Multimarker Responses of Zebrafish to the Effect of Ibuprofen and Gemfibrozil in

Environmentally Relevant Concentrations

Halina Falfushynska*, Nadiia Kasianchuk, Oksana Horyn, Oksana Bodnar

1
Ternopil Volodymyr Hnatiuk National Pedagogical University, 2, M. Kryvonosa Str., Ternopil,
Ukraine, 46027; E-Mails: falfushynska@tnpu.edu.ua (H.F.), kasianchuk.nadiia@lll.kpi.ua (N.K.),
horynoi@tnpu.edu.ua (O.H.), bodnar@tnpu.edu.ua (O.B.)

* Author to whom correspondence should be addressed; E-Mail: falfushynska@tnpu.edu.ua (H.F.);

Tel.: +380673506531.

Abstract:
Pharmaceutical pollution of water bodies belongs to the top-notch environmental health risk all over the
world. The aim of the present study was to investigate the effects of common pharmaceuticals namely
ibuprofen and gemfibrozil on Danio rerio in environmentally relevant concentrations. Gemfibrozil caused
a decrease in glutathione and glutathione transferase and an increase in catalase but had no effect on lipid
peroxidation and protein carbonylation in zebrafish liver. Ibuprofen altered antioxidant defense system,
promoted protein carbonylation in zebrafish liver, and increased vitellogenin-like protein in blood.
Ibuprofen and particularly gemfibrozil induced lysosomes biogenesis. Lactate dehydrogenase in blood was
also found to be higher in the both studied groups. Studied pharmaceuticals didn’t affect complex II of the
electron respiratory chain in zebrafish liver. Ibuprofen affects zebrafish health status more profound than
gemfibrozil. Our results showed that pharmaceuticals even in low environmentally realistic concentrations
induced profound changes in the stress-responsive systems of zebrafish.
Keywords: Danio rerio; gemfibrozil; ibuprofen; oxidative stress; endocrine disruption; protein carbonyls.
Introduction

Global consumption of pharmaceuticals is increasing rapidly as a consequence of the modern

lifestyle and Covid pandemic challenges. In particular, for the period of 2001-2020
pharmaceutical market revenue has increased from $ 390.2 billion to $ 1,265.1 billion US dollars
(Statista.com). Moreover, there is data that over 2021 medication consumption has increased by
another 2 % compared to 2020, and the market is expected to reach 1700.9 billion dollars in 2025
(Globenewswire.com). In recent decades pharmaceuticals have been determined as the emergent
pollutants because their increasing background concentrations in water bodies and potentials to
adversely affect aquatic life (Ebele et al. 2017).
Ibuprofen (IBU) is known to be one of the most widely used medications in the world
(Marketresearch.biz). Being conveyed in water bodies with runoff and municipal effluents, IBU
was found in water bodies within the concentration range 616 - 48,720 ngL-1 (Paíga P et al. 2013).
The permanent presence of IBU as the non-steroidal anti-inflammatory drug (NSAID) may pose a
potential threat to water animals and public health. IBU can affect water animals via triggering
oxidative stress, nephro- and hepatotoxicity (Mathias et al., 2018), impairment in cell energy
production, endocrine and immune disorders as well as attenuating adaptive capacity and stress-
responsive activity within a wide range of organisms (Constantine et al., 2020).
Following IBU and other NSAID, lipid lowering drugs (e. g. atorvastatin, clofibrate,
gemfibrozil) belong to the most commonly prescribed medications (Natesan and Kim, 2021).
Regarding gemfibrozil, the analysis of surface, wastewater and groundwater showed that its
content ranges from 0.08 to 63.8 μg∙L-1 (Fang, 2012). It is expected that such therapeutic
concentrations may affect key molecular and cellular events in non-target organisms, but the
appropriate information is limited. Also, responses of freshwater vertebrates to common
pharmaceuticals remain out of focus. To address these knowledge gaps, the aim of our research
was to study the effect of the common pharmaceuticals in the environmentally relevant
concentrations on oxidative stress, electron transport chain, and cytotoxicity in fish. Being
versatile and silent sentinel, zebrafish was chosen as the model organism. Studied biomarkers
have been selected based on general applicable criteria, our previous results, and probability for
biomarkers to be embedded conveniently in praxis and business (Falfushynska et al. 2017).

Experimental procedure

The study of the effect of common pharmaceuticals was performed on mature zebrafish Danio
rerio as the model. Animals were acclimated to laboratory conditions for 7 days. For the
experiment, the fish were placed in 10 liters volume tanks with the number of fish at the rate of 1
individual per 2 liters of water. Physicochemical conditions were maintained according to
generally accepted schemes of toxicological experiment.
Three groups were settled, one control and two experimental. In the water in which the animals
of the experimental groups were kept, the studied pharmaceuticals were added: 1) gemfibrozil
(GF) at the concentration of 1 μg∙L-1 and 2) ibuprofen (IBU) at the concentration of 25 μg∙L-1
which corresponded to the average level of these substances in wastewater (Dey et al., 2019). The
incubation lasted 14 days. Animal experiments were performed in accordance with the European
Convention for the Protection of Vertebrate Animals Used for Experimental and Scientific
Purposes (Strasbourg, 1986), resolutions of the First National Congress on Bioethics (Kyiv, 2000)
and decisions of the Ethics Commission of Ternopil V. Hnatiuk National Pedagogical University
(Protocol № 2, 2020). Animals were killed under ether narcosis. Tissue selection and processing
were performed at +4 oC. All reagents were from Sigma and Carl Roth and qualified as chemically
pure.
Oxidative stress markers
The content of reduced glutathione in liver tissue homogenates was determined using Elman's
reagent (Jollow et al., 1974) at a wavelength of 412 nm. Glutathione transferase activity (GST,
Е.С. 2.5.1.18) was determined in the supernatant fraction of liver tissue homogenate (1:10 w:v) by
the formation of products of 1-chloro-2,4-dinitrobenzene with glutathione at 340 nm (Habig et al.
1974). Catalase activity (CAT, E.C. 1.11.1.6) was determined using the protocol of (Aebi, 1974)
in the supernatant fraction of liver tissue homogenate (1:10 w:v) and presented in μmol per minute
per g of fresh weight.
The level of protein carbonyls (PC) was evaluated using 2,4-dinitrophenylhydrazine in liver
tissue homogenate which had been previously treated with trichloroacetic acid at 370 nm (Reznick
and Packer, 1994).
Cytotoxicity markers
To determine the lysosomal membranes stability, the neutral red retention assay was applied.
Crude lysosomal pellets were obtained from zebrafish hepatocytes using the protocol [22]. The
neutral red accumulated in the lysosomes was extracted with ethanol and acetic acid (1:1 v/v) and
measured at a wavelength of 550 nm (Falfushynska et al. 2019).
The activity of lactate dehydrogenase (LDH, EC 1.1.1.27) was measured in the blood serum
samples according to the protocol of Bergmeyer and Bernt (Bergmeyer and Bernt, 1974).
The concentration of vitellogenin-like proteins, the proved marker of endocrine disorders, was
determined as the concentration of alkali-labile phosphates in zebrafish blood by the method
(Nagler et al. 1987). The concentration of phosphates was determined by the phosphomolybdenum
colorimetric method.
Complex II of electron transport chain
Succinate dehydrogenase activity (SDH, E.C. 1.3.99.1) was determined in liver tissue
homogenate by a ferricyanide method based on the oxidation of succinate to potassium fumarate
hexacyanoferrate by succinate dehydrogenase (Dua and Gill, 2004). Enzyme activity was
expressed in nmol of succinate per g of fresh weight.
Statistical data processing
The normality and homogeneity of variances were checked using Kolmogorov-Smirnov and
Levine tests, respectively. When data deviated from a normal distribution or if the variances were
heterogeneous, Box-Cox transformation was used. The effects of the pharmaceuticals on the
biochemical and physiological parameters were tested using one-way ANOVA and Tukey's honest
Significant Difference (HSD) test. The principal component analysis (PCA) was used to select the
most relevant variance of the studied biological parameters based on the raw data. The index of
oxidative stress (IOS) was calculated using standardized data according the formula:
IOS = ((GSH+CAT+GST)) ⁄ ((TBARS+PC)). The IOS was expressed in percent deviation from
control value. The Index of biomarkers response (IBR) was calculated as the product of
multiplication of all studied biological traits after their standardization compared to the
correspondent control as the theoretical expected value.
All statistical operations were performed with Statistica v. 12.0 and Excel for Windows-2019.
Differences were considered significant if the probability of Type I error was <0.05. All data are
reported as the mean ± the standard error (S.E).
Results
Our results have shown that the IBU and GF didn’t affect the total antioxidant capacity, but
significantly induced CAT, the most responsive enzyme to H2O2 (Fig.1A,B). Also, studied
pharmaceuticals led to a decrease in the content of reduced glutathione in the zebrafish liver,
particularly after the effect of gemfibrozil (by 62.4 %). The changes in GST activity were drug-
dependent: IBU induced GST activity and GF oppressed it (Fig. 1 C,D).

A TAC B CAT
500 a 600
a
a b
400 molmin-1g-1
b
M Trolox

300 400

200
a
200
100

0 0
C IBU GF C IBU GF

C GSH
D
GST
5 80
nmol·min-1 ·mg-1 proteins

a b
4
60 a
b
mol g-1

3
40 c
2
c

20
1

0 0
C IBU GF C IBU GF

Fig. 1. The effect of common pharmaceuticals ibuprofen and gemfibrozil on oxidative stress parameters
in zebrafish liver. A, total antioxidant capacity, B, catalase, C, glutathione, D, glutathione S-transferase.
Columns that do not share the same letters represent significantly different values (P < 0.05). The means
are presented as M±SE, n=6.

IBU led to the five-fold increase in the PC levels but didn’t affect lipid peroxidation (Fig.2).
Markers of oxidative injury were not sensitive to gemfibrozil exposure (Fig. 2).
A TBARS B PC
150 a 4
a b
a 3
100
nmol·g-1

mol g-1

50 a
1 a

0 0
C IBU GF C IBU GF

Fig. 2. The effect of ibuprofen and gemfibrozil on oxidative injury indices in zebrafish liver. A, lipid
peroxidation TBARS, B, protein carbonyls. Columns that do not share the same letters represent
significantly different values (P < 0.05). The means are presented as M±SE, n=6.
 The NRR as the marker of the lysosomal membrane stability was observed to increase in
response to IBU and especially to GF (Fig. 3A). LDH increased in the blood of zebrafish
regardless of the acting pharmaceutical (Fig. 3B). Exposure to IBU led to elevated levels of Vtg-
LP in males as the proved indicator of endocrine disorders when GF didn’t affect it. No SDH
activation was observed in the experimental groups (Fig. 3D).
A NRR B LDH
10 c 15
b
b
8

molmin-1ml-1
10
D550mg-1

6
b a

4 a
5
2

0 0
C IBU GF C IBU GF

C Vtg-LP D SDH
300 b 60

nmol·min-1 ·mg-1 proteins

a
a a
a
a
g·g-1 FW

200 40

100 20

0 0
C IBU GF C IBU GF

Fig. 3. The effect of ibuprofen and gemfibrozil on markers of cytotoxicity in zebrafish liver and blood. A,
neutral red retention, B, lactate dehydrogenase, C, vitellogenin-like proteins, D, succinate
dehydrogenase. Columns that do not share the same letters represent significantly different values (P <
0.05). The means are presented as M±SE, n=6.

The calculation of the integrative index of oxidative stress and index of general biomarkers
response declares that IBU group which characterized by deep oxidative injury and general stress
(IBR (IBU) > IBR (GF)) was more affected when compared with GF group (Fig. 4).

A B C
Fig. 4.
The application of PCA to integrate the results of biochemical and physiological indices of
zebrafish treated with IBU and GF reflects the peculiarities of the response and the most sensitive
biomarkers specifically acted up to each of studied pharmaceuticals. The first two PCs account for
57 % of the total variation in the dataset. All studied groups mostly locate separately and GF takes
place the outland position regarding intact animals (Fig. 4B). Control group associated with
highest level of GSH and the lowest level of TBARS. The IBU group was characterized by step up
of endocrine disruption and carbonyl stress indices when the GF group was characterized by
cytotoxicity (lysosomal-related) and oxidative stress (peroxisomal-related) indices (Fig. 4 B,C).
The variability of biomarkers’ value was stronger in the IBU group than in the GF group. The
separate localization of zebrafish treated with IBU and GF in the plane of plot area of factor
analysis confirms the realization of different strategies of adaptation to NSAID and fibrates.
 Discussion
The toxicity of a number of environmental pollutants, including pharmaceuticals, is primarily
due to increased levels of intracellular reactive oxygen species and depletion of antioxidant
defense with the subsequent initiation of oxidative damage to biomolecules and loss of their
functions. The results of the present study showed that the effect of environmentally relevant
concentrations of fibrates (gemfibrozil) and especially nonsteroidal anti-inflammatory medications
(ibuprofen) initiate an imbalance of antioxidant defense system in the liver of zebrafish which
causes oxidative damage to proteins (protein carbonylation), and thus manifestations of
cytotoxicity, endocrine disorders which may subsequently reduce the animals' ability to adapt to
changing environmental conditions. In general, ibuprofen causes a more significant damaging
effect compared to gemfibrozil.
Ibuprofen is a non-steroidal anti-inflammatory pharmaceutical which ranks 29 in terms of the
worldwide number of prescriptions according to ClinCalc DrugStats (Clincalc.com). Steady
growth in its usage by people leads to a constant increase in IBU and its metabolites in the aquatic
environment. As for chemical nature, ibuprofen is a hydrophobic compound of crystalline
structure with low volatility and the ability to adsorb and interact with organic matter as well as a
long half-life in natural waters (up to 32 days) (Tixier et al. 2003). All of that contributes to its
bioaccumulation and bioconcentration by aquatic organisms throughout long time period
(Carlsson et al., 2006).
It is believed that NSAID can cause oxidative stress, cytotoxicity, and metabolic disorders in a
wide range of organisms. They are generally reported to activate the antioxidant defense system
and its key enzymes (Mathias et al. 2018; Islas-Flores et al. 2013). In particular, it has been shown
that among freshwater fish Rhamdia quelen ibuprofen in ecologically relevant concentrations (0.1
μg∙L-1, 1 μg∙L-1 and 10 μg∙L-1) induced antioxidant protection but led to a decrease in carbonic
anhydrase activity in the kidney and the number of blood leukocytes which generally indicates its
potential nephrotoxic and immunosuppressive effect (Mathias et al. 2018). The disruptive effect of
propionic acid derivate naproxen at a concentration of 1 μg∙L-1 on the expression of mRNA of
antioxidant enzymes and the UCP-2 gene (the corresponding protein that regulates transportation
processes in mitochondria) was detected in zebrafish as a model organism. Ibuprofen at a
concentration of 0.1-1.1 μg∙L-1 caused a significant increase in catalase activity in Danio rerio
(Sanchez-Aceves et al. 2021). Short-term exposure to ibuprofen at sublethal concentrations up to
10 mg∙L-1 caused in Cyprinus carpio a significant increase in catalase and glutathione peroxidase
activity (Islas-Flores et al. 2014). Our results concerning the imbalance of the antioxidant defense
system in zebrafish liver after GF and particularly IBU exposure are aligned with those in the
literature. It is highly likely that a partial compensatory reaction in catalase which increased by 2-3
times allowed total antioxidant capacity to be maintained at a stable level despite depletion of thiol
cellular pool and preventing oxidative damage to lipids and proteins as well (in the case of the GF
group).
It has been found that during the biotransformation of NSAID cytochrome P450 forms an
oxygenated intermediate product, oxycytochrome Р450 [P450 (Fe3+) O2*]. This complex is
thermodynamically unstable and capable of releasing superoxide anion and hydrogen peroxide
(Doi et al. 2002). Therefore, the activation of GST and catalase which play important roles in the
detoxification/decomposition of endogenously-derived ROS in the liver of zebrafish of the IBU
group can be considered an adequate adaptation response to pharmaceutical micropollutant
contamination.
Contrariwise to ibuprofen, gemfibrozil exposure significantly reduced GST activity in the liver
of zebrafish thereby highlighted different mechanisms of biotransformation/ detoxification
processes for fibrates and NSAID. Similar conclusions were previously made while using
mammals. In particular, gemfibrozil exposure (1000 and 16,000 p.p.m daily) up to 90 days
decreased glutathione S-transferase, glutathione reductase, and selenium-dependent glutathione
peroxidase activities in rats (O’Brien et al. 2001). Highly likely the amount of intracellular
electrophilic compounds was too low to modulate a set of cellular signaling processes and activate
glutathione-related enzymes.
Lysosomes play a crucial role in degradation of macromolecules and digestion pathogens using
more than 50 acid hydrolases accumulated internally (Papadopoulos and Meyer, 2017). The
lysosomal membrane protects cell compartments and organelles from leakage of degradative
enzymes. However, impairment of lysosomal membrane and disturbance of its functionality as a
result of xenobiotic effect have been linked to cytotoxicity and oxidative damage in mollusks and
fish, in some cases leading to cell death (Nazar et al., 2008; Zhang and Peterson, 2020). On the
other hand, it is proved that some substances are able to initiate lysosomal biogenesis and CLEAR
regulatory gene network with the master regulator TFEB is the most feasible in mammals (Skoupa
et al. 2020). In the present study, we have shown that both IBU and GF stimulated neutral red
uptake and obviously, induced lysosomes biogenesis. A recent studies suggested that
physiological doses of NSAID and particularly GF cause TFEB overexpression in mammalian
models (Chandra et al. 2018) that supports our speculation and points on the necessity of further
studies in the field.
It has found out that long-term (120 d) exposure of Oryzias latipes in environmentally relevant
concentration of ibuprofen (0.01 ∙g∙L-1-1000 ∙g∙L-1) led to prominent disturbances in fish
reproductive system (Han et al. 2010). In particular, vitellogenin concentration was noted to
increase and eggs hatching was noted to decrease (Han et al. 2010). This finding was also
supported by Morthorst et al. (2013) who emphasized on the prominent changes in reproduction of
ibuprofen-treated Danio rerio. Consistent with the literature, our research found that ibuprofen,
but not gemfibrozil induced vitellogenesis in male even in low concentration. It might be related
to the ability of ibuprofen as NSAID to induce estrogen receptors, G protein-coupled estrogen
receptor and to simultaneously inhibit testosterone and prostaglandin synthesis which play key
roles in fish reproduction (Banihani, 2019).
Succinate dehydrogenase (SDH), the complex II of the electron respiratory chain and its
response to common pharmaceuticals in non-targets have not been paid much attention to. It is
worth mentioning that some pharmaceuticals, e.g. fluoroquinolones, chloramphenicol succinate,
diazoxide, and anthracycline drugs, are complex II inhibitors for mammals (Scatena et al. 2008).
Also, some pharmaceuticals including gemfibrozil and ibuprofen in mammalian models both in
vivo and in vitro are able to inhibit membrane transporters, ADP/O (complex I) in liver, and state
3 in colon but induce changes in mitochondrial permeability and tissue-dependent mitochondrial
respiration as well as cause depolarization of membrane potential (Sandoval-Acuna et al. 2012;
Herminghaus et al. 2019). It is highly likely that the adverse effect of pharmaceuticals on electron
transport chain and mitochondria respiration depend on numerous factors including drug
concentration, tissue and species specificity. In our study, no changes were found in the succinate
dehydrogenase activity. Obviously, studied environmentally relevant concentrations of ibuprofen
and gemfibrozil are below threshold value for triggering changes in complex II of the electron
respiratory chain in zebrafish.
Conclusions
All in all, we have shown new findings about effects of commonly used and clinically
important drugs that belong to NSAID (ibuprofen) and lipid-lowering (gemfibrozil) classes, on
key parameters of oxidative stress, cytotoxicity, and mitochondrial respiration. This study’s results
reveal a rather negative effect of both ibuprofen and gemfibrozil on antioxidants, however, only
ibuprofen stimulated protein carbonylation. Furthermore, ibuprofen is claimed to be the endocrine
disruptor and cytotoxic agent to zebrafish. Meanwhile no strong variation in mitochondria
metabolic process was observed after the action of both studied pharmaceuticals. Ibuprofen and
particularly gemfibrozil had the positive effects on lysosomal biogenesis. All mentioned issues
raise concerns about the adverse effects of pharmaceutical effluents on the health status of fish.
Acknowledgements
 The work was in part supported by the Ministry of Education and Science of Ukraine (#MV-2),
National Research Foundation of Ukraine (#2020.02/0270), Alexander von Humboldt Foundation
and DAAD to HF. Authors would like to thank Dr. Yuliia Kostiuk for English proofreading.
Conflict of Interest
The authors declare no conflict of interest.

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