Am J Physiol Regul Integr Comp Physiol 306: R118 R123, 2014. First published December 4, 2013; doi:10.1152/ajpregu.00372.2013.

TRANSLATIONAL PHYSIOLOGY

Effects of varying doses of testosterone on atherogenic markers in healthy younger and older men
Christian K. Roberts,1 Brian H. Chen,1 Sandeep Pruthi,1 and Martin L. Lee2
Exercise and Metabolic Disease Research Laboratory, Translational Sciences Section, School of Nursing, University of California, Los Angeles, California; and 2Department of Biostatistics, School of Public Health, University of California, Los Angeles, California
Submitted 5 August 2013; accepted in nal form 29 November 2013
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Roberts CK, Chen BH, Pruthi S, Lee ML. Effects of varying doses of testosterone on atherogenic markers in healthy younger and older men. Am J Physiol Regul Integr Comp Physiol 306: R118 R123, 2014. First published December 4, 2013; doi:10.1152/ajpregu.00372.2013. Whether exogenous testosterone is proatherogenic remains controversial. We assessed the effects of graded doses of testosterone on serum markers of oxidative stress, chemotaxis, adhesion, and inammation in healthy younger and older men. In a double-blind, randomized trial, 121 eugonadal men (n 61, 18 35 years of age and n 60, 60 75 years of age) were randomized to one of ve groups to receive weekly injections of 25, 50, 125, 300, or 600 mg of testosterone enanthate for 20 wk, respectively, along with a long-acting gonadotropin-releasing hormone (GnRH) agonist. Energy and protein intakes were standardized and no resistance training was allowed. We measured plasma levels of the atherogenic biomarkers monocyte chemotactic protein-1 (MCP-1), soluble intracellular adhesion molecule-1 (sICAM-1), 8-isoprostane-PGF2 (8-iso-PGF2), and high-sensitivity C-reactive protein (hs-CRP) before and after the intervention. Administration of increasing doses of testosterone led to reduction in total 8-iso-PGF2 in the younger (p-trendYounger 0.01), but not older (p-trendOlder 0.79) men. No signicant linear associations were observed between testosterone dose and MCP-1, sICAM-1, or hs-CRP (all p-trend 0.20). In apparently healthy men, over a wide dose range, testosterone did not adversely affect atherogenic biomarkers. Long-term studies with larger sample sizes are warranted to determine whether testosterone supplementation affects atherosclerosis progression and cardiovascular risk. atherosclerosis; androgen; inammation; oxidative stress; adhesion; chemotaxis

needed to examine whether testosterone supplementation indeed leads to an increased risk of CAD. Previously, dose-dependent decreases in high-density lipoprotein (HDL) have been reported with testosterone administration (6, 7). Although circulating lipid levels are a good predictor of atherosclerotic risk, recent attention has focused on risk factors such as oxidative stress, inammation, and endothelial cell activation. For example, levels of the oxidative stress marker 8-isoprostane PGF2 (8-iso-PGF2) (33), chemokine monocyte chemotactic protein (MCP)-1 (28), and inammation-associated proteins high-sensitivity C-reactive protein (hs-CRP) and soluble intracellular adhesion molecule (sICAM)-1 (34) all independently predict risk of cardiovascular disease or acute myocardial infarction. The effects of graded doses of exogenous testosterone on these atherogenic markers in apparently healthy men are poorly understood. Thus the primary objective of the present study was to determine the dose-dependent effects of testosterone on these atherogenic markers. To evaluate whether differences may occur by age, we examined younger (18 35 years) and older (60 75 years) apparently healthy men randomized to one of ve testosterone dose regimens after endogenous testosterone production was suppressed with a long-acting gonadotropin-releasing hormone (GnRH) agonist. Based on previously reported dose-dependent decreases in HDL with testosterone in these men (6, 7), we hypothesized that there would be a dose-dependent increase in atherogenic marker concentrations.
METHODS

MEN EXHIBIT

more extensive coronary artery disease (CAD) compared with age-matched women, and consequently there is a widespread perception that testosterone increases the risk of atherosclerotic heart disease. However, this premise is not supported by available data (1, 16, 22, 35). Epidemiological studies suggest that serum total and free testosterone concentrations are inversely correlated with risk of CAD (1). Prospective population-based studies have also not found an association between total testosterone and cardiovascular mortality (4, 41). Because these epidemiological studies examined steadystate testosterone levels, randomized intervention trials are

Address for reprint requests and other correspondence: C. K. Roberts, Exercise and Metabolic Disease Research Laboratory, Translational Sciences Section, UCLA School of Nursing, Los Angeles, CA 90095 (e-mail: croberts@ucla.edu). R118

Experimental design. As described in detail previously (6, 7), this was a double-blind, randomized study that included a 20-wk treatment period. Each participant provided written, informed consent, approved by the institutional ethics and research review boards of Charles R. Drew University of Medicine and Science and Harbor-UCLA Research and Education Institute and all of the study protocols were performed according to the Declaration of Helsinki. A Data Safety Monitoring Board (DSMB) reviewed safety data every 3 mo. In brief, the subjects were healthy, eugonadal men, 18 35 and 60 75 years of age, with normal testosterone levels. Men with prostate cancer, American Urological Association symptom score 7, prostate-specic antigen level 4 ng/ml, hematocrit 48%, diabetes mellitus, congestive heart failure, severe sleep apnea, or myocardial infarction in the preceding 6 mo were excluded. These men had not used any androgenic steroids in the previous year, including dehydroepiandrosterone and androstenedione, or other anabolic agents. Men who participated in resistance exercise training or moderate to heavy endurance exercise training, dened as 2 days/wk of structured
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exercise, were also excluded. Study enrollment ended in 2003 and biomarker assays were completed in 2004. One-hundred and twenty-one eligible men (n 61 between 18 and 35 years of age, and n 60 between 60 and 75 years of age) were assigned to one of ve groups using a block randomization scheme (block size 4) within each age category (6, 7). All men received monthly injections of a long-acting GnRH agonist (Lupron Depot, 7.5 mg, TAP, North Chicago, IL) to suppress endogenous testosterone production and weekly injections of one of ve doses of testosterone enanthate (200 mg/ml; Delatestryl, Savient Pharmaceuticals, Iselin, NJ): 25 mg (12 younger men, 13 older men), 50 mg (12 younger men, 12 older men), 125 mg (12 younger men, 12 older men), 300 mg (12 younger men, 13 older men), or 600 mg (13 younger men, 10 older men). Testosterone enanthate was selected to raise testosterone concentrations into the supraphysiological range. The 25-mg dose was chosen because this was the smallest dose of testosterone that had been shown to maintain sexual function in men treated with a GnRH antagonist. The 600-mg dose was selected because this is the highest dose that had been administered safely to men in clinical trials (5, 6). The General Clinical Research Center staff administered all testosterone and GnRH agonist injections to assure compliance. Subject retention. As reported previously (6, 7), of 61 younger men enrolled, 54 completed the study: 11 in the 25-mg group, 8 in the 50-mg group, 12 in the 125-mg group, 10 in the 300-mg group, and 13 in the 600-mg group. One man discontinued treatment because of acne and 6 subjects were unable to meet the requirements of the protocol. Sixty older men were randomized; of these, 52 completed all phases of the study: 13 in the 25-mg group, 12 in the 50-mg group, 11 in the 125-mg group, 10 in the 300-mg group, and 6 in the 600-mg group. Eight men did not complete treatment, 6 because of serious adverse events (3 receiving 300 mg and 3 receiving 600 mg) (7), 1 who was lost to follow-up, and 1 subject in the 600-mg group was discontinued when DSMB stopped this study arm. Subsequent subjects were randomized to one of the other four doses. All subjects received 100% of their GnRH agonist injections, and only 1 younger man in the 125-mg group missed 1 testosterone injection. Nutritional intake and exercise. Energy and protein intakes were standardized at 36 kcalkg1day1 and 1.2 gkg1day1, respectively. A standardized diet, including recommendations for 1520% protein, 50 55% carbohydrate, and 30% fat was initiated 2 wk before treatment was started; dietary instructions were reinforced every 4 wk. Additionally, the participants were asked not to undertake resistance training or moderate-to-heavy endurance exercise during the study. These instructions were reinforced every 4 wk.

Outcome measures. Serum total testosterone was measured periodically (7 days after injection, nadir testosterone level) throughout the study with a radioimmunoassay that used iodinated testosterone as tracer (5, 39) and reported previously (6, 7). This assay has a sensitivity of 0.44 ng/dl and intra- and interassay coefcients of variation of 13.2% and 8.2%, respectively (39). Plasma 8-iso-PGF2 was measured in duplicate using an enzyme immunoassay kit (Cayman Chemical, Ann Arbor, MI). Plasma sICAM-1 and MCP-1 concentrations were measured in duplicate with specic ELISA sets according to the manufacturers instructions (BD Biosciences, San Jose, CA). Serum hs-CRP concentration was measured in duplicate with a specic ELISA kit (Diagnostic Systems Laboratories, Webster, TX), according to the manufacturers instructions. CRP was previously determined among the younger men in this trial (38). Statistical analysis. The distributions of variables were evaluated for normality. Differences in baseline characteristics across intervention groups were tested using one-way ANOVA and the Fishers exact test using an exact calculation of P value for continuous and categorical variables, respectively. Changes in atherogenic biomarker levels were computed as the postintervention levels minus the preintervention levels. Generalized linear models were used to compute change in biomarker levels across levels of testosterone. We conducted two complementary analyses: 1) intention-to-treat, which analyzes the change in biomarker levels according to assigned treatment; and 2) per-protocol, which examines the change in biomarker levels in relation to change in blood testosterone levels (15, 21). We excluded potential leverage points by excluding individuals whose biomarker levels were greater than 3 standard deviations from that of the entire study population in our per-protocol regression analyses. We conducted stratied analyses by the two age categories. Differences in trend between younger and older men were tested using a two-way ANOVA. Statistical signicance was dened as P 0.05. All analyses were conducted using SAS 9.2 (SAS Institute, Cary, NC) and R software.
RESULTS

Participant characteristics. The baseline characteristics of the subject population have been described previously (7). Across the ve treatment groups of younger or older men, baseline characteristics did not differ signicantly, with the exception of baseline MCP-1 levels, which differed across intervention groups among the younger men (Table 1). Prein-

Table 1. Baseline characteristics by testosterone treatment group in younger and older men
Testosterone Dose, im Characteristic 25 mg 50 mg 125 mg 300 mg 600 mg P Value*

Younger men (ages 1835 yr) Number of subjects (n) Age, years White race/ethnicity (n, %) MCP-1, pg/ml sICAM-1, ng/ml 8-Iso-PGF2, pg/ml Testosterone, ng/dl Older men (ages 6075 yr) Number of subjects (n) Age, years White race/ethnicity (n, %) MCP-1, pg/ml sICAM-1, ng/ml 8-Iso-PGF2, pg/ml Testosterone, ng/dl

12 27 5 7 (58%) 427 83 535 238 98 19 603 145 13 64 4 12 (92%) 672 78 365 418 95 33 349 107

12 27 5 9 (75%) 702 92 429 263 93 21 533 207 12 65 4 10 (83%) 740 81 1,315 435 82 34 322 69

12 28 4 8 (67%) 517 80 685 228 84 18 562 188 12 66 5 9 (75%) 674 81 212 435 72 34 363 105

12 24 4 9 (75%) 870 92 140 263 87 21 607 207 13 67 4 11 (85%) 843 78 407 417 66 33 305 116

13 25 4 9 (69%) 559 80 128 228 116 18 606 216 10 66 4 7 (70%) 660 88 1,162 476 50 37 326 78

0.19 0.99 0.01 0.38 0.76 0.89 0.46 0.68 0.45 0.27 0.91 0.60

Values are means SD. MCP-1, monocyte chemotactic protein-1; sICAM-1, soluble ICAM-1. *P value for one-way ANOVA testing differences of each continuous variable across intervention arms. P value for Fishers exact test of race/ethnicity across intervention arms. AJP-Regul Integr Comp Physiol doi:10.1152/ajpregu.00372.2013 www.ajpregu.org

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tervention testosterone levels were signicantly higher (P 0.0001) among younger men (means SD: 585 189 ng/dl) compared with older men (333 97 ng/dl). Additionally, there was no signicant change in daily caloric, protein, carbohydrate, or fat intake in any group during treatment (data not shown). Intention-to-treat analysis of atherogenic markers. With the exception of 8-iso-PGF2, which appeared to be inversely associated with assigned testosterone dose among younger men (p-trend 0.01) (Fig. 1A), no clear relationship between changes in 8-iso-PGF2 (p-trendOlder 0.79) (Fig. 1B), MCP-1 (p-trendYounger 0.20, p-trendOlder 0.70) (Fig. 2), or sICAM-1 (p-trendYounger 0.68, p-trendOlder 0.24) (Fig. 3) were detected across assigned testosterone doses (p-trend 0.20). We also did not detect any differences in the association between assigned testosterone dose and mean levels of these biomarkers by age groups (P 0.20). Additionally, no linear relation between assigned testosterone dose and levels of hs-CRP was noted for older men (Fig. 4). Similarly, no dose effects were observed for hs-CRP in younger men, as reported previously (38).

Fig. 2. Mean changes (and standard deviations) in monocyte chemotactic protein-1 (MCP-1) from baseline to week 20 for younger (A) and older (B) men in response to graded doses of testosterone enanthate. No signicant differences in the linear trends were detected between younger and older men (P 0.21).

Per-protocol analysis of atherogenic markers. Largely conrming the intention-to-treat analysis, a borderline inverse association was observed among younger men between change in 8-iso-PGF2 levels and change in blood testosterone levels8-iso-PGF2 levels decreased by 0.01 pg/ml per 1 ng/dl increase in blood testosterone levels (P 0.06). Similarly, a borderline inverse association was also observed among younger men between change in MCP-1 levels and change in blood testosterone levels (0.04 pg/ml decrease per 1 ng/dl increase in blood testosterone, P 0.07). All remaining associations were not statistically signicant.
DISCUSSION

Fig. 1. Mean changes (and standard deviations) in 8-iso-PGF2 from baseline to week 20 in younger (A) and older (B) men in response to graded doses of testosterone enanthate. No signicant differences in the linear trends were detected between younger and older men (P 0.32).

The perception that testosterone level and testosterone supplementation increases the risk of atherosclerotic heart disease is controversial (1). Administration of high doses of exogenous androgens as well as abuse of orally administered, nonaromatizable androgens results in insulin resistance, dyslipidemia, impaired endothelium-dependent vasodilation, ventricular hypertrophy, and an enhanced tendency for thrombosis (1). Isolated cases of cardiovascular events in bodybuilders using

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Fig. 3. Mean changes (and standard deviations) in soluble ICAM-1 (sICAM-1) from baseline to week 20 in younger (A) and older (B) men in response to graded doses of testosterone enanthate. No signicant differences in the linear trends were detected between younger and older men (P 0.31).

extremely high supraphysiological doses of anabolic steroids have also been reported (40). On the other hand, emerging data indicate that testosterone may be protective against atherosclerosis progression and CAD risk (4, 41). Much of this evidence is associative and comes from cross-sectional, case-control, and longitudinal studies. Of 32 cross-sectional studies, 16 demonstrated lower serum testosterone level in CAD patients compared with controls, and the other 16 showed no differences between cases and controls (41). Furthermore, prospective population-based studies (1) have not revealed an association between total testosterone levels and cardiovascular mortality. To determine whether testosterone is proatherogenic, randomized controlled trials are necessary. In light of the aforementioned controversy, the present study attempted to provide insight using samples from a randomized, double-blind study (6, 7) to investigate the effects of graded doses of testosterone on serum concentrations of markers of oxidative stress, inammation, and monocyte-endothelial interactions in younger and older men. The present study provided assessment of androgen-induced atherogenic marker changes in the controlled setting of a clinical research center that allowed standardization of energy intake. Combined administration of GnRH agonist

and testosterone suppressed luteinizing hormone and consequently endogenous testosterone production; this minimized heterogeneity in testosterone levels due to uneven suppression of endogenous testosterone production by exogenous androgen. Thus circulating testosterone concentrations during treatment were proportional to the administered dose of testosterone enanthate (7). This study provides evidence that different levels of circulating testosterone concentrations created by this regimen did not signicantly affect 8-iso-PGF2, MCP-1, sICAM-1, or hs-CRP in apparently healthy men. Several lines of evidence support the concept that low testosterone, generally 400 ng/dl (25, 37) is associated with increased CAD risk. First, data from cross-sectional and longitudinal studies reveal that low testosterone is associated with increased body mass index, systolic blood pressure, serum triglycerides (23), total cholesterol, low-density lipoprotein (LDL), apolipoprotein B (37), small dense LDL particles (13), low HDL (43) and apolipoprotein A1 (37), thrombotic factors (low tissue plasminogen activator, elevated plasminogen activator inhibitor-1 and brinogen) (8), endothelial dysfunction (10), insulin resistance (12), visceral adiposity (36), diabetes (11), and metabolic syndrome (19). Additionally, Yang et al. (42) noted increased levels of CRP, interleukin-6, sICAM-1, and soluble vascular cell adhesion molecule-1 (sVCAM-1), as well as elevated carotid plaque area in men with low free testosterone. Longitudinal studies in men failed to reveal a signicant association between serum testosterone levels and future risk of CAD events (41). An inverse correlation between testosterone levels and the degree of coronary artery (9), abdominal aortic (14), or carotid (29) atherosclerosis has also been reported. Furthermore, it has been suggested that low testosterone predicts increased risk of cardiovascular (18, 20) and all-cause (18) mortality. A large body of evidence from intervention studies with testosterone supplementation reveal reductions in visceral fat volume, serum glucose, insulin, lipids, blood pressure (27), lipoprotein(a), prothrombotic factors (2), carotid intima-media thickness and CRP (3), leptin (17), and improved insulin sensitivity (26). Previously, the effects of graded doses of testosterone on regional adipose tissue distribution were examined in these healthy younger and older men (7). The changes in whole body and intra-abdominal fat mass were inversely correlated with testosterone dose and concentration. Whole

Fig. 4. Mean changes (and standard deviations) in high-sensitivity C-reactive protein (hs-CRP) from baseline to week 20 in older men in response to graded doses of testosterone enanthate. No linear trend was detected in the older men (P 0.25).

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body and truncal fat mass increased signicantly in men who received 25 and 50 mg/wk of testosterone enanthate and decreased in those who received 300 and 600 mg/wk. The present study extends these data to markers of atherosclerotic cardiovascular disease, and suggests that over a wide range of doses, testosterone administration does not affect 8-iso-PGF2, MCP-1, sICAM-1, and hs-CRP. Few studies have investigated the effects of androgen supplementation on markers of atherosclerosis such as oxidative stress, inammation, and endothelial cell activation. Ng et al. (31) reported that neither dihydrotestosterone nor recombinant human chorionic gonadotropin administration for 3 mo altered CRP, sICAM-1, or sVCAM-1 in healthy older men with partial androgen deciency. Nakhai-Pour et al. (30) noted that 26 wk of 160 mg of testosterone undecanoate daily to older men with androgen deciency did not alter CRP levels. Testosterone also had no adverse effect on insulin sensitivity at any dose in younger men, plasma HDL cholesterol decreased in the 600-mg dose groups (7, 38), but changes in other lipids were not signicant. The present study was unable to assess whether the route of administration and aromatization modulated testosterones effects on these atherogenic markers or contributed to lack of proatherogenic effects of testosterone. Nevertheless, these data, when taken together with epidemiological data (1, 4, 35, 41), suggest that serum testosterone levels in the normal range for healthy younger men do not negatively impact cardiovascular risk proles in younger or older men. This study has several notable features: 1) the rst direct comparison of the effects of various doses of testosterone on atherogenic markers; 2) use of two age groups, younger and older men; and 3) use of a GnRH agonist to minimize differences in effects of exogenous testosterone on suppression of endogenous testosterone production. Additionally, there are limitations that warrant consideration. It is possible that effects on atherogenic markers may take 20 wk to occur. It is not known whether testosterone-induced effects translate into cardiovascular risk because these results were obtained in the controlled setting of a short-term clinical research trial. However, the biomarkers examined in this study represented established predictors of cardiovascular risk. It remains to be determined whether clinically signicant anabolic effects of longterm testosterone administration can be achieved without adverse effects on cardiovascular event rates (6). Thus no claims about the long-term risks of testosterone replacement in a community setting in older men or men with higher cardiovascular risk can be based on these results. In addition, because testosterone possesses immunomodulatory properties (24) the effects of testosterone in men with existing CAD or other chronic inammatory diseases may differ from those noted in apparently healthy men. Because of the very limited data on the effects of testosterone on the biomarkers measured in the present study, further studies are needed to determine the long-term effects of testosterone on oxidative stress, inammation, and endothelial cell activation. The present study also was not designed to examine subphysiological levels of testosterone, so our results cannot be extrapolated to hypogonadal men who may require supplementation in the absence of a GnRH agonist. Finally, because of potential interactions with diet and physical activity patterns, future studies should assess the interactions of these lifestyle factors with testosterone on the markers investigated in this study.

Perspectives and Signicance Over a wide dose range, 20 wk of testosterone supplementation did not adversely affect atherogenic biomarkers in apparently healthy younger and older men. These data suggest that serum testosterone concentrations in the normal range for healthy younger men may not negatively impact cardiovascular risk prole for men at any age. Given recent data that suggests higher testosterone is associated with lower cardiovascular events, even after adjusting for traditional risk factors (32), long-term studies with large sample sizes are needed to determine whether testosterone supplementation affects atherosclerosis progression and cardiovascular risk.
ACKNOWLEDGMENTS The authors acknowledge Dr. Shalender Bhasin, to whom we are grateful to for kindly providing us with the blood samples, nancial support for the ELISA assays of 8-isoPGF2, MCP-1, sICAM-1, and hs-CRP used in this study (NIA, 1R01AG14369), and his blessing for this paper. We are also grateful for the contributions of the other members of the study team at Charles R. Drew University of Medicine and Science and Harbor-UCLA Research and Education Institute who helped make this paper possible. GRANTS C. K. Roberts was supported by the American Heart Association (BGIA no. 0765139Y) during writing of this paper. DISCLOSURES No conicts of interest, nancial or otherwise, are declared by the author(s). AUTHOR CONTRIBUTIONS Author contributions: C.K.R. design of paper; C.K.R., B.H.C., and S.P. interpreted results of experiments; C.K.R. drafted manuscript; C.K.R. and B.H.C. edited and revised manuscript; C.K.R., B.H.C., S.P., and M.L.L. approved nal version of manuscript; B.H.C. and M.L.L. analyzed data; S.P. performed biomarker assays. REFERENCES 1. Alexandersen P, Haarbo J, Christiansen C. The relationship of natural androgens to coronary heart disease in males: a review. Atherosclerosis 125: 113, 1996. 2. Anderson RA, Ludlam CA, Wu FC. Haemostatic effects of supraphysiological levels of testosterone in normal men. Thromb Haemost 74: 693697, 1995. 3. Aversa A, Bruzziches R, Francomano D, Rosano G, Isidori AM, Lenzi A, Spera G. Effects of testosterone undecanoate on cardiovascular risk factors and atherosclerosis in middle-aged men with late-onset hypogonadism and metabolic syndrome: results from a 24-month, randomized, double-blind, placebo-controlled study. J Sexual Med 7: 34953503, 2010. 4. Bhasin S. Effects of testosterone administration on fat distribution, insulin sensitivity, and atherosclerosis progression. Clin Infect Dis 37, Suppl 2: S142S149, 2003. 5. Bhasin S, Storer TW, Berman N, Callegari C, Clevenger B, Phillips J, Bunnell TJ, Tricker R, Shirazi A, Casaburi R. The effects of supraphysiologic doses of testosterone on muscle size and strength in normal men. N Engl J Med 335: 17, 1996. 6. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Bhasin D, Berman N, Chen X, Yarasheski KE, Magliano L, Dzekov C, Dzekov J, Bross R, Phillips J, Sinha-Hikim I, Shen R, Storer TW. Testosterone doseresponse relationships in healthy young men. Am J Physiol Endocrinol Metab 281: E1172E1181, 2001. 7. Bhasin S, Woodhouse L, Casaburi R, Singh AB, Mac RP, Lee M, Yarasheski KE, Sinha-Hikim I, Dzekov C, Dzekov J, Magliano L, Storer TW. Older men are as responsive as young men to the anabolic effects of graded doses of testosterone on the skeletal muscle. J Clin Endocrinol Metab 90: 678 688, 2005. 8. Caron P, Bennet A, Camare R, Louvet JP, Boneu B, Sie P. Plasminogen activator inhibitor in plasma is related to testosterone in men. Metabolism 38: 1010 1015, 1989.

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