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1.
Oncogene ; 2021 Feb 24.
Artigo em Inglês | MEDLINE | ID: mdl-33627786

RESUMO

Protein ubiquitination is a critical regulator of cellular homeostasis. Aberrations in the addition or removal of ubiquitin can result in the development of cancer and key components of the ubiquitination machinery serve as oncogenes or tumour suppressors. An emerging target in the development of cancer therapeutics are the deubiquitinase (DUB) enzymes that remove ubiquitin from protein substrates. Whether this class of enzyme plays a role in cervical cancer has not been fully explored. By interrogating the cervical cancer data from the TCGA consortium, we noted that the DUB USP13 is amplified in ~15% of cervical cancer cases. We confirmed that USP13 expression was increased in cervical cancer cell lines, cytology samples from patients with cervical disease and in cervical cancer tissue. Depletion of USP13 inhibited cervical cancer cell proliferation. Mechanistically, USP13 bound to, deubiquitinated and stabilised Mcl-1, a pivotal member of the anti-apoptotic BCL-2 family. Furthermore, reduced Mcl-1 expression partially contributed to the observed proliferative defect in USP13 depleted cells. Importantly, the expression of USP13 and Mcl-1 proteins correlated in cervical cancer tissue. Finally, we demonstrated that depletion of USP13 expression or inhibition of USP13 enzymatic activity increased the sensitivity of cervical cancer cells to the BH3 mimetic inhibitor ABT-263. Together, our data demonstrates that USP13 is a potential oncogene in cervical cancer that functions to stabilise the pro-survival protein Mcl-1, offering a potential therapeutic target for these cancers.

2.
J Gen Virol ; 2021 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-33427604

RESUMO

Persistent infection with high-risk human papillomaviruses (HR-HPVs) is the causal factor in over 99 % of cervical cancer cases, and a significant proportion of oropharyngeal and anogenital cancers. The key drivers of HPV-mediated transformation are the oncoproteins E5, E6 and E7. Together, they act to prolong cell-cycle progression, delay differentiation and inhibit apoptosis in the host keratinocyte cell in order to generate an environment permissive for viral replication. The oncoproteins also have key roles in mediating evasion of the host immune response, enabling infection to persist. Moreover, prolonged infection within the cellular environment established by the HR-HPV oncoproteins can lead to the acquisition of host genetic mutations, eventually culminating in transformation to malignancy. In this review, we outline the many ways in which the HR-HPV oncoproteins manipulate the host cellular environment, focusing on how these activities can contribute to carcinogenesis.

3.
Cell Death Differ ; 2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-33303976

RESUMO

Human papillomaviruses (HPV) are a major cause of malignancy worldwide, contributing to ~5% of all human cancers including almost all cases of cervical cancer and a growing number of ano-genital and oral cancers. HPV-induced malignancy is primarily driven by the viral oncogenes, E6 and E7, which manipulate host cellular pathways to increase cell proliferation and enhance cell survival, ultimately predisposing infected cells to malignant transformation. Consequently, a more detailed understanding of viral-host interactions in HPV-associated disease offers the potential to identify novel therapeutic targets. Here, we identify that the c-Jun N-terminal kinase (JNK) signalling pathway is activated in cervical disease and in cervical cancer. The HPV E6 oncogene induces JNK1/2 phosphorylation in a manner that requires the E6 PDZ binding motif. We show that blockade of JNK1/2 signalling using small molecule inhibitors, or knockdown of the canonical JNK substrate c-Jun, reduces cell proliferation and induces apoptosis in cervical cancer cells. We further demonstrate that this phenotype is at least partially driven by JNK-dependent activation of EGFR signalling via increased expression of EGFR and the EGFR ligands EGF and HB-EGF. JNK/c-Jun signalling promoted the invasive potential of cervical cancer cells and was required for the expression of the epithelial to mesenchymal transition (EMT)-associated transcription factor Slug and the mesenchymal marker Vimentin. Furthermore, JNK/c-Jun signalling is required for the constitutive expression of HPV E6 and E7, which are essential for cervical cancer cell growth and survival. Together, these data demonstrate a positive feedback loop between the EGFR signalling pathway and HPV E6/E7 expression, identifying a regulatory mechanism in which HPV drives EGFR signalling to promote proliferation, survival and EMT. Thus, our study has identified a novel therapeutic target that may be beneficial for the treatment of cervical cancer.

5.
Ophthalmology ; 127(5): 668-678, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32081490

RESUMO

PURPOSE: To identify susceptibility genes associated with hereditary predisposition to uveal melanoma (UM) in patients with no detectable germline BAP1 alterations. DESIGN: Retrospective case series from academic referral centers. PARTICIPANTS: Cohort of 154 UM patients with high risk of hereditary cancer defined as patients with 1 or more of the following: (1) familial UM, (2) young age (<35 years) at diagnosis, (3) personal history of other primary cancers, and (4) family history of 2 or more primary cancers with no detectable mutation or deletion in BAP1 gene. METHODS: Whole exome sequencing, a cancer gene panel, or both were carried out. Probands included 27 patients with familial UM, 1 patient with bilateral UM, 1 patient with congenital UM, and 125 UM patients with strong personal or family histories, or both, of cancer. Functional validation of variants was carried out by immunohistochemistry, reverse-transcriptase polymerase chain reaction, and genotyping. MAIN OUTCOME MEASURES: Clinical characterization of UM patients with germline alterations in known cancer genes. RESULTS: We identified actionable pathogenic variants in 8 known hereditary cancer predisposition genes (PALB2, MLH1, MSH6, CHEK2, SMARCE1, ATM, BRCA1, and CTNNA1) in 9 patients, including 3 of 27 patients (11%) with familial UM and 6 of 127 patients (4.7%) with a high risk for cancer. Two patients showed pathogenic variants in CHEK2 and PALB2, whereas variants in the other genes each occurred in 1 patient. Biallelic inactivation of PALB2 and MLH1 was observed in tumors from the respective patients. The frequencies of pathogenic variants in PALB2, MLH1, and SMARCE1 in UM patients were significantly higher than the observed frequencies in noncancer controls (PALB2: P = 0.02; odds ratio, 8.9; 95% confidence interval, 1.5-30.6; MLH1: P = 0.04; odds ratio, 25.4; 95% confidence interval, 1.2-143; SMARCE1: P = 0.001; odds ratio, 2047; 95% confidence interval, 52-4.5e15, respectively). CONCLUSIONS: The study provided moderate evidence of gene and disease association of germline mutations in PALB2 and MLH1 with hereditary predisposition to UM. It also identified several other candidate susceptibility genes. The results suggest locus heterogeneity in predisposition to UM. Genetic testing for hereditary predisposition to cancer is warranted in UM patients with strong personal or family history of cancers, or both.


Assuntos
Genes Neoplásicos/genética , Predisposição Genética para Doença/genética , Melanoma/genética , Proteínas de Neoplasias/genética , Neoplasias Uveais/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , DNA de Neoplasias/genética , Feminino , Mutação em Linhagem Germinativa/genética , Humanos , Imuno-Histoquímica , Lactente , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética , Sequenciamento Completo do Exoma
6.
J Vet Pharmacol Ther ; 43(2): 162-170, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-32012314

RESUMO

In equine and racing practice, detomidine and butorphanol are commonly used in combination for their sedative properties. The aim of the study was to produce detection times to better inform European veterinary surgeons, so that both drugs can be used appropriately under regulatory rules. Three independent groups of 7, 8 and 6 horses, respectively, were given either a single intravenous administration of butorphanol (100 µg/kg), a single intravenous administration of detomidine (10 µg/kg) or a combination of both at 25 (butorphanol) and 10 (detomidine) µg/kg. Plasma and urine concentrations of butorphanol, detomidine and 3-hydroxydetomidine at predetermined time points were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The intravenous pharmacokinetics of butorphanol dosed individually compared with co-administration with detomidine had approximately a twofold larger clearance (646 ± 137 vs. 380 ± 86 ml hr-1  kg-1 ) but similar terminal half-life (5.21 ± 1.56 vs. 5.43 ± 0.44 hr). Pseudo-steady-state urine to plasma butorphanol concentration ratios were 730 and 560, respectively. The intravenous pharmacokinetics of detomidine dosed as a single administration compared with co-administration with butorphanol had similar clearance (3,278 ± 1,412 vs. 2,519 ± 630 ml hr-1  kg-1 ) but a slightly shorter terminal half-life (0.57 ± 0.06 vs. 0.70 ± 0.11 hr). Pseudo-steady-state urine to plasma detomidine concentration ratios are 4 and 8, respectively. The 3-hydroxy metabolite of detomidine was detected for at least 35 hr in urine from both the single and co-administrations. Detection times of 72 and 48 hr are recommended for the control of butorphanol and detomidine, respectively, in horseracing and equestrian competitions.

7.
Drug Test Anal ; 12(2): 215-229, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31654498

RESUMO

Boldenone (1-dehydrotestosterone) is an exogenous anabolic-androgenic steroid (AAS) but is also known to be endogenous in the entire male horse and potentially formed by microbes in voided urine, the gastrointestinal tract, or feed resulting in its detection in urine samples. In this study, equine fecal and urine samples were incubated in the presence of selected stable isotope labeled AAS precursors to investigate whether microbial activity could result in 1-dehydrogenation, in particular the formation of boldenone. Fecal matter was initially selected for investigation because of its high microbial activity, which could help to identify potential 1-dehydrogenated biomarkers that might also be present in low quantities in urine. Fecal incubations displayed Δ1-dehydrogenase activity, as evidenced by the use of isotope labeled precursors to show the formation of boldenone and boldione from testosterone and androstenedione, as well as the formation of Δ1-progesterone and boldione from progesterone. Unlabeled forms were also produced in unspiked fecal samples with Δ1-progesterone being identified for the first time. Subsequent incubation of urine samples with the labeled AAS precursors demonstrated that Δ1-dehydrogenase activity can also occur in this matrix. In all urine samples where labeled boldenone or boldione were detected, labeled Δ1-progesterone was also detected. Δ1-progesterone was not detected any non-incubated urine samples or following an administration of boldenone undecylenate to one mare/filly. Δ1-progesterone appears to be a candidate for further investigation as a suitable biomarker to help evaluate whether boldenone present in a urine sample may have arisen due to microbial activity rather than by its exogenous administration.


Assuntos
Anabolizantes/urina , Fezes/química , Cavalos/urina , Testosterona/análogos & derivados , Anabolizantes/análise , Anabolizantes/metabolismo , Animais , Cromatografia Líquida , Doping nos Esportes , Cavalos/fisiologia , Masculino , Detecção do Abuso de Substâncias , Espectrometria de Massas em Tandem , Testosterona/análise , Testosterona/metabolismo , Testosterona/urina
8.
J Clin Oncol ; 38(7): 674-685, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31841383

RESUMO

PURPOSE: To estimate age-specific relative and absolute cancer risks of breast cancer and to estimate risks of ovarian, pancreatic, male breast, prostate, and colorectal cancers associated with germline PALB2 pathogenic variants (PVs) because these risks have not been extensively characterized. METHODS: We analyzed data from 524 families with PALB2 PVs from 21 countries. Complex segregation analysis was used to estimate relative risks (RRs; relative to country-specific population incidences) and absolute risks of cancers. The models allowed for residual familial aggregation of breast and ovarian cancer and were adjusted for the family-specific ascertainment schemes. RESULTS: We found associations between PALB2 PVs and risk of female breast cancer (RR, 7.18; 95% CI, 5.82 to 8.85; P = 6.5 × 10-76), ovarian cancer (RR, 2.91; 95% CI, 1.40 to 6.04; P = 4.1 × 10-3), pancreatic cancer (RR, 2.37; 95% CI, 1.24 to 4.50; P = 8.7 × 10-3), and male breast cancer (RR, 7.34; 95% CI, 1.28 to 42.18; P = 2.6 × 10-2). There was no evidence for increased risks of prostate or colorectal cancer. The breast cancer RRs declined with age (P for trend = 2.0 × 10-3). After adjusting for family ascertainment, breast cancer risk estimates on the basis of multiple case families were similar to the estimates from families ascertained through population-based studies (P for difference = .41). On the basis of the combined data, the estimated risks to age 80 years were 53% (95% CI, 44% to 63%) for female breast cancer, 5% (95% CI, 2% to 10%) for ovarian cancer, 2%-3% (95% CI females, 1% to 4%; 95% CI males, 2% to 5%) for pancreatic cancer, and 1% (95% CI, 0.2% to 5%) for male breast cancer. CONCLUSION: These results confirm PALB2 as a major breast cancer susceptibility gene and establish substantial associations between germline PALB2 PVs and ovarian, pancreatic, and male breast cancers. These findings will facilitate incorporation of PALB2 into risk prediction models and optimize the clinical cancer risk management of PALB2 PV carriers.

9.
Steroids ; 146: 79-91, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-30951760

RESUMO

Boldenone is an anabolic-androgenic steroid that is prohibited in equine sports. Urine from the uncastrated male horse contains boldenone that is thought to be of endogenous origin and thus a threshold ('cut-off') concentration has been adopted internationally for free and conjugated boldenone to help distinguish cases of doping from its natural production. The testis is likely to be a source of boldenone. Qualitative analysis was performed on extracts of equine testicular homogenates (n = 3 horses) incubated non-spiked and in the presence of its potential precursors using liquid chromatography tandem mass spectrometry (LC-MS/MS) and LC high resolution mass spectrometry (LC-HRMS). Samples were analysed both underivatised and derivatised to increase the certainty of identification. In addition to previously reported endogenous steroids, analysis of non-spiked testicular tissue samples demonstrated the presence of boldenone and boldienone at trace levels in the equine testis. Incubation of homogenates with deuterium or carbon isotope labelled testosterone and androstenedione resulted in the matching stable isotope analogues of boldenone and boldienone being formed. Additionally, deuterium and carbon labelled 2-hydroxyandrostenedione was detected, raising the possibility that this steroid is a biosynthetic intermediate. In conclusion, boldenone and boldienone are naturally present in the equine testis, with the biosynthesis of these steroids arising from the conversion of testosterone and androstenedione. However, additional work employing larger numbers of animals, further enzyme kinetic experiments and pure reference standards for 2-OH androstenedione isomers would be required to better characterize the pathways involved in these transformations.


Assuntos
Testículo/metabolismo , Testosterona/análogos & derivados , Animais , Cavalos , Masculino , Testosterona/biossíntese , Testosterona/química , Testosterona/metabolismo
10.
Lancet Gastroenterol Hepatol ; 3(7): 489-498, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29706558

RESUMO

BACKGROUND: Germline pathogenic variants in the E-cadherin gene (CDH1) are strongly associated with the development of hereditary diffuse gastric cancer. There is a paucity of data to guide risk assessment and management of families with hereditary diffuse gastric cancer that do not carry a CDH1 pathogenic variant, making it difficult to make informed decisions about surveillance and risk-reducing surgery. We aimed to identify new candidate genes associated with predisposition to hereditary diffuse gastric cancer in affected families without pathogenic CDH1 variants. METHODS: We did whole-exome sequencing on DNA extracted from the blood of 39 individuals (28 individuals diagnosed with hereditary diffuse gastric cancer and 11 unaffected first-degree relatives) in 22 families without pathogenic CDH1 variants. Genes with loss-of-function variants were prioritised using gene-interaction analysis to identify clusters of genes that could be involved in predisposition to hereditary diffuse gastric cancer. FINDINGS: Protein-affecting germline variants were identified in probands from six families with hereditary diffuse gastric cancer; variants were found in genes known to predispose to cancer and in lesser-studied DNA repair genes. A frameshift deletion in PALB2 was found in one member of a family with a history of gastric and breast cancer. Two different MSH2 variants were identified in two unrelated affected individuals, including one frameshift insertion and one previously described start-codon loss. One family had a unique combination of variants in the DNA repair genes ATR and NBN. Two variants in the DNA repair gene RECQL5 were identified in two unrelated families: one missense variant and a splice-acceptor variant. INTERPRETATION: The results of this study suggest a role for the known cancer predisposition gene PALB2 in families with hereditary diffuse gastric cancer and no detected pathogenic CDH1 variants. We also identified new candidate genes associated with disease risk in these families. FUNDING: UK Medical Research Council (Sackler programme), European Research Council under the European Union's Seventh Framework Programme (2007-13), National Institute for Health Research Cambridge Biomedical Research Centre, Experimental Cancer Medicine Centres, and Cancer Research UK.


Assuntos
Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Predisposição Genética para Doença , Mutação em Linhagem Germinativa , Neoplasias Gástricas/genética , Adulto , Idoso , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Tomada de Decisão Clínica , Feminino , Mutação da Fase de Leitura , Humanos , Mutação com Perda de Função , Masculino , Pessoa de Meia-Idade , Proteína 2 Homóloga a MutS/genética , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , RecQ Helicases/genética , Sequenciamento Completo do Exoma , Adulto Jovem
11.
Drug Test Anal ; 10(9): 1469-1473, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-29757501

RESUMO

The effective analysis of anabolic-androgenic steroids in urine usually requires a suitable deconjugation method for the analysis of phase II metabolites such as sulphates and glucuronides. Acid hydrolysis using methanolysis is one adopted method of deconjugation that efficiently and rapidly cleaves both sulphates and glucuronides contemporaneously. The formation of artefactual by-products is a known disadvantage of this harsh method. However, the possible promotion of deuterium-hydrogen exchange of isotopically labelled internal standards has received little attention in the literature. This report demonstrates a complete deuterium-hydrogen exchange from deuterium labelled D9 -progesterone to progesterone driven by the acidic conditions of the methanolysis. The likely mechanisms of this exchange reaction are postulated, and the results compared to other deuterated steroids. This finding highlights the importance for careful consideration when selecting labelled internal standards in a conjunction with methanolysis.


Assuntos
Metanol/química , Congêneres da Testosterona/análise , Deutério , Doping nos Esportes , Cromatografia Gasosa-Espectrometria de Massas , Glucuronídeos/análise , Hidrólise , Indicadores e Reagentes , Isomerismo , Progesterona/análise , Padrões de Referência , Sulfatos/análise , Espectrometria de Massas em Tandem
12.
Drug Test Anal ; 10(10): 1518-1527, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29797687

RESUMO

The use of testosterone and its pro-drugs, such as dehydroepiandrosterone (DHEA), is currently regulated in horseracing by the application of international testosterone thresholds. However, additional steroidomic approaches, such as steroid ratios, to distinguish overall adrenal stimulation from drug administrations and an equine biological passport for longitudinal steroid profiling of individual animals could be advantageous in equine doping testing. Thus, DHEA concentrations and related ratios (testosterone [T] to DHEA and DHEA to epitestosterone [E]) were assessed in the reference population by quantitative analysis of 200 post-race gelding urine samples using liquid chromatography-tandem mass spectrometry. DHEA concentrations ranged between 0.9 and 136.6 ng/mL (mean 12.8 ng/mL), T:DHEA ratios between 0.06 and 1.85 (mean 0.43), and DHEA:E ratios between 0.21 and 13.56 (mean 2.20). Based on the reference population statistical upper limits of 5.4 for T:DHEA ratio and 48.1 for DHEA:E ratio are proposed with a risk of 1 in 10 000 for a normal outlier exceeding the value. Analysis of post-administration urine samples collected following administrations of DHEA, Equi-Bolic® (a mix of DHEA and pregnenolone) and testosterone propionate to geldings showed that the upper limit for T:DHEA ratio was exceeded following testosterone propionate administration and DHEA:E ratio following DHEA administrations and thus these ratios could be used as additional biomarkers when determining the cause of an atypical testosterone concentration. Additionally, DHEA concentrations and ratios can be used as a starting point to establish reference ranges for an equine biological passport.


Assuntos
Desidroepiandrosterona/urina , Cavalos/urina , Detecção do Abuso de Substâncias/métodos , Animais , Cromatografia Líquida/métodos , Doping nos Esportes , Epitestosterona/urina , Limite de Detecção , Masculino , Espectrometria de Massas em Tandem/métodos , Testosterona/urina
13.
Drug Test Anal ; 9(9): 1328-1336, 2017 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27717154

RESUMO

Detection of testosterone and/or its pro-drugs in the gelding is currently regulated by the application of an international threshold for urinary testosterone of 20 ng/mL. The use of steroid ratios may provide a useful supplementary approach to aid in differentiating between the administration of these steroids and unusual physiological conditions that may result in atypically high testosterone concentrations. In the current study, an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method was developed to quantify testosterone (T) and epitestosterone (E). The method was used to analyze 200 post-race urine samples from geldings in order to generate the ratios for the reference population. Following statistical analysis of the data, an upper limit of 5 for T:E ratio in geldings is proposed. Samples collected from 15 geldings with atypical urinary testosterone concentrations (>15 ng/mL) but otherwise normal steroid profile, had T:E ratios within those observed for the reference population. The applicability of an upper T:E ratio to detect an administration was demonstrated by the analysis of a selection of incurred samples from testosterone propionate, dehydroepiandrosterone (DHEA), and a mixture of DHEA and pregnenolone (Equi-Bolic®) administrations. These produced testosterone concentrations above the threshold of 20 ng/mL, but also T:E ratios above the proposed limit of 5. In conclusion, consideration of the T:E ratio appears to be a valuable complementary aid to evaluate whether an atypical testosterone concentration could be caused by a natural biological outlier as opposed to the administration of these steroids. Copyright © 2016 John Wiley & Sons, Ltd.


Assuntos
Líquidos Corporais/química , Desidroepiandrosterona/análise , Doping nos Esportes/estatística & dados numéricos , Epitestosterona/análise , Esteroides/análise , Espectrometria de Massas em Tandem/métodos , Testosterona/análise , Animais , Cromatografia Líquida , Desidroepiandrosterona/urina , Epitestosterona/urina , Cavalos , Humanos , Pró-Fármacos , Esteroides/urina , Detecção do Abuso de Substâncias , Testosterona/urina
14.
Drug Test Anal ; 3(10): 705-16, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21916022

RESUMO

Within horseracing, the detection of prohibited substance doping often requires urine analysis; hence, it is necessary to understand the metabolism of the drugs in question. Here, the previously unknown equine metabolism of eight sedatives is reported in order to provide information on target metabolites for use in doping control. Phase I metabolite information was provided by incubation with equine liver S9 fraction. In vitro techniques were chosen in order to reduce the ethical and financial issues surrounding the study of so many compounds, none of which are licensed for use in horses in the UK. Several metabolites of each drug were identified using liquid chromatography-high resolution mass spectrometric (LC-HRMS) analysis on an LTQ-Orbitrap. Further structural information was obtained by tandem mass spectrometry (MS/MS) analysis; allowing postulation of the structure of some of the most abundant in vitro metabolites. The most abundant metabolites of alpidem, etifoxine, indiplon, tiletamine, zaleplon, zolazepam, zolpidem, and zopiclone related to hydroxylation/N-oxidation, deethylation, demethylation, deethylation, hydroxylation/N-oxidation, demethylation, hydroxylation/N-oxidation and hydroxylation/N-oxidation, respectively. In many cases, further work would be required to fully elucidate the precise positioning of the functional groups involved. The results of this study provide metabolite information that can be used to enhance equine anti-doping screening methods. However, the in vitro metabolites identified are at present only a prediction of those that may occur in vivo. In the future, any positive findings of these drugs and/or their metabolites in horse urine samples could help validate these findings and/or refine the choice of target metabolites.


Assuntos
Depressores do Sistema Nervoso Central/metabolismo , Doping nos Esportes , Cavalos/metabolismo , Detecção do Abuso de Substâncias , Tiletamina/metabolismo , Zolazepam/metabolismo , Animais , Cromatografia Líquida/métodos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Detecção do Abuso de Substâncias/métodos , Espectrometria de Massas em Tandem/métodos
15.
Drug Test Anal ; 3(1): 74-87, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21322180

RESUMO

Detection of androgenic-anabolic steroid abuse in equine sports requires knowledge of the drug's metabolism in order to target appropriate metabolites, especially where urine is the matrix of choice. Studying 'designer' steroid metabolism is problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of toxicological data. In this study, the equine in vitro metabolism of eight steroids available for purchase on the Internet is reported; including androsta-1,4,6-triene-3,17-dione, 4-chloro,17α-methyl-androsta-1,4-diene-3,17ß-diol, estra-4,9-diene-3,17-dione, 4-hydroxyandrostenedione, 20-hydroxyecdysone, 11-keto-androstenedione, 17α-methyldrostanolone, and tetrahydrogestrinone. In order to allow for retrospective analysis of sample testing data, the use of a high-resolution (HR) accurate-mass Thermo LTQ-Orbitrap liquid chromatography-mass spectrometry (LC-MS) instrument was employed for metabolite identification of underivatized sample extracts. The full scan LC-HRMS Orbitrap data were complimented by LC-HRMS/MS and gas-chromatography-mass spectrometry (GC-MS) experiments in order to provide fragmentation information and to ascertain whether GC-MS was capable of detecting any metabolite not detected by LC-HRMS. With the exception of 20-hydroxyecdysone, all compounds were found to be metabolized by equine liver S9 and/or microsomes. With the exception of 17α-methyldrostanolone, which produced metabolites that could only be detected by GC-MS, the metabolites of all other compounds could be identified using LC-HRMS, thus allowing retrospective analysis of previously acquired full-scan data resulting from routine equine drug testing screens. In summary, while in vitro techniques do not serve as a replacement for more definitive in vivo studies in all situations, their use does offer an alternative in situations where it would not be ethical to administer untested drugs to animals.


Assuntos
Anabolizantes/análise , Androgênios/análise , Cromatografia Líquida/veterinária , Drogas Desenhadas/análise , Doping nos Esportes , Cavalos/metabolismo , Espectrometria de Massas/veterinária , Substâncias para Melhoria do Desempenho/análise , Esteroides/análise , Detecção do Abuso de Substâncias/veterinária , Anabolizantes/química , Anabolizantes/metabolismo , Androgênios/química , Androgênios/metabolismo , Animais , Biotransformação , Drogas Desenhadas/química , Drogas Desenhadas/metabolismo , Cromatografia Gasosa-Espectrometria de Massas/veterinária , Técnicas In Vitro , Espectroscopia de Ressonância Magnética , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Substâncias para Melhoria do Desempenho/química , Substâncias para Melhoria do Desempenho/metabolismo , Reprodutibilidade dos Testes , Especificidade da Espécie , Esteroides/química , Esteroides/metabolismo
16.
Drug Test Anal ; 3(1): 19-53, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20967889

RESUMO

A detailed understanding of equine drug metabolism is important for detection of drug abuse in horseracing and also in veterinary drug development and practice. To date, however, no comprehensive review of equine drug metabolism has been published. The majority of literature regarding equine drug metabolite profiles is derived from sports drug detection research and is generally targeted at detecting marker metabolites of drug abuse. However, the bulk of the literature on equine drug metabolism enzymology is derived from veterinary studies aimed at determining the molecular basis of metabolism. In this article, the phase 1 and 2 metabolisms of seven of the most important classes of drugs monitored in horseracing are reviewed, including: anabolic-androgenic steroids (AAS), ß2 -agonists, stimulants, sedatives/tranquilizers, local anesthetics, non-steroidal anti-inflammatory analgesics (NSAIDS)/cyclooxygenase-2 (COX-2) inhibitors, and opioid analgesics. A summary of the literature relating to the enzymology of drug metabolism in this species is also be presented. The future of equine drug metabolism in the area of doping research will be influenced by several factors, including: a possible move towards the increased use of blood and other alternative testing matrices; the development of assays based on intact drug conjugates; the increasing threat of 'designer' and herbal- based products; advances in the use of in vitro technologies; the increased use of liquid-chromatography/high-resolution mass spectrometry; and the possibility of screening using 'omics' approaches. Also, the recent cloning of a range of equine cytochrome P450 (CYP) enzymes opens up the potential for carrying out more detailed mechanistic pharmacological and toxicological veterinary studies.


Assuntos
Doping nos Esportes , Cavalos/metabolismo , Substâncias para Melhoria do Desempenho/análise , Detecção do Abuso de Substâncias/veterinária , Animais , Biotransformação , Substâncias para Melhoria do Desempenho/farmacocinética , Reprodutibilidade dos Testes , Especificidade da Espécie
17.
Bioanalysis ; 2(12): 1971-88, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-21110741

RESUMO

BACKGROUND: Within equine drug surveillance, there is significant interest in analyzing intact phase II conjugates of drugs in urine, but progress has been limited by a lack of reference material. METHOD: In this study, in vitro techniques using equine liver fractions were employed to produce glucuronide and sulfate conjugates of stanozolol, 16ß-hydroxystanozolol and nandrolone, the glucuronide conjugate of morphine and the glutathione metabolite of chlordinitrobenzene for the first time in equine sports drug surveillance. RESULTS: The glucuronide conjugate of the synthetic progestagen altrenogest was also produced in vitro, removing the requirement for sample hydrolysis during routine urinalyses. CONCLUSION: These results highlight the potential of in vitro studies for the production of phase II reference material, allowing the development of assays based on intact conjugates.


Assuntos
Anabolizantes/metabolismo , Doping nos Esportes , Glucuronídeos/metabolismo , Glutationa/metabolismo , Fígado/metabolismo , Esteroides/metabolismo , Detecção do Abuso de Substâncias/métodos , Anabolizantes/urina , Animais , Dinitroclorobenzeno/metabolismo , Dinitroclorobenzeno/urina , Glucuronídeos/urina , Glutationa/urina , Cavalos , Morfina/análise , Morfina/metabolismo , Nandrolona/metabolismo , Nandrolona/urina , Progestinas/metabolismo , Progestinas/urina , Estanozolol/análogos & derivados , Estanozolol/metabolismo , Estanozolol/urina , Esteroides/urina
18.
Drug Test Anal ; 2(1): 1-10, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20878880

RESUMO

The detection of drug abuse in horseracing often requires knowledge of drug metabolism, especially if urine is the matrix of choice. In this study, equine liver/lung microsomes/S9 tissue fractions were used to study the phase I metabolism of eight drugs of relevance to equine drug surveillance (acepromazine, azaperone, celecoxib, fentanyl, fluphenazine, mepivacaine, methylphenidate and tripelennamine). In vitro samples were analyzed qualitatively alongside samples originating from in vivo administrations using LC-MS on a high resolution accurate mass Thermo Orbitrap Discovery instrument and by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap.Using high resolution accurate mass full-scan analysis on the Orbitrap, the in vitro systems were found to generate at least the two most abundant phase I metabolites observed in vitro for all eight drugs studied. In the majority of cases, in vitro experiments were also able to generate the minor in vivo metabolites and sometimes metabolites that were only observed in vitro. More detailed analyses of fentanyl incubates using LC-MS/MS showed that it was possible to generate good quality spectra from the metabolites generated in vitro. These data support the suggestion of using in vitro incubates as metabolite reference material in place of in vivo post-administration samples in accordance with new qualitative identification guidelines in the 2009 International Laboratory Accreditation Cooperation-G7 (ILAC-G7) document.In summary, the in vitro and in vivo phase I metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment, refine and reduce the existing equine in vivo paradigm.


Assuntos
Cromatografia Líquida/métodos , Doping nos Esportes/métodos , Doping nos Esportes/prevenção & controle , Cavalos/metabolismo , Espectrometria de Massas/métodos , Preparações Farmacêuticas/metabolismo , Animais , Feminino , Guias como Assunto , Cavalos/urina , Inativação Metabólica , Masculino , Microssomos Hepáticos/metabolismo , Preparações Farmacêuticas/urina , Padrões de Referência , Detecção do Abuso de Substâncias/veterinária
19.
Steroids ; 75(10): 643-52, 2010 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-20381511

RESUMO

Effective detection of the abuse of androgenic-anabolic steroids in human and animal sports often requires knowledge of the drug's metabolism in order to target appropriate urinary metabolites. 'Designer' steroids are problematic since it is difficult to obtain ethical approval for in vivo metabolism studies due to a lack of a toxicological profile. In this study, the in vitro metabolism of estra-4,9-diene-3,17-dione is reported for the first time. This is also the first study comparing the metabolism of a designer steroid in the three major species subject to sport's doping control; namely the equine, canine and human. In order to allow the retrospective analysis of sample testing data, the use of a high-resolution (HR) accurate-mass Thermo LTQ-Orbitrap LC-MS instrument was employed for metabolite identification of underivatised sample extracts. The full scan HR-LC-MS Orbitrap data was complimented by several further experiments targeted at elucidating more detailed structural information for the most abundant metabolites. These included; HR-LC-MS/MS of the underivatised metabolites, functional group selective chemical derivatisation followed by full scan HR-LC-MS, enzyme inhibition experiments and full scan electron ionization GC-MS analysis of methoxyamine-trimethylsilyl derivatives. The major metabolite detected in all species, and therefore the most suitable candidate for screening of estra-4,9-diene-3,17-dione abuse, was proposed to be an isomer of 17-hydroxy-estra-4,9-dien-3-one. Less significant metabolic pathways in all species included hydroxylation and reduction followed by hydroxylation. Reductive metabolism in the canine was less significant than in the other two species, while the equine was unique in producing a di-reduced metabolite (proposed to be an isomer of estra-4,9-diene-3,17-diol) and also relatively large quantities of d-ring hydroxy and hydroxy-reduced metabolites.


Assuntos
Doping nos Esportes/prevenção & controle , Estrenos/química , Estrenos/metabolismo , Detecção do Abuso de Substâncias/métodos , Animais , Cromatografia Líquida , Cães , Cromatografia Gasosa-Espectrometria de Massas , Cavalos , Humanos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Espectrometria de Massas em Tandem
20.
Steroids ; 75(1): 57-69, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19854209

RESUMO

In this study, the use of equine liver/lung microsomes and S9 tissue fractions were used to study the metabolism of the androgenic/anabolic steroid stanozolol as an example of the potential of in vitro technologies in sports drug surveillance. In vitro incubates were analysed qualitatively alongside urine samples originating from in vivo stanozolol administrations using LC-MS on a high-resolution accurate mass Thermo Orbitrap Discovery instrument, by LC-MS/MS on an Applied Biosystems Sciex 5500 Q Trap and by GC-MS/MS on an Agilent 7000A. Using high-resolution accurate mass full scan analysis on the Orbitrap, equine liver microsome and S9 in vitro fractions were found to generate all the major phase-1 metabolites observed following in vivo administrations. Additionally, analysis of the liver microsomal incubates using a shallower HPLC gradient combined with various MS/MS functions on the 5500 Q trap allowed the identification of a number of phase 1 metabolites previously unreported in the equine or any other species. Comparison between liver and lung S9 metabolism showed that the liver was the major site of metabolic activity in the equine. Furthermore, using chemical enzyme inhibitors that are known to be selective for particular isoforms in other species suggested that an enzyme related to CYP2C8 may be responsible the production of 16-hydroxy-stanozolol metabolites in the equine. In summary, the in vitro and in vivo phase 1 metabolism results reported herein compare well and demonstrate the potential of in vitro studies to compliment the existing in vivo paradigm and to benefit animal welfare through a reduction and refinement of animal experimentation.


Assuntos
Doping nos Esportes , Estanozolol/análise , Estanozolol/urina , Detecção do Abuso de Substâncias/métodos , Anabolizantes/administração & dosagem , Anabolizantes/química , Anabolizantes/metabolismo , Androgênios/administração & dosagem , Androgênios/análise , Androgênios/metabolismo , Animais , Hidrocarboneto de Aril Hidroxilases/antagonistas & inibidores , Hidrocarboneto de Aril Hidroxilases/metabolismo , Cromatografia Gasosa , Cromatografia Líquida de Alta Pressão , Cavalos , Hidroxitestosteronas/química , Hidroxitestosteronas/metabolismo , Cetoconazol/farmacologia , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Quercetina/farmacologia , Reprodutibilidade dos Testes , Espectrometria de Massas por Ionização por Electrospray , Estanozolol/administração & dosagem
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