Estudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegans

ilustraciones, tablas

Autores:
Toscano Bayona, Lucía
Tipo de recurso:
Fecha de publicación:
2021
Institución:
Universidad Nacional de Colombia
Repositorio:
Universidad Nacional de Colombia
Idioma:
spa
OAI Identifier:
oai:repositorio.unal.edu.co:unal/79894
Acceso en línea:
https://repositorio.unal.edu.co/handle/unal/79894
https://repositorio.unal.edu.co/
Palabra clave:
610 - Medicina y salud
Doping en los Deportes
Doping in Sports
metandienona
Biotransformación
metabolismo
esteroides anabolizantes
Metabolism
Methandienone
Cunninghamella elegans
Anabolic steroids
Biotransformation
Rights
openAccess
License
Atribución-NoComercial-SinDerivadas 4.0 Internacional
id UNACIONAL2_86defa58f78a1cbb9ad65ee67afcbd26
oai_identifier_str oai:repositorio.unal.edu.co:unal/79894
network_acronym_str UNACIONAL2
network_name_str Universidad Nacional de Colombia
repository_id_str
dc.title.spa.fl_str_mv Estudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegans
dc.title.translated.eng.fl_str_mv Methandienone metabolism study through an in vitro incubation model with the fungus Cunninghamella elegans
title Estudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegans
spellingShingle Estudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegans
610 - Medicina y salud
Doping en los Deportes
Doping in Sports
metandienona
Biotransformación
metabolismo
esteroides anabolizantes
Metabolism
Methandienone
Cunninghamella elegans
Anabolic steroids
Biotransformation
title_short Estudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegans
title_full Estudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegans
title_fullStr Estudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegans
title_full_unstemmed Estudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegans
title_sort Estudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegans
dc.creator.fl_str_mv Toscano Bayona, Lucía
dc.contributor.advisor.none.fl_str_mv Chaves Silva, Diana Carolina
Cárdenas Cuadros, Paola Andrea
Martínez Ramírez, Jorge Ariel
dc.contributor.author.none.fl_str_mv Toscano Bayona, Lucía
dc.contributor.researchgroup.spa.fl_str_mv Sustancias Psicoactivas
dc.subject.ddc.spa.fl_str_mv 610 - Medicina y salud
topic 610 - Medicina y salud
Doping en los Deportes
Doping in Sports
metandienona
Biotransformación
metabolismo
esteroides anabolizantes
Metabolism
Methandienone
Cunninghamella elegans
Anabolic steroids
Biotransformation
dc.subject.decs.none.fl_str_mv Doping en los Deportes
Doping in Sports
dc.subject.proposal.spa.fl_str_mv metandienona
Biotransformación
metabolismo
esteroides anabolizantes
dc.subject.proposal.eng.fl_str_mv Metabolism
Methandienone
Cunninghamella elegans
Anabolic steroids
Biotransformation
description ilustraciones, tablas
publishDate 2021
dc.date.accessioned.none.fl_str_mv 2021-08-06T18:02:02Z
dc.date.available.none.fl_str_mv 2021-08-06T18:02:02Z
dc.date.issued.none.fl_str_mv 2021-05
dc.type.spa.fl_str_mv Trabajo de grado - Maestría
dc.type.driver.spa.fl_str_mv info:eu-repo/semantics/masterThesis
dc.type.version.spa.fl_str_mv info:eu-repo/semantics/acceptedVersion
dc.type.content.spa.fl_str_mv Text
dc.type.redcol.spa.fl_str_mv http://purl.org/redcol/resource_type/TM
status_str acceptedVersion
dc.identifier.uri.none.fl_str_mv https://repositorio.unal.edu.co/handle/unal/79894
dc.identifier.instname.spa.fl_str_mv Universidad Nacional de Colombia
dc.identifier.reponame.spa.fl_str_mv Repositorio Institucional Universidad Nacional de Colombia
dc.identifier.repourl.spa.fl_str_mv https://repositorio.unal.edu.co/
url https://repositorio.unal.edu.co/handle/unal/79894
https://repositorio.unal.edu.co/
identifier_str_mv Universidad Nacional de Colombia
Repositorio Institucional Universidad Nacional de Colombia
dc.language.iso.spa.fl_str_mv spa
language spa
dc.relation.references.spa.fl_str_mv 1. Barceloux DG. Anabolic-Androgenic Steroids. In: Medical Toxicology of Drug Abuse. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2012. p. 275–94.
2. Aguilar M, Muñoz-Guerra J, Plata M del M, Del Coso J. Thirteen years of the fight against doping in figures. Drug Test Anal. 2017;9(6):866–9.
3. Schänzer W, Geyer H, Donike M. Metabolism of metandienone in man: Identification and synthesis of conjugated excreted urinary metabolites, determination of excretion rates and gas chromatographic-mass spectrometric identification of bis-hydroxylated metabolites. J Steroid Biochem Mol Biol. 1991 Apr;38(4):441–64.
4. Hagedorn HW, Schulz R, Friedrich A. Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl. 1992;577(2):195–203.
5. Lootens L, Meuleman P, Pozo OJ, Van Eenoo P, Leroux-Roels G, Delbeke FT. uPA+/+-SCID Mouse with Humanized Liver as a Model for In Vivo Metabolism of Exogenous Steroids: Methandienone as a Case Study. Clin Chem. 2009;55(10):1783–93.
6. Khan NT, Zafar S, Noreen S, Al Majid AM, Al Othman ZA, Al-Resayes SI, et al. Biotransformation of dianabol with the filamentous fungi and β-glucuronidase inhibitory activity of resulting metabolites. Steroids. 2014;85:65–72.
7. Kicman AT. Pharmacology of anabolic steroids. Vol. 154, British Journal of Pharmacology. Wiley/Blackwell (10.1111); 2008. p. 502–21.
8. Morley JE. Anabolic Steroids and Frailty. J Am Med Dir Assoc. 2010 Oct;11(8):533–6.
9. Barceloux DG. MEDICAL TOXICOLOGY OF DRUG ABUSE. John Wiley & Sons, Inc.; 2012. 1041 p.
10. Kicman AT, Gower DB. Anabolic steroids in sport: biochemical, clinical and analytical perspectives. Ann Clin Biochem Int J Lab Med. 2003;40(4):321–56.
11. Link AF, Chemistry B. Drug Metabolism. Encycl Syst Biol. 2013;(March):618–618.
12. Repetto Jiménez M, Repetto Kuhn G. Toxicología Fundamental. Cuarta Edi. Sevilla: Díaz de Santos; 2009. 629 p.
13. Gallego Fernández A, de Sande García MA, Marín Fernández AM, Blanco Ramos S, González Galán MJ. Aspectos fundamentales del citocromo P450. Fundación Tejerina. 2011.
14. Asha S, Vidyavathi M. Cunninghamella – A microbial model for drug metabolism studies – A review. Biotechnol Adv. 2009 Jan;27(1):16–29.
15. Curtis D. Klaassen. Casarett and Doull’s Toxicology: The Basic Science of Poisons. Eighth Edi. Klaassen CD, editor. McGraw-Hill Education; 2013. 1454 p.
16. Schänzer W. Metabolism of anabolic androgenic steroids. Clin Chem. 1996;42(7):1001–20.
17. Hofmann FB, Beavo J a, Busch A, Ganten D, Michel MC, Page CP, et al. Doping in Sports. Thieme D, Hemmersbach P, editors. Berlin, Heidelberg: Springer Berlin Heidelberg; 2010. 274 p. (Handbook of Experimental Pharmacology; vol. 195).
18. Baggish AL, Weiner RB, Kanayama G, Hudson JI, Lu MT, Hoffmann U, et al. Cardiovascular Toxicity of Illicit Anabolic-Androgenic Steroid Use. Circulation. 2017 May 23;135(21):1991–2002.
19. White M, Brennan E, Mi Ren KY, Shi M, Thakrar A. Anabolic Androgenic Steroid Use as a Cause of Fulminant Heart Failure. Can J Cardiol. 2018;34(10):1369.
20. Montisci M, El Mazloum R, Cecchetto G, Terranova C, Ferrara SD, Thiene G, et al. Anabolic androgenic steroids abuse and cardiac death in athletes: Morphological and toxicological findings in four fatal cases. Forensic Sci Int. 2012;217(1–3):12–7.
21. Rocha M, Aguiar F, Ramos H. O uso de esteroides androgénicos anabolizantes e outros suplementos ergogénicos – uma epidemia silenciosa. Rev Port Endocrinol Diabetes e Metab. 2014;9(2):98–105.
22. Hartgens F, Kuipers H. Effects of Androgenic-Anabolic Steroids in Athletes. Sport Med. 2004;34(8):513–54. 23. Hall RCW, Hall RCW. Abuse of Supraphysiologic Doses of Anabolic Steroids. South Med J. 2005;98(5):550–6.
24. Christou MA, Christou PA, Markozannes G, Tsatsoulis A, Mastorakos G, Tigas S. Effects of Anabolic Androgenic Steroids on the Reproductive System of Athletes and Recreational Users: A Systematic Review and Meta-Analysis. Vol. 47, Sports Medicine. 2017. p. 1869–83.
25. Nieschlag E, Vorona E. Mechanisms in Endocrinology: Medical consequences of doping with anabolic androgenic steroids: Effects on reproductive functions. Eur J Endocrinol. 2015;173(2):R47–58.
26. Nieschlag E, Vorona E. Doping with anabolic androgenic steroids (AAS): Adverse effects on non-reproductive organs and functions. Vol. 16, Reviews in Endocrine and Metabolic Disorders. 2015. p. 199–211.
27. Schänzer W, Opfermann G, Donike M. 17-Epimerization of 17α-methyl anabolic steroids in humans: metabolism and synthesis of 17α-hydroxy-17β-methyl steroids. Steroids. 1992;57(11):537–50.
28. Schänzer W, Delahaut P, Geyer H, Machnik M, Horning S. Long-term detection and identification of metandienone and stanozolol abuse in athletes by gas chromatography-high-resolution mass spectrometry. J Chromatogr B Biomed Appl. 1996;687(1):93–108.
29. Kanayama G, Pope HG. History and epidemiology of anabolic androgens in athletes and non-athletes. Mol Cell Endocrinol. 2018;464.
30. Barrett-Connor EL. Testosterone and risk factors for cardiovascular disease in men. Diabete Metab. 1995;21(3):156–61. 31. U.S. DEPARTMENT OF JUSTICE • DRUG ENFORCEMENT ADMINISTRATION. Diversion Control Division [Internet]. [cited 2020 Sep 23]. Available from: https://www.deadiversion.usdoj.gov/pubs/brochures/steroids/public/
32. Goverment of Canada. Controlled Drugs and Substances Act [Internet]. 1996. Available from: http://laws-lois.justice.gc.ca/eng/acts/C-38.8/
33. Advisory Council on the Misuse of Drugs. Consideration of the Anabolic Steroids. 2010; Available from: https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/119132/anabolic-steroids.pdf
34. Australian Institute of Criminology. Illicict drugs and alcohol [Internet]. 2006 [cited 2020 Oct 2]. Available from: https://web.archive.org/web/20070405033442/http://www.aic.gov.au/research/drugs/types/steroids.html
35. Ministério da Saúde. Regulamento Técnico sobre substâncias e medicamentos sujeitos a controle especial [Internet]. Portaria no 344. 1998 [cited 2020 Sep 27]. Available from: http://bvsms.saude.gov.br/bvs/saudelegis/svs/1998/prt0344_12_05_1998_rep.html
36. WADA. International Standard for Laboratories. [Internet]. Canada; 2019 [cited 2020 Nov 5]. Available from: https://www.wada-ama.org/sites/default/files/resources/files/isl_nov2019.pdf
37. WADA. WADA Technical Document – TD2019MRPL. 2019.
38. Geyer H, Schänzer W, Thevis M. Anabolic agents: Recent strategies for their detection and protection from inadvertent doping. Br J Sports Med. 2014;48(10):820–6.
39. Schänzer W, Geyer H, Fußhöller G, Halatcheva N, Kohler M, Parr MK, et al. Mass spectrometric identification and characterization of a new long-term metabolite of metandienone in human urine. Rapid Commun Mass Spectrom. 2006;20(15):2252–8.
40. Piska K, |elaszczyk D, Jamrozik M, Kubowicz-Kwa|ny P, P|kala E. Cunninghamella Biotransformation - Similarities to Human Drug Metabolism and Its Relevance for the Drug Discovery Process. Curr Drug Metab. 2016 Jan;17(2):107–17.
41. Ahmad MS, Zafar S, Bibi M, Bano S, Atia-tul-Wahab, Atta-Ur-Rahman, et al. Biotransformation of androgenic steroid mesterolone with Cunninghamella blakesleeana and Macrophomina phaseolina. Steroids. 2014;82:53–9.
42. Choudhary MI, Khan NT, Musharraf SG, Anjum S, Atta-ur-Rahman. Biotransformation of adrenosterone by filamentous fungus, Cunninghamella elegans. Steroids. 2007;72(14):923–9.
43. Siddiqui M, Ahmad MS, Wahab A-T, Yousuf S, Fatima N, Shaikh NN, et al. Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites. Shahid M, editor. PLoS One. 2017;12(2).
44. Ma B, Huang H, Chen X, Sun Y, Lin L, Zhong D. Biotransformation of metoprolol by the fungus Cunninghamella blakesleeana. Acta Pharmacol Sin. 2007 Jul 1;28(7):1067–74.
45. Åberg AT, Löfgren H, Bondesson U, Hedeland M. Structural elucidation of N -oxidized clemastine metabolites by liquid chromatography/tandem mass spectrometry and the use of Cunninghamella elegans to facilitate drug metabolite identification. Rapid Commun Mass Spectrom. 2010;24(10):1447–56.
46. Zhong D-FF, Sun L, Liu L, Huang H-HH. Microbial transformation of naproxen by Cunninghamella species. Acta Pharmacol Sin. 2003 May;24(5):442–7.
47. Dumasia MC. In vivo biotransformation of 17α-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2003 Feb 28;17(4):320–9.
48. Ferris JP, Fasco MJ, Stylianopoulou FL, Jerina DM, Daly JW, Jeffrey AM. Monooxygenase activity in Cunninghamella bainieri: Evidence for a fungal system similar to liver microsomes. Arch Biochem Biophys. 1973;156(1):97–103.
49. Zhang D, Yang Y, Leakey JE., Cerniglia CE. Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics. FEMS Microbiol Lett. 1996 May 1;138(2–3):221–6.
50. Bhosale S, Saratale G, Govindwar S. Biotransformation enzymes in Cunninghamella blakesleeana (NCIM-687). J Basic Microbiol. 2006 Dec 1;46(6):444–8.
51. Smith R, Rosazza J. Microbial Models Aromatic of Mammalian Hydroxylation. Arch Biochem Biophys. 1974;161:551–8.
52. Summary E, Report S, Report TA, Analysis ABPR. Anti-Doping Testing Figures Executive Summary. 9:10–36.
53. World Anti-doping Agency. 2016 Anti-Doping Testing Figures Laboratory Report [Internet]. 2016. Available from: https://www.wada-ama.org/sites/default/files/resources/files/2018_testing_figures_report.pdf
54. Kam PCA, Yarrow M. Anabolic steroid abuse: Physiological and anaesthetic considerations. Vol. 60, Anaesthesia. 2005. p. 685–92.
55. Neri M, Bello S, Bonsignore A, Cantatore S, Riezzo I, Turillazzi E, et al. Anabolic Androgenic Steroids Abuse and Liver Toxicity. Mini-Reviews Med Chem. 2011;11(5):430–7.
56. Awai HI, Yu EL, Ellis LS, Schwimmer JB. Liver Toxicity of Anabolic Androgenic Steroid Use in an Adolescent With Nonalcoholic Fatty Liver Disease. J Pediatr Gastroenterol Nutr. 2014;59(3):e32–3.
57. Rockhold RW. Cardiovascular toxicity of anabolic steroids. AnnuRevPharmacolToxicol. 1993;33:497–520.
58. Santora LJ, Marin J, Vangrow J, Minegar C, Robinson M, Mora J, et al. Coronary calcification in body builders using anabolic steroids. Prev Cardiol. 2006;9(4):198–201.
59. Watanabe S, Kuzhiumparambil U, Winiarski Z, Fu S. Data on individual metabolites of synthetic cannabinoids JWH-018, JWH-073 and AM2201 by Cunninghamella elegans. Data Br. 2016 Jun;7:332–40.
60. Martínez-Ramírez JA, Walther G, Peters FT. Studies on drug metabolism by fungi colonizing decomposing human cadavers. Part II: biotransformation of five model drugs by fungi isolated from post-mortem material. Drug Test Anal. 2015 Apr;7(4):265–79.
61. Rydevik A, Lagojda A, Thevis M, Bondesson U, Hedeland M. Isolation and characterization of a β-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation. J Pharm Biomed Anal. 2014 Sep;98:36–9.
62. Martínez-Ramírez JA, Voigt K, Peters FT. Studies on the metabolism of five model drugs by fungi colonizing cadavers using LC-ESI-MS/MS and GC-MS analysis. Anal Bioanal Chem. 2012;404(5):1339–59.
63. Atlas RM. Handbook of Microbiological Media: Second Edition [Internet]. CRC-Press; 1996. Available from: https://books.google.com.co/books?id=u0HDQgAACAAJ
64. Martínez-Ramírez JA, Strien J, Walther G, Peters FT. Search for fungi-specific metabolites of four model drugs in postmortem blood as potential indicators of postmortem fungal metabolism. Forensic Sci Int. 2016
65. Baydoun E, Karam M, Atia-tul-Wahab, Khan MSA, Ahmad MS, Samreen, et al. Microbial transformation of nandrolone with Cunninghamella echinulata and Cunninghamella blakesleeana and evaluation of leishmaniacidal activity of transformed products. Steroids. 2014;88:95–100.
66. Rydevik A, Thevis M, Krug O, Bondesson U, Hedeland M. The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs). Xenobiotica. 2013;43(5):409–20.
67. Andrea E, Páez S. Estudio del metabolismo in vitro de catinonas sintéticas a través de hongos del género Cunninghamella. Universidad Nacional de Colombia; 2019.
68. Martinez-Brito D, de la Torre X, Parr MK, Botrè F. Mass spectrometric analysis of 7-oxygenated androst-5-ene structures. Influence in trimethylsilyl derivative formation. Rapid Commun Mass Spectrom. 2020;34(17).
69. Schänzer W, Donike M. Metabolism of anabolic steroids in man : synthesis and use of reference substances for identification of anabolic steroid metabolites. Anal Chim Acta. 1993;275:23–48.
70. Segura J, Ventura R, Jurado C. Derivatization procedures for gas chromatographic-mass spectrometric determination of xenobiotics in biological samples, with special attention to drugs of abuse and doping agents. J Chromatogr B Biomed Appl. 1998;713(1):61–90.
71. Jürgen H G. Mass Spectrometry. Third Edit. Switzerland: Springer International Publishing; 2017. 986 p.
72. WADA. WADA Technical Document - TD2021EAAS. Measurement and Reporting of Endogenous Anabolic Androgenic Steroid ( EAAS ) Markers of the Urinary Steroid Profile. 2021. p. 1–11.
73. Rydevik A, Bondesson U, Hedeland M. Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry. Rapid Commun Mass Spectrom. 2012;26(11):1338–46.
74. Mei J, Wang L, Wang S, Zhan J. Synthesis of two new hydroxylated derivatives of spironolactone by microbial transformation. Bioorg Med Chem Lett. 2014 Jul;24(14):3023–5.
75. Zafar S, Yousuf S, Kayani HA, Saifullah S, Khan S, Al-Majid AM, et al. Biotransformation of oral contraceptive ethynodiol diacetate with microbial and plant cell cultures. Chem Cent J. 2012;6(1):452.
76. Hooijerink D, Schilt R, Hoogenboom R, Huveneers-Oorsprong M. Identification of metabolites of the anabolic steroid methandienone formed by bovine hepatocytes in vitro†. Analyst. 1998;123(12):2637–41.
77. Hu S hui, Genain G, Azerad R. Microbial transformation of steroids: Contribution to 14α-hydroxylations. Steroids. 1995;60(4):337–52.
78. Rydevik A, Bondesson U, Thevis M, Hedeland M. Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO. J Pharm Biomed Anal. 2013;84:278–84.
79. Rydevik A, Hansson A, Hellqvist A, Bondesson U, Hedeland M. A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry. Drug Test Anal. 2015 Jul;7(7):626–33.
dc.rights.spa.fl_str_mv Derechos reservados al autor, 2021
dc.rights.coar.fl_str_mv http://purl.org/coar/access_right/c_abf2
dc.rights.license.spa.fl_str_mv Atribución-NoComercial-SinDerivadas 4.0 Internacional
dc.rights.uri.spa.fl_str_mv http://creativecommons.org/licenses/by-nc-nd/4.0/
dc.rights.accessrights.spa.fl_str_mv info:eu-repo/semantics/openAccess
rights_invalid_str_mv Atribución-NoComercial-SinDerivadas 4.0 Internacional
Derechos reservados al autor, 2021
http://creativecommons.org/licenses/by-nc-nd/4.0/
http://purl.org/coar/access_right/c_abf2
eu_rights_str_mv openAccess
dc.format.extent.spa.fl_str_mv 77 páginas
dc.format.mimetype.spa.fl_str_mv application/pdf
dc.publisher.spa.fl_str_mv Universidad Nacional de Colombia
dc.publisher.program.spa.fl_str_mv Bogotá - Medicina - Maestría en Toxicología
dc.publisher.department.spa.fl_str_mv Departamento de Toxicología
dc.publisher.faculty.spa.fl_str_mv Facultad de Medicina
dc.publisher.place.spa.fl_str_mv Bogotá, Colombia
dc.publisher.branch.spa.fl_str_mv Universidad Nacional de Colombia - Sede Bogotá
institution Universidad Nacional de Colombia
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spelling Atribución-NoComercial-SinDerivadas 4.0 InternacionalDerechos reservados al autor, 2021http://creativecommons.org/licenses/by-nc-nd/4.0/info:eu-repo/semantics/openAccesshttp://purl.org/coar/access_right/c_abf2Chaves Silva, Diana Carolina20e776318a9d5628459c32150f5783c2600Cárdenas Cuadros, Paola Andreaf718f1bf94bfc24b46db25d1c568a3ceMartínez Ramírez, Jorge Ariel17867d308b8a40fb2b3ed93fa4432138600Toscano Bayona, Lucía7f8b9cb9a4bc0dbcb368a30b79098019Sustancias Psicoactivas2021-08-06T18:02:02Z2021-08-06T18:02:02Z2021-05https://repositorio.unal.edu.co/handle/unal/79894Universidad Nacional de ColombiaRepositorio Institucional Universidad Nacional de Colombiahttps://repositorio.unal.edu.co/ilustraciones, tablasLa metandienona uno de los anabolizantes más reportado en los hallazgos analíticos adversos de los laboratorios de control al dopaje a nivel mundial. La molécula fue inicialmente usada para el tratamiento para el hipogonadismo, sin embargo, tiempo después su uso se expandió con el fin de obtener mayor masa muscular y un mejor rendimiento a nivel deportivo. Su metabolismo ha sido estudiado a través de diferentes modelos in vivo, con ratones y humanos e in vitro con fragmentos celulares del hígado, esto con el fin de establecer los metabolitos que indiquen su uso a corto y largo plazo que permitan su detección en las muestras de control al dopaje. Con este trabajo se busca estudiar el metabolismo de la metandienona a través de un modelo in vitro de biotransformación con el hongo Cunninghamella elegans, el cual ha sido utilizado con gran variedad de moléculas debido a que su sistema enzimático es similar al de los mamíferos. Con el desarrollo del modelo se proponen cuatro metabolitos monohidroxilados en las posiciones 6,7,14,15, esto se realizó a través de la técnica de cromatografía de gases acoplada a espectrometría de masas GC-MS/EI, se plantearon las posibles rutas de fragmentación y se compararon con los reportados para otros modelos de biotransformación.Methandienone is one of the most reported anabolics in the adverse analytical findings of doping control laboratories worldwide. The molecule was initially used for the treatment of hypogonadism, however, later its use expanded to obtain greater muscle mass and better performance at the sports level. Its metabolism has been studied through different in vivo models, with mice and humans and in vitro with liver cell fragments, this to establish the metabolites that indicate its use in the short and long term that allow its detection in the samples. doping control. This work seeks to study the metabolism of methandienone through an in vitro model of biotransformation with the fungus Cunninghamella elegans, which has been used with a great variety of molecules because its enzymatic system is like mammals. With the development of the model, four monohydroxylated metabolites were identified at positions 6,7,14,15, this was done through the gas chromatography technique coupled to mass spectrometry (GC-MS / EI), the possible routes of fragmentation and were compared with those reported for other biotransformation models.MaestríaMagíster en ToxicologíaComportamiento de variables toxicológicas y de salud relacionadas con el consumo de sustancias psicoactivas.77 páginasapplication/pdfspaUniversidad Nacional de ColombiaBogotá - Medicina - Maestría en ToxicologíaDepartamento de ToxicologíaFacultad de MedicinaBogotá, ColombiaUniversidad Nacional de Colombia - Sede Bogotá610 - Medicina y saludDoping en los DeportesDoping in SportsmetandienonaBiotransformaciónmetabolismoesteroides anabolizantesMetabolismMethandienoneCunninghamella elegansAnabolic steroidsBiotransformationEstudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegansMethandienone metabolism study through an in vitro incubation model with the fungus Cunninghamella elegansTrabajo de grado - Maestríainfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/acceptedVersionTexthttp://purl.org/redcol/resource_type/TM1. Barceloux DG. Anabolic-Androgenic Steroids. In: Medical Toxicology of Drug Abuse. Hoboken, NJ, USA: John Wiley & Sons, Inc.; 2012. p. 275–94.2. Aguilar M, Muñoz-Guerra J, Plata M del M, Del Coso J. Thirteen years of the fight against doping in figures. Drug Test Anal. 2017;9(6):866–9.3. Schänzer W, Geyer H, Donike M. Metabolism of metandienone in man: Identification and synthesis of conjugated excreted urinary metabolites, determination of excretion rates and gas chromatographic-mass spectrometric identification of bis-hydroxylated metabolites. J Steroid Biochem Mol Biol. 1991 Apr;38(4):441–64.4. Hagedorn HW, Schulz R, Friedrich A. Detection of methandienone (methandrostenolone) and metabolites in horse urine by gas chromatography-mass spectrometry. J Chromatogr B Biomed Sci Appl. 1992;577(2):195–203.5. Lootens L, Meuleman P, Pozo OJ, Van Eenoo P, Leroux-Roels G, Delbeke FT. uPA+/+-SCID Mouse with Humanized Liver as a Model for In Vivo Metabolism of Exogenous Steroids: Methandienone as a Case Study. Clin Chem. 2009;55(10):1783–93.6. Khan NT, Zafar S, Noreen S, Al Majid AM, Al Othman ZA, Al-Resayes SI, et al. Biotransformation of dianabol with the filamentous fungi and β-glucuronidase inhibitory activity of resulting metabolites. Steroids. 2014;85:65–72.7. Kicman AT. Pharmacology of anabolic steroids. Vol. 154, British Journal of Pharmacology. Wiley/Blackwell (10.1111); 2008. p. 502–21.8. Morley JE. Anabolic Steroids and Frailty. J Am Med Dir Assoc. 2010 Oct;11(8):533–6.9. Barceloux DG. MEDICAL TOXICOLOGY OF DRUG ABUSE. John Wiley & Sons, Inc.; 2012. 1041 p.10. Kicman AT, Gower DB. Anabolic steroids in sport: biochemical, clinical and analytical perspectives. Ann Clin Biochem Int J Lab Med. 2003;40(4):321–56.11. Link AF, Chemistry B. Drug Metabolism. Encycl Syst Biol. 2013;(March):618–618.12. Repetto Jiménez M, Repetto Kuhn G. Toxicología Fundamental. Cuarta Edi. Sevilla: Díaz de Santos; 2009. 629 p.13. Gallego Fernández A, de Sande García MA, Marín Fernández AM, Blanco Ramos S, González Galán MJ. Aspectos fundamentales del citocromo P450. Fundación Tejerina. 2011.14. Asha S, Vidyavathi M. Cunninghamella – A microbial model for drug metabolism studies – A review. Biotechnol Adv. 2009 Jan;27(1):16–29.15. Curtis D. Klaassen. Casarett and Doull’s Toxicology: The Basic Science of Poisons. Eighth Edi. Klaassen CD, editor. McGraw-Hill Education; 2013. 1454 p.16. Schänzer W. Metabolism of anabolic androgenic steroids. Clin Chem. 1996;42(7):1001–20.17. Hofmann FB, Beavo J a, Busch A, Ganten D, Michel MC, Page CP, et al. Doping in Sports. Thieme D, Hemmersbach P, editors. Berlin, Heidelberg: Springer Berlin Heidelberg; 2010. 274 p. (Handbook of Experimental Pharmacology; vol. 195).18. Baggish AL, Weiner RB, Kanayama G, Hudson JI, Lu MT, Hoffmann U, et al. Cardiovascular Toxicity of Illicit Anabolic-Androgenic Steroid Use. Circulation. 2017 May 23;135(21):1991–2002.19. White M, Brennan E, Mi Ren KY, Shi M, Thakrar A. Anabolic Androgenic Steroid Use as a Cause of Fulminant Heart Failure. Can J Cardiol. 2018;34(10):1369.20. Montisci M, El Mazloum R, Cecchetto G, Terranova C, Ferrara SD, Thiene G, et al. Anabolic androgenic steroids abuse and cardiac death in athletes: Morphological and toxicological findings in four fatal cases. Forensic Sci Int. 2012;217(1–3):12–7.21. Rocha M, Aguiar F, Ramos H. O uso de esteroides androgénicos anabolizantes e outros suplementos ergogénicos – uma epidemia silenciosa. Rev Port Endocrinol Diabetes e Metab. 2014;9(2):98–105.22. Hartgens F, Kuipers H. Effects of Androgenic-Anabolic Steroids in Athletes. Sport Med. 2004;34(8):513–54. 23. Hall RCW, Hall RCW. Abuse of Supraphysiologic Doses of Anabolic Steroids. South Med J. 2005;98(5):550–6.24. Christou MA, Christou PA, Markozannes G, Tsatsoulis A, Mastorakos G, Tigas S. Effects of Anabolic Androgenic Steroids on the Reproductive System of Athletes and Recreational Users: A Systematic Review and Meta-Analysis. Vol. 47, Sports Medicine. 2017. p. 1869–83.25. Nieschlag E, Vorona E. Mechanisms in Endocrinology: Medical consequences of doping with anabolic androgenic steroids: Effects on reproductive functions. Eur J Endocrinol. 2015;173(2):R47–58.26. Nieschlag E, Vorona E. Doping with anabolic androgenic steroids (AAS): Adverse effects on non-reproductive organs and functions. Vol. 16, Reviews in Endocrine and Metabolic Disorders. 2015. p. 199–211.27. Schänzer W, Opfermann G, Donike M. 17-Epimerization of 17α-methyl anabolic steroids in humans: metabolism and synthesis of 17α-hydroxy-17β-methyl steroids. Steroids. 1992;57(11):537–50.28. Schänzer W, Delahaut P, Geyer H, Machnik M, Horning S. Long-term detection and identification of metandienone and stanozolol abuse in athletes by gas chromatography-high-resolution mass spectrometry. J Chromatogr B Biomed Appl. 1996;687(1):93–108.29. Kanayama G, Pope HG. History and epidemiology of anabolic androgens in athletes and non-athletes. Mol Cell Endocrinol. 2018;464.30. Barrett-Connor EL. Testosterone and risk factors for cardiovascular disease in men. Diabete Metab. 1995;21(3):156–61. 31. U.S. DEPARTMENT OF JUSTICE • DRUG ENFORCEMENT ADMINISTRATION. Diversion Control Division [Internet]. [cited 2020 Sep 23]. Available from: https://www.deadiversion.usdoj.gov/pubs/brochures/steroids/public/32. Goverment of Canada. Controlled Drugs and Substances Act [Internet]. 1996. Available from: http://laws-lois.justice.gc.ca/eng/acts/C-38.8/33. Advisory Council on the Misuse of Drugs. Consideration of the Anabolic Steroids. 2010; Available from: https://assets.publishing.service.gov.uk/government/uploads/system/uploads/attachment_data/file/119132/anabolic-steroids.pdf34. Australian Institute of Criminology. Illicict drugs and alcohol [Internet]. 2006 [cited 2020 Oct 2]. Available from: https://web.archive.org/web/20070405033442/http://www.aic.gov.au/research/drugs/types/steroids.html35. Ministério da Saúde. Regulamento Técnico sobre substâncias e medicamentos sujeitos a controle especial [Internet]. Portaria no 344. 1998 [cited 2020 Sep 27]. Available from: http://bvsms.saude.gov.br/bvs/saudelegis/svs/1998/prt0344_12_05_1998_rep.html36. WADA. International Standard for Laboratories. [Internet]. Canada; 2019 [cited 2020 Nov 5]. Available from: https://www.wada-ama.org/sites/default/files/resources/files/isl_nov2019.pdf37. WADA. WADA Technical Document – TD2019MRPL. 2019.38. Geyer H, Schänzer W, Thevis M. Anabolic agents: Recent strategies for their detection and protection from inadvertent doping. Br J Sports Med. 2014;48(10):820–6.39. Schänzer W, Geyer H, Fußhöller G, Halatcheva N, Kohler M, Parr MK, et al. Mass spectrometric identification and characterization of a new long-term metabolite of metandienone in human urine. Rapid Commun Mass Spectrom. 2006;20(15):2252–8.40. Piska K, |elaszczyk D, Jamrozik M, Kubowicz-Kwa|ny P, P|kala E. Cunninghamella Biotransformation - Similarities to Human Drug Metabolism and Its Relevance for the Drug Discovery Process. Curr Drug Metab. 2016 Jan;17(2):107–17.41. Ahmad MS, Zafar S, Bibi M, Bano S, Atia-tul-Wahab, Atta-Ur-Rahman, et al. Biotransformation of androgenic steroid mesterolone with Cunninghamella blakesleeana and Macrophomina phaseolina. Steroids. 2014;82:53–9.42. Choudhary MI, Khan NT, Musharraf SG, Anjum S, Atta-ur-Rahman. Biotransformation of adrenosterone by filamentous fungus, Cunninghamella elegans. Steroids. 2007;72(14):923–9.43. Siddiqui M, Ahmad MS, Wahab A-T, Yousuf S, Fatima N, Shaikh NN, et al. Biotransformation of a potent anabolic steroid, mibolerone, with Cunninghamella blakesleeana, C. echinulata, and Macrophomina phaseolina, and biological activity evaluation of its metabolites. Shahid M, editor. PLoS One. 2017;12(2).44. Ma B, Huang H, Chen X, Sun Y, Lin L, Zhong D. Biotransformation of metoprolol by the fungus Cunninghamella blakesleeana. Acta Pharmacol Sin. 2007 Jul 1;28(7):1067–74.45. Åberg AT, Löfgren H, Bondesson U, Hedeland M. Structural elucidation of N -oxidized clemastine metabolites by liquid chromatography/tandem mass spectrometry and the use of Cunninghamella elegans to facilitate drug metabolite identification. Rapid Commun Mass Spectrom. 2010;24(10):1447–56.46. Zhong D-FF, Sun L, Liu L, Huang H-HH. Microbial transformation of naproxen by Cunninghamella species. Acta Pharmacol Sin. 2003 May;24(5):442–7.47. Dumasia MC. In vivo biotransformation of 17α-methyltestosterone in the horse revisited: identification of 17-hydroxymethyl metabolites in equine urine by capillary gas chromatography/mass spectrometry. Rapid Commun Mass Spectrom. 2003 Feb 28;17(4):320–9.48. Ferris JP, Fasco MJ, Stylianopoulou FL, Jerina DM, Daly JW, Jeffrey AM. Monooxygenase activity in Cunninghamella bainieri: Evidence for a fungal system similar to liver microsomes. Arch Biochem Biophys. 1973;156(1):97–103.49. Zhang D, Yang Y, Leakey JE., Cerniglia CE. Phase I and phase II enzymes produced by Cunninghamella elegans for the metabolism of xenobiotics. FEMS Microbiol Lett. 1996 May 1;138(2–3):221–6.50. Bhosale S, Saratale G, Govindwar S. Biotransformation enzymes in Cunninghamella blakesleeana (NCIM-687). J Basic Microbiol. 2006 Dec 1;46(6):444–8.51. Smith R, Rosazza J. Microbial Models Aromatic of Mammalian Hydroxylation. Arch Biochem Biophys. 1974;161:551–8.52. Summary E, Report S, Report TA, Analysis ABPR. Anti-Doping Testing Figures Executive Summary. 9:10–36.53. World Anti-doping Agency. 2016 Anti-Doping Testing Figures Laboratory Report [Internet]. 2016. Available from: https://www.wada-ama.org/sites/default/files/resources/files/2018_testing_figures_report.pdf54. Kam PCA, Yarrow M. Anabolic steroid abuse: Physiological and anaesthetic considerations. Vol. 60, Anaesthesia. 2005. p. 685–92.55. Neri M, Bello S, Bonsignore A, Cantatore S, Riezzo I, Turillazzi E, et al. Anabolic Androgenic Steroids Abuse and Liver Toxicity. Mini-Reviews Med Chem. 2011;11(5):430–7.56. Awai HI, Yu EL, Ellis LS, Schwimmer JB. Liver Toxicity of Anabolic Androgenic Steroid Use in an Adolescent With Nonalcoholic Fatty Liver Disease. J Pediatr Gastroenterol Nutr. 2014;59(3):e32–3.57. Rockhold RW. Cardiovascular toxicity of anabolic steroids. AnnuRevPharmacolToxicol. 1993;33:497–520.58. Santora LJ, Marin J, Vangrow J, Minegar C, Robinson M, Mora J, et al. Coronary calcification in body builders using anabolic steroids. Prev Cardiol. 2006;9(4):198–201.59. Watanabe S, Kuzhiumparambil U, Winiarski Z, Fu S. Data on individual metabolites of synthetic cannabinoids JWH-018, JWH-073 and AM2201 by Cunninghamella elegans. Data Br. 2016 Jun;7:332–40.60. Martínez-Ramírez JA, Walther G, Peters FT. Studies on drug metabolism by fungi colonizing decomposing human cadavers. Part II: biotransformation of five model drugs by fungi isolated from post-mortem material. Drug Test Anal. 2015 Apr;7(4):265–79.61. Rydevik A, Lagojda A, Thevis M, Bondesson U, Hedeland M. Isolation and characterization of a β-glucuronide of hydroxylated SARM S1 produced using a combination of biotransformation and chemical oxidation. J Pharm Biomed Anal. 2014 Sep;98:36–9.62. Martínez-Ramírez JA, Voigt K, Peters FT. Studies on the metabolism of five model drugs by fungi colonizing cadavers using LC-ESI-MS/MS and GC-MS analysis. Anal Bioanal Chem. 2012;404(5):1339–59.63. Atlas RM. Handbook of Microbiological Media: Second Edition [Internet]. CRC-Press; 1996. Available from: https://books.google.com.co/books?id=u0HDQgAACAAJ64. Martínez-Ramírez JA, Strien J, Walther G, Peters FT. Search for fungi-specific metabolites of four model drugs in postmortem blood as potential indicators of postmortem fungal metabolism. Forensic Sci Int. 201665. Baydoun E, Karam M, Atia-tul-Wahab, Khan MSA, Ahmad MS, Samreen, et al. Microbial transformation of nandrolone with Cunninghamella echinulata and Cunninghamella blakesleeana and evaluation of leishmaniacidal activity of transformed products. Steroids. 2014;88:95–100.66. Rydevik A, Thevis M, Krug O, Bondesson U, Hedeland M. The fungus Cunninghamella elegans can produce human and equine metabolites of selective androgen receptor modulators (SARMs). Xenobiotica. 2013;43(5):409–20.67. Andrea E, Páez S. Estudio del metabolismo in vitro de catinonas sintéticas a través de hongos del género Cunninghamella. Universidad Nacional de Colombia; 2019.68. Martinez-Brito D, de la Torre X, Parr MK, Botrè F. Mass spectrometric analysis of 7-oxygenated androst-5-ene structures. Influence in trimethylsilyl derivative formation. Rapid Commun Mass Spectrom. 2020;34(17).69. Schänzer W, Donike M. Metabolism of anabolic steroids in man : synthesis and use of reference substances for identification of anabolic steroid metabolites. Anal Chim Acta. 1993;275:23–48.70. Segura J, Ventura R, Jurado C. Derivatization procedures for gas chromatographic-mass spectrometric determination of xenobiotics in biological samples, with special attention to drugs of abuse and doping agents. J Chromatogr B Biomed Appl. 1998;713(1):61–90.71. Jürgen H G. Mass Spectrometry. Third Edit. Switzerland: Springer International Publishing; 2017. 986 p.72. WADA. WADA Technical Document - TD2021EAAS. Measurement and Reporting of Endogenous Anabolic Androgenic Steroid ( EAAS ) Markers of the Urinary Steroid Profile. 2021. p. 1–11.73. Rydevik A, Bondesson U, Hedeland M. Structural elucidation of phase I and II metabolites of bupivacaine in horse urine and fungi of the Cunninghamella species using liquid chromatography/multi-stage mass spectrometry. Rapid Commun Mass Spectrom. 2012;26(11):1338–46.74. Mei J, Wang L, Wang S, Zhan J. Synthesis of two new hydroxylated derivatives of spironolactone by microbial transformation. Bioorg Med Chem Lett. 2014 Jul;24(14):3023–5.75. Zafar S, Yousuf S, Kayani HA, Saifullah S, Khan S, Al-Majid AM, et al. Biotransformation of oral contraceptive ethynodiol diacetate with microbial and plant cell cultures. Chem Cent J. 2012;6(1):452.76. Hooijerink D, Schilt R, Hoogenboom R, Huveneers-Oorsprong M. Identification of metabolites of the anabolic steroid methandienone formed by bovine hepatocytes in vitro†. Analyst. 1998;123(12):2637–41.77. Hu S hui, Genain G, Azerad R. Microbial transformation of steroids: Contribution to 14α-hydroxylations. Steroids. 1995;60(4):337–52.78. Rydevik A, Bondesson U, Thevis M, Hedeland M. Mass spectrometric characterization of glucuronides formed by a new concept, combining Cunninghamella elegans with TEMPO. J Pharm Biomed Anal. 2013;84:278–84.79. Rydevik A, Hansson A, Hellqvist A, Bondesson U, Hedeland M. A novel trapping system for the detection of reactive drug metabolites using the fungus Cunninghamella elegans and high resolution mass spectrometry. Drug Test Anal. 2015 Jul;7(7):626–33.GeneralEstudio del metabolismo de la metandienona a través de un modelo in vitro de incubación con el hongo Cunninghamella elegansConvocatoria Invitación a presentar proyectos de investigación Ministerio del Deporte y Universidad Nacional de ColombiaLICENSElicense.txtlicense.txttext/plain; charset=utf-83964https://repositorio.unal.edu.co/bitstream/unal/79894/1/license.txtcccfe52f796b7c63423298c2d3365fc6MD51ORIGINAL1032432126.2021.pdf1032432126.2021.pdfTesis de Maestría en Toxicologíaapplication/pdf4926543https://repositorio.unal.edu.co/bitstream/unal/79894/2/1032432126.2021.pdf9c7912cf3111c7d03259a0fe2ab49ab0MD52CC-LICENSElicense_rdflicense_rdfapplication/rdf+xml; charset=utf-8805https://repositorio.unal.edu.co/bitstream/unal/79894/3/license_rdf4460e5956bc1d1639be9ae6146a50347MD53THUMBNAIL1032432126.2021.pdf.jpg1032432126.2021.pdf.jpgGenerated Thumbnailimage/jpeg4666https://repositorio.unal.edu.co/bitstream/unal/79894/4/1032432126.2021.pdf.jpg6112eaee564da5793babde62e6ac7dd9MD54unal/79894oai:repositorio.unal.edu.co:unal/798942023-07-24 23:04:40.662Repositorio Institucional Universidad Nacional de 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GVyZWNob3MgZGUgYXV0b3IgcXVlIGNvbmxsZXZlIGxhIGRpc3RyaWJ1Y2nDs24gZGUgZXN0b3MgYXJjaGl2b3MgeSBtZXRhZGF0b3MuCkFsIGhhY2VyIGNsaWMgZW4gZWwgc2lndWllbnRlIGJvdMOzbiwgdXN0ZWQgaW5kaWNhIHF1ZSBlc3TDoSBkZSBhY3VlcmRvIGNvbiBlc3RvcyB0w6lybWlub3MuCgpVTklWRVJTSURBRCBOQUNJT05BTCBERSBDT0xPTUJJQSAtIMOabHRpbWEgbW9kaWZpY2FjacOzbiAyNy8yMC8yMDIwCg==

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