Erythropoietin Abuse and Erythropoietin Gene Doping - Springer Link

Sports Med 2005; 35 (10): 831-840 0112-1642/05/0010-0831/$34.95/0

CURRENT OPINION

 2005 Adis Data Information BV. All rights reserved.

Erythropoietin Abuse and Erythropoietin Gene Doping Detection Strategies in the Genomic Era Evanthia Diamanti-Kandarakis,1 Panagiotis A. Konstantinopoulos,2 Joanna Papailiou,1 Stylianos A. Kandarakis,1 Anastasios Andreopoulos1 and Gerasimos P. Sykiotis3 1 2 3

Department of Medicine, Endocrine Section, Medical School, University of Athens, Athens, Greece Department of Medicine, SUNY Upstate Medical University, Syracuse, New York, USA Department of Biomedical Genetics, University of Rochester Medical Center, Rochester, New York, USA

Abstract

The administration of recombinant human erythropoietin (rhEPO) increases the maximum oxygen consumption capacity, and is therefore abused as a doping method in endurance sports. The detection of erythropoietin (EPO) abuse is based on direct pharmacological and indirect haematological approaches, both of which have several limitations. In addition, current detection methods cannot cope with the emerging doping strategies of EPO mimicry, analogues and gene doping, and thus novel detection strategies are urgently needed. Direct detection methods for EPO misuse can be either pharmacological approaches that identify exogenous substances based on their physicochemical properties, or molecular methods that recognise EPO transgenes or gene transfer vectors. Since direct detection with molecular methods requires invasive procedures, it is not appropriate for routine screening of large numbers of athletes. In contrast, novel indirect methods based on haematological and/or molecular profiling could be better suited as screening tools, and athletes who are suspect of doping would then be submitted to direct pharmacological and molecular tests. This article reviews the current state of the EPO doping field, discusses available detection methods and their shortcomings, outlines emerging pharmaceutical and genetic technologies in EPO misuse, and proposes potential directions for the development of novel detection strategies.

But when now they were running the last part of the course, straightway Odysseus made prayer in his heart to flashing-eyed Athene: “Hear me, goddess, and come a goodly helper to my feet.” So spake he in prayer, and Pallas Athene heard him, and made his limbs light, his feet and his hands above. But when they were just about to dart forth to win the prize, then Aias slipped as he ran – for Athene hampered him… So then much-enduring,

goodly Odysseus took up the bowl, seeing he came in the first, and glorious Aias took the ox.[1] This passage from the Illiad is likely the oldest documentation of an athlete winning a competition ‘with a little help from his friends’. The ancient Greeks indeed believed that the winner of an athletic contest was largely decided by the gods. But in our era, athletes determined to win at all costs do not rely solely on divine intervention; biotechnology

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offers them much more tangible options.[2] The abuse of recombinant erythropoietin (EPO) as a doping agent is an important problem for endurance sports, especially because new methods of EPO abuse are anticipated in the coming years. 1. Physiological Role, Therapeutic Use and Athletic Abuse of Erythropoietin (EPO) EPO is the primary regulator of erythropoiesis; it is a glycoprotein hormone produced initially in the fetal liver, and thereafter in the kidneys.[3,4] EPO acts synergistically with other cytokines to promote the proliferation, differentiation and survival of progenitor cells of the erythroid lineage, and thus boosts the production of red blood cells. EPO does not mediate the adoption of an erythroid fate by the early pluripotent stem cells; it acts later on colonyforming units erythroid cells to prevent their apoptosis and induce the expression of erythroidspecific proteins. Given its crucial role in physiology, it is not surprising that EPO was also the first haematopoietic growth factor to be cloned, no less than 20 years ago. Recombinant human EPO (rhEPO) is now widely used in the treatment of anaemia associated with various pathologies, such as chronic renal insufficiency, HIV disease, haematological malignancies, chemotherapy, and premature birth, as well as to minimise allogeneic blood transfusions after major surgical procedures. Interestingly, the biological activity of EPO extends well beyond erythropoiesis, and encompasses diverse physiological processes, ranging from angiogenesis (formation of capillaries from pre-existing blood vessels) and vasculogenesis (formation of capillaries from undifferentiated endothelial cells), to the regulation of vascular resistance, and even neuroprotection.[5] It is well established that the administration of rhEPO increases the body’s maximum oxygen consumption capacity, and thus increases endurance; this knowledge has prompted the misuse of EPO as a pharmacological ergogenic aid in endurance sports.[6] In addition to violating athletic ethical standards, this practice can cause serious adverse effects, including hyperviscosity, thrombosis and  2005 Adis Data Information BV. All rights reserved.

hypertension.[7] Although not yet documented in athletes, red blood cell aplasia with resultant transfusion-dependent anaemia has been reported in patients with renal insufficiency on long-term rhEPO treatment. In 1990, the International Olympic Committee (IOC) prohibited the use of EPO in sports. However, the definite proof of EPO abuse by an athlete remains challenging, for two reasons: (i) it has been extremely difficult to discriminate between the natural endogenous EPO and recombinant exogenous hormone; and (ii) EPO has a relatively short half-life (the half-life of rhEPO-a is 8.5 ± 2.4 hours when administered intravenously, and 19.4 ± 10.7 hours when administered subcutaneously), which also varies significantly among individuals. The half-life of endogenous EPO, and thus its effects, are largely dependent upon its glycosylation.[8] The removal of sialic acid residues from native EPO decreases its half-life significantly, due to its rapid clearance by the asialoglycoprotein receptor in the liver. It has been shown that chemical modifications that increase EPO half-life are able to overcome associated decreases in receptor affinity, leading to superior biological activity.[9] Therefore, the biological activity of EPO depends much more on its halflife than on its affinity for the EPO receptor. There are currently both direct and indirect methods to detect EPO abuse, but both of these approaches have inherent limitations. 2. Direct Methods of EPO Detection Approaches to directly detect EPO misuse rely on the physicochemical properties of the hormone. While the peptide portion of EPO has a stable amino acid sequence, the carbohydrate component is highly variable, a feature referred to as ‘glycosylation microheterogeneity’. The EPO molecule comprises three asparagine (N-linked) sugar chains, and a single serine/threonine (O-linked) sugar chain.[9] The N-linked carbohydrate chains may contain two, three or four branches, each of which is terminated with sialic acid, a negatively charged sugar molecule. The O-linked chain can comprise only two sialic acids. Therefore, endogenous EPO is actually a compendium of different isoforms that can have Sports Med 2005; 35 (10)

Erythropoietin Abuse in the Genomic Era

from 8 to 14 sialic acid residues, and as a consequence bear a different net negative charge. Similar to the endogenous hormone, recombinant EPO is also a pool of many different isoforms with different net negative charge. The total number of sialic acid residues has a major impact on the biological activity and pharmacokinetic properties of both endogenous and exogenous EPO. The basis of direct EPO detection is that the different carbohydrate components of recombinant and endogenous hormones confer different net electrical charges and thus distinguishable isoelectric points. This is the underlying principle of the only direct method of rhEPO detection that has been approved by the Court of Arbitration for Sport; this method was described by Lasne and de Ceaurriz in 2000.[10] It uses electrophoretic techniques to separate the isoform profiles of recombinant and endogenous EPO in the urine according to their isoelectric points. The Lasne test can detect various forms of rhEPO in urine samples, including rhEPO-a, rhEPO-b and rh-EPO-w. rhEPO-a and rhEPO-b are isolated from Chinese hamster ovary cells; they have minimal structural differences and comparable physiological effects. rhEPO-w is a sialoglycoprotein isolated from baby hamster kidney cells; it has different physicochemical properties but similar efficacy and adverse effects as rhEPO-a and rhEPO-b. Today there is no proof that the Lasne test is able to detect EPOω in human urine. However, it is true that this test is able to show the difference between the EPOω and the human endogenous EPO. Another recombinant EPO, termed ‘gene-activated EPO’ (GA-EPO) or rhEPO-d,[11] is produced in human cell lines by inducing the endogenous EPO gene. Since GA-EPO is not produced by renal cells, its glycolylation pattern will likely be different than that of the endogenous EPO. Therefore, Lasne’s method might prove able to detect GA-EPO, although no data on this matter have yet become available. Unfortunately, the short half-life of EPO makes it undetectable in the urine as soon as 3–4 days after an injection. As a result, the Lasne method will fail to detect EPO abuse if a urine sample is harvested >3–4 days after the last rhEPO injection. It is also challenging to use  2005 Adis Data Information BV. All rights reserved.

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this method to screen large numbers of athletes, because it requires highly trained technicians and standardisation between laboratories. 3. Indirect Methods of EPO Abuse Detection While direct detection methods aim to detect the doping agent itself, many indirect approaches have also been developed that attempt to infer doping from changes in haematological parameters. In addition to the Lasne urine test, athletes participating in the 2000 Sydney Olympic Games were subjected to a blood test that measured five haematological parameters:[12] (i) the haematocrit; (ii) the reticulocyte haematocrit (fractional volume of the reticulocyte pool in the bloodstream, which equals the product of the number of reticulocytes and their mean corpuscular volume – Ret × MCVRet); (iii) the percentage of macrocytes (macrocytic erythrocytes); (iv) the concentration of EPO; and (v) the concentration of soluble transferrin receptors in the serum.[13,14] These parameters were used to construct two statistical models for EPO misuse detection: (i) the ON model is indicative of the accelerated erythropoiesis that occurs during rhEPO use, and can thus identify current rhEPO users; and (ii) the OFF model was designed to differentiate between recent rhEPO users and non-users; it relies upon three parameters (low serum EPO, low reticulocyte haematocrit, and high haematocrit) consistent with the down-regulation of erythropoiesis that occurs following the discontinuation of rhEPO. Other indirect methods of EPO abuse detection that utilise various haematological markers have also been described, but are not considered as reliable and feasible as the ON and OFF multiparametric methods. These models are continuously being revised, and simpler secondgeneration blood tests have been proposed.[15] There are currently two ‘ON’ models: the ‘HE model’ (haemoglobin and serum EPO concentration), and the ‘HES model’ (haemoglobin, serum EPO concentration and transferrin soluble receptor), and two ‘OFF’ models: the ‘HR model’ (haemoglobin and reticulocytes) and the ‘HRE model’ (haemoglobin, reticulocytes and serum EPO concentration). Sports Med 2005; 35 (10)

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Unfortunately, these indirect detection methods have several limitations. Numerous factors can affect the measured haematological parameters, such as ethnicity, exercise, altitude exposure, and various diseases, thus creating elevated ON or OFF scores. Multiple common haematological conditions and inter-individual genetic variations associated with extreme haematological profiles can also obscure the specificity of these indirect detection methods.[16] For example, the Finnish cross-country skier Eero Mantyranta who won two gold medals in the 1964 Winter Olympics was later identified to have a mutation in the EPO receptor (EPO-R) gene that caused sustained activation of EPO signalling. Mantyranta’s oxygen carrying capacity was increased by 25–50%; if he were tested using the indirect methods, he would definitely have had elevated ON scores. Therefore, the current indirect detection tests are unable to differentiate naturally occurring genetic polymorphisms from EPO abuse. In addition to such rare cases of false-positives, it is also very likely that athletes abusing EPO may manipulate the administration scheme and dosage, in conjunction with close medical monitoring, to ensure that their haematological parameters will remain within levels ‘acceptable’ by the indirect detection tests, leading to false-negative results. 4. Novel EPO Doping Strategies: Analogues, Mimetics, Cell Encapsulation and Gene Doping Together with the limitations of current direct and indirect detection methods, novel EPO abuse approaches further threaten the integrity and credibility of athletic competitions. These novel doping techniques need to be appreciated in order to facilitate the generation of innovative detection methods that will be capable of addressing all methods of EPO misuse. 4.1 EPO Analogues

In pursuit of an EPO molecule with enhanced biological activity and extended half-life, novel EPO analogues have been generated that can potentially be used in doping. It has been demonstrated  2005 Adis Data Information BV. All rights reserved.

that the number of sialic acids in the EPO molecule correlates positively with its serum half-life and in vivo biological activity, and negatively with its affinity for the EPO receptor.[9] Based on this finding, additional N-linked carbohydrate chains were added to the EPO molecule, creating darbepoetin-α (also known as novel erythropoiesis stimulating protein [NESP]), which contains five (instead of three) Nlinked sugar chains. Darbepoetin can have up to 22 sialic acid residues (as opposed to 14 of rhEPO), and thus has an extended half-life (48.8 + 5.2 hours when administered subcutaneously). Although its affinity for the EPO receptor is reduced, darbopoetin has increased biological activity, presumably because the increase in serum half-life overcomes the handicap in receptor binding. The resulting clinical advantage of darbepoetin is that it can be administered once per week, whereas rhEPO is injected 2–3 times per week. Importantly, since NESP has a different glycosylation profile and thus a different electrical charge from endogenous EPO, it can be unequivocally detected using Lasne’s method.[17] Another EPO analogue that could be used for doping is synthetic erythropoiesis protein (SEP).[18] This agent possesses a potent biological activity and a significantly prolonged duration of action. SEP consists of a polypeptide chain similar to that of EPO and two covalently attached, branched polymer moieties that bear a total of eight negative charges. These polymers enhance the molecule’s stability by protecting it from proteolytic cleavage. Importantly, SEP is less immunogenic than recombinant EPOs because it is chemically synthesised and is therefore free of contaminating antigens. Although it is currently unknown whether the unchanged SEP molecule exists in the urine, it has been demonstrated that SEP can be detected by the Lasne method.[18] 4.2 EPO Mimetics

Hormone mimicry is another novel approach of generating EPO substitutes.[19] The costs and difficulties associated with chronic parenteral EPO administration prompted extensive research into the development of oral EPO analogues. EPO mimetics are small molecules capable of activating the EPO Sports Med 2005; 35 (10)


receptor in a similar way as EPO.[20] Upon binding to a specific domain of the EPO-R, EPO and EPO mimetics cause the receptor to dimerise and activate multiple cellular signalling pathways; ultimately, multiple genes are transcriptionally induced to mediate the proliferative, antiapoptotic and erythopoietic effects of EPO.[21] In developing novel EPO mimetics,[22] initially the peptide domains of the EPO molecule that mediate activation of the EPO-R are identified, and subsequently non-peptide smallmolecule analogues of these domains are synthesised. Such analogues must be resistant to proteolytic digestion and possess good membrane permeability, so as to be suitable for oral administration.[23,24] Although no oral EPO mimetic is currently commercially available, significant advancement has been made towards this goal. Hematide 1 is a synthetic dimeric peptide that has no sequence identity to natural human EPO but is a potent stimulator of erythropoiesis with prolonged serum half-life.[25] Antibodies generated experimentally against the Hematide peptide do not cross-react with recombinant human EPO, and therefore Hematide is unlikely to cause aplastic anaemia. Pharmacokinetic studies in rats, dogs and monkeys have demonstrated the extended plasma half-life of Hematide. The erythropoietic activity of Hematide has been demonstrated in normocythemic mice, rats, dogs, and monkeys, and in nephrectomised rats. No antibodies to Hematide were detectable in these preclinical models. Hematide has now entered phase I clinical trials; it is anticipated that its unique pharmacokinetic properties will permit dosing intervals of 3–4 weeks. An alternative approach of augmenting the action of EPO mimetics and endogenous EPO[22] could be the inhibition of haematopoietic cell phosphatase (HCP), an indigenous negative regulator of the EPO-EPO-R signalling cascade.[26] It is possible that the combination of EPO mimetics with HCP inhibitors could provide an oral substitute of endogenous EPO with equivalent potency. This field of research into oral EPO agonists with equivalent potency to EPO has clearly important clinical and commercial 1

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implications, but at the same time the possibility of their illicit use for doping should not be overlooked. 4.3 EPO Delivery by Cell Encapsulation

The transplantation of genetically engineered cells as a method of systemic EPO delivery has also been investigated.[27,28] In this method, the modified cells are enclosed inside semi-permeable membrane polymers that isolate the encapsulated cells and thus prevent antigen recognition and immune rejection. Cell encapsulation as a form of immunoprotection has been shown to enhance erythropoiesis in healthy subjects.[28] Rinsch et al.[29] have successfully demonstrated the efficacy of this method of EPO delivery in a mouse model of severe anaemia that reflects the blood haematocrit levels found in patients with chronic renal failure. 4.4 EPO Gene Doping

The potential use of gene doping to enhance athletic performance is another major challenge to current anti-doping strategies. Gene doping is defined as the transfer of genetic material to improve athletic performance.[30] In 2003, the IOC and the World Anti-Doping Agency incorporated gene doping into their list of prohibited practices. Multiple approaches to EPO gene transfer have been described.[30,31] The two principal strategies are either in vivo gene transfer through intramuscular injection of a virus containing a gene encoding EPO,[31] or ex vivo gene transfer into cells that are subsequently transplanted into the recipient organism. The feasibility and effectiveness of these approaches has been clearly demonstrated. Svensson et al.[31] reported that a single intramuscular injection of an adenovirus encoding EPO into mice resulted in elevation of haematocrits from 49% to 81%, and these effects persisted after 1 year. Similar results were reported in monkeys (haematocrit elevations from 40% to 70%, stable for 84 days). In addition to viruses, other possible vectors for in vivo gene delivery include plasmid DNA, liposomes, and protein-DNA conjugates; alternatively, direct injec-

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tion of EPO gene into the muscles could also be effective.[32] The major challenge in EPO gene therapy (and doping) is the ability to modulate the expression of the transgene. Continuous unregulated expression of the EPO transgene would likely cause toxic phenomena from excessive erythropoiesis, such as thrombosis, hypertension, hyperviscosity and heart failure. Modulating transgene expression is an area of extensive research, and many regulation systems have been proposed. Some rely on the additional administration of a small molecule that induces the expression of the EPO transgene.[33] A mifepristonedependent GeneSwitch system can regulate the expression of transgenes encoding vascular endothelial growth factor and EPO in mice.[34] Another system relies on the administration of rapamycin to reconstitute two chimeric, human-derived proteins into a transcription factor complex in mice and monkeys.[35] Repoxygen is a recently developed drug that consists of a viral gene delivery vector carrying the human EPO gene under the control of a hypoxia response element.[36] The drug is administered intramuscularly, and the expression of the EPO transgene can then be switched on in response to low oxygen levels. This strategy aims at inducing controlled release of EPO in response to low oxygen levels, leading to a self-regulated production of red blood cells that mimics the natural regulation of erythropoiesis in the kidneys. Preclinical unpublished data suggest that this method is successful in mouse models of anaemia.[36] Although repoxygen would probably be unsuitable for doping, its development provides the proof of principle for selfregulated EPO gene transfer methods. The time to clinical application of these models of EPO gene therapy is unknown; however, experts on gene therapy and sports organisations alike consider that gene doping may be a reality at the 2008 Olympic Games.[37] Apart from the health risks resulting from excessive haematocrit levels, the general clinical safety issues of gene therapy are not yet completely resolved.[38] It is possible that the integrated gene may affect the organism in unexpected ways, for example, by mutating genes or regulatory  2005 Adis Data Information BV. All rights reserved.


sequences at the site of its insertion into the host genome. Another major concern in gene therapy is that effectiveness may diminish over time due to the innate immune response, and thus more frequent administrations of the transgene may be required; without close medical surveillance, this might further complicate the safety issues of gene doping. 5. Strategies for Detection of EPO Analogues and Mimetics and EPO Gene Doping The impending advent of EPO analogues, mimetics and genes to the doping arena, necessitates that direct pharmacological approaches and indirect markers of erythropoiesis be supplemented with novel molecular anti-doping methods. The development of such strategies could take advantage of the differential molecular profiles and gene expression patterns associated with cellular exposure to EPO. 5.1 Direct Methods

Direct detection methods for EPO abuse can be either pharmacological approaches that identify exogenous substances based on their physicochemical properties, or molecular methods that recognise the gene transfer vectors or the transgenes themselves. EPO mimetics and HCP inhibitors are xenobiotics, and therefore direct pharmacological tests that detect their presence in the blood or urine could be developed, provided that the structure of these drugs is known to anti-doping authorities. However, there are important obstacles to this optimistic scenario. First, the rate of production of such substances can probably outreach the ability of anti-doping authorities to identify them, list them as prohibited, and develop direct tests to detect them. Secondly, some of these novel agents may have a very short half-life but a substantial duration of biological action; in that case, their rapid clearance from the serum and urine could hamper direct detection approaches. Although EPO analogues are also xenobiotics, they bear very similar peptidic and carbohydrate structure to the endogenous hormone, thus compromising the ability of direct pharmacological tests to distinguish between them. rhEPO-d (GA-EPO) and Sports Med 2005; 35 (10)


SEP, which can mimic the glycosylation of endogenous EPO and possess almost identical net negative charges, are examples of EPO analogues whose direct detection may prove extremely challenging. It can also be envisaged that the future development of EPO analogues that are nearly or completely identical to endogenous EPO will surpass the discriminating ability of direct pharmacological tests. EPO gene doping detection using pharmacological approaches is challenging. Since the glycosylation of EPO depends on the cell where it is produced, EPO gene doping could be detectable. In this vein, it has been demonstrated that recombinant EPO from genetically engineered muscle is not identical to the endogenous hormone and can be detected via Lasne’s method.[39] On the other hand, although skeletal muscle is an easily accessible and efficiently transduced tissue, this does not eliminate the possibility that other tissues can be targets of gene transfer strategies for doping purposes. There are currently no data about whether these gene transfer strategies (where the skeletal muscle is not the target) could be detectable via direct methods. Alternatively, molecular approaches like the direct detection of the artificial transgene itself could lead to unequivocal proof of EPO gene doping. In turn, this requires that the sequence of the transgene is known and that a sample of the tissue that contains it can be obtained. A major disadvantage of such an approach is that it would be invasive, involving muscle or other tissue biopsies, which will likely be unacceptable to athletes. Thus, such methods might be employed as a last resort, when doping is strongly suspected but remains unproven by other methods. Importantly, the direct molecular detection of gene doping has an important advantage over indirect approaches: since the transgene is integrated into the athlete’s genome, its detection will not be limited to a narrow window of time, as is the case after an EPO injection, for example. If invasive techniques can be employed, gene doping could also be exposed by the direct detection of the vector (plasmid and viral vectors, liposomes and protein-DNA conjugates) that were used as the gene delivery system. Another option would be to label all EPO gene transfer  2005 Adis Data Information BV. All rights reserved.

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products with genetic ‘barcodes’; this solution would definitely facilitate the direct detection of an EPO transgene. Genetic barcodes are characteristic 20 base pair-long fragments of DNA that can be inserted into gene transfer products.[40] These barcodes could then be subsequently detected using molecular approaches,[40] and thus would serve as proxies for the EPO transgene itself. Obviously, this detection approach would require the cooperation of multiple authorities and parties including government agencies, scientists, athletes and ethicists. 5.2 Indirect Methods

In the era of the biotechnology revolution, EPO misuse detection cannot rely solely upon direct pharmacological approaches. Direct detection based on genomic methods is definitely an exciting alternative, but requires invasive approaches and is therefore not appropriate for routine screening of large numbers of athletes. Instead, novel indirect methods could be better suited as screening tools. Athletes who are suspect of doping according to these indirect methods could then be submitted to the direct pharmacological and molecular tests, which would then serve as unequivocal evidence of EPO abuse. The currently available indirect methods (ON and OFF models) can be suggestive of EPO gene doping, but may be unable to differentiate gene doping from naturally occurring genetic polymorphisms. An alternative approach could be the implementation of the ‘haematological passport’.[41] According to this concept, the haematological parameters can be monitored sequentially for all athletes, and thus subject-specific reference ranges can be defined for the haematocrit and haemoglobin of each athlete. It is estimated that five determinations are required to reliably define such subject-specific reference ranges.[41] Athletes who have not used doping and have normal baseline haematological parameters have a width of fluctuation of haemoglobin and haematocrit values of 5%. Increases in haematocrit or haemoglobin >10% should be considered abnormal, and in this case athletes should be excluded from competition and regarded Sports Med 2005; 35 (10)

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as suspect of doping. Since this method creates reference range for each athlete (a personal haematological ‘passport’ or ‘identity’) it can obviously detect EPO abuse even in cases of athletes who possess uncommon genetic mutations/polymorphisms, or experience common or rare haematological conditions. It should be noted, however, that the haematological passport can only be implemented for athletes who have normal baseline haematological parameters. Similar to EPO abuse detection by haematological parameters, EPO doping detection by genomic approaches can be obscured by significant individual variations in the expression levels of EPO target genes and interferences with altitude stay and/or diseases. To address this problem, individual ‘molecular passports’ could be created. Sequential determinations of the expression levels of certain EPO target genes could define athlete-specific reference ranges for the level of expression of these genes. Athletes with gene expression levels above or below their personal range would be considered suspicious for doping. Alternatively, the expression levels of these genes could be weighed and ON and OFF models predicting EPO abuse could be constructed. To identify sets of genes whose expression is modified after EPO treatment, expression profiling by DNA microarray analysis could be employed. DNA microarrays can simultaneously evaluate the level of expression of thousands of genes, based solely on sequence information.[42] With this methodology it was documented that exposure of erythroid progenitor cells to EPO leads to up-regulation of 54 genes and down-regulation of 36 genes.[43] Quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) can be used to quantify the transcript levels of such genes. The peripheral blood mononuclear cells (PBMCs) contain erythroid progenitors expressing EPO-R,[44] and could therefore be used as a surrogate tissue to assess the transcriptional effects of EPO exposure. Because PBMCs are readily accessible, their gene expression profiles have been extensively mined to identify biomarkers of exposure to drugs,[45] as well as dis 2005 Adis Data Information BV. All rights reserved.


ease biomarkers (especially in oncology).[46] This approach has been successful even in cases where PBMCs are not the direct target tissue of the drug or disease (most notably, in the case of renal cell carcinoma).[46] Such methods might be able to detect changes in EPO target genes resulting from the inducible expression of EPO transgenes, thus differentiating gene doping from naturally occurring gene polymorphisms, which should not be associated with significant fluctuations in gene expression levels. Moreover, the EPO gene could be a prime candidate for inclusion into such profiling strategies. Increased EPO transcript levels would be anticipated in cases of EPO gene doping, and suppressed levels could result from the administration of EPO analogues and mimetics. Finally, in the case of inducible transgenes,[34] exposure of cells derived from muscle biopsies to inducers like mifepristone should lead to detectable expression of the EPO transgene and its targets, providing indirect but clear evidence of EPO gene doping. 6. Conclusion The misuse of biotechnology methods to enhance athletic performance provides athletes with novel EPO abuse strategies that will be virtually undetectable by current approaches in the future. EPO analogues and mimetics and EPO gene doping may be a reality in the next Olympic Games, and anti-doping authorities should be prepared to cope with this unethical and dangerous malpractice. Innovative genomic approaches can serve as powerful tools to detect (and thus also deter) EPO misuse, as well as the abuse of other hormones (e.g. growth hormone) and cytokines. Novel indirect methods are required that will be suitable for screening large numbers of athletes before more sophisticated and/or invasive direct detection methods are applied. Acknowledgements No sources of funding were used to assist in the preparation of this manuscript. The authors have no potential conflicts of interest that are directly relevant to the contents of this manuscript.

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Correspondence and offprints: Dr Evanthia DiamantiKandarakis, First Department of Medicine, Medical School, Laiko Hospital, University of Athens, 1A-Zefyrou, Ekali, Athens, 145-78, Greece. E-mail: [email protected]

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