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A Race to the Starting Line

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Science  30 Jul 2004:
Vol. 305, Issue 5684, pp. 632-635
DOI: 10.1126/science.305.5684.632

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Scientists are scrambling to devise new methods for snaring athletes who cheat with steroids, hormones, and, someday, even extra genes

KREISCHA, GERMANY—Tucked on the wooded edge of this village in the Saxon hills south of Dresden is a drab, single-story office building with a sinister past. Until 1989 officials of the German Democratic Republic tested their athletes here to certify them as drug-free before international competitions. But it was all a charade. Many of the East German athletes, both men and women, were systematically doped up with testosterone and other anabolic steroids, often without their knowledge. It was the Kreischa lab's responsibility to ensure that the regimen was suspended long enough before a competition to flush out any traces of drugs, explains Klaus Müller, director of the Institute for Doping Analysis and Sport Biochemistry that today occupies the building. Sometimes the drug docs cut it too close. “You would hear that a certain famous athlete couldn't travel to a competition because of a ‘sudden illness,’” says Müller, whose institute is part of a worldwide antidoping network. “We all knew what that meant.”

That crooked chapter in German sport is over, but the practice of doping appears to be more widespread than ever. Last month world champion sprinter Kelli White received a 2-year ban from competition after admitting to having taken banned steroids and the hormone erythropoietin (EPO), which boosts red blood cell counts. Other clients of the Bay Area Laboratory Co-operative (BALCO) nutrition center in Burlingame, California, were also implicated in the scandal; as Science went to press, U.S. officials were investigating evidence that Olympic gold medalists Marion Jones and Chryste Gaines and world-record sprinter Tim Montgomery had been treated with banned steroids and hormones by the same lab.

In the privileged world of elite sports, avarice and the pursuit of glory continue to lead coaches and chemists astray and tempt athletes to risk health and medals. “Sport can be so magnificent and so powerful precisely because humans play the key role,” says Andrew Pipe, a physician at the University of Ottawa Heart Institute and former chief medical officer to Canada's Olympic team. “It can be so depressing and sordid for exactly the same reason.”

Dopers are getting better at covering their tracks, forcing researchers to invent new techniques to detect ever more subtle uses of synthetic chemicals or proteins that boost the body's ability to build muscle, shed fat, or carry oxygen. What was once the exclusive domain of analytical chemists—who searched for steroids in urine samples—now involves endocrinologists and geneticists as authorities attempt to clamp down on what could become the next illicit frontier: doping with genes for muscle building. “Testing gets better and better, but the opposition gets better and better too,” says Don Catlin, director of the University of California, Los Angeles (UCLA), Olympic Analytical Laboratory.

Back-alley chemistry

Athletes have been seeking an artificial edge since at least the late 1800s, when runners and long-distance bicyclists used nitroglycerin and even cocaine to boost stamina and block pain. But although authorities began testing for banned substances in the 1970s, their efforts had little impact, says Peter Sonksen of St. Thomas' Hospital in London and a former member of the International Olympic Committee's (IOC's) medical commission. “For a long time there was a feeling that many sporting bodies were protecting their players,” he says.

A lack of vigilance created an environment for blatant cheating. For example, a series of astounding world-record performances in the 1980s, especially in power sports such as the shot put or hammer throw, were almost certainly fueled by testosterone and other prohibited anabolic steroids, Müller says.

There is little doubt that steroids help athletes beef up. By targeting the same receptor as testosterone does, they boost the body's capacity for building muscle and erode its capacity for breaking it down. But they have manifold side effects. Although women produce some testosterone naturally, ratcheting up levels even slightly leads to increased body hair and acne and can wreak havoc with the reproductive system. In men, taking steroids suppresses natural production of testosterone, which can lead to bigger breasts, shrunken testicles, and infertility. In both sexes, high doses of the drugs damage the liver and the cardiovascular system.

As testing for steroids began to be enforced more strictly in the 1990s, use of the drugs plummeted —and the pace of record-breaking tapered off. The antidoping forces seemed to have the upper hand until 2002, when the sport world was rocked by revelations that a pair of so-called designer steroids—drugs with no legitimate medical use—had been synthesized, apparently to elude doping testers.

In one case Catlin's team detected unusually low levels of natural steroids such as testosterone, epitestosterone, and androsterone in the urine of a female cyclist, a sign that something was amiss. Probing further, his group found traces of norbolethone, an androgen developed by Wyeth in the 1960s. In animal tests, Catlin says, norbolethone appeared to be a very effective muscle builder while having relatively few masculinizing side effects. It was tested in short children and underweight patients, but Wyeth shelved the compound, apparently because of toxic side effects. Evidently, someone was cooking up a new supply.

A whistleblower made the second discovery possible. In June 2003 Catlin received the residue from a used but empty syringe from the U.S. Anti-Doping Agency. A track coach had sent it to the authorities, suggesting that they take a careful look. Within a few weeks, Catlin and his colleagues had identified tetrahydrogestrinone (THG). The new chemical, which had never before been described, resembles two steroids banned for use by professional athletes: gestrinone, prescribed occasionally for the treatment of endometriosis, and trenbolone, which has some uses in veterinary medicine. Both steroids have powerful anabolic effects, and the UCLA team quickly suspected that the derivative had been designed to activate the same receptors while foiling standard screens for known steroids. When authorities tested urine stored from previous competitions, they found at least a dozen THG-tainted samples, many from athletes who had connections to BALCO.

Because routine screening would never have caught THG, doping testers were confronted with the prospect of having to develop ways to detect an incalculable array of steroids and other chemicals that might play a similar performance-enhancing role. “The THG story tells us very convincingly that there are people out there who are scheming to develop new entities to give to athletes,” says Catlin. “We've studied the chemistry, and there's essentially no end to the possibilities. Are there others out there? There certainly are.” They just haven't been identified yet, he adds.

Some labs are hoping to defeat dopers at their own game. Wilhelm Schänzer and his colleagues at the Institute of Biochemistry of the German Sport University Cologne have begun churning out more than a dozen potential designer agents by tinkering with existing steroids. “We're trying to think in the same way as those who are trying to make new compounds,” Schänzer says. His group uses mass spectrometry to profile the concoctions and identify signals that might betray illicit compounds in bodily fluids.

THG presented a legal challenge as well. Lawyers for athletes who tested positive argued that the authorities couldn't demonstrate that the substance is an anabolic steroid, and therefore it could not be classified as a banned substance. Indeed, the chemical's effects in animals—much less humans—had never been characterized in a legitimate lab; standard animal tests take many months. Under court- imposed time constraints, scientists resorted to a quicker solution, a test originally designed to ferret out environmental pollutants that mimic hormones. The test uses yeast cells altered to make the human version of the testosterone receptor as well as a luminescent protein that glows when the receptor is activated. Using the test, David Handelsman of the ANZAC Research Institute in Concord, Australia, found that THG lights up the cells more brightly than standard anabolic steroids such as trenbolone and even testosterone.

The confirmation came just in time to support the case against European champion sprinter Dwain Chambers, who had tested positive for THG in August 2003. (Chambers has said that he ingested the compound unknowingly in a supplement provided by BALCO.) In February, U.K. Athletics banned him from running in competitions for 2 years. Chambers had been considered a favorite for the gold medal this summer in Athens, but according to British Olympic Association rules, he is banned from the Athens games.

The bioassays may soon join a growing arsenal that scientists are assembling to thwart the use of new designer steroids, says Handelsman. He and his colleagues, for example, are working on a simple test to compare the amount of testosterone normally present in the urine of men and women with the total steroid load, as measured by the bioassays. “If there's a gap, then that suggests there's an unidentified substance there,” Handelsman says.

The workhorse of steroid detection, the mass spectrometer, could also be put to innovative use. Even if an analysis fails to flag unexpected side chains or telltale peaks, it can reveal subtle differences in the ratio of carbon isotopes that can help identify the origin of organic molecules. An unusual ratio of carbon-12 to carbon-13 in certain molecules can raise a red flag in a doping test. If a steroid molecule has a ratio typical of a plant rather than an animal, it is a sign that it comes from an outside source, says Schänzer.

Banned.

Dwain Chambers, who tested positive for the steroid THG, is barred from the British Olympic team.

CREDIT: MIKE HEWITT/GETTY IMAGES

In pursuit of oxygen

Unknown steroids are hard enough to pin down; injections of naturally occurring hormones are even more elusive. Hormone levels fluctuate from hour to hour and from person to person, so measuring absolute amounts can't nail a doper. To do that, scientists must find secondary signals indicating that the body's normal chemistry has been tampered with.

For years, some athletes took advantage of the dearth of detection methods to pump themselves up with EPO. The hormone, produced mainly in the kidneys, stimulates the body's production of red blood cells so that the blood carries more oxygen. People living at high altitudes produce more EPO naturally to compensate for the lower oxygen concentration in the air. Athletes often take advantage of that trick, training at high altitudes for competitions held nearer sea level. But when recombinant EPO, used to treat anemia, became available in the late 1980s, it spawned a doping epidemic.

The practice is dangerous. If blood has too many red blood cells, it can become too viscous for the heart to pump effectively. EPO is thought to have played a role in the deaths of more than a dozen Dutch and Belgian cyclists who died of sudden heart attacks in the 1980s, just after EPO became available in Europe. Despite the risks, EPO's use was apparently widespread in the 1990s as scientists raced to figure out how to detect its use.

The first EPO tests, introduced a decade ago, set a limit for hematocrit, the percentage of red blood cells in the blood. But that test is flawed, as it cannot tell whether an athlete has used EPO to boost his or her hematocrit to a level just below the allowed limit.

In 2000, in time for the Olympic Games in Sydney, Australia, the IOC introduced a combined blood and urine test for EPO. The blood test measures, among other things, the concentration of hemoglobin and the level of reticulocytes—immature red blood cells—in the blood. Testers look for unusually high levels or sudden changes from previous tests to tip them off to possible dopers. The test has one major advantage: It can detect signs of EPO use weeks after an athlete takes it. But because it does not measure illegal EPO directly, it cannot prove a doping allegation.

Take a deep breath.

Blood samples will be collected from athletes in Athens to check for erythropoietin and a range of other substances.

CREDIT: GETTY IMAGES

A second method allows testers to spot traces of recombinant EPO directly in urine. Because the recombinant version is produced in animal cells, it carries slightly different sugars in its side chains than the natural version. These differences show up in electrophoresis, which measures the distance proteins chug through a gel under the influence of electricity. The concentration of EPO in urine is fairly low, however, so the test could be foiled if an athlete takes diuretics or other urine-increasing drugs.

The bottom line is that the current tests simply don't cut it. “Athletes are getting around the EPO tests all the time,” Catlin says. Officials of the World Anti-Doping Agency (WADA) agree. “We need cheaper and more sensitive tests for EPO,” says Olivier Rabin, WADA's scientific director.

WADA is also funding projects to tackle an old-fashioned doping technique that the organization claims is back in vogue since the introduction of EPO tests. Called blood doping, it involves an athlete either receiving blood transfusions—enriched in red blood cells—from donors, or removing an athlete's own blood, spinning it to concentrate the red blood cells, then reinfusing it right before competition. Although the techniques don't involve foreign chemicals, they are banned by sports organizations on safety grounds.

A growing threat

One of the compounds that BALCO clients are accused of abusing is something that doesn't show up in any standard doping tests: human growth hormone (hGH). The protein is part of a biochemical cascade that spurs muscle buildup and the shedding of fat. It's used legitimately to treat children who lack the protein and are unusually short. But like EPO and legitimate steroids, it too has been hijacked for use in athletes. Although its effects in healthy athletes are unclear, doping experts suspect that its use is widespread—especially because authorities have not yet introduced an official test for the compound.

That's a high priority, however, and scientists say they have several tests ready for the Athens Games. WADA officials are circumspect about whether they will use any of the tests for hGH in August. “Athletes know it is on the banned substances list,” says Rabin, and should expect to be tested.

Detecting hGH is even harder than detecting EPO, because it doesn't have telltale sugars to betray artificial versions. But in a lucky break for doping sleuths, the pituitary gland's production of growth hormone is rather messy. The gland makes a mixture of variations of the protein as well as protein fragments. The manufactured version, on the other hand, is much cleaner, consisting chiefly of one of the heavier versions, so when someone shoots up with the recombinant protein, the ratio of the different forms is skewed. Endocrinologist Christian Strasburger of the Charité University Clinics in Berlin and his colleagues at the Medizinische Klinik Innenstadt at the University of Munich have developed an immunoassay that measures the ratio of the two forms. The test seems extremely reliable, Strasburger says.

Another group led by Sonksen of St. Thomas' Hospital has developed a method to measure the effects of growth hormone on the production of other proteins, including insulin-like growth factor-1 (IGF-1) and collagen. The test is not as clear-cut as that developed by Strasburger and his colleagues, but it can detect the effects of hGH weeks after someone has injected it. The Strasburger method works best 24 to 36 hours after injection.

Those who go to the trouble and expense—a month's dose costs more than $2500—may not be getting their money's worth. “It looks as though growth hormone is fool's gold,” says Ken Ho, an endocrinologist at the Garvan Institute of Medical Research near Sydney. “In the normal person with normal levels of growth hormone, adding extra has not been shown to confer a benefit.” Yet, Ho says, “at the end of the day, if a 0.01% advantage is the difference between winning and losing,” a minuscule boost from growth hormone—even if it's purely psychological—might help an athlete to victory.

Tackling this murky question, Ho's group is giving growth hormone to healthy volunteers both to screen for biochemical changes that might be picked up in a doping test and to look for performance- enhancing effects. Whether the benefit is real or not, the hormone is on the list of banned substances, and athletes caught using it will forfeit any medals they receive next month in Athens.

Self-assembled superathletes

In a case that made headlines this summer, doctors described a young boy in Berlin who seems destined for athletic greatness. The boy was born with a mutation that turns off the gene for myostatin, which in animals seems to block the activation of muscle stem cells. Mice and cattle that carry myostatin mutations have twice as much muscle as normal animals. At 4-and-a-half years old, the boy had the physique of a mini-bodybuilder and could hold out two 3-kg dumbbells with his arms extended, his doctors reported in the 24 June New England Journal of Medicine. Some experts are thrilled: They suggest that the mutation could be exploited as a treatment for muscle-wasting diseases.

Natural boost.

A Berlin child who carries a mutation in the myostatin gene has had bodybuilder muscles since birth.

CREDIT: THE NEW ENGLAND JOURNAL OF MEDICINE, 2004

But antidoping officials are cringing. They fear that gene therapy could soon be the next fad among athletes. Their nightmare scenario is athletes injecting a retrovirus bearing a myostatin-blocking gene or another muscle-building gene such as IGF-1 (see sidebar on p. 633). Once the gene is incorporated into cells, it begins to pump out its products and build muscle, but the illicit source would be extremely difficult to trace.

Clinical trials of gene therapy for fatal diseases have been fraught with problems, including the death of one volunteer and the development of leukemia in other patients. But that might not stop some athletes. “These [gene therapy] methods remain extremely risky,” says Rabin. “But on the other hand we know that some athletes are willing to take incredible risks. THG went straight from the test tube to the athletes, with no proper testing.”

WADA is funding efforts to detect gene doping, either through traces of retrovirus vectors or by spotting indirect effects of gene boosting, Rabin says. Almost any gene doping would influence a wide range of other genes, and changes in those might be traceable with ever more sensitive tests that can flag gene expression, he says.

As doping sprouts more Medusa-like heads, authorities may be forced to develop personalized tests. Ideally, Rabin says, each athlete would submit a biological “passport” containing highlights of their blood chemistry. “If we then saw an abnormal change, we could follow it up,” he says. Microarrays that measure the expression levels of thousands of genes at once could betray blips that might result from gene doping. Ideally, he says, “in a single drop of blood, we'll be able to detect changes based on any genes that are modified.” For now, though, such tests would cost thousands of dollars per athlete, prohibitively expensive for most testing organizations.

Müller doesn't foresee a breakthrough in antidoping efforts anytime soon. His experience as a medical scientist in East Germany, where athletes were lavishly funded as an international propaganda tool, left him questioning the value of top-level sports. “We are not dealing here with problems of human existence or survival,” he says. “The world will not come to an end if dopers go uncaught.” His motivation, he says, is the example that elite athletes set for millions of amateurs: “It's important for people to be able to understand that you can do amazing things without doping.”

Unfortunately, many elite athletes don't buy that message. “We're never going to eliminate [doping] completely,” says the University of Ottawa's Pipe. The contest will continue, with both sides intent on raising their game to the next level.

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