Autores
dos Santos, L. (UFRJ) ; Matos, R.R. (UFRJ) ; Pereira, H.M.G. (LBCD-UFRJ)
Resumo
The in vivo Zebrafish Water Tank (ZWT) model is an emerging model to assess the
metabolism of xenobiotics. The class of selective estrogen receptor modulator
(SERM) has not yet been evaluated by this model. This work aimed to evaluate the
applicability of the ZWT model to study the tamoxifen metabolism, as a
representative of the SERM. Tamoxifen was administered to a 200 mL tank
containing water and 8 adult zebrafish at 32 ± 1°C for 8 hours. The water tank
samples collected throughout the experiment were analyzed both with and without
pretreatment by LC-HRMS/MS. ZWT was able to reproduce the human metabolism of
tamoxifen, with the main phase I metabolites being confirmed. Hence, ZWT could
be applied to investigate other doping agents with similar structure in a very
straightforward way.
Palavras chaves
ZWT 's model; anti-doping science; Tamoxifen
Introdução
The experimental design so-called “Zebrafish Water Tank” (ZWT) model already
showed success in reproducing qualitatively the human metabolism of xenobiotics
from different classes of substances for purposes of anti-doping science. Based
on the high physiological and genetic homology with humans, ZWT has already
contemplated the study of anabolic agents, cannabimimetics, stimulants and
glucocorticoids (ANSELMO et al., 2017; SARDELA et al., 2020; DE ARAUJO et al.,
2021; MATOS et al., 2021; PRADO et al., 2021). However, the class of selective
estrogen receptor modulator (SERM) has not yet been evaluated by this model.
Tamoxifen (2-[4-[(Z)-1,2-diphenylbut-1-enyl]phenoxy]-N,N-dimethylethanamine) is
a SERM with is used by athletes to compensate for the AAS extensive abuse side
effects (MAZZARINO, BIAVA et al., 2013; DOMÍNGUEZ-ROMERO et al., 2015). Clinical
studies demonstrated that tamoxifen promote a moderate increase in luteinizing
hormone (LH), follicle-stimulating hormone (FSH), testosterone (MAZZARINO et
al., 2011). This work aimed to evaluate the applicability of the ZWT model to
study the tamoxifen metabolism, as a representant of the SERM with diphenylbut-
1-enyl-phenoxy skeleton.
Material e métodos
The ZWT protocol used in previous studies was adopted. Tamoxifen was administered
to a 200 mL tank containing water and 8 adult zebrafish at 32 ± 1°C. Experiments
were performed in triplicate and interrupted after 8 hrs. Additional tanks without
fish with drug and without the drug with fish were used as controls. The water
tank samples collected throughout the experiment were analyzed both with and
without pretreatment (i.e., dilute-and-shoot, and LLE with and without hydrolysis)
by Liquid Chromatography coupled with High-Resolution Mass Spectrometry (LC-
HRMS/MS) in positive ionization mode.
Resultado e discussão
No zebrafish ortholog of human CYP2D6, a critical enzyme of tamoxifen
metabolism, has been reported in the literature yet. However, the 4-hydroxy-
tamoxifen, N-desmethyl-tamoxifen and endoxifen, the main phase I metabolites
observed in humans, were identified in the samples using reference material,
according to the World Anti-Doping Agency criteria. The profile of the
metabolite’s generation could also be evaluated by the bioaccumulation curves
(metabolite to IS area ratio x time). Using this approach, the conversion of the
4-hydroxy-tamoxifen and N-desmethyl-tamoxifen to endoxifen was clearly observed.
Comparison of results from sample preparation with and without hydrolysis step
presented an indirect evidence of phase II metabolites formation. The results
obtained showed that these TMX’s metabolites were excreted mainly as free and
gluco-conjugated, whereas only small amounts were excreted as sulphate. The same
behavior is observed for humans (MAZZARINO et al., 2013). Due to the lack of
reference material, several other putative phase I metabolites, preliminary
identified by the exact mass, were only suggested. Some of the chemical
reactions proposed for TMX’s biotransformation pathways include hydroxylation in
different position(s), N-demethylation, N-oxidation, methoxylation and
combinations of them. Primary standards would be required for further
confirmation of the metabolite’s structure.
Conclusões
According to the results obtained, ZWT was able to reproduce the human metabolism
of tamoxifen, with the main phase I metabolites been confirmed in the water tank
using HRMS and reference material. Hence, ZWT could be applied to investigate
other doping agents with similar structure in a very straightforward way.
Agradecimentos
We wish to express our gratitude to the UFRJ’s Institute of Chemistry and the
financial support from the Brazilian research funding agency CNPq and the
Brazilian Authority for Doping Control (ABCD).
Referências
DE ARAUJO, A. L. D., NUNES, I. K. da C., SARDELA, V. F., et al. "Is zebrafish (Danio rerio) water tank model applicable for the assessment of glucocorticoids metabolism? The budesonide assessment", Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences, v. 1179, n. May, p. 122826, 2021. DOI: 10.1016/j.jchromb.2021.122826. Disponível em: https://doi.org/10.1016/j.jchromb.2021.122826.
DE SOUZA ANSELMO, C., SARDELA, V. F., MATIAS, B. F., et al. "Is zebrafish (Danio rerio) a tool for human-like metabolism study?", Drug Testing and Analysis, v. 9, n. 11–12, p. 1685–1694, 2017. DOI: 10.1002/dta.2318. .
DOMÍNGUEZ-ROMERO, J. C., GARCÍA-REYES, J. F., BENEITO-CAMBRA, M., et al. "Study of tamoxifen urinary metabolites in rat by ultra-high-performance liquid chromatography time-of-flight mass spectrometry", Biomedical Chromatography, v. 29, n. 8, p. 1220–1228, 2015. DOI: 10.1002/bmc.3411. .
MATOS, R. R., ANSELMO, C. de S., SARDELA, V. F., et al. "Phase II stanozolol metabolism study using the zebrafish water tank (ZWT) model", Journal of Pharmaceutical and Biomedical Analysis, v. 195, 2021. DOI: 10.1016/j.jpba.2020.113886. .
MAZZARINO, M., BIAVA, M., DE LA TORRE, X., et al. "Characterization of the biotransformation pathways of clomiphene, tamoxifen and toremifene as assessed by LC-MS/(MS) following in vitro and excretion studies", Analytical and Bioanalytical Chemistry, v. 405, n. 16, p. 5467–5487, 2013. DOI: 10.1007/s00216-013-6961-7. .
MAZZARINO, M., BRAGAN, M. C., DE LA TORRE, X., et al. "Relevance of the selective oestrogen receptor modulators tamoxifen, toremifene and clomiphene in doping field: Endogenous steroids urinary profile after multiple oral doses", Steroids, v. 76, n. 12, p. 1400–1406, 2011. DOI: 10.1016/j.steroids.2011.06.005. Disponível em: http://dx.doi.org/10.1016/j.steroids.2011.06.005.
PRADO, E., MATOS, R. R., DE LIMA GOMES, G. M., et al. "Metabolism of synthetic cathinones through the zebrafish water tank model: a promising tool for forensic toxicology laboratories", Forensic Toxicology, v. 39, n. 1, p. 73–88, 2021. DOI: 10.1007/s11419-020-00543-w. Disponível em: https://doi.org/10.1007/s11419-020-00543-w.
SARDELA, V. F., SARDELA, P. D. O., LISBOA, R. R., et al. "Comprehensive Zebrafish Water Tank Experiment for Metabolic Studies of Testolactone", Zebrafish, v. 17, n. 2, p. 104–111, 2020. DOI: 10.1089/zeb.2019.1791.
