F. BOTRE'

BOTRE', Francesco

Archivio della ricerca- Università di Roma La Sapienza

Drugs of abuse and abuse of drugs in sportsmen: the role of in vitromodels to study effects and mechanismsFrancesco Botrè*Department CGMIA, University of Rome ‘‘La Sapienza’’ and Laboratorio Antidoping FMSI, Largo Giulio Onesti 1, 00197 Rome, ItalyAccepted 15 May 2003AbstractA particular field of analytical chemistry applied to forensic toxicology is represented by the anti-doping analysis, where biolo-gical samples (urine and in some instances blood) collected, either ‘‘in competition’’ or ‘‘out of competition’’, from athletes ruled fornational/international sport federations, are analyzed to detect the putative use of prohibited substances and methods. Togetherwith the official anti-doping activity to test the athletes (i.e. who engages in competitive sport) for the non-physiological enhance-ment of sport performance, it is mandatory to activate a new strategy of doping control, that should necessarily comprise a deepand exhaustive toxicological evaluation of the entire spectrum of doping substances and methods. An outline of the present statusand of the future trends of the antidoping research is here presented, showing that most of the new tasks could greatly benefit froman approach based on in vitro methods, ranging from specific toxicity studies to the possible detection of new forms of doping.# 2003 Elsevier Ltd. All rights reserved.1. The anti-doping tests: an outline of the presentsituationThe use of performance-enhancing substances andmethods in sport threatens not only the meaning andthe ethical value of the sport itself, but also the health ofthe athletes. So far, the fight against doping in sport hasbeen carried out mostly by the sport authorities, andprimarily among them by the International OlympicCommittee (IOC). According to the definition of theIOC, ‘‘doping is (i) the use of an expedient (substance ormethod) which is potentially harmful to athlete’s healthand/or capable of enhancing their performance, or (ii)the presence in the athlete’s body of a prohibited sub-stance or evidence of the use thereof or evidence of theuse of a prohibited method’’ (International OlympicCommittee, 1999).The efforts of the IOC are being integrated by, andsomehow progressively transferred to, the activity of theWorld Anti Doping Agency (WADA), constituted inLausanne in 1999 and formed by both sport and poli-tical components, stressing the fact that doping is notonly a problem of fair competition, but more generally aworldwide health issue.Regardless the international authority responsible for theanti-doping control, the current anti-doping regulation isstill basedona list of prohibited classes of substances andofprohibited methods, outlined in Table 1 (InternationalOlympic Committee Medical Commission, 2002): this listhas been, and will continue to be, periodically updated (upto now by the IOC, in the future by theWADA), followingthe development of new possible doping agents.In principle, all substances and methods included inthe list should be tested for by the anti-doping labora-tories; but a number of doping strategies, and especiallythose based on the administration of arificial hormones,whose structures are virtually identical to the natural,endogenous ones, are not yet detectable. Nonetheless,tests currently available permit the detection of most ofthe forbidden substances in urine, and the efforts of theanti-doping laboratories are aimed towards the devel-opment of reliable analytical methods to further reducethe number of doping agents that are still ‘‘invisible’’ atthe tests (Cowan and Kicman, 1997; Honor, 1996;Hemmersbach and de la Torre, 1996).In practice, over 150,000 anti-doping tests are carriedout yearly by the anti-doping laboratories accreditatedby the IOC (Table 2). Basically, the research activitycarried out by the IOC laboratories is focused on the0887-2333/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.doi:10.1016/S0887-2333(03)00120-6Toxicology in Vitro 17 (2003) 509–513www.elsevier.com/locate/toxinvit* Corresponding author. Tel.: +39-06-36859179; fax: +39-06-8078971.E-mail address: botre@uniroma1.it (F. Botrè).improvement of the analytical methods and procedures,aiming both to increase the number of detectable ana-lytes, as well as to identify new potential doping/mask-ing agents, to reduce the gap between testers andcheaters. Since the result of a positive test constitutesthe basis for the possible sanctioning of the athlete, allefforts are devoted to guarantee the maximum of solid-ity of the experimental results (Jiménez et al., 2002): ISO17025 accreditation has been imposed since the year2000 as a further prerequisite of an IOC anti-dopinglaboratory, and criteria for reporting a positive samplesare more severe than those established for the analysisof food residues within the European Union (Table 3).The increasing demands for anti-doping tests also on‘‘amateurs’’, i.e. on sportsmen not officially ruled forsport federations, also impose a thoughtful optimizationof the available human and instrumental resources.Fig. 1 gives an outline of the ‘‘pathway’’ of a urinesample, from its collection to the communication of theresults of the anti-doping analysis: each sample is testedfor the presence of about 200 drugs/metabolites, dividedin several preliminary screening procedures, run in par-allel, with subsequent confirmation procedures to becarried out on suspect samples only.It is self evident that there is no room, at present, forany toxicological evaluation. The function of an anti-doping test is indeed markedly different from that ofdiagnostic tests (Badia et al., 1998): Table 4 summarizesthe main differences between clinical chemistry and anti-doping laboratories. The only way of ensuring a uni-form control policy and an acceptable decisional strat-egy is indeed to follow the principle of strict liability:this means that the burden of the proof is reversed, sincethe simple presence of a forbidden drug/metabolite inthe athlete’s body is sufficient to support the sanction-ing, unless the athlete is able to prove his/her innocence.With such a forensic objective in mind, it is unavoid-able that most of the resources are devoted to strenghtenthe reliability of the experimental results, with the con-sequence that some key aspects of the anti-dopingTable 1The new list of prohibited classes and methods (effective January 1st2003)Prohibited classes of substancesStimulantsNarcoticsAnabolic agentsAnabolic androgenic steroidsOther anabolic agents (beta-2-agonists)DiureticsPeptide hormones, mimetics and analoguesAgents with anti-oestrogenic activityMasking agentsProhibited methodsEnhancement of oxygen transferPharmacological, chemical and physical manipulationGene dopingClasses of prohibited substances in certain sportsAlcoholCannabinoidsLocal anaestheticsGlucocorticoidsBeta-blockersTable 2The 30 anti-doping laboratories accreditated by the International Olympic Committee (last update: July 2002)Africa: South Africa (Bloemfontein), Tunisia (Tunis)Americas: Brazil (Rio de Janeiro), Canada (Montreal), Colombia (Bogota), United States (Los Angeles)Asia: China (Beijing), Korea (Seoul), Japan (Tokyo), Malaysia (Penang), Thailandia (Bangkok)Europe: Austria (Seibersdorf), Belgium (Ghent), Czech Republic (Prague), Finland (Helsinki), France (Paris), Germany (Cologne, Kreischa), Greece(Athens), Italy (Rome), Norway (Oslo), Portugal (Lisbon), Russian Federation (Moscow), Spain (Barcelona, Madrid), Sweden (Stockholm),Switzerland (Lausanne), Turkey (Ankara), United Kingdom (London)Oceania: Australia (Sydney)Table 3Criteria for confirmation in GC-MS and GC-MSn (expressed as maximum differences between sample and reference standard)European Community Reference Laboratories(CRL) for residues:Relative abundance (% of base peak) GC-MS EI (relative) GC-MS CI, GC-MS-MSn,LC-MS, LC-MS-MSn (relative)>50% 10% 20%20–50% 15% 25%10–20% 20% 30%<10% 50% 50%Guidelines of the IOC Medical Commissionfor reporting a positive result:Whichever is greater between: GC-MS, LC-MS GC-MS-MSn, LC-MS-MSnAbsolute abundance 5% 10%Relative abundance 20% 25%510 F. Botrè / Toxicology in Vitro 17 (2003) 509–513activity are not adequately managed by the IOClaboratories. The potential toxicological risks of abuseof performance enhancing substances and methodscannot indeed be fully evaluated by a single measure-ment of urinary concentration values of drugs, metabo-lites and/or other representative indicators ofadministration. Implementing novel diagnostic approa-ches is therefore mandatory to fully accomplish therequirements of an effective anti-doping strategy.As of now, and to speak in the language of toxicol-ogy, the antidoping laboratories search for markers ofexposure, not for markers of effect.1.1. Future challenges of the anti-doping fight: the roleof in vitro methodsAt least four different doping strategies can be expec-ted to be followed in the future for the illegal enhance-ment of sport performance:1. the use of ‘‘doping designer drugs’’ (i.e. sub-stances that are very similar to the ones presentlysearched for by the antidoping laboratory);2. the administration based on the knowledge of thepharmacokinetic properties of the selected drug,so to appear ‘‘clean’’ at the time of producing thebiological sample;3. the use of ‘‘natural’’ hormones and/or pre-cursors, that cannot yet be distinguished from theendogenous ones;4. gene doping, included for the first time amongthe prohibited methods (see again Fig. 1).Tentatively, some of the most effective counter-measures should include the following strategies:(a) to develop analytical methods based on struc-ture-activity relationships;(b) to intensify the number of out-of-competition, noadvance notice tests, possibly considering also theanalysis of alternative biological matrices (not onlyblood, but also saliva, sweat, hair, expired air);Table 4Main differences between clinical and antidoping testsClinical chemistry:Matrices: the most suitable biological materialAim: diagnostic teststest report can be based on:detection and quantitation of diagnostic markersand/ormultiparametric profilesa doubtful sample can be treated as a positive one(activating further investigation)Doping control analysis:Matrices: only urine for officially recognized testsAim: to supply evidencetest report based on:unambiguous identification of specific drugs/metabolitesa doubtful sample is generally treated as a negative one(no further investigation)Fig. 1. The general process of an anti-doping analysis (based on the procedure followed by the anti-doping laboratory of Rome).F. Botrè / Toxicology in Vitro 17 (2003) 509–513 511(c) to exploit the real potential of advanced techni-ques of instrumental analytical chemistry (GC-IRMS, LC-MSn and LC-HRMS) and molecularbiology [for instance isoelectric focusing follow-ing the so called ‘‘double blotting’’ (Lasne,2001)];(d) to enter in the field of molecular diagnostics.As it can be seen, even considering simply the ‘‘faircompetition’’ side of doping, the need for in vitrotests is going to become more urgent in the nearfuture. Should the gene doping become somethingmore than a hypothetical menace, it is clear that thetesting strategies of the anti-doping laboratories willhave to embrace a wider range of professionalexpertises.The situation appears even more appealing to in vitrotoxicologists if the evaluation of the possible healthrisks of doping is also considered. In terms of the cur-rent pharmacologic regulation, doping is a form of ‘‘offlabel’’ experimentation of drugs, with active principlesgenerally administered at higher doses than those usedin therapy, and very often in association with otherdrugs, either legal or illegal: the result is that the final,desired effect is generally very different from the ‘‘offi-cial’’ one. From the point of observation of the officialpharmacological research, these results show a generallack of scientifically acceptable information, also con-sidering that the ‘‘doping experimenters’’ do not publishtheir results: it is therefore almost impossibile to verifythe solidity of the claimed results, as well as, mostimportant, of the adverse side effects. To repeat thesame ‘‘studies’’, but following the fundamentals of acorrect pharmacological research on humans, is abso-lutely unacceptable, so that most of the putative effectsof doping substances and methods remain confined,with few exceptions [for instance as far as the effect onthe respiratory system is concerned (Dekhuijzen et al.,1999)], within the field of anecdotal results. The reducedsignificance of data obtained on animal models, as wellas of epidemiological studies (the number of officialpositive cases is — luckily — still too limited) do nothelp to shed light on this aspect of the problem. In thiscontext, in vitro toxicology methods could represent anextraordinary research tool.By dedicated in vitro methods it would indeed bepossible to reconsider the effects and mechanisms ofdoping substances and methods that have not yet beendealt with in sufficient details. In general, the followingareas of study could greatly benefit from the support ofin vitro toxicology studies: Assessment of toxicity (general, selective, geno-toxicity, cell- and tissue-specific function toxicity) Determination of the LOEL (lowest observedeffect level) for specific doping substances Toxicokinetic modeling for sportsmen, includingthe study of the presumptive effect of training/exercise on the biophysico-chemical processes ofuptake, metabolism and excretion, moving fromPB-TK (physiology-based toxicokinetics) toPOEB-TK (physiology of exercise-based tox-icokinetics) Evaluation of the consequences of non ther-apeutic strategies (mainly overdosage and unu-sual drug associations), under the effects ofphysical stress (transient tissue hypoxygenation,dehydration, hemolysis. . .) Description of any unknown effects on sportperformance, leading to the inclusion in the anti-doping lists of new banned substances andmethodsSpecific application to sport medicine, allowing thedevelopment of mechanism-based evaluations in ‘‘nor-mal’’ subjects as well as in sportsmen (healthy, trainedhumans) could also be extended to drugs and methodsnot included in the anti-doping list. An effective anti-doping strategy should also consider the study ofallowed substances, stressing the fact that a correctinformation can discourage the use of drugs without arational therapeutic need (Monaghan, 2002), and thatmost of the risks derive from their improper use ratherthan from their intrinsic toxicity.2. ConclusionsIf doping is to be considered not only a ‘‘fair compe-tition’’ problem, but also a real health threateningmenace, then the ‘‘traditional’’ antidoping tests (foren-sic urinalysis) can no longer be considered as the uniquetool of an effective control strategy, but they should beintegrated with other toxicologic investigations.As already mentioned, gene doping has been formallyincluded in the anti-doping list, among the prohibiteddoping methods. This definition opens new perspectivestowards the detection of doping by the IOC labora-tories. The ultimate goal will continue to be the ‘‘urin-ary marker’’, but indirect evidence could be accepted assatisfying [as it has been for blood doping and abnormalhaematological parameters (Ashenden, 2002)], provideda risk for the health can be proven.At present, none appropriate laboratory tests exist toevaluate the possible toxicological risks associated withthe abuse of drugs, either legally or illegally, by sports-men, especially concerning the ‘‘unknown’’ risks deriv-ing from the combination of two or more dopingagents. Following the example of other branches ofanalytical toxicology, like for instance in the field offood control (Eisenbrand et al., 2002), in vitro methods(primarily among the techniques based on cell toxicity512 F. Botrè / Toxicology in Vitro 17 (2003) 509–513tests, but also those involving inhibition biosensors,simulation studies, and in silico methods) could provideextremely useful information, covering most of thoseaspects of doping control activity, and especially thetoxicokinetics, that are not covered by the ‘‘traditional’’analytical results supplied by the antidoping labora-tories. Such an integrated approach could allow to shiftthe real focus of the doping control activity from thesimple identification of markers of ‘‘exposure’’ to amore complete assessment of markers of ‘‘effect’’ ofdoping agents.ReferencesAshenden, M.J., 2002. A strategy to deter blood doping in sport.Haematologica 87, 225–232.Badia, R., de la Torre, R., Corcione, S., Segura, J., 1998. Analyticalapproaches of European Union laboratories to drugs of abuse ana-lysis. Clin. Chem. 44, 790–799.Cowan, D.A., Kicman, A.T., 1997. Doping in sport: misuse, analyticaltests, and legal aspects. Clin. Chem. 43, 1110–1113.Dekhuijzen, P.N.R., Machiels, H.A., Heunks, L.M.A., van der Heij-den, H.F.M., van Balkom, R.H.H., 1999. Athletes and doping:effects of drugs on the respiratory system. Thorax 54, 1041–1046.Eisenbrand, G., Pool-Zobel, B., Baker, V., Balls, M., Blaauboer, B.J.,Boobis, A., Carere, A., Kevekordes, S., Lhuguenot, J.-C., Pieters,R., Kleiner, J., 2002. Methods of in vitro toxicology. Food Chem.Toxicol. 40, 193–236.Hemmersbach, P., de la Torre, R., 1996. Stimulants, narcotics andbeta-blockers: 25 years of developments in analytical techniques fordoping control. J. Chromatogr. B 687, 221–238.Honor, J.W., 1996. Testing for drug abuse. Lancet 348, 41–43.International Olympic Committee, Olympic Movement AntidopingCode, Lausanne, 1999.International Olympic Committee Medical Commission, IOC List ofProhibited Classes of Substances and Prohibited Methods, OlympicMovement Antidoping Code, Appendix A, International OlympicCommittee, Lausanne, October 2002.Jiménez, C., Ventura, R., de la Torre, X., Segura, J., 2002. Strategiesfor internal quality control in antidoping analyses. Anal. Chim.Acta 460, 289–307.Lasne, F., 2001. Double-blotting: a solution to the problem of non-specific binding of secondary antibodies in immunoblotting proce-dures. J. Immunol. Methods 253, 125–131.Monaghan, L.F., 2002. Vocabularies of motive for illicit steroid useamong bodybuilders. Social Science & Medicine 55, 695–708.F. Botrè / Toxicology in Vitro 17 (2003) 509–513 513

Drugs of Abuse and Abuse of Drugs in Sportsmen: The Role of In Vitro Methods to Study Effect and Mechanism

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