DHT and IGF-1 in peripheral blood lymphocytes: new markers for the biological passport of athletes

We performed a pilot study using human peripheral blood lymphocytes (PBL) as a novel system to identify new biomarkers of dihydrotestosterone (DHT) and insulin-like growth factor-1 (IGF-1) abuse in sport. First, to obtain a gene signature, we treated cultures of lymphocytes from sedentary males with three doses of 0.237 microg/ml DHT, each of which is 80-fold the physiological concentration in young adult male serum, at days 0, 2 and 4, or with a single dose of 1.25 microg/ml IGF-1, which is 5-fold the physiological concentration in young adult male serum. We then used the Human Genome U133 Plus 2.0 microarray to identify a gene signature related to DHT or IGF-1 administration. Gene expression was evaluated after 7 and 21 days of DHT treatment, and after 24 h, 72 h and 7 days of IGF-1 treatment. Microarray analysis yielded a list of genes whose expression was altered after DHT or IGF-1 treatment. Among these we selected the genes that are most representative of the pathways associated with skeletal and muscular disorders using the IPA bioinformatics tool. We identified six (IDO1, CXCL13, CCL1, GZMB, VDR and IL2RA) and two (FN1 and RAB31) genes that were up-regulated in lymphocytes from sedentary subjects after 7 days of DHT and IGF-1 treatment, respectively. The expression of these genes in lymphocytes from differently trained athletes was either down-regulated or similar to that in lymphocytes from sedentary subjects. This finding suggests that up-regulation was due to the drug and not to physical exercise. In conclusion, we demonstrate that PBL can be useful in anti-doping checks, and we describe new biomarkers of DHT and IGF-1 abuse which can be included in the Athlete's Biological Passport.