E-mail: andrebairros yahoo. Gene doping is characterized by non-therapeutic use of cells, genes and genetic elements, or modulation of gene expression with the aim to increase sports performance. This can only be accomplished through gene manipulation. This doping practice is characterized as virtually "undetectable", which represents new challenges for analytical detection.

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In the past few years considerable progress regarding the knowledge of the human genome map has been achieved. In vitro studies improve the production of human recombinant proteins, such as insulin INS , growth hormone GH , insulin-like growth factor-1 IGF-1 and erythropoietin EPO , which could have therapeutic application.

Unfortunately, genetic methods developed for therapeutic purposes are increasingly being used in competitive sports. Some new substances e. The use of these substances may cause an increase of body weight and muscle mass and a significant improvement of muscle strength.

At the same time, anti-doping research is undertaken in many laboratories around the world to try to develop and refine ever newer techniques for gene doping detection in sport. Thanks to the World Anti-Doping Agency WADA and other sports organizations there is a hope for real protection of athletes from adverse health effects of gene doping, which at the same time gives a chance to sustain the idea of fair play in sport.

With the development of science, athletes enjoy the more modern methods and pharmacological agents supporting their physical fitness, muscle strength and improving athletic skills. Now, with the completion of the Human Genome Project HUGO Project and the development of gene therapy in medicine, there has been dynamic progress of research on gene doping and gene delivery technologies to improve athletic performance in various sports.

In WADA clarified the type of manipulation of genetic material prohibited in sport as the transfer of nucleic acids or their analogues into cells and the use of genetically modified cells [ 9 ].

Genetic material can be introduced into a cell either in vivo or ex vivo. The in vivo strategy is direct gene delivery into the human body, i. In indirect DNA transfer strategy, i. In gene therapy and, similarly, in gene doping the genetic material is delivered into cells and tissues using various carriers that can be viral or non-viral [ 10 ]. Using viral vectors attenuated retroviruses, adenoviruses or lentiviruses a transgene is released in target cells and is expressed using cell replication machinery.

Some of these viruses, such as retroviruses, integrate their genetic material with chromosomes of a human cell. Other viruses, such as adenoviruses, introduce the transgene into the cell nucleus without chromosomal integration [ 11 ].

In some cases, however, irreversible side effects, such as unexpected endogenous virus recombination, may occur. It leads to the rapid transformation of normal cells in vitro as well as initiating tumours in vivo via amplification of the host proto-oncogene sequences in the viral genome [ 10 — 13 ]. Additionally, viral vectors can be recognized by the host immune system, resulting in an increased immune response.

This effect reduces the effectiveness of the transfection efficiency by reducing the efficiency of the subsequent transgene delivery. The most important biological properties of the viral vectors used in gene therapy, including the treatment of sports injuries, are shown in Table 1.

Non-viral gene delivery techniques are less effective methods of introducing genetic material into human cells, though characterized by low cytotoxicity. Non-viral gene delivery systems may cause an increased immune response [ 19 — 20 ]. Physical methods of gene delivery allow DNA transfer into the cell cytoplasm or nucleus, through local and reversible damage of the cell membrane.

The most common physical technique is electroporation, based on the application of a high voltage electrical pulse to the cells, leading to the formation of hydrophilic pores in the cell membrane, of several nanometres in diameter [ 15 ]. Electroporation is a very effective method, and one of its strengths is the protection of cells against the introduction of undesirable substances during the transgene delivery.

Nowadays, electroporation is the most frequently used method to introduce DNA into skin cells or liver cells. Biochemical methods involve the use of chemical carriers, which form complexes with nucleic acids to neutralize their negative charge.

Such complexes are introduced into the cell by phagocytosis, and less frequently by fusion with the cell membrane. Some of the chemical carriers facilitate the release of nucleic acid into the cytoplasm from the endosome, and protect it from cellular nucleases [ 15 ].

The main difficulty in the application of gene transfer in gene doping is to achieve a long-lasting effect, as well as monitoring the changes induced in the genome. A long-lasting effect can be achieved by multiple repeated gene doping applications or by the integration of a transgene into the chromosome.

However, it should be emphasized that the integration of gene transfer vectors is associated with a risk of undesirable side effects, including insertional mutagenesis. Integration of the transgene at the wrong site may lead to the development of cancerous cells [ 21 ]. Research on gene doping, which has been carried out mainly in animal models, but also more and more often as gene therapy in humans, has brought many successes. It was reported that injection of a plasmid with a vascular endothelial growth factor VEGFA gene into the muscle of patients with chronic critical limb ischaemia led to improved distal flow [ 22 ].

In rats, the introduction of the insulin-like growth factor-1 Igf1 gene in a recombinant viral vector led to an increase in muscle mass and strength and to increase in endurance [ 24 ].

Transfer of the phosphoenolpyruvate carboxykinase Pck1 gene resulted in increased activity of the transgenic mice, increased strength and speed during a race, and additionally, the mice were characterized by lower mass and fat content as compared to control mice [ 25 ]. In other studies on animals, gene therapy was used to increase the production of growth hormone GH.

By intramuscular injection of a plasmid containing the somatoliberin Ghrh gene, under the control of a muscle-specific gene promoter, increased concentrations of GH and IGF-1, and improvement of anabolic and haematological parameters were achieved. Moreover, the obtained results persisted for over one year [ 26 ]. The results of such studies are a major cause for concern over the direct threat of the spread of gene doping in competitive sports. Another problem — which is a priority for sport organizations — is the difficulty in detecting gene doping.

So far, the attempts to standardize the ideal test that could be used to detect gene doping have failed [ 5 — 6 ]. It should be emphasized, however, that several intensive studies on a number of promising strategies are being carried out e.

Lack of tests to detect gene doping is associated with the fact that the protein produced by the foreign gene or genetically manipulated cells will be structurally and functionally very similar to the endogenous proteins. Most transgenic proteins, especially those that enhance muscle strength, are produced locally in the injected muscle and may be undetectable in blood or urine.

The only reliable method would require a muscle biopsy, but such an approach is virtually impossible to use in sport. Furthermore, gene expression can be modulated as desired using the appropriate pharmacotherapy.

It is also associated with a high risk of danger to the health of athletes. Of course, the full list is much longer. Functional protein products of those genes are related to specific increase of endurance, physical strength, redistribution of fat or increase of muscle mass.

In addition, gene doping takes into account the genes encoding the peptides that relieve pain e. The EPO gene encodes a glycoprotein hormone that increases the number of red blood cells and the amount of oxygen in the blood, thereby increasing the oxygen supply to the muscles [ 29 , 43 ].

The expected effect of the physiological expression of the EPO gene would be increased endurance. For gene doping, an additional copy of the EPO gene may be introduced into the athlete's body using a viral vector, thus leading to the overexpression of EPO , increased production of red blood cells in the liver and kidneys, and to increased oxygen binding capacity of the blood. Physiologically dangerous side effects of doping with EPO transfer are primarily an increase in haematocrit, which may enhance the likelihood of stroke, myocardial infarction, thrombosis and an increase in total peripheral vascular resistance [ 29 ].

In , the British pharmaceutical company Oxford BioMedica developed Repoxygen as a potential drug for the treatment of anaemia associated with chemotherapy used in kidney cancer. The drug is administered intramuscularly, and consists of a viral vector transferring the modified human EPO gene under the control of genes encoding proteins of oxygen homeostasis e. EPO transgene is expressed in response to low levels of oxygen, and is turned off when the oxygen concentration reaches the correct value.

In , Repoxygen attracted the attention of the world of sports, when in Germany it began to be administered to young female runners to maintain constant expression of EPO in muscle cells.

Erythropoietin was the first recombinant haematopoietic growth factor produced and available commercially as a recombinant protein drug [ 30 ]. The Sydney Olympics marked the beginning of the use of effective methods to detect injected rEPO. This method would be relatively safe, because the effect of its actions would be limited to the target muscles.

It has been shown that overexpression of IGF1 and its protein product combined with increased resistance training induced greater muscle hypertrophy [ 24 ].

Additionally, studies have shown that IGF1 gene transfer enabled the regeneration of skeletal muscle following injury and was more efficient than systemic administration of its protein product [ 33 ].

IGF1 expression is associated with increased muscle size and weight, thereby increasing muscle strength [ 2 ]. However, IGF1 delivery may lead to profound hypoglycaemia, similar to the administration of insulin. IGF1 protein, also known as somatomedin C, belongs to the group of polypeptide hormones, which are essential for proper development of the fetus. In the mature organism it is involved in the regeneration of tissues, especially connective tissue, and also exhibits insulin-like activity, e.

IGF1 mediates some anabolic processes of growth hormone. One of the main functions of GH — mediated also by IGF1 — is the stimulation of body growth and body weight.

GH also affects carbohydrate metabolism stimulation of glycogenolysis and increased glucose release from the liver , fat metabolism increased lipolysis and decreased lipogenesis and protein metabolism increased protein synthesis [ 38 ].

There are only a few published reports confirming the enhancing effects of GH on muscle strength and cardiovascular and respiratory functions in trained healthy individuals [ 39 — 40 ]. On the other hand, evidence of the health risks associated with the use of GH e.

GH overexpression is associated with intracranial hypertension, headache, peripheral oedema, carpal tunnel syndrome, joint and muscle pain, or cardiomegaly in trained persons [ 41 ]. However, there is anecdotal evidence that recombinant GH rGH is commonly abused by athletes. The HIF-1 gene encodes proteins involved in the process of hypoxia, angiogenesis and erythropoiesis activation or regulation of glucose metabolism.

Doping associated with stimulation of HIF-1 expression under normal conditions of oxygen supply, e. On the other hand, it affects mitochondrial oxygen metabolism, while also stimulating genes associated with metabolic adaptation of cells e. These molecular changes in the cells may result in myocardial infarction, stroke or cancer.

HIF-1 regulates oxygen homeostasis, thus facilitating the cell's adaptation to low oxygen conditions. HIF-1 also affects erythropoiesis, iron metabolism, pH regulation, apoptosis, cell proliferation and intracellular interactions. Hypoxia itself regulates the expression of genes involved, among others, in cell energy metabolism, glucose transport and angiogenesis [ 42 ]. Thus, gene therapy using the HIF-1 gene or protein may result in physiological changes at many levels in the body.

Research is being conducted with the use of HIF-1 in the treatment of cardiovascular diseases. Based on the animal studies and early clinical trials in humans, it is believed that HIF-1 administered as gene therapy effectively induces neovascularization in ischaemic tissues [ 43 — 45 ]. Experiments have shown that the activation of PPARD reduces weight gain, increases skeletal muscle metabolic rate and endurance, and improves insulin sensitivity. It was further found that the increase in PPARD expression suppresses atherogenic inflammation [ 47 ].

Nuclear hormone receptor protein is associated with de novo formation of skeletal muscle fibres of type I slow-twitch fibres and their transformation from type II fibres fast-twitch fibres , which determine the athlete's endurance and speed. It controls the body's energy balance. Thus, this protein plays an important role in the control of body weight [ 50 ]. It is recognized that GW improves the exercise capacity of trained animals [ 51 ].

However, there are no published data on the ergogenic effects of GW in healthy and trained people. GW is an experimental drug that has been used in the treatment of obesity, metabolic syndrome, and type 2 diabetes in some clinical trials [ 52 ]. This molecule is included in the WADA prohibition list and there are reports that some athletes have already been caught using such doping.

Studies have shown that activated AMPK enzyme may reduce the level of anabolic processes, including synthesis of fatty acids and proteins, and increase the level of catabolic pathways such as glycolysis and fatty acid oxidation [ 53 ]. It is believed that the ergogenic effect is achieved by the mutual interaction between AICAR and training, and their effect on the activation of many genes, determining the exertion efficiency [ 51 , 54 ].


Dopaje genético: transferencia génica y su posible detección molecular

In the past few years considerable progress regarding the knowledge of the human genome map has been achieved. In vitro studies improve the production of human recombinant proteins, such as insulin INS , growth hormone GH , insulin-like growth factor-1 IGF-1 and erythropoietin EPO , which could have therapeutic application. Unfortunately, genetic methods developed for therapeutic purposes are increasingly being used in competitive sports. Some new substances e. The use of these substances may cause an increase of body weight and muscle mass and a significant improvement of muscle strength.


2007, Number 2

ISSN Genetic engineering has brought possibilities before unimaginable, in which not long ago was only seen in movies. Of gene therapy, aiming at a correction or cure a disease, pass the possibility of genetic improvement, currently glimpsed in the sports world with gene doping. But, gene doping would not be violating the right to genetic patrimony unmodified?

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