title: Dual-color bioluminescent bioreporter for forensic analysis: evidence of androgenic and anti-androgenic activity of illicit drugs.
abstract: Bioassays represent promising complementary techniques to conventional analytical approaches used in doping analysis to detect illicit drugs like anabolic-androgenic steroids (AAS). The fact that all AAS share a common mechanism of action via the human androgen receptor (hAR) enables the use of bioassays, relying on the activation of hAR as antidoping screening tools. Previously, we developed a dual-color bioreporter based on yeast cells engineered to express hAR and androgen response elements driving the expression of the bioluminescent (BL) reporter protein Photinus pyralis luciferase. A second reporter protein, the red-emitting luciferase PpyRE8, was introduced in the bioreporter as internal viability control. Here, we report the first forensic application of a straightforward, accurate, and cost-effective bioassay, relying on spectral resolution of the two BL signals, in 96-microwell format. The bioreporter responds to dihydrotestosterone as reference androgen in a concentration-dependent manner from 0.08 to 1,000 nM with intra- and inter-assay variation coefficients of 11.4 % and 13.1 %, respectively. We also demonstrated the suitability of this dual-color bioreporter to assess (anti)-androgenic activity of pure AAS, mixtures of AAS, and other illicit drugs provided by the Scientific Police. Significant anti-androgenic activity was observed in samples labeled as marijuana and hashish, containing Δ(9)-tetrahydrocannabinol as major constituent.
authors.affiliation: INBB, Istituto Nazionale di Biostrutture e Biosistemi, Rome, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Cevenini
authors.forename: Elisa
authors.initials: E
authors.lastname: Michelini
authors.forename: Marcello
authors.initials: M
authors.lastname: D'Elia
authors.forename: Massimo
authors.initials: M
authors.lastname: Guardigli
authors.forename: Aldo
authors.initials: A
authors.lastname: Roda
mesh_terms: D000726:Androgen Antagonists / Q000032:analysis* / Q000652:urine; D000728:Androgens / Q000032:analysis* / Q000652:urine; D015374:Biosensing Techniques / Q000379:methods*; D005554:Forensic Medicine / Q000379:methods*; D017930:Genes, Reporter; D006801:Humans; D013287:Illicit Drugs / Q000032:analysis* / Q000652:urine; D008156:Luciferases / Q000032:analysis / Q000652:urine; D008163:Luminescent Measurements / Q000379:methods*; D008297:Male; D011944:Receptors, Androgen / Q000235:genetics / Q000378:metabolism; D013256:Steroids / Q000032:analysis / Q000652:urine
chemical_list: D000726:Androgen Antagonists; D000728:Androgens; D013287:Illicit Drugs; D011944:Receptors, Androgen; D013256:Steroids; D008156:Luciferases