness.’ There is also good documentationof the effects on many other parts of the body, such as theliver, the cardiovascular system, and the male and femalereproductive systems. Premature epiphyseal closure has alsobeen reported in youths.’

Little is known about the effects of anabolic steroids onconnective tissue. The collagen network of skeletal muscledistributes the forces of muscle contraction.21 A muscle isattached to its tendon at the end of the fibers, where thedifferent collagen types of muscle tissue fuse or interdigitatewith collagen fibrils of the tendon.’ Collagen is known toadapt to different levels of physical activity, 18,29 and itsbiosynthesis is coupled to muscular growth.18,33 Collagenbiosynthesis is characterized by the presence of a largenumber of posttranslational modifications of the polypep-tide chains that affect the quality and stability of the colla-gen molecule. Prolyl 4-hydroxylase and galactosylhydroxy-lysyl glucosyltransferase (GGT) are enzymes that catalyzetwo of these posttranslational modifications of collagen bio-synthesis. Activity levels of these enzymes generally increaseand decrease with the rates of collagen biosynthesis. Assaysof their activities have been used for estimating the rate ofcollagen synthesis in different experimental and physiologicconditions.14,15,18,29 Hydroxyproline is an imino acid exclu-sive to collagen. Its concentration is relatively constant incollagen and can be used as an indicator of collagen concen-tration.15

t Address correspondence and reprint requests to: Jarmo Karpakka, MD,Department of Sports Medicine, Deaconess Institute of Oulu, SF-90100 Oulu,Finland.

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Because there is a lack of information on the effects ofanabolic steroids on collagen biosynthesis, this study wasdesigned to examine the biosynthesis of collagen in rat soleusmuscle and Achilles tendon during anabolic steroid treat-ment using a therapeutic dOSage34,35,37 and a dosage that wasfive times the therapeutic level.

MATERIALS AND METHODS

Fifty-six adult male Sprague-Dawley rats, weighing 321 ±16 g, were randomly divided into seven groups of eight each.The four experimental groups received intramuscular injec-tions of nandrolone-decanoate (Deca-Durabolin, Organon,Oss, Holland) twice a week for 1 or 3 weeks. At 1 and 3weeks two groups were evaluated, one group had received atherapeutic dose (TD group) of 1.0 mg x kg per week andthe other group received a high dose (HD group) of 5.0 mgx kg-1 per week. This corresponded to 3 and 15 mg x kg 1in the groups examined at 3 weeks. Control groups wereevaluated at 0, 1, and 3 weeks. All controls received equalvolumes of arachidonic oil containing 10% benzyl alcohol.All rats received standard rodent chow (Astra-Evos, S6der-talje, Sweden) and water ad libitum. Exercise was confinedto cage activity.The rats were decapitated and the soleus muscle and

Achilles tendon from the right leg were excised and thesoleus muscle was weighed. Muscle and tendon samples werefrozen in liquid nitrogen and stored at -70°C until analyzed.The muscle and tendon samples were homogenized with

an Ultra-Turrax (Janke & Kunkel, Staufen, Germany) ho-mogenizer in two (muscle) or six (tendon) bursts of 5 secondsin a cold solution containing 0.2 M NaCI, 0.1% (muscle) or0.5% (tendon) wt/vol Triton X-100, 0.01% wt/vol soy beantrypsin inhibitor, 0.1 M glycine, 50 uM dithiothreitol, and0.2 M Tris-HCI buffer. This solution was pH adjusted to 7.5at 4°C. The homogenates (6% to 10% wt/vol) were centri-fuged at 15,000 x g for 30 minutes (muscle) or 11,000 x gfor 20 minutes (tendon) at 4°C and the supernatants takenfor the assays of the enzyme activities and protein concen-tration. Pellets were used for hydroxyproline analysis.

Assays

The assay for prolyl 4-hydroxylase was based on measure-ment of the labeled hydroxyproline formed from peptide-bound prolyl residues of unhydroxylated labeled protocolla-gen substrate. 14 Freshly isolated chick-embryo cells wereused to prepare [14C]proline-labeled protocollagen sub-strate. 15 Activity of GGT was assayed by determining theamount of radioactive glucosylgalactosylhydroxylysineformed in a heat-denatured, gelatinized calf skin collagensubstrate.25 Hydroxyproline content was measured using themethod of Kivirikko et a1.13 after hydrolysis for 16 hours in6 M HCI at 120°C. Protein content was assayed as describedby Bradford.5 The biochemical values are given per gram ofsoluble (supernatant) protein. The ratio of supernatant pro-tein to muscle wet weight was constant throughout theexperiment.

Statistical evaluation of the results were performed usingthe one-way analysis of variance and Student’s t-test.

RESULTS

Body and soleus muscle weights

There was a significant (P < 0.05) but transient increase inthe average group body weight in the TD group after 1 week(Table 1). After 3 weeks, the average group body weights inthe TD and HD groups were slightly below control value,but the changes were not significant. The average weightgain was significantly higher in the TD (P < 0.01) and HD(P < 0.05) groups after 1 week, but after 3 weeks the averageweight gain in the control group was higher than in the TD(no significance) and the HD (P < 0.01) groups. The averagewet weight of the soleus muscle in the TD and HD groupswas slightly, but insignificantly, above control level.

Prolyl 4-hydroxylase, GGT, and hydroxyproline in soleusmuscle

The specific prolyl 4-hydroxylase and GGT activities were36% (P < 0.01) and 27% (P < 0.05), respectively, below thecontrol level after 1 week in the TD group, and 38% (P <

0.01) and 32% (no significance), respectively, below controllevel in the HD group (Fig. 1). The values were at the controllevel after 3 weeks. There were no significant changes inhydroxyproline concentration.

Prolyl 4-hydroxylase, GGT, and hydroxyproline in Achillestendon

The specific activity of prolyl 4-hydroxylase was 29% (P <

0.05) below the control level in the HD group after 3 weeks.At the same time hydroxyproline concentration per wetweight was decreased by 13% (P < 0.05) in the same group.There were no significant changes in GGT activity (Fig. 2).

DISCUSSION

A transient increase in weight gain was observed after 1week in the treatment groups. The use of a large dose of

TABLE 1The effects of anabolic steroid treatment on body and muscle

weight in male rats (mean ± SD)

° C, control; TD, therapeutic dose; HD, high dose.’’P<0.05,’P<0.01,~P<0.001.

264

Figure 1. Effects of anabolic steroid treatment on the activitiesof prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glu-cosyltransferase (GGT) and 4-hydroxyproline (HYP) concen-tration in rat soleus muscle (S-M). PH activity is 10s dpm/gsoluble protein, GGT activity is 105 dpm/g soluble protein.Hyp concentration is milligrams per gram wet weight. 0,control; 0, therapeutic dose (TD); ~, high dose (HD). Theresults are mean ± SE from eight animals. *P < 0.05 (TDversus controls) , **P < 0.01, ++P < 0.01 (HD versuscontrols).

anabolic steroid for 3 weeks seemed to significantly diminishthe weight gain. In other studies, there has been either adecrease3,26,41 or no change2,26,39 in body weights of steroid-treated rats compared to the controls in male rats. Femalerats2,7,28 or castrated male rats 16 seem to have an increase inbody weight during treatment. Anabolic steroids are said tocause water retention, but its significance in body composi-tion and weight parameters is controversia1.3~9 It has beenshown that the effects of anabolic steroids on body weightcould be dose-dependent. Kochakian et al. 17 found that malerats treated with low doses of testosterone propionate had

Figure 2. Effects of anabolic steroid treatment on the activitiesof prolyl 4-hydroxylase (PH) and GGT and hydroxyproline(HYP) concentration in rat Achilles tendon (A-T). PH activityis 107 dpm/g soluble protein, GGT activity is 106 dpm/gsoluble protein. Hyp concentration is milligrams per gram wetweight. +P < 0.05. For details, see Figure 1.

increased weight gain, while those treated with high dosesdecreased. It seems that male rats lose some of their appetiteduring treatment.26,41 It is also possible that there is a greateruse of body fat during treatment,41 which could partiallyexplain the decrease in body weight.

It has been shown that anabolic steroids can promoteprotein synthesis at both transcriptional and translationallevels.&dquo; The anabolic steroid used in this study, nandrolone,has been shown to bind strongly to the androgen receptorsin rat skeletal muscle, the relative binding affinity beinggreater than that of testosterone.2’ We observed only a slightand statistically insignificant increase in muscle wet weightafter treatment with anabolic steroids for 1 or 3 weeks.Other studies have also failed to show increased wet muscle

weights in male rats.2,3,41 On the other hand, muscle wetweights have been shown to increase in female rats treated

265

with anabolic steroids.28,31,32 SalmonS28 has suggested thatall muscles are not equally responsive to anabolic treatment.He also suggested that anabolic effects might be manifestedonly in especially active muscles. The soleus muscle is anantigravity muscle, therefore it is constantly active duringcage activity.

Anabolic steroid effects have been suspected to be fiber-type specific with selective effects on fast-twitch muscles. 1,21The soleus muscle consists mainly of type I muscle cells. Inrabbits, the concentration of androgen receptors in slow-twitch soleus muscle has been shown to be similar to thatin fast-twitch gastrocnemius muscle when expressed per mgofDNA. 21 Witzmann3a found that anabolic steroid treatmentduring immobilization of the soleus muscle in a lengthenedposition increased the muscle weight significantly more thanimmobilization in the same lengthened position alone; dur-ing immobilization in a shortened position, anabolic steroidsdid not have any effect. Therefore, he concluded that ana-bolic steroids also have an influence on the slow-twitchmuscle fibers. But it is also possible that the degree of stretchmight have influenced the results to some degree.

Anabolic steroid treatment seemed to cause significantbut transient decreases in the activities of prolyl 4-hydrox-ylase and GGT in soleus muscle after 1 week. After 3 weeks,their activities were at the control level. In states of altered

collagen biosynthesis, the change in prolyl 4-hydroxylase isgenerally greater than that of GGT in skeletal muscle, 14,29,30as in this study. There was no change in the concentrationof hydroxyproline during this short experiment period, prob-ably because of the slow turnover of collagen, although thereseemed to be a significant decrease in its biosynthesis.The total collagen consists of the insoluble (mature) and

soluble (newly synthesized) collagen. The concentration ofnewly synthesized collagen constitutes only a minor portionof the total collagen,ll therefore it was not measured in thisstudy. However, the changes in the concentration of soluble,newly synthesized collagen have been shown to be relatedto the changes in prolyl 4-hydroxylase activity,&dquo;, 18 whichreflects the changes in the fractional synthesis rate. It is

also known that prolyl 4-hydroxylase activity in crude tissueextracts generally correlates with the in situ measured ra-dioactive proline incorporation into collagen hydroxypro-line. Thus, prolyl 4-hydroxylase activity has been used toestimate collagen synthesis in a number of experimental andclinical investigations.&dquo;, 14,15,18,29, 30,&dquo; No dose-dependencywas observed in the activities of these marker enzymes.

There are several studies on the effects of anabolic steroidsin tendons .22-24,39 Tendons consist almost entirely of type Icollagen,’ whereas in muscle, types I and III are the majortypes2° and types IV and V are also found. It was originallysuggested by Michna and Stang-Voss24 that anabolic steroidscould cause a predisposition to tendon ruptures. Clinicalreports also seem to confirm this.19 In his later studies,Michna22,23 has shown that anabolic steroid treatmentcauses alterations in collagen fibril architecture in tendonsby increasing the number of dysplastic collagen fibrils. Woodet al. 39 found biomechanical changes in tendons after treat-

ment that might also predispose the tendon to injuries. Inthe current study, a high-dose treatment seemed to cause adecrease in prolyl 4-hydroxylase activity and hydroxyprolineconcentration after 3 weeks, suggesting that anabolic ste-roids might have adverse effects on the biosynthesis ofcollagen in tendon.

In fact, these results contradict our hypothesis that col-lagen synthesis increases during anabolic steroid treatmentbecause fibroblast are shown to possess receptor machineryfor androgens. 4,11 Michna23 has found an anabolic steroid-induced increase in collagen by comparing the morphometricand stereologic results in female rat tendon. Again, as withmuscle tissue there could be a sex difference.

CONCLUSION

Anabolic steroid treatment may, at least transiently, de-crease collagen biosynthesis in muscle and tendon. In ten-don, the effects are seen with high doses. It must be kept inmind that athletes are known to use higher doses for longperiods of time. Because of the relatively short follow-upperiod in this study, definitive conclusions cannot be made.However, the trend seems to indicate that the possibility ofadverse effects of anabolic steroids on collagen in musclesand tendons must be taken into consideration. There is aneed to study the combined effects of anabolic steroids andexercise on collagen synthesis in an appropriate model.

ACKNOWLEDGEMENTS

This study was supported by the Ministry of Education,Finland. We thank Mrs. Marja Arbelius for her experttechnical assistance.

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COMMENTARY

Herbert A. Haupt, MD, St. Louis, Missouri: This paperemploys a very original approach to the study of the effectsof anabolic steroids on muscle and tendon. Investigating theeffects of steroids on markers of collagen synthesis in muscleand tendon would appear to be an excellent way of assessingthe potential adverse or beneficial effects of steroids to thesestructures. Unfortunately, the short experimental durationof just 3 weeks makes it difficult to really be comfortablewith the results presented. Other studies that have investi-gated muscle and tendon effects of steroids have been of atleast 6 weeks’ duration.As the authors note in their closing paragraph, other

animal studies that have shown profound changes in thetendons of experimentally treated animals also employed anexercise regimen. The combination of exercise and steroiduse seems to cause the most profound changes. I feel it wouldbe very important to assess the effect of exercise using thesecollagen synthesis markers in any future studies.Regarding the results of this study, only the Achilles

tendon demonstrated significant changes in collagen biosyn-thesis at the end of 3 weeks. In the soleus muscle, whilethere was a transient decrease in the collagen markers, thesehad returned to normal at the end of the study. Therefore,the study suggests a potential alteration in the collagenbiosynthesis only in the tendon model, not the muscle model.There is certainly question as to whether the changes in thecollagen markers in the tendon model may have been tran-sient, as they were in the muscle model. Only a study oflonger duration will clarify this.

In summary, while I feel this paper suggests some inter-esting trends, I do not feel that the short experimentalduration would allow this data to be considered truly mean-ingful. I would strongly suggest to the authors that theyrepeat the experiment using the innovative techniques thatthey have employed on this experiment, but investigatingthe effects over a period of at least 6 weeks. In addition, Iwould strongly suggest that they employ at least one animalmodel that included an exercise regimen to assess this effectas well.