Background: To date, even if anabolic androgenic steroid (AAS) abuse is clearly associated with a broad spectrum of collateral effects, adolescents and athletes frequently use a large group of synthetic derivatives of testosterone, both for esthetical purposes and for improving their performance. AAS use is frequently associated with different substances such as energy drinks and dietary supplements. The combined use of different substances, such as AASs, energy drinks, and dietary supplements, could worsen each adverse effect, worsening the risk for several pathologies such as cardiovascular disease. Moreover, the presence of other silent substrates (for example, the genetic predisposition to cardiovascular diseases, the presence of the Metabolic Syndrome or physical inactivity) combined with the use of this kind of substance can represent an explosive mix for the users, causing severe consequences and, in particular cases, death. Open questions: One common factor between detecting each of these performance- enhancing drugs is that the traditional methods of drug testing were taken at a single time point. An enhanced method of detection has been required to counter the increasingly sophisticated doping regimens and the ongoing development of new substances. The contemporary use of these substances could represent an important warning for public health, worsening the cost of health care. Aims: The goal of this PhD project was to analyze several important aspects linked to the use of AASs, defining the following: 1 - organ damage; 2 - new molecular biomarkers; 3 - epidemiology; 4 - pathology; 5 - toxicology. Research plan: (i) During the project, two complete literature reviews were performed using the most used scientific databases. (ii) All samples were selected analyzing the documentation of all autopsies performed by the Institute of Legal Medicine of Foggia from 2001 to July 2018 (about 1700 autopsies): - 5 cases of young men positive for AAS use were selected for the “AAS group”; - 6 cases of men who died of heart failure were enrolled as the "HF (heart failure) group", with the aims to evaluate the heart damages; - 15 cases composed the three groups enrolled to analyze brain injury (5 for the “Stroke group (SG)”, 5 for the “Drug group (DG)”, and 5 for the “Aging group (AG)”); - 5 cases of men who died of cardiac arrest who had suffered from cirrhosis caused by non-alcoholic fatty liver disease (NAFLD group), were selected to evaluate the liver damages; - 5 cases of men who had died of cardiac arrest after a long period (more than 10 years) of Chronic kidney disease (CKD group), were enrolled to evaluate kidney damages; - 6 cases of healthy men, who had died in car accidents were enrolled to compose the control groups: 4 cases (age mean 28 ± 7.4 with negative anamnesis for heart suffering and negative for the toxicological examination were selected as Control Group in the Quantitative Real-Time PCR (qRT-PCR) reactions for the miRNA tested in cardiac and musculoskeletal tissues, and 2 cases (mean age 41.5 ± 2.12 years), who died in car accidents for causes other than brain trauma, NAFLD, CKD and negative for toxicological examination were selected as Control group in the qRT-PCR reactions performed on brain, liver, and kidney. (iii) Histological and toxicological examinations were performed in all groups; (iv) miRNA quantification through qRT-PCR was performed; (v) for each miRNA the prediction effect analysis was carried out through both with the literature review and “in silico”, using several informatics tools; (vi) two surveys using online data collection tools were performed: “Multidimensional Body-Self Relations Questionnaire (MBSRQ)” and “in house” questionnaire to define the voluptuary habits of the interviewees; (vii) Statistical analysis was carried out through the software Statistica for Windows. Results: The literature reviews were very important to define the main adverse effects linked to AAS use/abuse and to identify the “candidate miRNAs” to test on the tissue samples of the selected cases. The main adverse effects concerning AAS use were strictly linked to the reproductive systems, particularly in adolescents. Moreover, other organs that are severely damaged by AAS use were heart, kidney, liver, and brain. For these reasons, several miRNAs related to these organs were selected and tested in the selected groups. For heart and musculoskeletal tissues, the miRNA expression values of miRNA has-133a-3p, hsa- miR-208a-3p, hsa-miR-499a-3p, and hsa-miR-1-3p were tested. The expression levels of miR-133 in heart tissue (HF and AAS groups) and muscle tissue (AAS group) resulted upregulated compared to controls. The expression levels of miR-208 in heart tissue (HF and AAS groups) resulted upregulated compared to controls. These results were not confirmed in muscle tissue (AAS group), where expression levels were similarly to controls. The expression levels of miR-499 resulted upregulated in the HF group, while in the AAS group, the expression levels of this miRNA were similar to controls. The expression levels of miR- 1_3 both in HF and AAS groups resulted upregulated compared to controls. Moreover, this miRNA resulted overexpressed in the muscle (vastus lateralis) of the AAS group. In kidney tissue, miR-21 and miR-205 were tested. The expression values of miR-21 in kidney tissues were upregulated compared to controls, even if the CDK values were significantly higher than the AAS group. The expression levels of miR-205 were higher in the CKD group compared to the AAS group, even if they were overexpressed in both groups compared to controls. Analyzing the results on the liver tissue, comparing the expression values of miR-21 in the two tested groups (AAS vs NAFDL), even if the values were higher compared to controls, this miRNA resulted overexpressed in the AAS group with a statistical significant difference. Concerning the expression levels of miR-122 in each group, even if the values were overexpressed compared to controls, the NAFDL values were significantly higher than the AAS group. Analyzing the expression levels of miRNA-132, there were no significant differences comparing the AAS group to the NAFDL group, even if both miRNAs were higher compared to controls. The same results were obtained analyzing the data about miR- 155. On brain tissue, several miRNAs were tested (miR-21, miR-34, miR-124, miR-132, and miR-200b) in four groups (SG, AG, DG, and AAS groups). For miR-21 the expression values of the AAS group were similar to the expression values of the SG and AG groups and was significantly higher compared to DG. For miR-34, the AAS group showed expression levels of this miRNA similar to the DG group, resulting higher compared to the AG and SG groups. The tissue levels of miR-124 were higher in the AAS and SG groups compared to the DG and AG groups. miR-132 was higher in the DG and AAS groups compare to the AG and SG groups. Finally, miR-200b was significantly overexpressed in the AAS group, even if this miRNA was higher in all groups than in controls. The interpretation of miRNA expression was performed through several web tools (“in silico” analysis) ascertaining the possible effects of the aberrant expression at cellular levels. In this way, it was possible to achieve the main goal of this project, to ascertain the pivotal roles for several miRNAs expressed in specific tissue, linking them to specific organ damage. Finally, from the analysis of the questionnaires it was possible to create the first Italian map on the phenomenon linked to the use of several substances, such as AASs, Energy Drinks and Supplements. The collected data demonstrated that there was a significant association (p < 0.05) between the use of anabolic steroids and consumption of other substances, such as energy drinks and supplements. The potential health risks related to heavy consumption of these products have largely gone unaddressed. For these reasons, the effort of the scientific community both for a better understand and to better communicate the risks linked to the use of these substances should be improved. Significance and Impact of research: In the light of the results discussed in this project, it would appear that concerns in the scientific community and among the public regarding the potential adverse health effects of the increased consumption of energy drinks, supplements and AASs are broadly valid. As extensively discussed, AAS use is not limited to athletes, but concerns young people who use these kinds of substances for esthetical purposes. For these reasons, this ongoing field of research is very challenging for the scientific community, in particular for the forensic field, involving a large number of people. As demonstrated in this PhD project, the main organs involved as targets of the adverse effects of AAS use are heart, kidney, liver, and brain; in several cases, the pathologies could be fatal for the abusers as described in the discussed cases. The identification of new molecular biomarkers can be considered of interest for the scientific community, not only for anti-doping purposes but for public health, too. Indeed, considering the results of the questionnaires, the combined use of AASs with other substances such as supplements or energy drinks, is significantly higher compared to non- users, demonstrating the idea of polypharmacy for AAS users. These substances can be anonymously supplied by the internet, where they are sold with alleged effects on mental, physical and sexual performance without any real evidence-based research. Obviously, the adverse effects of each substance are combined with each other, increasing the health risks for users and the public health cost for assistance in severe cases such as cardiovascular diseases. Future trends: The potential health risks related to a heavy consumption of these products have largely gone unaddressed. For these reasons, the effort of the scientific community both for a better understanding and to better communicate the risks linked to the use of these substances should be improved.
Introduzione. Anche se l'abuso di steroidi anabolizzanti (SA) è chiaramente associato a un ampio spettro di effetti collaterali, gli adolescenti e gli atleti usano frequentemente diversi derivati sintetici di testosterone, sia a fini estetici che per migliorare le loro prestazioni. L'uso di SA è spesso associato a diverse sostanze come bevande energetiche e integratori alimentari. L'uso combinato di diverse sostanze, come SA, bevande energetiche e integratori alimentari, può peggiorare i singoli effetti collaterali di ciascuna sostanza, aggravando il rischio di diverse patologie correlate alla loro assunzione, come per esempio le patologie cardiovascolari. Pertanto, l'utilizzo contemporaneo di più sostanze può incrementare ogni singolo effetto collaterale, divenendo un importante problema per la salute pubblica, aggravando il costo dell'assistenza sanitaria. Inoltre, l’eventuale presenza di altri substrati ignoti (ad esempio, la predisposizione genetica alle malattie cardiovascolari, la presenza della sindrome metabolica o l'inattività fisica) combinata all'uso di queste sostanze, può rappresentare un mix esplosivo per i consumatori, generando gravi conseguenze per la salute individuale e, nei casi più gravi, la morte. Infine, un problema comune legato all’utilizzo di queste sostanze è quello relativo al loro monitoraggio, sia che si tratti di integratori alimentari o che si tratti di SA. Pertanto la messa a punto di test di rilevazione di ultima generazione è molto importante al fine di contrastare regimi di doping sempre più sofisticati agevolati anche dal continuo sviluppo di nuove sostanze dagli effetti simili ma dalla composizione chimica differente. Obiettivi: questo lavoro di dottorato mirava ad analizzare diversi aspetti importanti legati all'uso di SA, definendo: 1 - danni d'organo correlati; 2 –determinazione di nuovi potenziali biomarcatori molecolari; 3 - epidemiologia; 4 - patologia; 5 - tossicologia. Programma di ricerca: (i) Durante il progetto di dottorato, sono state eseguite due revisioni complete della letteratura utilizzando i database scientifici più diffusi. (ii) Tutti i campioni testati in questo lavoro sono stati selezionati analizzando la documentazione di tutte le autopsie eseguite presso l'Istituto di medicina legale dell’Università degli studi di Foggia dal 2001 a luglio 2018 (circa 1700 autopsie): - 5 casi di giovani positivi per l'uso di SA sono stati selezionati per il “gruppo AAS “; - 6 casi di uomini morti per insufficienza cardiaca sono stati arruolati come "gruppo HF (insufficienza cardiaca)" per la valutazione del danno cardiaco; - 15 casi hanno composto i tre gruppi arruolati per analizzare il danno d’organo encefalico: 5 per il gruppo "Stroke (SG)", 5 per il gruppo "Drug (DG)" e 5 per il gruppo "Aging (AG)"; - sono stati selezionati 5 casi di uomini deceduti per arresto cardiaco 3 che avevano sofferto, per un periodo pari o superiore a 10 anni, di cirrosi generata da steatosi epatica non alcolica (gruppo NAFLD) per la valutazione del danno epatico; - 5 casi di uomini deceduti per arresto cardiaco dopo un periodo pari o superiore a 10 anni di malattia renale cronica (gruppo CKD) per la valutazione del danno renale; - infine, 6 casi di uomini sani, deceduti in incidenti stradali sono stati arruolati come controlli: 4 casi (età media 28 ± 7,4) di soggetti con anamnesi negativa per patologie cardiache e negativi per l'esame tossicologico sono stati selezionati come gruppo di controllo nei test di alcuni miRNAs nei tessuti cardiaci e muscoloscheletrici; Inoltre, sono stati arruolati altri 2 casi (età media 41,5 ± 2,12 anni) di soggetti deceduti in incidenti, privi di danno encefalico e con esame tossicologico negativo. Questi ultimi due controlli sono stati utilizzati nelle reazioni di quantificazione eseguite su cervello, fegato e rene. (iii) Esami istologici ed esami tossicologici sono stati eseguiti in tutti i casi selezionati; (iv) la quantificazione dei miRNAs per tutti i gruppi ed i tessuti testati è avvenuta mediante “quantitative real-time PCR” (qRT- PCR); (v) infine per la predizione degli effetti di ciascun miRNA, sono stati utilizzati diversi tool per l’analisi in silico, capaci di predire i geni target. Inoltre, il “pathway” di ciascun miRNA è stato analizzato anche un’attenta revisione della letteratura al fine di identificare il suo meccanismo di azione all’interno del contesto abusers; (vi) Sono stati inoltre utilizzati due tipi di questionari come strumenti di raccolta dati mediante moduli on line: 1- “Multidimensional Body-Self Relations Questionnaire (MBSRQ)”; 2 - questionario “in house” per definire le abitudini voluttuarie degli intervistati; (vii) Infine l'analisi statistica è stata effettuata attraverso il software Statistica per Windows. Risultati: entrambi i lavori di revisione della letteratura scientifica sono stati molto importanti per definire i principali effetti avversi associati all'uso/abuso di SA e per identificare i "miRNA candidati"; questi lavori hanno consentito di sviluppare i successivi steps del lavoro di dottorato. In base ai dati di letteratura scientifica, i principali effetti avversi legati all’uso di SA sono prevalentemente a carico dei sistemi riproduttivi maschile e femminile, in particolare nel caso queste sostanze siano assunte da adolescenti. Inoltre, cuore, rene, fegato e cervello sono risultati essere gli altri organi bersaglio legati all'uso di SA. Pertanto, i miRNA sono stati selezionati e testati sugli organi bersaglio: cuore, fegato, rene, encefalo. Grazie al lavoro di revisione scientifica è stato possibile individuare una batteria di miRNA candidati legati al relativo danno d’organo. Tali miRNA sono stati testati sia nei soggetti del gruppo AAS che negli altri gruppi. Per il tessuto cardiaco e muscoloscheletrico, 4 sono stati testati i valori di espressione dei seguenti miRNA: hsa-miR-133a-3p, hsa-miR- 208a-3p, hsa-miR-499a-3p e hsa-miR-1-3p. I livelli di espressione di miR-133 nel tessuto cardiaco nei gruppi HF e AAS e nel tessuto muscolare (solo per il gruppo AAS) sono risultati aumentati rispetto ai controlli. I livelli di espressione di miR-208 nel tessuto cardiaco (gruppi HF e AAS) sono risultati up-regolati rispetto ai controlli. Questi risultati non sono stati confermati nel tessuto muscolare (gruppo AAS), dove i livelli di espressione rilevati sono risultati essere simili ai controlli. I livelli di espressione di miR-499 sono risultati up-regolati nel tessuto cardiaco nel gruppo HF. Nel cuore e nei tessuti muscolari del gruppo AAS, i livelli di espressione erano simili ai controlli. I livelli di espressione di miR-1_3 nel tessuto cardiaco (gruppi HF e AAS) e nel tessuto muscolare (gruppo AAS) sono risultati up-regolati rispetto ai controlli. Questo miRNA risultava over espresso nel muscolo (vastus lateralis) del gruppo AAS. Nel tessuto renale sono stati testati il miR-21 e il miR-205. I valori di espressione di miR- 21 nei tessuti renali sono risultati up-regolati rispetto ai controlli, anche se i valori nel gruppo CDK erano significativamente più alti rispetto al gruppo AAS. I livelli di espressione del miR-205 erano più alti nel gruppo CKD rispetto al gruppo AAS, anche se erano over espressi in entrambi i gruppi rispetto ai controlli. Nel tessuto epatico, confrontando i valori di espressione di miR-21 nei due gruppi testati (AAS vs NAFDL), anche se i valori erano più alti rispetto ai controlli in entrambi i gruppi, questo miRNA risultava over espresso nel gruppo AAS con una differenza statistica significativa. Analizzando i livelli di espressione di miR-122 in ciascun gruppo, anche se i valori erano up-regolati rispetto ai controlli, i valori nel gruppo NAFDL erano significativamente più alti rispetto al gruppo AAS. Analizzando i livelli di espressione di miRNA-132, non sono state rilevate differenze significative confrontando il gruppo AAS con il gruppo NAFDL, anche se entrambi i miRNA erano più alti rispetto ai controlli. Lo stesso trend è emerso analizzando i dati sui livelli di espressione del miR-155. Sul tessuto cerebrale sono stati testati diversi miRNA (miR-21, miR-34, miR-124, miR-132 e miR-200b) in quattro gruppi (gruppi SG, AG, DG e AAS). Per miR-21 i valori di espressione del gruppo AAS erano simili ai valori di espressione dei gruppi SG e AG ed erano significativamente più alti rispetto al gruppo DG. Per il miR-34, il gruppo AAS ha mostrato livelli di espressione simili al gruppo DG, risultando inoltre più elevati rispetto ai gruppi AG e SG. I livelli di espressione tessutale di miR-124 erano più alti nei gruppi AAS e SG confrontandoli con quelli dei gruppi DG e AG. miR-132 era più alto nei gruppi DG e 5 AAS rispetto ai gruppi AG e SG. Infine, miR-200b era significativamente over espresso nel gruppo AAS, anche se i livelli di espressione tissutale di questo miRNA erano più alti in tutti i gruppi rispetto ai livelli di espressione tissutale dei controlli. L'interpretazione dell'espressione dei miRNA è stata eseguita attraverso diversi software di predizione online (analisi “in silico”) al fine di individuare i possibili meccanismi di azione derivanti dell'espressione aberrante a livello cellulare dei miRNA testati. In questo modo, è stato possibile raggiungere l'obiettivo principale di questo progetto, cioè accertare i ruoli cardine per diversi miRNA, collegandoli al danno d'organo specifico. Infine, dall'analisi dell’indagine eseguita mediante i due sopracitati questionari è stato possibile creare la prima mappa italiana sul fenomeno legato all'uso di diverse sostanze (SA, bevande energetiche e integratori), correlandoli anche allo stato psicologico individuale dei soggetti intervistati. Tra i dati ottenuti, risulta senza dubbio importante evidenziare l'esistenza di un'associazione significativa (p <0,05) tra l'uso di steroidi anabolizzanti e il consumo di altre sostanze (bevande energetiche e integratori). Infatti gli utilizzatori di SA (circa il 4% degli intervistati) ha dichiarato di utilizzare contemporaneamente anche integratori e bevande energetiche. Questi dati hanno confermato l’idea già presentata in altri report realizzati in altri stati europei dell’utilizzo combinato tra SA, integratori e altre sostanze. In questo contesto, i potenziali rischi per la salute legati al consumo combinato di questi prodotti sono allo stato attualo ancora pochi investigati. Per tale ragione, dovrebbe essere migliorato lo sforzo della comunità scientifica per comprendere e comunicare meglio i rischi legati all'uso di queste sostanze. Significato e impatto della ricerca: alla luce dei risultati presentati in questo progetto, emerge che le recenti preoccupazioni della comunità scientifica internazionale in merito ai potenziali effetti negativi sulla salute dei consumatori legati al consumo di bevande energetiche, integratori e SA sono ampiamente valide. Come ampiamente discusso, l'uso di SA non si limita agli atleti, ma riguarda i giovani che usano questo tipo di sostanze anche per fini estetici. Per questi motivi, questo campo di ricerca è molto impegnativo per la comunità scientifica, in particolare per il settore forense, che vede coinvolte diverse figure professionali. Come dimostrato dai dati ottenuti dal presente progetto di dottorato, i principali organi bersaglio legato ai danni collaterali derivanti dall'uso di SA sono cuore, rene, fegato e cervello; in diversi casi, le patologie potrebbero essere fatali per gli utilizzatori come descritto nei casi presentati in questo lavoro di tesi. L'identificazione di nuovi biomarcatori 6 molecolari può essere considerata di interesse per la comunità scientifica, non solo per la definizione di metodi antidoping innovativi, ma anche per la salute pubblica. In effetti, considerando i risultati dei questionari, l'uso combinato di SA con altre sostanze come integratori o bevande energetiche, è significativamente più alto rispetto ai non utilizzatori, confermando l'idea del “polypharmacy” per gli utilizzatori SA. Queste sostanze possono essere acquistate anonimamente da Internet, dove vengono vendute con presunti effetti sulle prestazioni mentali, fisiche e sessuali nonostante non vi siano evidenze scientifiche su tali effetti. Inoltre, quando gli effetti negativi di ciascuna sostanza sono combinati tra loro, aumentando i rischi per la salute degli utenti, con la possibilità di incrementare i costi per l’assistenza sanitaria. Tendenze future: i rischi per la salute legati a un aumentato e combinato consumo di questi prodotti non sono stati completamente chiariti. Per questi motivi, anche alla luce dei risultati presentati in questo lavoro di tesi di dottorato, gli sforzi della comunità scientifica internazionale dovrebbero essere aumentati al fine sia di migliorare la comprensione del fenomeno che la comunicazione relativa ai rischi derivanti dall’uso singolo o combinato di queste sostanze.
Life style, medicine and anabolic steroids: organ damages, new molecular biomarkers, epidemiology, pathology and toxicology / Sessa, Francesco. - (2020). [10.14274/sessa-francesco_phd2020]
Life style, medicine and anabolic steroids: organ damages, new molecular biomarkers, epidemiology, pathology and toxicology
Sessa, Francesco
2020-01-01
Abstract
Background: To date, even if anabolic androgenic steroid (AAS) abuse is clearly associated with a broad spectrum of collateral effects, adolescents and athletes frequently use a large group of synthetic derivatives of testosterone, both for esthetical purposes and for improving their performance. AAS use is frequently associated with different substances such as energy drinks and dietary supplements. The combined use of different substances, such as AASs, energy drinks, and dietary supplements, could worsen each adverse effect, worsening the risk for several pathologies such as cardiovascular disease. Moreover, the presence of other silent substrates (for example, the genetic predisposition to cardiovascular diseases, the presence of the Metabolic Syndrome or physical inactivity) combined with the use of this kind of substance can represent an explosive mix for the users, causing severe consequences and, in particular cases, death. Open questions: One common factor between detecting each of these performance- enhancing drugs is that the traditional methods of drug testing were taken at a single time point. An enhanced method of detection has been required to counter the increasingly sophisticated doping regimens and the ongoing development of new substances. The contemporary use of these substances could represent an important warning for public health, worsening the cost of health care. Aims: The goal of this PhD project was to analyze several important aspects linked to the use of AASs, defining the following: 1 - organ damage; 2 - new molecular biomarkers; 3 - epidemiology; 4 - pathology; 5 - toxicology. Research plan: (i) During the project, two complete literature reviews were performed using the most used scientific databases. (ii) All samples were selected analyzing the documentation of all autopsies performed by the Institute of Legal Medicine of Foggia from 2001 to July 2018 (about 1700 autopsies): - 5 cases of young men positive for AAS use were selected for the “AAS group”; - 6 cases of men who died of heart failure were enrolled as the "HF (heart failure) group", with the aims to evaluate the heart damages; - 15 cases composed the three groups enrolled to analyze brain injury (5 for the “Stroke group (SG)”, 5 for the “Drug group (DG)”, and 5 for the “Aging group (AG)”); - 5 cases of men who died of cardiac arrest who had suffered from cirrhosis caused by non-alcoholic fatty liver disease (NAFLD group), were selected to evaluate the liver damages; - 5 cases of men who had died of cardiac arrest after a long period (more than 10 years) of Chronic kidney disease (CKD group), were enrolled to evaluate kidney damages; - 6 cases of healthy men, who had died in car accidents were enrolled to compose the control groups: 4 cases (age mean 28 ± 7.4 with negative anamnesis for heart suffering and negative for the toxicological examination were selected as Control Group in the Quantitative Real-Time PCR (qRT-PCR) reactions for the miRNA tested in cardiac and musculoskeletal tissues, and 2 cases (mean age 41.5 ± 2.12 years), who died in car accidents for causes other than brain trauma, NAFLD, CKD and negative for toxicological examination were selected as Control group in the qRT-PCR reactions performed on brain, liver, and kidney. (iii) Histological and toxicological examinations were performed in all groups; (iv) miRNA quantification through qRT-PCR was performed; (v) for each miRNA the prediction effect analysis was carried out through both with the literature review and “in silico”, using several informatics tools; (vi) two surveys using online data collection tools were performed: “Multidimensional Body-Self Relations Questionnaire (MBSRQ)” and “in house” questionnaire to define the voluptuary habits of the interviewees; (vii) Statistical analysis was carried out through the software Statistica for Windows. Results: The literature reviews were very important to define the main adverse effects linked to AAS use/abuse and to identify the “candidate miRNAs” to test on the tissue samples of the selected cases. The main adverse effects concerning AAS use were strictly linked to the reproductive systems, particularly in adolescents. Moreover, other organs that are severely damaged by AAS use were heart, kidney, liver, and brain. For these reasons, several miRNAs related to these organs were selected and tested in the selected groups. For heart and musculoskeletal tissues, the miRNA expression values of miRNA has-133a-3p, hsa- miR-208a-3p, hsa-miR-499a-3p, and hsa-miR-1-3p were tested. The expression levels of miR-133 in heart tissue (HF and AAS groups) and muscle tissue (AAS group) resulted upregulated compared to controls. The expression levels of miR-208 in heart tissue (HF and AAS groups) resulted upregulated compared to controls. These results were not confirmed in muscle tissue (AAS group), where expression levels were similarly to controls. The expression levels of miR-499 resulted upregulated in the HF group, while in the AAS group, the expression levels of this miRNA were similar to controls. The expression levels of miR- 1_3 both in HF and AAS groups resulted upregulated compared to controls. Moreover, this miRNA resulted overexpressed in the muscle (vastus lateralis) of the AAS group. In kidney tissue, miR-21 and miR-205 were tested. The expression values of miR-21 in kidney tissues were upregulated compared to controls, even if the CDK values were significantly higher than the AAS group. The expression levels of miR-205 were higher in the CKD group compared to the AAS group, even if they were overexpressed in both groups compared to controls. Analyzing the results on the liver tissue, comparing the expression values of miR-21 in the two tested groups (AAS vs NAFDL), even if the values were higher compared to controls, this miRNA resulted overexpressed in the AAS group with a statistical significant difference. Concerning the expression levels of miR-122 in each group, even if the values were overexpressed compared to controls, the NAFDL values were significantly higher than the AAS group. Analyzing the expression levels of miRNA-132, there were no significant differences comparing the AAS group to the NAFDL group, even if both miRNAs were higher compared to controls. The same results were obtained analyzing the data about miR- 155. On brain tissue, several miRNAs were tested (miR-21, miR-34, miR-124, miR-132, and miR-200b) in four groups (SG, AG, DG, and AAS groups). For miR-21 the expression values of the AAS group were similar to the expression values of the SG and AG groups and was significantly higher compared to DG. For miR-34, the AAS group showed expression levels of this miRNA similar to the DG group, resulting higher compared to the AG and SG groups. The tissue levels of miR-124 were higher in the AAS and SG groups compared to the DG and AG groups. miR-132 was higher in the DG and AAS groups compare to the AG and SG groups. Finally, miR-200b was significantly overexpressed in the AAS group, even if this miRNA was higher in all groups than in controls. The interpretation of miRNA expression was performed through several web tools (“in silico” analysis) ascertaining the possible effects of the aberrant expression at cellular levels. In this way, it was possible to achieve the main goal of this project, to ascertain the pivotal roles for several miRNAs expressed in specific tissue, linking them to specific organ damage. Finally, from the analysis of the questionnaires it was possible to create the first Italian map on the phenomenon linked to the use of several substances, such as AASs, Energy Drinks and Supplements. The collected data demonstrated that there was a significant association (p < 0.05) between the use of anabolic steroids and consumption of other substances, such as energy drinks and supplements. The potential health risks related to heavy consumption of these products have largely gone unaddressed. For these reasons, the effort of the scientific community both for a better understand and to better communicate the risks linked to the use of these substances should be improved. Significance and Impact of research: In the light of the results discussed in this project, it would appear that concerns in the scientific community and among the public regarding the potential adverse health effects of the increased consumption of energy drinks, supplements and AASs are broadly valid. As extensively discussed, AAS use is not limited to athletes, but concerns young people who use these kinds of substances for esthetical purposes. For these reasons, this ongoing field of research is very challenging for the scientific community, in particular for the forensic field, involving a large number of people. As demonstrated in this PhD project, the main organs involved as targets of the adverse effects of AAS use are heart, kidney, liver, and brain; in several cases, the pathologies could be fatal for the abusers as described in the discussed cases. The identification of new molecular biomarkers can be considered of interest for the scientific community, not only for anti-doping purposes but for public health, too. Indeed, considering the results of the questionnaires, the combined use of AASs with other substances such as supplements or energy drinks, is significantly higher compared to non- users, demonstrating the idea of polypharmacy for AAS users. These substances can be anonymously supplied by the internet, where they are sold with alleged effects on mental, physical and sexual performance without any real evidence-based research. Obviously, the adverse effects of each substance are combined with each other, increasing the health risks for users and the public health cost for assistance in severe cases such as cardiovascular diseases. Future trends: The potential health risks related to a heavy consumption of these products have largely gone unaddressed. For these reasons, the effort of the scientific community both for a better understanding and to better communicate the risks linked to the use of these substances should be improved.File | Dimensione | Formato | |
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Tesi dottorato Francesco SESSA- IMAEV (ciclo XXXII).pdf
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