Diuretics and other masking agents
Peptide hormones, growth factors, and related substances
Hormone and metabolic modulators
Enhancement of oxygen transfer
Chemical and physical manipulation
9.2 Substances Prohibited at All Times
9.2.1 Non-approved Substances
Athletes are warned against the use of any substances that are not registered (or with expired/lost registration) for human therapeutic use. This includes also agents under evaluation in clinical trials.
9.2.2 Anabolic Agents
184.108.40.206 Anabolic-Androgenic Steroids (AAS)
AAS are probably the most common performance enhancers easily available all over the world. They are used by both elite and recreational athletes. There is no doubt that individuals keen on extreme disciplines may misuse AAS as well.
It is estimated that 2.4 % of Australian students report lifetime AAS use , while in Sweden between 10,000 and 100,000 subjects may be exposed to AAS every year . Data from other countries and continents suggest that AAS users can be counted in millions [5–7]. The situation is emerging as a public health concern .
The most coveted by athletes effect of AAS is muscle hypertrophy. However, one cannot forget about side effects of these compounds. They are common and involve diverse body organs and systems [9–11]. One of the most prominent AAS effects in men is suppression of the hypothalamo-pituitary-gonadal (HPG) axis leading to decreased production of testosterone and spermatozoa. AAS abusers are at risk of acne, baldness, gynecomastia, cardiovascular diseases, lipid profile changes, liver tumors, and peliosis hepatis. Women may suffer from masculinization: decrease of the breasts size, changes in fat distribution and skin structure, hirsutism, losing scalp hair, deepening of the voice, and enlargement of the clitoris. Adolescents using ASS do not achieve the expected height due to premature epiphyseal closure.
AAS effects are not limited to the anabolic ones, but they exert also direct psychoactive actions. There is evidence that prone individuals may develop psychiatric dysfunction while using steroids. A number of studies link AAS abuse with increased risk of mania, anxiety, aggression, violence, or paranoia [12, 13].
Clinicians often observe AAS dependence in recreational or elite athletes. The long-term use of steroids, their higher doses, and greater dissatisfaction with body image are factors that increase such a risk. There are attempts to explain it by “myoactive” and psychoactive effects of steroid compounds . Another important issue is depression or suicidal attempts following the withdrawal of AAS .
AAS may modulate neurotransmission in concert with other drugs of abuse. AAS are, e.g., used concomitantly with opioids. It is interesting that animal models show that AAS overdose induces changes similar to those observed after opioids . Testosterone acts as a partial opioid agonist. AAS increase beta-endorphin levels in the ventral tegmental area and the thalamus. Nandrolone use is associated with decreased levels of kappa receptors in the nucleus accumbens and increased mu, delta, and kappa receptor binding in the hypothalamus, striatum, and midbrain periaqueductal gray [16, 17].
The classical pathways of AAS effects in the brain comprise androgen and estrogen receptors (alpha and beta) which are present in highest concentrations in basal telencephalon and diencephalon. The enzymes that play an important role here are 5α-reductase, aromatase, 3α-HSD, 3β-HSD, and 17β-HSD. AAS are thought to induce transcription and synthesis of new proteins .
Apart from genomic effects, AAS modulate kinase activity, ion channels, and G-protein second messenger systems. Some of these actions are much quicker than those induced through transcription factors .
Aggression is indicated as one of the most prominent behavioral traits in AAS abusers. It is observed even after discontinuation of AAS use. Animal studies prove that it can be attenuated by application of, e.g., selective serotonin reuptake inhibitors . AAS reduce the expression of serotonin receptors in the anterior hypothalamus (1A), globus pallidus (1B), or hippocampus [19, 20]. Anabolic-androgenic steroids decrease serotonin concentration in basal forebrain and dorsal striatum  but increase in the cerebral cortex . For example, methyltestosterone injections associate with rise in energy, sexual arousal, and shorter sleep. It is probably caused by elevation of serotonin within the cerebral cortex what was monitored by an increase of 5-hydroxyindoleacetic acid in the cerebrospinal fluid .
AAS are also likely to modify the mesolimbic dopamine system by stimulation of dopamine release and synthesis . It has been shown in human volunteers that nandrolone injections increase the serum levels of dopamine metabolite – homovanilic acid .
Another central neurotransmission system that is involved in AAS actions is GABA system. Androgen derivatives diminish concentration of GABA receptors and thus reduce fear in animals . It stands in line with observations of, e.g., male users of AAS.
220.127.116.11 Other Anabolic Agents
Clenbuterol is a beta-2 agonist used as a bronchodilator in asthma. It seems that dopers combine testosterone with clenbuterol more often than with GH, levothyroxine, EPO, or insulin . Animal studies showed that clenbuterol increases muscle mass and decreases fat deposits. Catabolism is reduced up to 18 % what leads to a raise of the total protein content by 6 %.
Due to the use of clenbuterol in fattened animals (the procedure forbidden in EU and the USA), there is a risk of positive anti-doping testing after consumption of contaminated meat.
9.2.3 Peptide Hormones, Growth Factors, and Related Substances
Erythropoietin (EPO) is responsible for the oxygen-carrying capacity of the blood. When it is used in therapeutic doses, it may increase red blood cells and hemoglobin by 6–11 % and lead to raises in VO2max. Sportsmen use EPO to increase aerobic power and endurance. The substance is applied as subcutaneous, intravenous, or intraperitoneal injections.
Compared with the first-generation recombinant human EPO, the second-generation products (e.g., darbepoetin) have extended half-life. Continuous erythropoiesis receptor activators (CERAs) are called the third-generation agents. The second- and third-generation EPO can be applied less frequently as their half-life reaches 140 h. A disadvantage of the newer forms of EPO – from the point of view of a doper – is longer period in which they can be detected. Cheating athletes may undertake training at higher altitudes or use altitude tents to mask EPO doping. The latter forms of performance enhancement are not forbidden. Thus increases of hematocrit could be attributed to the latter “procedures” rather than to doping with EPO.
Another problem of the recent years is availability of products biosimilar to EPO. Their structures and properties are different to the medically approved form, and they might not be detected by standard anti-doping tests .
Side effects of EPO comprise arterial hypertension, increased risk of arterial thrombosis, and venous thromboembolism but also flu-like symptoms (fever, arthralgias, muscle pains, conjunctivitis), skin allergic reactions, seizures, changes of serum potassium, urea, and phosphorus concentrations. EPO doping poses a special risk for dehydrated athletes, e.g., triathlonist or ultramarathon runners (a rise in the hematocrit can be augmented and reach even 80 %). One cannot exclude mitogenic effects of EPO when used in supraphysiological doses.
The first suspected cases of EPO doping were several cyclists who died suddenly in the late 1980s. The first documented darbapoietin dopers were four medalists of Salt Lake City 2002 Winter Olympics. The winner of the 2004 Hawaii Ironman Triathlon (Triathlon World Championship) Nina Kraft was stripped of the title by World Triathlon Corporation being found positive for EPO.
hCG stimulates synthesis of testosterone in the testes. It is not very popular, and it is used by male athletes only. The side effects are similar to those of AAS . There is no scientific rationale for the use of gonadotrophins as “protection” for gonads during AAS doping.
Growth hormone (GH), GH secretagogues, IGF-I, and its analogues began to gain their reputation in the sport world from the Los Angeles 1984 Summer Olympic Games. GH is desired for its anabolic and lipolytic properties. It is often applied together with AAS. In an anonymous American survey, 25 % of AAS buyers reported concomitant use of GH .
Our knowledge on GH effects is based mainly on studies in subjects with GH deficiency. In such cases, positive effects of GH administration on body composition and performance are well documented. GH is to increase VO2max and exercise time. What stands in contrast – in patients with acromegaly (a model of GH excess) – one finds reduced aerobic fitness and reduced left ventricle ejection fraction.
GH decreases body fat, increases cardiac output, and enhances wound healing. Observed effects of GH in muscles comprise:
↑ diameter of muscle fibers
↑ muscle protein content
↑ number of muscle cell nuclei
↑ glucose uptake
↑ protein synthesis
↓ muscle protein degradation
↑ myoblast proliferation
↓ myoblast apoptosis
The scientifical evidence for effectiveness of GH as a performance-enhancing agent in healthy individuals is poor. Athletes administer 3–8 mg of GH/24 h on 3–4 days of every week (mean daily dose of GH is 1–2 mg). It is 2–3 × higher than physiological pituitary secretion of GH . In one double-blind, placebo-controlled study in the elderly testosterone combined with GH in a higher dose was less effective in changing muscle strength than testosterone with a lower dose of GH.
One must keep in mind that prolonged use of GH/IGF-1 in high doses is associated with a range of serious side effects. Among the most typical ones are edema, muscle and joint pain, arterial hypertension, headache, vertigo, tinnitus, nausea, vomitus, gynecomastia, insulin resistance, goiter, and mitogenesis (colon cancer).
Anti-doping laboratories developed techniques to detect GH/IGF-1 abuse; however, it still poses a challenge .
9.2.4 Beta-2 Agonists
Beta-2 agonists are the first-line therapeutics in bronchial asthma. The evidence seems to exclude their ergogenic effects (if inhaled). For example, there was no improvement in 5 km time-trial performance following the inhalation of up to 1600 μg of salbutamol in non-asthmatic athletes . Nevertheless, it is intriguing that prevalence of asthma is several times higher in elite athletes (Olympic medalists) than in general population.
Dopers are supposed to use beta-2 agonists in doses exceeding recommended levels by several times. Agents such as salbutamol, salmeterol, and fenoterol applied in high doses increase glycogenolysis, lipolysis, and muscle contractility. They stimulate insulin and growth hormone secretion. Animal studies show that beta-2 agonists decrease degradation of proteins and stimulate muscle mass gain. Such effects have been not unequivocally confirmed in humans; however, it is suspected that beta-2 agonists may enhance muscle strength and endurance in mechanisms not elucidated yet. Typical signs of intoxication are headaches, vertigo, chest pain, dyspnoe, tremor, sweating, tachycardia, hypotonia, hyperglycemia, hypokalemia, and myocardial damage (leading to heart infarcts).
9.2.5 Hormone and Metabolic Modulators
The list includes aromatase inhibitors (e.g., anastrozole), selective estrogen receptor modulators (e.g., raloxifene), other anti-estrogenic substances (e.g., clomiphene), agents modifying myostatin function (e.g., myostatin inhibitors), and metabolic modulators (e.g., insulin).
Aromatase inhibitors (aminoglutethimide, anastrozole, letrozole, testolactone) inhibit the synthesis of estrogens from AAS or testosterone. They are registered for the treatment of breast cancer. They may stimulate LH secretion and further increase production of testosterone. Similar effects are observed during the application of clomiphene, which is used in ovulatory dysfunction in infertile women. Selective estrogen receptor modulators (SERMs) may behave as agonists or antagonists of the estrogen receptor (depending on the tissue). They oppose bone loss, and they are used to prevent osteoporosis in postmenopausal women.
Myostatin is a negative regulator of skeletal muscle mass. It is a member of transforming growth factor family. Animal and human observations indicate that mutations of the myostatin gene result in muscle hypertrophy. In the absence of myostatin, muscle fibers show hypertrophy, hyperplasia, changes of glucose, and fat metabolism. Myostatin inhibitors have a potential to be used by athletes to increase their muscle mass. Among such inhibitors one can find antibodies or proteins directed against myostatin. So far neither of these substances has been approved for the treatment of humans.
Insulin has potent anabolic properties. It acts synergistically with growth hormone and androgens. Insulin increases the uptake of glucose into adipose/muscle tissues and stimulates glycogenesis what improves postexercise recovery. Apart from the impact on glucose metabolism, insulin inhibits proteolysis and thus enables muscle mass gain. During the application of insulin, there are observed improvements of endurance. Tissue repair processes are facilitated as well. A dangerous side effect of insulin use is the risk of hypoglycemia. Athletes using insulin may experience hypoglycemia even long hours after its application. As a growing number of sportspersons use AAS, glucocorticosteroids, or GH, they may develop insulin resistance what in turn may require insulin therapy. There are some unanswered questions in regard to diagnosing and treating of diabetes in athletes (potential doping properties).