Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46

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Comparative Biochemistry and Physiology, Part C

journal homepage: www.elsevier.com/locate/cbpc

Review

Zebrafish (Danio rerio): A valuable tool for predicting the metabolism of T

xenobiotics in humans?
⁎
Carina de Souza Anselmoa,b, , Vinicius Figueiredo Sardelaa,c, Valeria Pereira de Sousab,
Henrique Marcelo Gualberto Pereiraa
a
Federal University of Rio de Janeiro, Institute of Chemistry, LBCD-LADETEC, Av Horácio Macedo, 1281, 21941-598, Polo de Química, bloco C, Cidade Universitária,
Rio de Janeiro, RJ, Brazil
b
Federal University of Rio de Janeiro, Department of Drugs and Pharmaceutics, Faculty of Pharmacy, LabCQ, Av Carlos Chagas Filho, 373, 21941-902, Bss36, Cidade
Universitária, Rio de Janeiro, RJ, Brazil
c
Federal University of Rio de Janeiro, Institute of Chemistry, LPDI-LADETEC, Av Horácio Macedo, 1281, 21941-598, Polo de Química, bloco C, Cidade Universitária, Rio
de Janeiro, RJ, Brazil

A R T I C LE I N FO A B S T R A C T

Keywords: Zebrafish has become a popular model organism in several lines of biological research sharing physiological,
Zebrafish (Danio rerio) morphological and histological similarities with mammals. In fact, many human cytochrome P450 (CYP) en-
Xenobiotic metabolism zymes have direct orthologs in zebrafish, suggesting that zebrafish xenobiotic metabolic profiles may be similar
Cytochrome P450 to those in mammals. The focus of the review is to analyse the studies that have evaluated the metabolite
Non-human model
production in zebrafish over the years, either of the drugs themselves or xenobiotics in general (environmental
Drug discovery
pollutants, natural products, etc.), bringing a vision of how these works were performed and comparing, where
Toxicology
possible, with human metabolism. Early studies that observed metabolic production by zebrafish focused on
environmental toxicology, and in recent years the main focus has been on toxicity screening of pharmaceuticals
and drug candidates. Nevertheless, there is still a lack of standardization of the model and the knowledge of the
extent of similarity with human metabolism. Zebrafish screenings are performed at different life stages, typically
being carried out in adult fish through in vivo assays, followed by early larval stages and embryos. Studies
comparing metabolism at the different zebrafish life stages are also common. As with any non-human model, the
zebrafish presents similarities and differences in relation to the profile of generated metabolites compared to that
observed in humans. Although more studies are still needed to assess the degree to which zebrafish metabolism
can be compared to human metabolism, the facts presented indicate that the zebrafish is an excellent potential
model for assessing xenobiotic metabolism.

1. Introduction
Fish are the most numerous and phylogenetically diverse group of
vertebrates and are useful in the study of fundamental processes in
vertebrate evolution, development, toxicology and disease processes
(Garcia et al., 2016; Langheinrich, 2003; Spitsbergen and Kent, 2003).
In particular, zebrafish (Danio rerio), which is a tropical fish of the
Cyprinidae family, has been a prominent genetic model since 1981
(Streisinger et al., 1981), when its genetic amenability allowed the first
genetic screening for mutations that affect organ development in a
vertebrate (Kamel and Ninov, 2017). Since then, zebrafish has become
a popular model organism in several lines of biological research beyond
genetics, including developmental biology (Carten et al., 2011; Ho
et al., 2003), toxicology (Thompson et al., 2010; Weigt et al., 2011;
Zhang et al., 2016), drug discovery (Fleming et al., 2005; Kithcart and
MacRae, 2017), disease models (Brugman, 2016; Capiotti et al., 2014)
and neurobiology (Pinho et al., 2016; Shams et al., 2017). Therefore,
zebrafish represents a viable alternative to the classical mammalian
models currently used in biological research because it has some unique
properties: it is an intact organism with similar development, anatomy
and physiology to higher vertebrates (Otte et al., 2017); it is easier and
less expensive than popular rodent models; it is a genetically tractable
vertebrate model that can be easily injected with modified genes and
can absorb chemical mutagens through water; embryos are transparent
and develop externally, allowing the use of noninvasive imaging tech-
niques; embryos develop very rapidly compared to mammalian models,

⁎
Corresponding author at: LBCD-LADETEC, Institute of Chemistry, Federal University of Rio de Janeiro, Av. Horácio Macedo, 1281, bloco C - Cidade Universitária, RJ, 21941-598 Rio
de Janeiro, Brazil.
E-mail address: carinaanselmo@iq.ufrj.br (C. de Souza Anselmo).

https://doi.org/10.1016/j.cbpc.2018.06.005
Received 8 May 2018; Received in revised form 25 June 2018; Accepted 27 June 2018
Available online 30 June 2018
1532-0456/ © 2018 Elsevier Inc. All rights reserved.
C. de Souza Anselmo et al.
potentially reducing the experimentation time (Garcia et al., 2016); and
it has a high reproduction capacity, where a pair of zebrafish can
generate hundreds of fertilized eggs, which develop rapidly into larvae
with functional metabolic organs (Kamel and Ninov, 2017).
Although there are some major differences resulting from adapta-
tion to aquatic life, most zebrafish organs perform the same functions as
their human counterparts and exhibit well-conserved physiology
(MacRae and Peterson, 2015). In fact, zebrafish share physiological,
morphological and histological similarities with mammals (Diekmann
and Hill, 2013) and its genome has been sequenced, revealing that
~70% of human genes have a zebrafish orthologue and approximately
82% of potential human disease-related genes have at least one obvious
zebrafish orthologue (Howe et al., 2013). Not surprisingly, in terms of
publication, zebrafish are one of the fastest growing model organisms
(Garcia et al., 2016). Furthermore, chemical-genetic screens in zebra-
fish have already rendered the drug ProHema, which is undergoing
phase II clinical trials for improving hematopoietic stem cell engraft-
ment and expansion (Kamel and Ninov, 2017; North et al., 2007). As-
sessing the toxicity of a compound is a critical step in the discovery of
new drugs. In forensic toxicology, it is also very important to assess the
toxicity of a compound, since the knowledge of its metabolism will be
fundamental for determining the analytical targets to be monitored as
the emergence of ever newer designer drugs is an ongoing challenge for
analytical toxicologists in forensic as well as clinical toxicology (Peters
and Martinez-Ramirez, 2010). To understand or predict the toxic po-
tential of a compound, knowledge of the absorption, distribution, me-
tabolism, and elimination of the compound is required (Garcia et al.,
2016).
Notably, extensive metabolism of a xenobiotic, an external and
foreign compound to the organism, could lead to reduced activity,
whereas creation of new active metabolites could lead to increased
toxicity (Diekmann and Hill, 2013). Zebrafish embryos represent an
attractive model for studies of developmental toxicity of xenobiotics
both for human and environmental risk assessment (Carlsson et al.,
2013). The reason for the preference in the use of embryos instead of
adult fish is because in Europe, zebrafish embryos are not considered
laboratory animals until the independent feeding stage (European
Commission, 2016), which makes them ideal candidates for several
chemical screenings. On the other hand, in the United States of America
fish are not protected for research use, while in Brazil, China and India,
all animals are protected for use in research (Sneddon et al., 2017). In
fact, the zebrafish embryo has already been accepted as a validated
alternative for the acute fish toxicity test (OECD TG236) (OECD, 2013).
In addition, toxicology may be the most prevalent use of zebrafish in
the industry, with the majority of large pharmaceutical companies re-
porting some use of zebrafish for toxicology (MacRae and Peterson,
2015). The metabolism of the substance as a critical step in the tox-
icological evaluation. Therefore, the purpose of this review is to analyse
publications involving xenobiotic metabolism studies at various stages
in the zebrafish's evolution over the years. Considerations will also be
made on the development of zebrafish, enzymes involved in the me-
tabolism of xenobiotics, and analytical methods for the detection of
metabolites, highlighting advantages and limitations of zebrafish as a
model for the evaluation of xenobiotic metabolism.
2. Development
The genetic signals and responses that drive early embryonic de-
velopment are fundamentally similar between zebrafish and mamma-
lian embryogenesis (Sipes et al., 2011). The formation of different
organ systems can be followed under the microscope during the de-
velopmental period, offering many opportunities for the study of or-
ganogenesis (Dawid, 2004). The zebrafish has a rapid development,
progressing from zygote to larvae in 72 h. During the early embryonic
stages, zebrafish are covered by a chorionic membrane (egg shell)
which will be lost between 3 or 4 dpf (days post-fertilization) (Kimmel
35
Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46
et al., 1995). The chorion is an acellular envelope containing pores that
are approximately 0.5 μm in diameter with 2 μm spacing (Lee et al.,
2009). A limitation to using the zebrafish embryo is its need to take up
substances through the chorion and other membranes, because the gills
of the adult fish are not yet developed (Vallverdú-Queralt et al., 2015).
Therefore, the chorion can significantly confound the early life zebra-
fish toxicity assay by leading to false positives because of size-depen-
dent limitations in the uptake of large compounds (Langheinrich et al.,
2003; Mandrell et al., 2012). The chorion may be removed but not
without potentially affecting embryo integrity and behavior (Mandrell
et al., 2012; Sipes et al., 2011).
The morphogenesis of the liver, which is an organ that plays a key
role in the biotransformation of xenobiotics, starts at approximately
24 hpf (hours post-fertilization), and liver-specific markers occur even
earlier (16 hpf) (Tao and Peng, 2009). By 5 dpf, the heart, liver, brain,
pancreas, and other organs are developed (Garcia et al., 2016; Kimmel
et al., 1995). The zebrafish larvae begin independent feeding at ap-
proximately 6 dpf, growing rapidly and developing into juveniles at
approximately 30 dpf, and subsequently into adults at approximately
90 dpf (Kamel and Ninov, 2017). Similar to the mammalian liver, the
zebrafish liver is an essential organ in the body and performs several
vital activities, including metabolism, detoxification and homeostasis
(Menke et al., 2011; Tao and Peng, 2009). However, zebrafish have a
unique hepatic anatomy and cellular architecture, despite the high
conservation of cell types within the liver, compared with mammals
(Goessling and Sadler, 2015; Tao and Peng, 2009).
3. Enzymes involved in the metabolism of xenobiotics
Drug-metabolizing enzymes play central roles in the metabolism,
elimination and/or detoxification of xenobiotics introduced into the
organism (Xu et al., 2005). Zebrafish have the ability to perform both
phase I (oxidation, N-demethylation, O-demethylation and N-deal-
kylation) and phase II (sulfation and glucuronidation) metabolism re-
actions, and the metabolic enzymes responsible for these reactions are
highly conserved in relation to mammals (Brox et al., 2016; Chng et al.,
2012; Diekmann and Hill, 2013). Phase I enzymes consist primarily of
the cytochrome P450 (CYP) superfamily of microsomal enzymes, which
are found abundantly in the liver, gastrointestinal tract, lung and
kidney, catalyzing the oxidative and reductive metabolism of many
chemicals and endogenous compounds (Tseng et al., 2005; Xu et al.,
2005). In humans, it is believed that five CYP gene families, namely,
CYP1, CYP2, CYP3, CYP4 and CYP7, play crucial roles in hepatic as well
as extra-hepatic metabolism and elimination of xenobiotics and drugs,
as CYP1A2, CYP2C9, CYP2D6 and CYP3A4/5 are responsible for the
oxidative biotransformation of approximately 70% of most clinically
used drugs (Saad et al., 2017; Xu et al., 2005). Therefore, CYP families
1-3 are the main metabolizing enzymes responsible for xenobiotic
metabolism (Guengerich, 2008; Saad et al., 2016).
Many human CYP enzymes have direct orthologs in zebrafish
(Goldstone et al., 2010), suggesting that zebrafish metabolic profiles
may be similar to those of mammals. In addition, there are other CYPs
described in fish that lack orthologs in humans, such as CYP1C, CYP2AE
and CYP2X (Table 1) (Goldstone et al., 2010). Nevertheless, as CYP
activity is not necessarily correlated with its gene expression or even
protein levels, more focus is needed on CYP activity (Saad et al., 2017).
Like the human CYP3A genes, CYP3A65 transcription in the foregut
region was enhanced by treatment of the zebrafish larvae with dex-
amethasone and rifampicin (Tseng et al., 2005). On the other hand, 7-
benzyloxy-4-(trifluoromethyl)coumarin, a vertebrate CYP3A substrate
not usually associated with CYP1 activity, was metabolized more effi-
ciently by CYP1A than CYP3A65 in zebrafish (Scornaienchi et al.,
2010). That is, a zebrafish CYP that has an ortholog in human will not
necessarily have the same behavior on a certain substrate. Another
interesting case is the metabolism of dextromethorphan in zebrafish;
although no ortholog of human CYP2D6 has been found in the fish
C. de Souza Anselmo et al. Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46

Table 1 abundant), showing close similarities between mammalian and zebra-

Synteny comparison between zebrafish and the major fish Ugt1 and Ugt2 families (Huang and Wu, 2010; Christen and Fent,
human CYPs involved in the metabolism of xenobiotics. 2014). All the isoforms of UGTs have tissue-, sex- and developmental-
Source: Adapted from Goldstone et al. (2010). specific expression patterns. UGTs expression level dropped at 2 dpf and
Zebrafish Human increased again from 3 to 5 dpf and features high expression of Ugt1a
and Ugt1b in the liver and intestine of female and male zebrafish
CYP1A CYP1A1/1A2
(Christen and Fent, 2014).
CYP1B1 CYP1B1
CYP1C1,2 –
SULTs have also been identified in zebrafish being divided into five
CYP1D1 CYP1D1P families SULT 1, SULT 2, SULT 3 and SULT 6. Similar to the human
CYP2Ks CYP2W1 SULTs, the majority of the zebrafish SULTs that have been cloned be-
CYP2N13 CYP2J2 long to the SULT1 gene family (Liu et al., 2010; Mohammed et al.,
CYP2Ps CYP2J2
2012). The amino acid sequence alignment demonstrates that the pu-
CYP2R1 CYP2R1
CYP2U1 CYP2U1 tative zebrafish Naa10 (catalytic subunit Nα-acetyltransferase from
CYP2V1 CYP2J2 NAT) is highly similar to human Naa10 (Ree et al., 2015). Phylogenetic
CYP2X1-10 – analysis revealed a great diversity of fish GSTs, with 27 members found
CYP2Y3,4 CYP2A/B/F/S
in zebrafish (Glisic et al., 2015). Glisic et al. (2015) suggest involve-
CYP2AA1-12 –
– CYP2D
ment of GST Pi class, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b in the
– CYP2C biotransformation of xenobiotics on their functional similarity to the
CYP2AD2,3,6 CYP2J2 human orthologs in respect to the xenobiotic metabolism. Other work
CYP2AE1,2 – showed further the considerable conservation with regard to the critical
CYP3A65 CYP3A-se1,-se2a
amino acid residues between the zebrafish and mammalian COMTs
CYP3C1-4 CYP3A3,4,7
CYP4F43 CYP4F (Alazizi et al., 2011). In fact, many studies have revealed the presence
CYP4V7,8 CYP4V2 of phase II metabolites produced by zebrafish at different life stages
CYP4T8 – (Alderton et al., 2010; Anselmo et al., 2017; Brox et al., 2016; Shen
CYP5A1 CYP5A1 et al., 2017).
CYP7A1 CYP7A1
CYP7B1 CYP7B1
CYP7C1 – 4. Metabolism of xenobiotics

The zebrafish is increasingly used as a vertebrate model for several

(Table 1) (Goldstone et al., 2010). Saad et al. (2017) showed that
zebrafish do metabolize dextromethorphan to dextrorphan, in addition
to 3-methoxymorphinan, but the ratio between both metabolite con-
centrations was different than in man. This indicates that not having an
ortholog of human CYP in the zebrafish does not necessarily mean that
there will be no biotransformation of xenobiotics.
In several mammalian species including man, CYP-related drug
metabolism tends to be significantly lower during early life stages, and
the same was observed in vitro in zebrafish (Saad et al., 2017), despite
several studies having already observed the production of metabolites
in the early larval stages (2 to 7 dpf) (Alderton et al., 2010; Chng et al.,
2012; Hu et al., 2012; Pardal et al., 2014) and embryos (fertilized eggs
exposed right after being collected) (Brox et al., 2016; Vallverdú-
Queralt et al., 2015; Wiegand et al., 2001). Regarding the embryos, the
biotransformation capability was five-fold higher in the older fish em-
bryos (50 hpf) than in the younger embryos (2 to 26 hpf) (Kühnert
et al., 2017). Concerning CYP3A65, the strength of transcription in-
creased with stages of development; CYP3A65 mRNA first appeared at
the 72 hpf stage, and in adult fish, it was intensively transcribed in liver
and intestine (Chang et al., 2013). The same is true for zebrafish
CYP1A, where expression is low during the early stages of development
and increases dramatically after hatching at approximately 72 hpf (Saad
et al., 2016). Overall, the expression of zebrafish CYPs appears to
broadly increase throughout normal embryonic development, with
higher expression observed in larvae posthatching (Jones et al., 2010).
During phase II drug metabolism, the drugs or metabolites from
phase I pathways are enzymatically conjugated with a hydrophilic en-
dogenous compound by the most common transferase enzymes: UDP-
glucuronosyltransferases (UGTs), sulfotransferases (SULTs), N-acetyl-
transferases (NATs), glutathione S-transferases (GSTs), thiopurine S-
methyltransferases (TPMTs), and catechol O-methyltransferases
(COMTs) (Almazroo et al., 2017; Xu et al., 2005). Glucuronidation is
the major phase II drug metabolism pathway, with approximately 40%
to 70% of human endogenous and exogenous compounds conjugated to
glucuronidated forms to be excreted from the body (Almazroo et al.,
2017). In zebrafish, a total of 45 UGT genes were identified that can be
divided into three families consisting of Ugt1s, Ugt2s and Ugt5s (most
studies, including in vivo toxicological screenings, because it combines
the scale and throughput of in vitro systems with the physiological
complexity of a vertebrate whole animal (Garcia et al., 2016). Several
studies using zebrafish have observed the formation of metabolites of
the substances administered in the fish, with several goals in several
areas of interest, such as pollutants of the environment, drug discovery
and toxicological evaluation. However, there is still a lot to be under-
stood about the model and several particularities to be discussed.
Through an advanced search in the Science Direct database (https://
www.sciencedirect.com/science/search), searching for “zebrafish and
metabolism or metabolite” in the abstract, title and keywords fields, the
result presented in Fig. 1 was obtained. As can be observed, the subject
in question presented a considerable growth in the number of pub-
lications overtime, especially after 2005. After evaluating the results of
the search and withdrawing the studies that did not focus on the ob-
servation of the metabolism of exogenous substances by the fish, the
data presented in Table 2 were obtained, which included studies that,
with different aims, evaluated the production of xenobiotic metabolites
in zebrafish.
Most of the studies evaluated the metabolism of xenobiotics through
in vivo assays, four used in vitro assays, and two compared in vitro with
in vivo (Table 2; Fig. 2a). Zebrafish screenings involving metabolite
analysis are performed at different life stages, typically being carried
out in adult fish, followed by early larvae stages and embryos (Table 2;
Fig. 2b). Studies comparing metabolism at the different zebrafish life
stages are also common (Chng et al., 2012; Hertl and Nagel, 1993; Saad
et al., 2017). In cases where adult fish were used, most studies used
both sexes, and some did not cite this type of information (Table 2). The
in vitro studies were performed using liver homogenate, liver micro-
somes or whole zebrafish, whereas in vivo assays generally directly
exposed the fish to water or medium containing the substances, with
two exceptions in the routes of administration: spiked food (Wen et al.,
2015) and intraperitoneal injection (Troxel et al., 1997). The number of
animals used in the studies was widely variable, as generally when
using embryos or larvae the number tends to be higher (25 (Brox et al.,
2016), 60 (Vallverdú-Queralt et al., 2015) and 200 (Hertl and Nagel,
1993) embryos; 20 (Chng et al., 2012), 30 (Jones et al., 2012), 40

36
C. de Souza Anselmo et al.
Fig. 1. Number of publications over the years mentioning the terms “zebrafish” and “metabolism” or “metabolite” in the title, abstract and/or keywords.
Source: https://www.sciencedirect.com/science/search.
(Alderton et al., 2010) and 100 (Hertl and Nagel, 1993) larvae) than
when using adult animals (6 (Shen et al., 2017), 12 (Anselmo et al.,
2017), 16 (Wen et al., 2015) and 20 animals (Kasokat et al., 1987,
1989)), but the number of animals used is not always provided.
Another factor that has been shown to be quite variable is the ex-
posure time of the fish to the substances tested: in the in vitro assays the
exposure time tends to be shorter and less variable (from 45 min to
24 h) than in the in vivo tests (from 3 h to 20 days) (Fig. 2c). The mean
fish maintenance temperature, in the case of the in vivo experiments and
the in vitro assays, ranged from 23 to 28.5 °C, except for an in vitro study
that used a temperature of 35 °C (Wiegand et al., 2001) (Table 2).
Zebrafish are classified as eurythermal, as they exhibit a tolerance for
wide temperature ranges, and usually a temperature of 28.5 °C is con-
sidered optimal and recommended (Kimmel et al., 1995; Lawrence,
2007), although OECD guidelines also suggest ranges between 21 and
25 °C (OECD, 1992) and 26 ± 1 °C (OECD, 2013). Overall, adult zeb-
rafish were cultured in a 12 h:12 h (Shen et al., 2017; G. Wang et al.,
2017; Zok et al., 1991) or 14 h:10 h (Saad et al., 2017; Troxel et al.,
1997) day/night cycle as recommended (12–16 h photoperiod) (OECD,
2013). Concerning the use of organic solvents to increase the solubility
of tested compounds, several studies used DMSO at different con-
centrations (0.01 to 1%, v/v); two used methanol (0.1 or 1%, v/v),
ethanol (0.01 or 0.0015%, v/v) or toluene (0.007%, v/v); and less than
half of the studies did not use any type of solvent (Fig. 2d). It is im-
portant to note that even low concentrations of organic solvents may
influence the activity of zebrafish phase I and II enzymes (David et al.,
2012), and some studies have used high concentrations of DMSO and
methanol (1%, v/v) (Table 2).
The first publications that verified the generation of metabolites by
zebrafish were focused on environmental toxicology, since the knowl-
edge of biotransformation pathways of xenobiotics plays a fundamental
role in environmental monitoring programs. To the best of our knowl-
edge, the pioneering work that evaluated xenobiotics metabolites in
zebrafish was conducted in 1987 (Kasokat et al., 1987) (Table 2), in
which the metabolism of phenol and substituted phenols was studied in
adult zebrafish following in vivo exposure in the water. On this occa-
sion, phenyl glucuronide, phenyl sulfate, and quinol sulfate were
identified as metabolites of phenol; 2-cresyl glucuronide, 2-cresyl sul-
fate, and 2-hydroxybenzoic acid were determined to be metabolites of
2-cresol; and only the glucuronide and sulfate conjugates were detected
as metabolites of 4-nitrophenol, 4-chlorophenol and penta-
chlorophenol. Overall, no significant differences from related fresh-
water fish species were found, with the exception of the considerably
lower oxidation rates which were observed in the case of phenol and 2-
37
Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46
cresol (Kasokat et al., 1987). Following this line of study, the metabo-
lism of chlorobenzene (CB) and hexachlorobenzene (HCB) was also
evaluated in adult zebrafish in a very similar way to the previous work
(Kasokat et al., 1989). The metabolites found for CB were isomeric
chlorophenols (phase I products) and the corresponding glucuronide
and sulfate conjugates (phase II products), and no metabolites were
detected for HCB. The results for CB were in agreement with those
observed in other freshwater fish species. However, other fish species
have already been shown to metabolize HCB, which may indicate that
failure to observe metabolites in this work may be due to the exposure
time, mode of exposure or use of the organic solvent toluene.
Continuing to use the zebrafish for xenobiotic analysis with impact
on the environment, Gorge and Nagel (1990) evaluated the hazards of
the pesticides lindane and atrazine to know whether if and to what
extent substances are incorporated, bioconcentrated and eliminated
when exposure is stopped. None of the metabolites were identified, but
it was possible to observe that atrazine was metabolized to a small
extent (4%) and that only three metabolites were formed. In contrast,
50% of lindane was converted to twelve more polar metabolites than
lindane, which must be dechlorination products of the compound. Zok
et al. (1991) conducted an experiment to evaluate the toxicokinetics of
nine anilines in adult zebrafish following in vivo exposure in water. The
corresponding acetanilides were the only metabolites identified in
water, and no biotransformation occurred at all for 2- and 4-nitroani-
line, suggesting that substitution in the ortho position of the aniline
sterically hinders acetylation. The acetylation reaction is catalyzed by
an acetyltransferase that depends on acetyl-CoA, which exists in zeb-
rafish (Ree et al., 2015). No hydroxylation of the aromatic ring was
observed for the anilines in zebrafish, which was not in agreement with
results observed in humans, where N-acetyl-4-aminophenol was the
predominant urinary aniline metabolite and acetanilides were found
only in minor amounts (Modick et al., 2016).
Hertl and Nagel (1993) evaluated the in vivo metabolism of 3,4-
dichloroaniline, which is an intermediate product of the degradation of
several herbicides, at different life stages of zebrafish. All life stages of
zebrafish possess the capability to metabolize 3,4-dichloroaniline to
3,4-dichloroacetanilide, and acetanilide formation appeared to be
higher in the more advanced stages of the fish. In this work, although
without confirmation, there is evidence of the presence of the hydro-
xylated metabolite for 3,4-dichloroaniline. Other studies with en-
vironmental pollutants were performed, such as the evaluation of the
metabolism of atrazine and bisphenol A in zebrafish. Atrazine is a
widely used herbicide, as the induction of GST provides an effective
resistance against atrazine in plants, and it was evaluated whether if the
 Table 2
Publications involving the study of the metabolism of xenobiotics in zebrafish.
Author Zebrafish's life stage Brief Experimental design Analytical technique Results Observations

Kasokat et al., 1987 Adult (female; age not The metabolism of phenol and In vivo. Exposure in water with ethanol HPLC-UV Several phenol and substituted Overall, the metabolites recovered
informed) substituted phenols in zebrafish was 0.01% (v/v) for some compounds. phenols metabolites have been were higher for substituted phenols
studied Water analyzed. ETa: 48 h/35 h Tb: identified, with phase II being than or phenol itself indicating that
C. de Souza Anselmo et al.

26 °C more common than phase I some substituted phenols are more

metabolites. rapidly metabolized
Kasokat et al., 1989 Adult (female; age not The metabolism of chlorobenzene (CB) In vivo. Exposure in water with ethanol HPLC-UV Hydroxylate and conjugate Zebrafish is able to hydroxylate
informed) and hexachlorobenzene (HCB) in 0.01/toluene 0.007% (v/v). Water and metabolites were detected for aromatic compounds. The finding
zebrafish was studied fish analyzed. ETa: 48 h Tb: 26 °C CB. Turning to HCB, no was in contrast to published work
metabolites could be detected concerning the metabolism of HCB
under the experimental in fish.
conditions
Gorge and Nagel, Larvae (28 dpf) Study of the metabolism of two In vivo. Exposure in water. Water HPLC-UV Presence of 12 metabolites of Lindane more metabolized than
1990 radiolabelled pesticides, lindane and analyzed. ETa: 40 h/Tb: (26 ± 1)°C lindane and 4 metabolites of atrazine.
atrazine atrazine
Zok et al., 1991 Adult (both sexes; Biotransformation products of anilines In vivo. Exposure in water. Water and HPLC-UV Corresponding acetanilides Substitution in the ortho position of
12 weeks) in the zebrafish were analyzed by fish analyzed. ETa: 24 h Tb: were the only metabolites the aniline sterically hinders
HPLC-UV (26.5 ± 1)°C identified in water. No acetylation. No ring hydroxylation of
biotransformation for 2- and 4- the anilines was observed.
nitroaniline.
Hertl and Nagel, 1993 Embryos (4 hpf), larvae Metabolism of 3,4-dichloroaniline In vivo. Exposure in water. Fish HPLC-UV All life stages of zebrafish There is evidence of hydroxylation
(4 dpf and 17 dpf) and (3,4-DCA) analyzed. ETa: 48 h Tb: 26 °C possess the capability to product of the aniline.
adult (female; 24 weeks) generate the metabolite 3,4-
dichloroacetanilide
Troxel et al., 1997 Adult (sexually mature, Characterize the in vivo metabolism In vivo/in vitro. Intraperitoneal HPLC-UV The major metabolites of AFB1 The results demonstrate that
both sexes) and hepatic DNA adduction of the injection.. Water analyzed. ET: 24 h/T: recovered in the water were zebrafish have the capacity for both

38
carcinogenic aflatoxin B1 (AFB1) in the
zebrafish
Wiegand et al., 2001 Embryos (fertilized eggs; Characterize glutathione S-transferases
prim 6 stage embryos) (GST)-formed atrazine metabolites
Lindholst et al., 2003 Adult (both sexes; age Metabolism of bisphenol A in zebrafish
not informed) (Danio rerio)
Li et al., 2009 Larvae (2 dpf) Zebrafish CYP3A4 and CYP2D6
functional activity assays for assessing
drug metabolism
Alderton et al., 2010 Larvae (7 dpf) Accumulation and metabolism of drugs
commonly used as probes for human
cytochrome P450s (CYPs) in zebrafish
larvae
Jones et al., 2010 Larvae (4 dpf) Identify genes homologous to
mammalian CYP's and UDP-UGT genes
and assess the ability of zebrafish to
metabolize substrates for these
enzymes
Smith et al., 2010
(26 ± 1)°C. Zebrafish liver
homogenate. ETa: 45 min/Tb: 28 °C
In vitro. Enzyme assay with zebrafish's
embryos. ETa: 24 h/Tb: 35 °C
In vivo. Exposure in water. Water and
tissue. ETa: 7 days Tb: (27 ± 1)°C
In vitro. Microplate whole zebrafish
assay with 0.1% DMSO. ETa: 24 h Tb:
Not informed
In vivo. Exposure in medium with
DMSO 1% (v/v). Medium and larvae
analyzed. ETa: 4 h Tb: (28.5 ± 0.5)°C
In vivo Exposure in water with 0.1% of
DMSO ETa: 10 h Tb: (28 ± 1)°C
identified as aflatoxicol and
aflatoxicol-glucuronide
HPLC-UV-ESI-QqQ Zebrafish embryos conjugate
atrazine to glutathione by the
GSTs in the same way that
plants do
HPLC-ESI-MSD Zebrafish produced the
metabolites bisphenol A
glucuronic acid (BPAGA) and
bisphenol A sulfate
CYP's specific CYP's functional activity was up
chemiluminogenic and downregulated in zebrafish
substrate similar to the response in
humans.
HPLC-ESI-MS/MS Cisapride, chlorpromazine,
verapamil, testosterone, and
dextromethorphan showed that
the zebrafish larvae catalyze a
range of phase I and phase II
reactions.
Fluorescence ECODc, Zebrafish larvae express genes
ERODd and OOMRe similar to mammalian CYP and
assay UGT isoforms throughout early
development and have
activities toward model CYP
substrates
HPLC-APCI-MS/MS
phase I and phase II metabolism of
AFB1
Phase I metabolism has not been
evaluated, although in animals,
hydroxylation and N-dealkylation
are described as the main
metabolism pathway.
The high concentration of BPAGA
found in the bile indicates that
biliary excretion into the intestines is
a major route of elimination.
These findings underscore the high
degree of CYP conservation in
zebrafish.
Metabolism of amodiaquine,
midazolam, felodipine, and S-
mephenytoin could not be detected.
Both similarities and differences in
the metabolism were observed in
zebrafish when compared to
mammals
The continued use of these model
organisms in toxicity testing is
supported by this study.
(continued on next page)
Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46
 Table 2 (continued)
Author Zebrafish's life stage Brief
Adult (sexually mature, In vitro hepatic fluoxetine metabolism
both sexes) was determined in several model fish
species: rainbow trout, goldfish,
zebrafish and killifish.
Chen et al., 2012 Embryos (2 hpf) Determine if zebrafish larvae can be
used to evaluate thyroid endocrine
disruption of BDE-209 and if it
bioaccumulates and is metabolized.
Chng et al., 2012 Larvae (5 dpf) and adult Investigation of the bioactivation
(age not informed; potential and metabolism profile of
female) zebrafish versus human using
acetaminophen (APAP) and
testosterone
Hu et al., 2012 Larvae (2 dpf) Characterize the metabolic profile of
calycosin in a zebrafish model
Jones et al., 2012 Larvae (3 dpf) Determine if zebrafish larvae could
39
metabolize ibuprofen (CYP2C
substrate) that has well characterised
metabolism in mammals
Carlsson et al., 2013 Embryos (fertilized eggs Potential risk to fish embryos of
15 min after collection) veterinary pharmaceuticals were
investigated and metabolism by the
embryos was studied for albendazole
(AL), febantel, fenbendazole and
oxfendazole.
Pardal et al., 2014 Larvae (3 dpf) Metabolism of resveratrol and its
glucoside (piceid) in zebrafish
Wen et al., 2015 Adults (20 weeks, both Evaluate potential risks posed by BDE-
sexes) and eggs 47 and their analogs in aquatic
(collected every environment, distribution among
morning before feeding) tissues of adult fish and transfer from
females to eggs
Vallverdú-Queralt Embryos (fertilized eggs; To provide accurate and
et al., 2015 24 hpf) comprehensive identification of the
phenolic constituents found in wine
extracts and zebrafish embryos
Brox et al., 2016
Experimental design
a b
In vitro. Liver microsomes ET : 1 h T :
25 °C
In vivo. Exposure in BDE solution with
0.01% of DMSO. Larvae analyse. ETa:
14 days Tb: (28 ± 0.5)°C
In vitro/in vivo. Human (HLM) and
adult zebrafish's (ZLM) liver
microsomes; larvae exposure in
medium with methanol 0.1% (v/v) ETa
(in vitro): 2 h/Tb: 28.5 °C. ETa (in vivo):
3 h/Tb: 28.5 °C.
In vivo. Exposure in medium with 0.1%
DMSO. Medium and larvae analyzed.
ETa: 24 h Tb: 28.5 °C.
In vivo. Exposure in water with ethanol
0.0015% (v/v). Water and fish
analyzed ETa: 24 h Tb: (28 ± 1)°C
In vivo. Exposure in medium with 0.1%
DMSO. Medium analyzed. ETa: 6 days
Tb: (25.1 ± 0.4)°C
In vivo. Exposure in medium with 1%
DMSO. Medium and larvae analyzed.
ETa: 48 h Tb: 27 °C
In vivo. Adult exposure by spiked food.
Adults and eggs analyzed. ETa: 20 days
Tb: (28 ± 1)°C
In vivo. Exposure in medium. Embryos
analyzed ETa: 24 h Tb: (27 ± 1)°C
Analytical technique Results
Fluoxetine metabolism in fish is
much less than that seen in
mammals. The major
mammalian metabolite,
norfluoxetine, is not the
primary metabolite in fish
GC–MS Bioconcentration of BDE-209 in
the larvae was evident, and
BDE-209 could be metabolized.
Both T4 and T3 levels were
changed by BDE-209 exposure
in the larvae.
HPLC-MS/MS/HPLC- NAPQI (APAP's reactive
QTOF/MS/MS metabolite) was generated by
ZLM at lower levels than HLM.
Several metabolites for
testosterone were identified in
ZLM and larvae.
HPLC-MS/MS A total of ten calycosin
metabolites formed from
glucuronidation, glucosylation,
sulfation, oxidation,
demonstrating existence of both
phase I and phase II metabolic
activities.
HPLC-LTQ-orbitrap- Hydroxy-ibuprofen was also
MS/MS identified in larvae extracts and
water
HPLC-ESI-MS/MS AL was metabolized efficiently
into albendazole sulfoxide,
resulting in reduced toxicity.
The prodrug febantel was
metabolized to fenbendazole
which is further metabolized to
oxfendazole in various species.
HPLC-UV-MS/MS The principal metabolites found
were monoglucoronide and
monosulfate forms of
resveratrol. Other metabolites
found in the literature were not
detected in zebrafish larvae
GC–MS/MS Concentrations of compounds
in the liver of males were
higher than in females.
Significant transfer of all
compounds from adult females
to eggs was observed. Several
metabolites formed.
HPLC-LTQ-Orbitrap- Quercetin-O-glucoronide and
MS anthocyanin metabolites was
detected in embryos.
HPLC-QTOF-MS
Observations
The low level of metabolism is likely
the reason that fish bioaccumulate
fluoxetine in vivo. After killifish,
zebrafish was the fish model with
C. de Souza Anselmo et al.
higher rates of metabolism of
fluoxetine.
The limited metabolites of BDE-209
detected compared with previous
studies suggest lower metabolic
capability in larvae,
The ZLM overall metabolite profile
of testosterone had more
hydroxylated metabolites compared
with HLM. The zebrafish larvae
produced only two hydroxilated
metabolites
The predominant phase II
conjugation of calycosin observed in
zebrafish larvae were similar to the
metabolic profiles of other
flavonoids in mammals
Zebrafish larvae (72–96 hpf) can
absorb, oxidize and excrete
ibuprofen, similar to that identified
in mammalian systems.
The results also demonstrate the
great value of including sublethal
endpoints and chemical analysis to
the zebrafish embryo test for hazard
identification and risk assessment
Besides the metabolites found in this
study for piceid, it's already been
identified glucuronidated and
sulfated piceid in rat urine after oral
administration.
Transfer of residues from adult
females to eggs was thought to be
one of the main factors for the sex-
dependent difference in
concentrations in liver.
This research constitutes the first
comprehensive identification of
phenolic compounds in zebrafish by
HPLC-HRMS
(continued on next page)
Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46
 Table 2 (continued)

Author Zebrafish's life stage Brief Experimental design Analytical technique Results Observations

Embryos (fertilized eggs; Study of xenobiotic metabolism of
incubated until 72 hpf zebrafish embryo (ZE) using clofibric
before use) acid and comparing it with the
metabolism in other primarily higher
In vivo. Exposure in medium. Medium Eighteen metabolites was
and embryos analyzed. ETa: 24 h Tb: detected formed by phase I and
(26 ± 1)°C phase II metabolism.
Transformation of clofibric acid
The results of this study outline the
high metabolic potential of the ZE
with respect to the transformation of
xenobiotics. Similarities but also
C. de Souza Anselmo et al.

organisms. involves conjugation with differences to other test systems

Anselmo et al., 2017 Adult (age and sex not The ability of adult zebrafish to
informed) produce metabolites of sibutramine
and stanozolol, substances with a well-
known metabolism that are widely
used as doping agents in sports, was
evaluated.
Shen et al., 2017 Adult (24–40 weeks; Metabolite profiles of ginsenosides Rk1
both sexes) and Rg5 from red ginseng or red
notoginseng in zebrafish were
qualitatively analyzed
Saad et al., 2017 Different developmental Drug metabolism human CYP-specific
stages (5 and 24 hpf, 2, substrates: dextromethorphan (DXM),
3, 4 and 5 dpf) and adult diclofenac (DIC), testosterone (TST)
In vivo. Exposure in water. Water
analyzed ETa: 7 days Tb: (26 ± 2)°C
In vivo. Exposure in water 0.5% DMSO.
Medium and embryos analyzed. ETa:
24 h Tb: (23 ± 1)°C
In vitro. Liver microsomes with < 0.5%
(v/v) DMSO. ETa: 2 h Tb: 28.5 °C
sulfate or glucuronic acid,
carnitine, taurine, and
aminomethanesulfonic acid.
HPLC-QExactive- Adult zebrafish could produce
Orbitrap-MS several sibutramine and
stanozolol metabolites,
including demethylated,
hydroxylated, dehydroxylated,
and reduced derivatives, all of
which have already been
detected in human
HPLC-QTOF-MS Several metabolites of Rk1 (4)
and Rg5 (7), were identified.
There are glucuronidation,
sulfation, dihydroxylation and
dehydroxy-methylation
products in metabolites of Rk1
and/or Rg5.
HPLC-MS/MS Adult zebrafish produced the
two major human metabolites
of DIC and DXM. For DIC the
were observed.
This study demonstrates that adult
zebrafish can absorb, oxidize, and
excrete several metabolites in a
manner similar to humans.
Desugarization, glucuronidation,
sulfation, and dehydroxy-
methylation was observed.
Dehydroxylation and loss of C-17
residue were also metabolic
pathways of ginsenoside Rg5
No sex-related differences were
detected, except for the higher TST
depletion rate in adult females.

40
(age not informed; both and midazolam (MDZ) was
sexes) investigated in adult (both sexes),
embryos and larvae
G. Wang et al., 2017 Adult (20 weeks; both The metabolism of six
sexes) organophosphate (OP) flame
retardants was investigated in adult
zebrafish
C. Wang et al., 2017 Larvae (7 dpf) Zebrafish as the biotransformation
system for rapid identification of
metabolites derived from herbal
compounds
In vivo. Exposure in water 0.01%
DMSO. Water and fish analyzed ETa:
19 days Tb: (24 ± 1)°C
In vivo. Exposure in water 0.5% DMSO.
Water analyzed. ETa: 24 h Tb: 28.5 °C
metabolite ratio was similar to
that in man, whereas it was
different for DXM. For TST, the
major human metabolite could
not be detected and MDZ was
not metabolized.
HPLC-QTOF-MS Twenty main metabolites were
detected in the liver of OPs-
exposed zebrafish using high
resolution mass spectrometry
HPLC-QTOF-MS 46 metabolites screened from
water samples of zebrafish
treated with total Epimedium
flavonoids (EFs) could be
matched with their
corresponding parent
compounds.
Zebrafish embryos and larvae
showed no or low biotransformation
capacity.
The reaction pathways involving
scission of the ester bond
(hydrolysis), cleavage of the ether
bond, oxidative hydroxylation,
dechlorination, and coupling with
glucuronic acid
37 of the metabolites were identified
and validated. 75% of the identified
EFs metabolites were successfully
detected in urine samples of rats

a
Exposure time.
b
Temperature; dpf: days post-fertilization; hpf: hours post-fertilization. ECOD.
c
ECOD Assay: The metabolism of 7-ethoxycoumarin to 7-hydroxycoumarin as a general marker for CYP2 activities in mammals; EROD.
d
EROD Assay: The metabolism of 7-ethoxyresorufin to resorufin as a general marker for CYP1 activities in mammals; OOMR.
e
OOMR Assay: The conversion of octyloxymethylresorufin to resorufin was used as a substrate for CYP3 activities in mammals.
Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46
C. de Souza Anselmo et al.
Fig. 2. Overview of zebrafish experiments in relation to the type of experiment (a), life stage (b), exposure time (c) and use of organic solvents (d).
zebrafish was capable of performing this reaction (Wiegand et al.,
2001). In vitro experiments were performed with enzyme extracts from
zebrafish embryos and atrazine being showed that atrazine-glutathione
conjugates products were formed. In this work, phase I metabolism had
not been evaluated, although in animals, hydroxylation and N-deal-
kylation are described as the main metabolic pathways. A study of the
metabolism of bisphenol A, an endocrine disrupter which is used in the
production of plastics, has shown that the zebrafish produced the phase
II metabolites bisphenol A glucuronic acid and bisphenol A sulfate after
exposure of adult animals in water (Lindholst et al., 2003). Another
toxicological analysis performed on zebrafish was with carcinogenic
agents. Zebrafish were used as alternative in vivo and in vitro models for
chemical carcinogenesis studies of aflatoxin B1 (AFB1) (Troxel et al.,
1997). The major metabolites of AFB1 recovered in the water at various
time points after adult intraperitoneal injection were identified as
aflatoxicol (predominant metabolite, produced by a cytosolic reductase
reaction) and aflatoxicol-glucuronide. The in vitro assay assessing AFB1
metabolism again demonstrated the proficiency of AFL formation in
zebrafish and produced AFB1-8,9-epoxide, which is responsible for the
carcinogenic effects. Male zebrafish did appear to have lower activity
toward the formation of the AFB1–epoxide than females. This pub-
lication demonstrated not only the ability of zebrafish to generate phase
I and II metabolites but also sex differences in relation to the metabo-
lism of xenobiotics.
Due to their low cost and high performance, polybrominated di-
phenyl ethers (PBDEs) have been widely used for many years as flame
retardants in various commercial products and are an environmental
problem. As PBDEs have similar structures to those of thyroid hormones
(THs), they have the potential to disrupt thyroid endocrine activities;
thus, it is important to understand their in vivo metabolism (Chen et al.,
2012; Wen et al., 2015). After 14 days of exposure, the bioconcentra-
tion of BDE-209 in the larvae was evident and BDE-209 could also be
metabolized, with the main metabolite being nona-BDE. Other BDE
metabolites have already been identified in zebrafish, including tri-
41
Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46
BDE, tetra-BDE, penta-BDE, hex-BDE, hepta-BDE and octa-BDE after
exposure for 5 months (He et al., 2011). The limited production of BDE-
209 metabolites in this study (Chen et al., 2012) compared with He
et al. (2011) can suggest lower metabolic capability in larvae, but may
also be due to the different exposure times and species used. Both T4
and T3 levels were changed by BDE-209 exposure in the larvae, sug-
gesting thyroid disruption (Chen et al., 2012). Other work evaluated
maternal transfer, distribution, and metabolism of BDE-47 and its re-
lated hydroxylated, methoxylated analogs in zebrafish, because analogs
may be more toxic than PBDEs (Wen et al., 2015). Unlike the other
studies, adult fish were exposed by spiked food in this study. The results
indicated that the concentrations of all compounds in the liver of male
zebrafish were significantly greater than those in the liver of females,
suggesting transfer of the residues from adult females to eggs, with
several metabolites formed in adult fish. More recently, G. Wang et al.
(2017) evaluated the in vivo metabolism of six typical organophosphate
(OP) flame retardants using adult zebrafish. In this work, they found
several phase I and phase II metabolites, including OP diesters, hy-
droxylated OP triesters, hydroxylated OP diesters, and glucuronic acid
conjugated metabolites, after hydroxylation.
In 2009, the first study to evaluate the zebrafish CYP response to
drugs used to treat humans was carried out, seeking an in vivo com-
parison between the species. Li et al. (2009) developed a microplate
whole zebrafish larvae (2 dpf) assay to evaluate zebrafish CYP3A4 and
CYP2D6 functional activity with various specific substrates of these
enzymes. The overall prediction success rate was 86% for CYP3A4 in-
hibition and induction in zebrafish, 100% for CYP3A4 inhibition and
71% for CYP3A4 induction (lovastatin and phenytoin did not induce
CYP3A4 in zebrafish as they do in humans); zebrafish CYP2D6 was
upregulated by treatment with dexamethasone, 2,4-di-
chlorophenoxyacetic acid and ethanol. These results highlight the high
degree of CYP conservation in zebrafish. After this work, several others
have succeeded in evaluating drug metabolism in zebrafish.
Alderton et al. (2010) examined the accumulation and metabolism
C. de Souza Anselmo et al.
of a number of drugs and commonly used probes for human CYPs in
zebrafish larvae (7 dpf) under conditions relevant to pharmacological
and toxicological assays. Metabolism studies with cisapride, chlorpro-
mazine, verapamil, testosterone, and dextromethorphan showed that
the zebrafish larvae catalyze several phase I (oxidation, N-demethyla-
tion, O-de-ethylation, hydroxylation and N-dealkylation) and phase II
(sulfation and glucuronidation) reactions, where both similarities and
differences in the metabolic pathways were observed in zebrafish larvae
when compared to mammals. Additionally, the metabolism of phena-
cetin to paracetamol and dextromethorphan to dextrorphan (metabolic
reactions catalyzed by CYP1A2 and CYP2D6 in humans, respectively)
was observed in the zebrafish larvae. Metabolism of amodiaquine,
midazolam, felodipine, and S-mephenytoin could not be detected under
the conditions used in these experiments. When zebrafish larvae were
exposed to cisapride, the only metabolite observed was cisapride N-
sulfate, which is a minor metabolite in mammalian species observed
only in the dog. Although some hydroxylation of testosterone (very
low) was observed in the current experiments, the predominant meta-
bolic pathway of testosterone observed in zebrafish larvae was glu-
curonidation. In addition to that, the amount of any specific metabolite
formed was always much lower than that of the parent compound. All
these factors may indicate that the high amount of DMSO used (1%) in
this work may have influenced the results, since it has already been
demonstrated that low concentrations of organic solvents may influence
the activity of zebrafish phase I and II enzymes (David et al., 2012).
Jones et al. (2010) have assessed the expression of genes that have
been identified as similar to mammalian CYP1A, CYP2B6, CYP3A5, and
UGT1A1, which are considered key mammalian families responsible for
the oxidative metabolism of a variety of pharmaceuticals. Overall, this
study provided further evidence that zebrafish larvae express genes and
proteins with activities that are responsible for xenobiotic metabolism
with both oxidative and conjugative metabolism similar to those of
mammalian systems. Another study evaluated the in vitro hepatic
fluoxetine metabolism of adult zebrafish compared with several others
model fish: rainbow trout, goldfish and killifish (Smith et al., 2010).
After killifish, zebrafish was the fish model with the highest rates of
metabolism of fluoxetine. In humans, fluoxetine is primarily metabo-
lized into norfluoxetine (major metabolite) through demethylation by
CYP2D6 (has no ortholog in zebrafish, Table 1) (Goldstone et al., 2010)
and to a lesser extent, by CYPs 2C9, 3A4 and 2C19. In zebrafish, the loss
of fluoxetine is much greater than the production of the demethylated
metabolite, norfluoxetine, suggesting that there may be production of
multiple fluoxetine metabolites in fish and that norfluoxetine is unlikely
to be the primary fluoxetine metabolite, as it is in humans (Smith et al.,
2010).
Chng et al. (2012) progressed forward and investigated the bioac-
tivation potential and metabolism profile of zebrafish (in vitro and in
vivo) versus human (in vitro) using acetaminophen (APAP) and testos-
terone. The hepatotoxicity of APAP is attributed to its reactive meta-
bolite, NAPQI (N-acetyl-p-benzoquinone imine), that was generated by
adult zebrafish liver microsomes (ZLM) (possibly by CYP3A65), albeit
at lower levels than by human liver microsomes (HLM). Several hy-
droxylated metabolites of testosterone were identified, including 2α-,
6β-, and 16β hydroxytestosterone and three putative metabolites (M2,
M3, and M7) in the ZLM and only 6β-hydroxytestosterone, testosterone
glucuronide, and M8 in zebrafish larvae homogenate and media. Al-
though the main metabolite formed by zebrafish was 6β-hydro-
xytestosterone, the overall metabolite profile was distributed across
more hydroxylated metabolites compared with that of HLM, and a
number of unique hydroxytestosterone metabolites were observed only
in zebrafish. This result suggests a difference in either the function or
the expression of the drug-metabolizing enzymes between zebrafish
larvae and adult zebrafish, in addition to demonstrating differences
between human and zebrafish in vitro testosterone metabolism.
Another study evaluated the metabolism of ibuprofen in zebrafish
larvae (3 dpf) after in vivo exposure in water with ethanol 0.0015% (v/
42
Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46
v) (Jones et al., 2012). Ibuprofen metabolism is well described in hu-
mans, involving oxidation of the parent compound to hydroxy-ibu-
profen and carboxy-ibuprofen by CYP2C8/9, followed by conjugation
with glucuronic acid. Although no CYP2C-corresponding orthologs
have been identified in zebrafish, it has been able to generate hydroxy-
ibuprofen and other hydroxylated derivatives. However, the presence of
carboxy-ibuprofen and glycoconjugate derivatives was not observed,
revealing differences between zebrafish and human metabolism of
ibuprofen.
Several other studies have observed the in vivo production of phase I
and II metabolites during different life stages of zebrafish using the
following various substances, showing once again that the zebrafish is a
promising model that shares similarities and differences with mammals:
flavonoids (Hu et al., 2012), veterinary pharmaceuticals (albendazole,
febantel, fenbendazole and oxfendazole) (Carlsson et al., 2013), re-
sveratrol (Pardal et al., 2014), phenolic constituents found in wine
(Vallverdú-Queralt et al., 2015), clofibric acid (Brox et al., 2016) and
ginsenosides Rk1 and Rg5 (Shen et al., 2017). Pardal et al. (2014)
evaluated the metabolism of the phenolic compounds resveratrol and
its glucoside derivative (piceid) in vivo after exposure of zebrafish
larvae in 1% (v/v) DMSO medium. The principal metabolites found in
zebrafish larvae were monoglucuronide and monosulfate forms of re-
sveratrol (the most abundant metabolite), which is in agreement with
other mammals, including human. In the case of the piceid group, the
most abundant metabolite was the sulfated form of resveratrol, even if
it appeared later. In this work, it was not possible to identify glucur-
onidated or sulfated piceid and other metabolites found in rat urine,
possibly for one of the following reasons: inability of the zebrafish
model to generate these metabolites; minor compounds that cannot be
detected under the experimental conditions; high amount of DMSO
used that may have interfered in the metabolism.
Brox et al. (2016) evaluated the metabolism of clofibric acid using
zebrafish embryos and compared it with the metabolism in other, pri-
marily higher organisms. A total of 18 metabolites of clofibric acid were
detected, including reactions of hydroxylation, demethylation, hydro-
lysis, methylation, decarboxylation and conjugation with sulfate and
glucuronic acid. In addition, conjugation with taurine, aminometha-
nesulfonic acid, and carnitine represents new phase II conjugates that
have not been described in zebrafish before this work. In humans, the
acyl glucuronide of clofibric acid (also generated by zebrafish) re-
presents the major metabolite, and taurine-conjugated clofibric acid
was previously detected in dogs, cats, and ferrets, and now in zebrafish
but not in humans.
Anselmo et al. (2017) evaluated the ability of adult zebrafish to
produce metabolites of sibutramine and stanozolol after in vivo ex-
posure, with the focus of using the model to search for analytical targets
for the detection of doping abuse. Several sibutramine and stanozolol
metabolites were identified, including demethylated, hydroxylated,
dehydroxylated, and reduced derivatives and stanozolol conjugates, all
of which have already been detected in human urine. This study sug-
gests that adult zebrafish can absorb, oxidize, and excrete several me-
tabolites in a manner similar to humans.
Saad et al. (2017) made an important contribution by comparing
the in vitro drug metabolism of human CYP-specific substrates at dif-
ferent life stages of zebrafish with humans. Several CYP-related reac-
tions in adult zebrafish using human CYP2- and CYP3-specific sub-
strates were detected. No in vitro biotransformation of the tested
substrates was observed during zebrafish embryonic development until
96 hpf (end of organogenesis). Unlike other studies, the bio-
transformation rates were predominantly higher in zebrafish compared
to humans, except for CYP2D6-like activity. Sex-related differences
were only present for testosterone. Diclofenac biotransformation in
zebrafish into 4- and 5-OH-diclofenac reveals similar biotransformation
pathways compared to humans by the production of similar major
metabolites with similar proportions in both species. However, dex-
tromethorphan was also biotransformed into similar main metabolites
C. de Souza Anselmo et al.
as in humans but with altered ratios as shown (dextrorphan/3-meth-
oxymorphinan). In this study, no production of the main testosterone
metabolite was observed, which was already reported in another work
(Chng et al., 2012). For midazolam, neither consumption nor metabo-
lites could be detected in ZLM, whereas this substrate was largely me-
tabolized into 1-OH-midazolam and, to a much lesser extent, into 4-OH-
midazolam in HLM. As a result, midazolam, a typical substrate for
CYP3A in man, appeared to have no affinity for any of the zebrafish
CYPs. Therefore, the in vitro CYP-mediated drug metabolism can be
substantially different in zebrafish compared with man.
C. Wang et al. (2017) proposed a rapid identification of metabolites
derived from herbal compounds after in vivo exposure of zebrafish
larvae (7 dpf), compared with metabolism in rats, a representative
mammalian model. This work identified 46 potential metabolites that
could be matched with their corresponding parent compounds, and 37
of them were identified and validated by the known metabolic path-
ways and fragmentation patterns. Among the 37 metabolites identified
in zebrafish, 28 were found in rat urine. This result encourages re-
searchers to use zebrafish larvae as the biotransformation system for
preliminary metabolism study.
5. Metabolites analysis
The first studies that carried out the detection of xenobiotic meta-
bolites in zebrafish used HPLC (high-performance liquid chromato-
graphy) with diode-array detection (DAD) analysis (Gorge and Nagel,
1990; Hertl and Nagel, 1993; Kasokat et al., 1987, 1989; Troxel et al.,
1997; Zok et al., 1991). HPLC-DAD provides a means for analysing
various compounds, but is limited by the non-specific nature of UV
detection. On the other hand, gas chromatography–mass spectrometry
(GC–MS), together with detailed GC–MS spectral libraries, is a very
useful tool for toxicological analysis, but non-volatile, polar and ther-
mally labile compounds are difficult or impossible to analyse without a
long derivatization procedure (Couchman and Morgan, 2011).
An important contribution for analytical toxicology was the devel-
opment of the electrospray ionization (ESI) (Fenn, 2002), which was
responsible for the coupling of liquid chromatography (LC) and mass
spectrometry (MS), expanding the detection range of new metabolites,
and increasing the detection capacity of substances even at low con-
centrations. Triple quadrupole (QqQ) instruments operating in selected
reaction monitoring (SRM) mode are the most common mass analysers
used in bioanalysis (Núñez et al., 2013), as can be observed in several
studies (Alderton et al., 2010; Carlsson et al., 2013; Chng et al., 2012;
Saad et al., 2017). However, for the detection by tandem-MS techni-
ques, as the triple quadrupole, the potential metabolites must be pre-
viously known or need to share a high degree of structural similarity
with the parent compound to allow their determination by neutral loss
or precursor ion scan. However, these conditions are not necessarily
possible in metabolism studies (Brox et al., 2016).
An emerging approach for toxicological analysis is that of the de-
termination of exact mass with high-resolution (HRMS). Through a full-
scan HRMS experiment at high mass accuracy (mass errors below
6 ppm), it is possible to very precisely filter the full-scan data and ex-
tract analyte chromatograms with very low background noise, which
allows the identification of metabolites by knowledge of the elemental
composition alone, without the need for reference material (Couchman
and Morgan, 2011). Time-of-flight (TOF) and Orbitrap-based technol-
ogies are currently the most common analysers used in LC–HRMS
(Núñez et al., 2013). In fact, studies using LC-HRMS to search for xe-
nobiotic metabolites have been able to identify and sometimes char-
acterize a greater number of substances (Anselmo et al., 2017; Brox
et al., 2016; Shen et al., 2017; C. Wang et al., 2017) compared to other
techniques. The major challenge in the identification of metabolites by
LC-MS is the detection and structural elucidation of trace levels of
unknown metabolites in the presence of large amounts of complex in-
terfering ions of endogenous components, called matrix effect (Zhu
43
Comparative Biochemistry and Physiology, Part C 212 (2018) 34–46
et al., 2011). Thus, a major advantage of using the in vivo zebrafish
model for predicting metabolism is that the matrix is cleaner than urine,
for example, widely used in other animal models, which reduces the
matrix effect.
6. Zebrafish considerations for metabolite screening
In general, the studies that evaluated the production of xenobiotic
metabolites in zebrafish observed several different phase I and II me-
tabolites, demonstrating the utility of the model in the prediction of the
metabolism of several substances. Therefore, there is further evidence
that zebrafish express genes and proteins with activities responsible for
xenobiotic metabolism with both oxidative and conjugative metabolism
similar to mammalian systems. Regarding the zebrafish CYP orthologs
in humans, it is important to highlight that three cases were observed:
no orthology with production of metabolites (fluoxetine (Smith et al.,
2010), CYP2D6; and ibuprofen (Jones et al., 2012), CYP2C); presence of
orthology without production of metabolites (midazolam (Saad et al.,
2017), CYP3A) and presence of orthology with production of metabo-
lites (sibutramine and stanozolol (Anselmo et al., 2017), CYP2B6 and
CYP3A4, respectively), which is the ideal case and the most often
found. Even in cases where human orthologs of CYP are present in
zebrafish, divergences may occur in relation to the major metabolites
generated or the production ratio of these metabolites in comparison
with humans (Alderton et al., 2010; Chng et al., 2012; Jones et al.,
2012). So, further studies are needed to understand to what extent the
zebrafish is able to reproduce the metabolism observed in humans.
Nevertheless, it is important to highlight that even the mouse, a classic
model for prediction of toxicity and evaluation of metabolites, presents
some differences in relation to human metabolism (Lootens et al.,
2009). Therefore, this issue does not prevent zebrafish from being
considered a model with great potential for studying the metabolism of
xenobiotics.
Zebrafish larvae represent a very interesting alternative model to
animal experimentation, although it is important to remember that
early life stages (3 to 5 dpf) are still undergoing embryogenic processes
and that their metabolic status is different from that of the young or
adult zebrafish (Pardal et al., 2014). In addition, when using zebrafish
embryos, there is a need for them to take up substances through the
chorion and other membranes (Vallverdú-Queralt et al., 2015), which
can generate results compromised by the differentiated absorption at
this life stage. Moreover, it has already been observed that the activity
of most CYPs involved in xenobiotic metabolism is increased dramati-
cally after hatching approximately 3 dpf (Saad et al., 2016) and that the
liver is fully formed at approximately 5 dpf (Kimmel et al., 1995). Al-
though several studies have observed the production of metabolites in
the embryonic and larval stages of zebrafish (Table 2), Saad et al.
(2017) observed no in vitro biotransformation during zebrafish em-
bryonic development until 96 hpf. Therefore, the age of the zebrafish is
a controversial issue and should be taken into account during experi-
mental design and when comparing the results with other mammals.
In zebrafish in vivo screening models, the main route of exposure is
dermal until approximately 5 dpf when the mouth is opened and both
dermal and enteral routes exist from 5 to 14 dpf while the embryo is
free-feeding (Garcia et al., 2016; Sipes et al., 2011). Usually, in con-
ventional in vivo experiments, a known and measurable amount of drug
is administered to the animal. However, in the zebrafish in vivo models,
the uptake from the water into the zebrafish is non-uniform and un-
predictable, which can make it difficult to interpret screening results
and to make comparisons between compounds (Fleming and Alderton,
2013).
Another important issue to be discussed in relation to zebrafish
screening is the use of organic solvents to increase the solubility of the
substances tested and their impact on metabolism enzymes. David et al.
(2012) observed that metabolism of ethoxyresorufin in zebrafish larvae
was significantly reduced by dimethylsulfoxide (DMSO) and methanol
C. de Souza Anselmo et al.
(0.1% and 0.05% v/v, respectively) after 24 h of exposure. The de-
creases in activity of ethoxyresorufin were accompanied by decreased
expression of various genes coding for drug metabolizing enzymes
corresponding to CYP1, CYP2, CYP3 and UGT family enzymes (David
et al., 2012). Thus, relatively low concentrations of organic solvents
may impact the biotransformation of certain xenobiotics in zebrafish
larvae, and this deserves consideration when evaluating substances for
the study of metabolism and toxicity in this species. This fact, together
with the age of the fish, the amount of substance used in the experiment
and the unpredictable uptake from the exposure water, may be a de-
terminant in the metabolic profile that will be found and should be
taken into account when designing and interpreting results from me-
tabolite screening.
7. Conclusion
In the last 30 years, the zebrafish has been used to predict the me-
tabolism of xenobiotics, with different focuses. In the early years, stu-
dies focused on environmental toxicology, and in the possible impact of
the generated metabolites on increasing the toxicity of the con-
taminants. Currently, several studies have evaluated the metabolism of
drugs aiming to establish zebrafish as a model for toxicological
screening, either for the development of new drugs or for analytical
targets in anti-doping control. These studies have shown that the zeb-
rafish enzymes responsible for metabolism are analogous with those in
humans, performing several phase I and II reactions. Some factors need
to be considered for the use of the model, since they can directly in-
fluence the results, such as: fish age, impact of the use of organic sol-
vents and time of exposure. In addition, a limitation of the in vivo model
of zebrafish is that it is not possible to control the amount of substance
administered by each fish and can generate non-reproducible results.
As with any non-human model, zebrafish model presents similarities
and differences in relation to the profile of metabolites generated
compared to that observed in humans. All the facts presented indicate
that zebrafish is a valuable potential model for the evaluation of xe-
nobiotic metabolism, but further studies are still needed to assess the
degree to which it can be compared to human metabolism.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
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