Autores
dos Santos, L. (UFRJ) ; de Aguiar, P.F. (LABQUIM - 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. This work aimed to evaluate the model’s environmental
parameters to allow the use of experiments with a duration longer than that
studied (7 days). The parameters (pH, temperature, NH3, NO2- and NO3-) were
evaluated through a multiparameter meter and colorimetric tests. The experiment
lasted 21 days without the use of partial water changes. Deaths began to be
observed after 13 days mainly along a process of pH and NH3 concentration decay
and an increase in NO2- and NO3- concentrations. The implementation of protocols
and development of studies to maintain and control the pH and the production of
toxic species in an adequate range are essential for ZWT model experiments over
7 days.
Palavras chaves
ZWT 's model; anti-doping science; environmental parameters
Introdução
In routine doping controls, the majority of test samples consist of urine
samples and therefore the knowledge about drug metabolism and excretion becomes
crucial. Another important aspect is the fact that several classes of drugs are
considered prohibited at all times by the World Anti-Doping Agency (WADA).
Consequently, the use of long-term experiments becomes necessary and the
analytical doping control assays require in-depth investigations with a focus on
so-called Long-Term Metabolites (LTMs) (THEVIS et al., 2021). The study of the
metabolic profiles of drugs involving human exposure to potentially harmful
agents to health in which there is no complete understanding of their toxicity
is questionable from an ethical point of view (PRADO et al., 2021). Therefore,
an emerging in vivo model for metabolism assessment of controlled substances for
doping control analysis is the evaluation of a zebrafish water tank (ZWT) in the
presence of a drug. This experimental setup is supported by the presence of
enzymes in zebrafish with action highly comparable to humans, capable of
performing all the fundamental steps for the metabolism, elimination and/or
detoxification of administered xenobiotics. Other advantages in using the ZWT
model are the small size of the animal, cheap maintenance and the matrix
analyzed is much cleaner than other biological matrices, making zebrafish a
viable alternative to classical mammalian models (MATOS et al., 2021; SARDELA et
al., 2020). Despite the success in producing the main metabolites of different
classes of substances typically used as dopants in short-term experiments (8
hours and 7 days), the ZWT model has a limitation, the low conversion rate of
dopants into metabolites. Therefore, the proposed experimental design does not
include partial water changes in the tanks, allowing the bioaccumulation of
metabolites. One approach that has not yet been investigated for the ZWT model
is the implementation of long-term experiments. This procedure could allow a
greater accumulation of metabolites in the tanks and the formation of LTMs.
However, studies involving zebrafish and long-term experiments in the literature
mostly involve the use of partial water changes (JIA et al., 2020; MAO et al.,
2020). Moreover, there is a close relationship between the parameters of the
aquatic environment and fish metabolism. Changes in pre-established standards
and ranges for environmental parameters can cause considerable impacts on
animals. Thus, water quality represents the main factor to be regularly
monitored regarding its physicochemical properties to enable the use of long-
term experiments as its impact on the animal's well-being and, consequently, on
the animal's ability to metabolize (HAMMER, 2019). This work aimed to increase
the duration of a ZWT model experiment, investigating how environmental
parameters are changed, as well as their impact on welfare and, consequently, on
the animal's ability to metabolize.
Material e métodos
The environmental conditions (pH, temperature, ammonia, nitrite and nitrate) of
the ZWT model were monitored in a 21-day experiment. The pH and temperature
parameters were monitored by sensors coupled to a multiparameter meter from
Hanna Instruments. The monitoring of these factors was performed at 0h, 1h, 3h,
6h and 12h, and then an interval of 24h was adopted, counting from the beginning
of the experiment. Nitrogen residues (Total Ammonia Nitrogen - TAN, nitrite and
nitrate) were evaluated by aquarium kits, colorimetric tests that estimate the
concentration of these chemical species in the aquarium. On the first day of the
experiment, the collection points for these factors were at 0h, 6h and 12h and
then an interval of 48 h was adopted. Based on the measured pH and temperature
data, it was possible to calculate the ammonia acidity constant for each
measurement. And, with these data, the concentration of non-ionized ammonia,
which represents a greater toxicity in relation to the ammonium ion (NH4+), was
calculated. The experiment was submitted to the Ethics Committee on the Use of
Animals in Research at UFRJ (protocol 032/21). Furthermore, the experiment was
carried out in triplicate and following some pre-established conditions for the
7-day ZWT model. The temperature was controlled with the aid of a device in the
room intended for the experiment and the sensor present in the multiparameter
meter. The stocking density for each 4 L tank was 18 fishes, feeding was done
once a day to attenuate the accumulation of organic materials. The light cycle
was 12h light: 12h dark.(ANSELMO et al., 2017; SARDELA et al., 2018; MATOS et
al., 2020). During the experiment, the environmental conditions of the aquarium
were monitored as well as excessive stress, changes in behavior and wounds in
the fish, which were considered as end point criteria of the experimental. The
physiological and/or behavioral parameters that were used to assess the fish
welfare on a daily basis were: observation of damage and change in skin color,
morphological abnormalities and visible behavioral changes (lethargy, immobility
and poor response to stimulus) (HAPPER & LAWRENCE, 2010).
Resultado e discussão
The status of the animals was monitored daily, checking for any morphological
and/or behavioral anomalies. None of these signals were observed for aquarium 1
(AQ1) and aquarium 3 (AQ3). In the case of aquarium 2 (AQ2), however, it was
found that the animals showed lethargy in the face of the stimulus of feeding
from 13 days. The lethargy symptom was interpreted as a situation of mild
absence of well-being. However, as there was no evolution to the state of
immobility or any other symptom, it was decided to continue the experiment.
Based on the motivations of the study, and on literature data, that included
long periods of experimentation and partial changes of water, the duration of
the experiment was initially set at 28 days (JIA et al., 2020; MAO et al.,
2020). However, the experiment was shortened to 21 days. The justification for
this decision considered the number of deaths observed during the experiment. In
AQ1, no deaths were verified and, in AQ3, only the death of 2 animals was
observed on day 13 of the experiment. For AQ2, however, 12 deaths were recorded
in total, from the 15th to the 18th day of the experiment, representing almost
70% of the number of animals that were in the aquarium. Thus, it was understood
that the stress situation for the animals, which was monitored in parallel with
changes in environmental conditions, had increased. Moreover, as established in
the ZWT model, no partial water change could be carried out to regularize the
aquarium situation. At the beginning of the experiment, temperatures very close
to or above the upper limit of the range considered adequate (26 - 30 °C) were
observed, directly impacting the evaporation of water from the aquariums.
Maintaining high temperatures is capable of inducing an increase in the
metabolic rate of zebrafish (LÓPEZ-OLMEDA & SÁNCHEZ-VÁZQUEZ, 2011), which could
favor the metabolism of doping agents in later experiments. Nonetheless, with
increasing temperature, there is a greater demand for oxygen by fishes (HAMMER,
2019). Furthermore, combined with the proposal of the study, which consists of a
long-term experiment, elevated temperatures may represent a pro-oxidant stressor
for animals (PARK et al., 2020). In this way, after 6 days, there was a more
rigorous control regarding the temperature of the environment, keeping it at the
lower limit of the range. Up to approximately 120h (5 days), the tanks showed pH
values within the appropriate range and in a similar and increasing behavior.
After reaching a pH equal to 7.45, AQ1 and AQ3, presented a pH decay of
approximately 1 unit, which lasted 4 days and 7 days, respectively. However, in
AQ2, the increasing behavior lasted up to 12 days of experiment, which indicated
the increase of the ammonia species in the medium, being one of the
justifications for the change in behavior observed in this aquarium. Then, the
pH in AQ2 suffered a sudden drop (72h) of approximately 2 units and, right
after, 12 deaths occurred in this aquarium. This is another indication that
aquatic life has difficulties in adjusting to the sudden changes and keeping
acid-base and ionic regulation and ammonia excretion (ZAHANGIR et al., 2015).
With the observation, for AQ2, of an increase in pH for a long period (12 days),
for example, it was also possible to verify an accumulation of NH3, above the
recommended (0.05 mg.L-1). After 12 days, both pH and ammonia concentration
decreased, indicating the beginning of the oxidation process and production of
nitrite and nitrate ions, which showed an increase. The deaths, in turn,
occurred during this process of NO2- and NO3- increase, which presented values
far above the tolerable values for zebrafish 0.02 mg.L-1 and 50 mg.L-1,
respectively (HAMMER, 2019). Based on the acquired results, it was possible to
observe the behavior of the aquatic systems in all experimentation tanks for the
elimination of NH3, promoting its oxidation, through nitrifying bacteria, to the
nitrate ion (NO3-) and having the intermediate product as NO2-. However, in the
case of AQ2, the accumulation of NH3 greater than in the others resulted in an
increase in NO2- and a decrease in pH in a shorter period of time. This faster
deterioration of environmental conditions for this tank may have made it
possible for so many deaths to occur. Furthermore, the correlation between pH
and nitrogen residues, mainly NH3, can be confirmed, reaffirming the need of its
control, for example, with the addition of conditioners to control the pH (acids
and/or bases), and monitoring in long-term experiments. The three aquariums,
which represented replicates of the experiment, were handled and monitored in
the same way throughout the entire experiment. Even so, the different behaviors
observed also demonstrate the need to increase the control of these
environmental parameters in the ZWT model. Finally, a systematic study that
includes the variation and control of pH, in parallel with the monitoring of
nitrogen residues, is necessary to verify the possibility of controlling the
production of toxic species (NH3 and NO2¬-) to zebrafish in the medium, allowing
experiments with a longer duration.
Conclusões
A rigorous temperature control proved to be essential to avoid excessive water
loss and factors that could represent some stress to the animals throughout the
experiment. In all, 14 deaths were observed in the tanks, 12 in aquarium 2 and 2
in aquarium 3. Deaths were mainly observed along the process of decreasing pH and
ammonia concentration and increasing concentration of nitrate and nitrite, the
latter being the most toxic nitrogen residue for fish. Some signs were identified
during the experiment that indicate deterioration in aquarium water quality. For
instance, the increase in pH along with the concentration of NH3, above the
allowed value (0.05 mg.L-1), followed by a decrease, in a short period (72h) with
pH values below the recommended (6.5 - 8.5). In addition, the increase in the
concentration of NO3- species and, mainly, NO2- above the permitted values
represents a risk to the health of zebrafish. Carrying out experiments lasting
longer than 7 days requires some adaptations, such as the addition of conditioners
to control the pH (acids and/or bases) and the monitoring of environmental
conditions in long-term experiments. Finally, a systematic study that includes the
variation and control of pH, in parallel with the monitoring of nitrogen residues,
is necessary to verify the possibility of controlling the production of toxic
species (NH3 and NO2-) to zebrafish in the medium, allowing experiments with a
longer duration.
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 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. .
HAMMER, H. S. "Water quality for zebrafish culture", The Zebrafish in Biomedical Research: Biology, Husbandry, Diseases, and Research Applications, p. 321–335, 2019. DOI: 10.1016/B978-0-12-812431-4.00029-4. .
JIA, D., LI, X., DU, S., et al. "Single and combined effects of carbamazepine and copper on nervous and antioxidant systems of zebrafish (Danio rerio)", Environmental Toxicology, v. 35, n. 10, p. 1091–1099, 2020. DOI: 10.1002/tox.22945. .
LÓPEZ-OLMEDA, J. F., SÁNCHEZ-VÁZQUEZ, F. J. "Thermal biology of zebrafish (Danio rerio)", Journal of Thermal Biology, v. 36, n. 2, p. 91–104, 2011. DOI: 10.1016/j.jtherbio.2010.12.005. .
MAO, L., JIA, W., ZHANG, L., et al. "Embryonic development and oxidative stress effects in the larvae and adult fish livers of zebrafish (Danio rerio) exposed to the strobilurin fungicides, kresoxim-methyl and pyraclostrobin", Science of the Total Environment, v. 729, p. 139031, 2020. DOI: 10.1016/j.scitotenv.2020.139031. Disponível em: https://doi.org/10.1016/j.scitotenv.2020.139031.
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. .
MATOS, R. R., MARTUCCI, M. E. P., DE ANSELMO, C. S., et al. "Pharmacokinetic study of xylazine in a zebrafish water tank, a human-like surrogate, by liquid chromatography Q-Orbitrap mass spectrometry", Forensic Toxicology, v. 38, n. 1, p. 108–121, 2020. DOI: 10.1007/s11419-019-00493-y. Disponível em: https://doi.org/10.1007/s11419-019-00493-y.
PARK, K., HAN, E. J., AHN, G., et al. "Effects of thermal stress-induced lead (Pb) toxicity on apoptotic cell death, inflammatory response, oxidative defense, and DNA methylation in zebrafish (Danio rerio) embryos", Aquatic Toxicology, v. 224, n. March, p. 105479, 2020. DOI: 10.1016/j.aquatox.2020.105479. Disponível em: https://doi.org/10.1016/j.aquatox.2020.105479.
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, Vinicius Figueiredo, ANSELMO, C. de S., NUNES, I. K. da C., et al. "Zebrafish (Danio rerio) water tank model for the investigation of drug metabolism: Progress, outlook, and challenges", Drug Testing and Analysis, v. 10, n. 11–12, p. 1657–1669, 2018. DOI: 10.1002/dta.2523. .
SARDELA, Vinícius Figueiredo, 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. .
THEVIS, M., PIPER, T., THOMAS, A. "Recent advances in identifying and utilizing metabolites of selected doping agents in human sports drug testing", Journal of Pharmaceutical and Biomedical Analysis, v. 205, p. 114312, 2021. DOI: 10.1016/j.jpba.2021.114312. Disponível em: https://doi.org/10.1016/j.jpba.2021.114312.
ZAHANGIR, M., HAQUE, F., MOSTAKIM, G. M., et al. "Secondary stress responses of zebrafish to different pH : Evaluation in a seasonal manner", Aquaculture Reports, v. 2, p. 91–96, 2015. DOI: 10.1016/j.aqrep.2015.08.008. Disponível em: http://dx.doi.org/10.1016/j.aqrep.2015.08.008.
