| Abstract |
In the present PhD thesis, the in-vivo toxicity of anabolic steroid, nandrolone on the cardiovascular system was studied. Specifically, we evaluated the effects of the prolonged use of the most commonly used injectable ester of nandrolone, (nan-drolone decanoate), on the cardiovascular system of the experimental animals, both at a macroscopic and cellular level. Furthermore, the possibility of reversible, or not, side effects from the anabolic substance after a time period of detoxification (wash-out period, 4 months) was additionally evaluated. To achieve the above, an ultra-sound scan as well as a histological estimation of the animal’s cardiac tissue was done. Besides the cardiological aspect, telomerase activity in peripheral blood mono-cytes and heart tissue, as well as measurements of biochemical indicators of oxida-tive stress in the blood were determined. To ensure these results, an analytical method was developed and applied to determine the parent compound and its me-tabolites in biological matrices (urine, hair) sampled from the study animals.
At the same time of the study, possible toxic effects on the kidney function was estimated conducting histological tests and measuring telomerase activity, oxi-dative stress indicators as well as kidney function biochemical markers.
Anabolic Androgenic Steroids (AAS) are synthetic derivatives of the natural hormone testosterone and exert two main functions, them being to promote the de-velopment of the male reproductive system and to enhance muscle growth. Although AAS was initially used in various situations as a medical treatment, non-medical use of AAS followed in order to improve athlete’s capabilities regarding weight lifting and sports that would require increased muscle strength. Νowdays, despite the fact that the AAS is a banned substance of W.A.D.A (World Antidoping Agency), they are still the leading group of positive results during doping control.
In the recent years, anabolic substance abuse has increased rapidly and has spread into areas beyond sports, such as for social (i.e. entertainment purposes in combination with alcohol) and cosmetic reasons (e.g. improved appearance, acquir-ing a muscular body type). Another interesting aspect is that the intake doses have increased over the years with doses taken by the athletes to be 10-100 times higher than the acceptable level needed for a therapeutic purpose.
Although nandrolone was synthesized in the early 1950s, it is one of the most used anabolic steroids in sports and clinically applied in catabolic states, such as se-vere burns, cancer, AIDS, aplastic anemia, chronic liver failure and osteoporosis. The chemical structure is similar to that of testosterone, with the only difference being the absence of the methyl group at position 19, thus characterized as 19-nortestosterone. Nandrolone is available either in the form of injectable esters, or as prohormones, administered orally. All forms of nandrolone are converted almost completely into phase I metabolites and excreted as conjugates of norandrosterone (3a-hydroxy-5a-estr-17-one, 19-NA) and noretiocholanolone (3a-hydroxy-5-estr-17-one, 19-NE).
The in vivo experiment consisted of rabbits of the same age and sex. The an-imals were divided into 4 groups: Control, High dose Intramuscular (HDIM), Low dose Intramuscular (LDIM) and High Dose Subcutaneous (HDSC). In the HDIM and HDSC groups the dose was 10 mg/kg and in the LDIM group the dose was 4 mg/kg, administered twice a week for a six-month period. The experimental scheme of ad-ministration was selected in such a way so as to simulate an athlete’s use. It consist-ed of two periods, the administration period that lasted six months and the period of detoxification (wash-out period) that lasted for four months. During the experiment, biological matrixes (blood, hair, urine) were collected at the start of the experiment and at the end of each two-month period of administration and detoxification. Sam-pled blood was used for estimating biochemical indicators, telomerase activity and oxidative stress markers, whereas urine was used to study the metabolism of AAS and hair was used to study possible deposition.
For the detection of nandrolone and its metabolites, in hair and urine, an ana-lytical method was developed based on liquid chromatography-mass spectrometry system (LC-MS). The analysis was conducted in a Discovery HS C18 (25cm x 4.6cm x 5μm) column, the mobile phase was gradient, consisting of 0.1% formic acid and methanol and APCI source was used. For the evaluation of the proposed methods, the following analytical parameters: linearity, accuracy, recovery, precision, LOD and LOQ were measured. The retention time for nandrolone was 14.93 min and the ex-amined ions were 275.15 and 307.25. The retention time for 19-NE was 16.13 min and the examined ions were 259.25 and 241.10. The retention time for 19-NA was 16.33 min and the examined ions were 259.15 and 277.15. The retention time for turinabol (I.STD) was 15.73 min and the examined ions were 317.73 and 335.25.
In hair samples, LOD of the method regarding the parent compound was 1.2 pg/mg, while for the two major metabolites 19-NE and 19-NA it was 2.8 pg/mg and 6.2pg/mg respectively. The LOQ for the parent compound was 4.0 pg/mg, while for the two major metabolites 19-NE and 19-NA were 9.3 pg/mg and 20.8 pg/mg, re-spectively. In hair samples, the average recovery of nandrolone was 92% and 119% for the metabolites. The accuracy of the method for nandrolone was 99%, while for 19-NE was 98% and 19-NA 102%. The % RSD in the hair samples was 14% for nandrolone, 29% for 19-NE and 17% for the 19-NA The linearity of the method was satisfactory for both the parent compound as well as for its metabolites (΄&γτ 0.99) in the concentration range studied (0-1000 pg/mg). In hair samples, only the parent com-pound was detected where the average concentration of the high dose group was 33,9 ± 5,9 pg/mg and the average concentration for the low dose group was 26,7 ± 1,0 pg/mg.
In the urine samples, LOD of the method regarding the parent substance was 0.9 ng/ml, while the two major metabolites, 19-NE and 19-NA were 1.8 ng/ml and 1.7 ng/ml respectively. The LOQ for the parent substance was 3.1 ng/ml, while the two major metabolites, 19-NE and 19-NA were 8.7 ng / ml and 5.5 ng /ml, respectively. The mean recovery for nandrolone was 75%, for 19-NE 101% and for 19-NA 114%. The accuracy of the method in urine samples for nandrolone was 104% and for 19-NE 93% and for 19-NA 98%. The % RSD was 27% for nandrolone, 25% for the 19-NE and 30% for 19-NA. The linearity of the method was satisfactory for both the par-ent substance as also for the metabolites (΄&γγγγτ 0.99) in the concentration range studied (0-1000 ng/ml). Urine samples of the exposed animals were found to be positive only to nandrolone metabolites with the high dose group having a mean concentration of 6.9 ± 4.0 ng/ml for 19-NE and respectively, the average concentration of 19-NA was 8.9 ± 4.5 ng/ml.
In the histological evaluation of the cardiac tissues, focal fibrosis and mild chronic inflammation in the high dose groups (HDIM, HDSC) was observed although mild focal fibrosis was also observed in the low dose group (LDIM). In addition to this, the HDSC group also had a noticeable edema. No statistically significant changes were observed in both the weight of the heart and the total weight of the animal body in which the anabolic substance was administered (p΄&γτ 0,05).
Animals that were exposed to the anabolic substance had a tendency for non-significant increased values of the myocardial mass (p=0.340), which was associated with the deterioration of the indices of total myocardial performance (MPI-PW: treated animals 0.73 ± 0.16 vs control group 0.52 ± 0.07, p = 0.026; MPI-TDI: treated ani-mals 0.91 ± 0.09 vs control group 0.63 ± 0.02, p = 0.001). The systolic function showed no change or trend for deterioration in the exposed animals. Moreover, ani-mals that received a high dose of anabolic substance showed a more pronounced increase in myocardial mass (myocardial mass-mmode: high-dose treated animals 6.0 ± 1.4 g vs low-dose treated animals 4.9 ± 0.31 g, p = 0.343) as well as more sig-nificant deterioration in the indices of total myocardial function compared with the low-dose group.
Measurement of oxidative stress markers (TBARS, carbonyls, TAC, catalase) showed a significant increase in the levels of TBARS (p ΄&λτ0,05) in the two high dose groups (HDIM, HDSC) and non-substantially reduction of catalase levels (p = 0,237, p = 0,238 respectively). Of particular interest is the fact that during the detoxification period, levels of TBARS and catalase returned to normal in the HDIM group but for the HDSC group, although the levels of catalase did return to normal, the level of TBARS increased (p = 0.01). In the low dose group (LDIM) levels of oxidative stress indicators remained practically constant. The levels of carbonyls and TAC did not show any significant differences in any treatment group.
Measurement of telomerase activity in heart tissues during the exposure peri-od significantly increased in all administration groups (LDIM 230% vs HDIM 552%, p = 0,004; and HDSC 212%). The intramuscular administration route may have con-tributed to the increased inflammation, as shown by the relative activity of telomerase in peripheral blood monocytes (HDIM 652% vs HDSC 312%, p = 0,003; LDIM 330% vs HDSC 312%, p = 0,20). During the detoxification period, all telomerase activity levels remained stable in the two high dose groups (p ΄&λτ0,05).
Measured levels of biochemical markers that are linked with cardiovascular function showed an inconsistent significant (p΄&γτ 0.05) increase in CPK levels in all administration groups (control: 332 ± 253 U / L, LDIM: 645 ± 442 U / L, HDIM: 1149 ± 733 U / L, HDSC: 461 ± 314 U / L) while a mild increase in LDH levels in the intra-muscular administration group was also noticed. A significant increase was observed for Troponin I levels in the HDSC group (p = 0,024) during the administration period. Elevated Troponin I levels were observed during the detoxification period for the aforementioned group as well as a sudden increase was observed in the BNP levels (7.3 ± 3.6 pg / mg).
Additionally, the measured levels of urea and creatinine in the serum showed an increase in all groups, with significant differences (p ΄&λτ0.05) only in the HD (urea: control: 13.8 ± 6.4 ng / dl, LDIM: 20.0 ± 5.7 ng / dl, HDIM: 21.6 ± 9.1 ng/dl, HDSC: 16.7 ± 7.3 ng / dl, creatinine: control: 0.37 ± 0.37 mg / dl, LDIM: 0.46 ± 0.27 mg / dl , HDIM: 0.55 ± 0.27 mg / dl, HDSC: 0.36 ± 0.26 mg / dl). Values of creatinine for the HDIM group decreased during the detoxification period by 20%, while values of urea significantly increased in the HDIM and HDSC groups (56%, p = 0,034 and 21%, p = 0,047, respectively) and non-significant changes occurred during the detoxification period.
There was also a change of oxidative stress indicators (TBARS, GSH) in kid-ney tissue in the HDIM and HDSC groups (control: TBARS: 17.7 ± 4.6 nmol / mg pro-tein, HDIM: 35.4 ± 7.2 nmol / mg protein, HDSC: 42 ± 29 nmol / mg protein, GSH: control: 0.138 ± 0.004 mol / mg protein, HDIM: 0.063 ± 0.020 mol / mg protein, HDSC: 0.098 ± 0.012 mol / mg protein). The levels of GSH decreased significantly (p = 0.018) by 50% in the HDIM group and by 29% in HDSC group (p = 0,046). During the period of detoxification, levels of oxidative stress markers practically remained unchanged with a non-significant (p΄&γτ 0,05) increased levels of TBARs and carbonyls in the HDSC group.
Measurements of telomerase activity in the renal tissues showed a significant increase only in the HDIM group (p = 0,020) throughout the administration period. During the detoxification period, relative telomerase activity decreased non-significantly in HDIM and HDSC groups (12% and 26%, respectively).
From the above results it appears that nandrolone is responsible for the dete-rioration in biochemical markers of renal function along with alterations in the kid-neys, presumably as a result of increased oxidative stress.
This protocol is an innovative research effort to study the side effects of the abuse of anabolic steroids as it examines diverse and innovative parameters so as to make an assessment of the extent and mechanism of nandrolone cardiotoxicity. In conclusion, prolonged use of nandrolone affects diastolic function, as shown by the indicators of total myocardial function. The increased levels of Troponin and BNP are an important indications of initial heart failure. The fact that there is an increase of Troponin during the detoxification period is a concerning sign for prolonged or de-layed toxic effects of the anabolic to the heart. In addition, histopathological findings demonstrate the local damage of heart tissue as well as that the increased activity of telomerase probably functions as a counteract mechanism due to the increase of ox-idative stress. Concluding, high dose groups are by far affected more compared to the low dose groups. The intramuscular administration of high doses seems to affect the cardiovascular system in relation to the corresponding low dose intramuscular administration. However, subcutaneous administration appears to cause the most invariant effects on the cardiovascular system.
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