https://doi.org/10.52973/rcfcv-e34505
Received: 13/08/2024 Accepted: 05/10/2024 Published: 24/11/2024
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Revista Científica, FCV-LUZ / Vol. XXXIV, rcfcv-e34505
ABSTRACT
Non–steroidal anti–inflammatory drugs (NSAIDs) are a widely
prescribed medication class that is used in the treatment of
numerous conditions worldwide. Of these drugs, naproxen is the
most commonly used NSAID. Following administration, non–steroidal
anti–inammatory drugs (NSAIDs) such as naproxen are eliminated
from the body in either their original chemical form or as metabolites,
ultimately entering the aquatic environment. The current study sought
to show the impacts of naproxen on the oxidant/antioxidant status
of Gammarus pulex, an aquatic invertebrate (Amphipoda). Gammarus
pulex were exposed to sublethal concentrations of naproxen (3.44,
6.87 and 13.75 mg·L
-1
) for 96 hours (h). Whole body tissue samples were
collected after 24, 48 and 96 h of exposure and analysed to determine
the oxidant/antioxidant status by quantifying malondialdehyde (MDA)
and total glutathione levels (GSH), and superoxide dismutase (SOD),
and catalase (CAT) activities of the G. pulex. The level of MDA exhibited
a remarkable increase, while the endogenous GSH level showed a
signicant depletion in tested whole body tissues in a time–dependent
manner after naproxen treatment of G. pulex. In G. pulex exposed to
the highest dose of naproxen; decreases in GSH activity, SOD and CAT
activities were observed.The SOD activity did not show a discernible
rise in statistics after 24 and 48 h of exposure, however, a difference
was observed after 96 h compared to the control group (P<0.05).
The ndings of this study demonstrated the capacity of naproxen
to initiate oxidative stress and elevate MDA levels in G. pulex, even
at remarkably low concentrations. This study emphasizes that it is
essential to develop effective methodologies to impede naproxen
entry into the aquatic environment.
Key words: Gammarus pulex, naproxen, oxidative stress, antioxidant
status
RESUMEN
Los antiinamatorios no esteroideos (AINEs) son medicamentos
ampliamente prescritos en todo el mundo y pertenecen a una clase
terapéutica destacada. De estos fármacos, el naproxeno es un AINE
de uso común. Después de la administración, los AINEs como el
naproxeno se eliminan del cuerpo ya sea en su forma química original
o como metabolitos, y nalmente pasan al medio acuático. El presente
estudio buscó demostrar los impactos del naproxeno en el estado
oxidante/antioxidante de Gammarus pulex, un invertebrado acuático
(anfípodos). Los G. pulex estuvieron expuestos a concentraciones
subletales de naproxeno (3,44, 6,87 y 13,75 mg·L
-1
) durante 96 horas(h).
Se recogieron muestras de tejido corporal entero después de 24,
48 y 96 h de exposición y se analizaron para determinar el estado
oxidante/antioxidante cuanticando los niveles de malondialdehído
(MDA) y glutatión total (GSH), y las actividades de superóxido
dismutasa (SOD) y catalasa (CAT) de G.pulex. El nivel de MDA mostró
un aumento notable, mientras que el nivel de GSH endógeno
mostró una disminución significativa en los tejidos corporales
completos analizados de manera dependiente del tiempo después
del tratamiento con naproxeno de G. pulex. En G. pulex expuesta a
la dosis más alta de naproxeno; Se observaron disminuciones en
la actividad de GSH, SOD y CAT. La actividad de SOD no mostró un
aumento perceptible en las estadísticas después de 24 y 48 h de
exposición, sin embargo, se observó una diferencia después de 96
h en comparación con el grupo de control (P<0,05). Los hallazgos de
este estudio demostraron la capacidad del naproxeno para iniciar el
estrés oxidativo y elevar los niveles de MDA en G. pulex, incluso en
concentraciones notablemente bajas. Este estudio enfatiza que es
esencial desarrollar metodologías efectivas para impedir la entrada
de naproxeno en el ambiente acuático.
Palabras clave: Gammarus pulex, naproxeno, estrés oxidativo,
estado antioxidante
Effect of naproxen on oxidative stress biomarkers in Gammarus pulex
Efecto del naproxeno sobre los biomarcadores de estrés oxidativo en Gammarus pulex
Engin Seker
Tunceli Munzur University, Pertek Sakine Genç Vocational School Laborant Veterinary Health Department, Pertek, Tunceli, Türkiye.
*Corresponding author: enginseker@munzur.edu.tr
Effects of Naproxen, in Gammarus pulex / Seker ____________________________________________________________________________________
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INTRODUCTION
The Gammarus family, Gammaridae: Amphipoda, drew the attention
of experts in taxonomy and ecology but in recent years, professionals
in molecular biology, genetics, agriculture, and notably, toxicology
also have been interested. Gammarus species have rapidly gained
use in ecotoxicology due to their high abundance [1], distinct sexual
dimorphism, ease of collection, and tolerance to a wide range of toxic
substances [2, 3]. While the primary focus of research within the
domain of aquatic toxicology currently centers on identifying sensitive
long–term markers for a variety of test species including gammarids,
the role in oxidative stress of acute toxicity exposure continues its
popularity in elucidating the effects of toxins [1, 4, 5]. The assessment
of chemical screening and the compliance of wastewater with
discharge regulations is primarily based on physiological toxicity
data. This approach plays a critical role in determining the potential
effects of wastewater on human health and the environment.
The common use of non–steroidal anti–inflammatory drugs,
including naproxen and other chemicals has been documented in
various regions all around the world [6]. Naproxen, the propionic
acid derivative, is a non–steroidal anti–inammatory drug (NSAID),
widely used for the treatment of primary dysmenorrhoea, rheumatoid
arthritis, osteoarthritis, ankylosing, tendinitis, bursitis, acute gout
and juvenile arthritis in human [7, 8]. These applications have
disrupted the balance within aquatic ecosystems, containing rivers,
streams, lakes, estuaries, and coastal and deep oceans. Although
it is known the pollution of NSAIDs, their potential toxic impacts
on aquatic organisms have become the subject of research and
experiment recently. Whereas the mechanisms of action of these
substances are well–established in humans and other vertebrates
[9], they remain largely unknown in aquatic invertebrates, that are
continuously exposed to these compounds. As a result, there is
considerable uncertainty regarding the environmental risks related
to their presence [10]. The primary way NSAIDs work is by inhibiting
the enzyme cyclooxygenase (COX). This enzyme is essential for
converting arachidonic acid into thromboxanes, prostaglandins,
and prostacyclins. The therapeutic benets of NSAIDs result from
the reduced production of these eicosanoids [9]. Studies on the
effects of naproxen on aquatic organisms have primarily concentrated
on planktonic species such as water fleas (Daphnia magna and
Moina macrocopa) and sh (Oryzias latipes and Danio rerio) [11, 12,
13]. Exposure to naproxen has been shown to affect the genetic
material, inammatory processes, and metabolic processes of aquatic
organisms [13]. Additionally, it has been reported that naproxen poses
an ecological risk to Daphnia manga [14].
Nonsteroidal anti–inammatory drugs (NSAIDs) which encompass
analgesic compounds constitute a highly notable category of
pharmaceuticals globally with an approximate annual production
of multiple kilotons [15]. Until now, the studies have indicated
the presence of NSAIDs in the aquatic ecosystem, specically in
wastewater and surface water [16]. Naproxen is a kind of NSAID
drugs. Ever since its introduction to the market in 1976, naproxen
has consistently remained highly popular [17]. Induction of oxidative
stress and the impact of detoxification mechanisms by various
NSAIDs in aquatic organisms have also been evaluated through the
measurement of activities of enzymes taking part in a variety of
biochemical pathways [18, 19, 20, 21, 22, 23].
Oxidative stress happens as a consequence of an imbalance
between the generation of reactive oxygen species (ROS) and the
detoxication of these reactive compounds within an organism. ROS
are naturally produced as byproducts of various essential biochemical
reactions that play crucial roles in energy transfer, cellular defense,
and cell signaling [24]. ROS have the potential to harm vital biological
macromolecules and induce peroxidation of membrane lipids in
biological systems, leading to the disruption of membrane structure
and function. The exposure to environmental stressors can trigger
a disproportionate increase in ROS levels, resulting in cellular and
tissue damage [25]. Maintaining a balance between oxidants and
antioxidants is a crucial for cellular homeostasis.
Gammarus pulex is a tiny amphipod crustacean that inhabits
freshwater environments throughout Europe. This species is well–
suited for biomonitoring studies due to its notable role in freshwater
ecosystems. G. pulex serves as a vital food source for various
invertebrates, sh, and birds, making it an important link in the food
chain [26]. This organism has also been widely utilized in monitoring
contaminants, including toxicity tests for a range of pollutants like
metals, PAHs/PCBs, and pharmaceutical substances. This highlights
its crucial role in assessing environmental risks [ 27, 28, 29, 30].
The current investigation aims to contribute to the assessment
of aquatic toxicity tests employed for evaluating the potential
toxicological inuence of naproxen on aquatic organisms. Regarding
that all effects are not necessarily detrimental, the primary objective
of these tests is to identify chemicals that may have adverse impacts.
The bioassay tests will yield a database that can be utilized to evaluate
the risks associated with a given scenario.
MATERIALS AND METHODS
Living material
A model invertebrate species, G. pulex (L., 1758), was used for the
exposure experiment. G. pulex samples were collected from the
Munzur River (39.156820 N, 39.499640 E) in Tunceli province, from
the slower owing and relatively deeper parts of the source, using
hand nets from under leaves and stones. G. pulex samples, which
were quickly brought alive to the laboratory in air–reinforced plastic
bottles, were placed in two 80×40×25 cm aerated stock aquariums
prepared similar to their natural environment. Rested water taken
from the natural environments where the samples were obtained was
placed in the aquariums, and 50% of the water in the aquariums was
replaced with rested water once a week.
Experimental setup and naproxen exposure
Before being used in experiments, G. pulex samples were fed with
rotten willow tree leaves collected from their natural habitat in a
room kept at a constant temperature of 18°C, in a 12:12 h light:dark
cycle, for 15 days [28]. During adaptation, the feeding and mobility
of the creatures were observed.
In the study, after 15 days of adaptation, G. pulex samples selected
from stock aquariums; Care was taken to ensure that they were male
individuals who had reached sexual maturity, completed their molting,
were in good health, and had an average weight of approximately
0.350–0.400 g.
Gammarus pulex samples were divided into four groups after
a two–week adaptation period to laboratory conditions. The
experimental setup included a control and three experimental
groups, each consisting of three replicates. 2 liter glass jars were
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used in the experiments. 1 liter of water was placed in these glass
jars and ventilated with air engines. 60 live G. pulex were used for all
concentration groups and replicates, including the control group.
Sublethal concentrations of naproxen were selected based on the
96–hour median lethal concentration (LC50) value previously reported
for G. pulex, which was found to be 110 mg·L
-1
by [31]. Naproxen was
dissolved in 0.9% NaCl [32]. G. pulex was exposed to sublethal
concentrations of naproxen at approximately 1/32, 1/16, and 1/8 of
the 96–hour LC50 of naproxen (3.44; 6.87 and 13.75 mg·L
-1
) for 24, 48,
and 96 h. Experimental aquariums were ventilated during the exposure
period and feeding was not done during the experiments. No deaths
in animals were observed during these exposures.
• (Control Group), the control group drug–free.
• (A Group), 1/8 ratio of the LC50 value of naproxen.
• (B Group), 1/16 ratio of the LC50 value of naproxen.
• (C Group), 1/32 ratio of the LC50 value of naproxen.
Test organisms were exposed to naproxen for 24, 48 h and 96 h. At
the end of these periods, 20 G. pulex from each experimental group
were selected by random sampling for biochemical analyses.
Preparation of G. pulex homogenates and Biochemical estimations
At the end of the test, G. pulex were anaesthetized in icecold chilled
water and whole bodies were isolated for biomarker analysis.
After rinsing with cold 0.09% NaCl solution and ltering to remove
the liquid, 5 g of 20 G. pulex samples were weighed and used to
prepare homogenate. Preparation of G. pulex homogenates involved
homogenizing the tissues using a Teflon–glass homogenizer in a
buffer solution containing 1.15% KCl, resulting in a 1:10 (w·v
-1
) whole
homogenate. Subsequently, the homogenates were centrifuged
(Nüve, NF800R, Turkey) at 18000 G for 30 min at 4°C. The resulting
supernatant was used for the analysis of malondialdehyde (MDA) and
reduced glutathione (GSH) concentrations, as well as the determination
of catalase (CAT), and superoxide dismutase (SOD) activities.
MDA level
MDA was measured in accordance with the method of Ohkawaetal.
[33]. The MDA level was assessed using the thiobarbituric acid
reactive substances (TBARS) method based on the reaction between
MDA and thiobarbituric acid. To start the procedure, 0.1 mL of the
extract was mixed with 0.15 mL of 0.8% thiobarbituric acid. Then,
0.04 mL of 8.1% sodium dodecyl sulphate and 0.15 mL of acetic acid
were added sequentially. The resulting mixture was brought to a total
volume of 0.5 mL with distilled water and then incubated (Nüve EN
400, Turkey) in a hot water bath at 95°C for 1 h. After cooling, 0.1 mL
of distilled water and 0.5 mL of a mixture of n–butanol and pyridine
(15:1 ratio, v·v
-1
) were added. The mixture was vortexed (Nüve VM
02,Turkey) vigorously and after centrifugation at 4000 r·min
−1
for
10min, the absorbance of the organic layer (top layer) was measured
at 532 nm using a spectrophotometer (Thermo Scientic, Multiskan
GO, USA), distilled water was used as a blank. The amount of MDA
was calculated using an extinction coecient of 1.56 mM
−1
·cm
−1
and
expressed in nmoles MDA·mg
-1
protein.
GSH activity
GSH level will be determined using the method reported by Ellman
[34]. Accordingly, Ellman’s reagent and disodium hydrogen phosphate
(Na
2
HPO
4
) will be added to the supernatants obtained from tissue
samples centrifuged with precipitant solution (metaphosphoric acid,
ethylenediaminetetetraacetic acid (EDTA), sodium chloride (NaCl) at
3000 rpm for 20 min. The mixture will be read in a spectrophotometer
against the blank at 412 nm and and glutathione content was calculated
from the standard curve.
CAT activity
The method described by Aebi [35] was employed to determine
catalase activity. For this, 0.2 mL of tissue samples were taken, and
1 mL of hydrogen peroxide was added. The difference in absorbance
at 0 and 30 s was measured at 240 nm. CAT activity was expressed
as units·mg
-1
protein·min
-1
.
SOD activity
The Beauchamp and Fridovich [36] standard superoxide dismutase
assay was performed using xanthine oxidase in combination with
nitroblue tetrazolium (NBT). In addition, catalase was added at the
rate of one unit per millilitre. The concentration of xanthine oxidase
was meticulously adjusted to ensure roughly equivalent initial rates
of blue formazan production under different experimental conditions.
SOD activity was expressed in U·mL
−1
of protein. U—one unit of SOD
activity—corresponds to the amount of enzyme causing 50% inhibition
of the rate of NBT reduction reaction.
Protein concentration, for calculating biomarker data were
measured according to Lowry et al. [37].
Statistical analysis
All data are given as mean and standard deviation. Analysis of
variance (ANOVA) was utilized to examine the obtained data and Tukey
test, and differences between groups were determined. Statistical
differences in all analyzes were determined at the 95% condence
interval. Statistical analyzes were performed using a commercial
statistical program package (SPSS 25.0). To reduce the impact of
random error on the results, the same protein preparation was used
in all experiments. This ensured that any deviation from perfect
linearity in the original preparation would be consistent across all
trials. Thus, differences between data sets would more accurately
reect their inherent effects (if any) on each test.
RESULTS AND DISCUSSION
The results of MDA are shown in TABLE I. The TABLE I showed that
there was a noticeable dose–dependent rise in the concentration
of lipid peroxidation products (MDA) when G. pulex were exposed
to naproxen. Notably, remarkable changes in the levels of lipid
peroxidation products were monitored in contrast to the control
group (P<0.05). Regarding the products of lipid peroxidation, the
highest dosage (13.75 mg·L
-1
, Group 4) showed a signicant elevation
after 24, 48 and 96 h of exposure compared to the control group
measurement (P<0.05).
TABLE II provided a clear evidence of a conspicuous change in
the activity of superoxide dismutase (SOD) in response to naproxen
exposure in G. pulex. The whole body tissue of G. pulex exhibited a
dose–dependent increase in SOD activity, and a remarkable change
compared to the control group was observed at the applied dose of
13.75 mg·L
-1
naproxen (P<0.05).It is vital that no deaths were recorded
among G. pulex throughout the entire treatment period at this dose.
TABLE I
Malondialdehyde (µmol·g
–1
protein, mean ± standard deviation) in
24–96 hours naproxen exposed and controls Gammarus pulex
24 hours 48 hours 96 hours
Control Group 0.48 ± 0.02 0.63 ± 0.07 0.54 ± 0.04
A Group 0.68 ± 0.07
a
0.70 ± 0.04
a
0.84 ± 0.02
a
B Group 0.61 ± 0.00
a
0.81 ± 0.06
a,b
0.93 ± 0.05
a,b,c
C Group 0.74 ± 0.03
a
0.95 ± 0.18
a,b
0.79 ± 0.02
a
Different letters express significative differences (P<0.05).
a, b, c
: A statistically
signicant dierence exists between the mean (± SE) values represented by dierent
letters on the same column. (
P<0.05).
a
: Control Group,
b
: A Group,
c
: B Group
TABLE II
Superoxide dismutase activity (U mL
–1
, mean ± standard deviation)
in 24–96 hours naproxen exposed and controls Gammarus pulex
24 hours 48 hours 96 hours
Control Group 6.28 ± 0.33 5.93 ± 0.44 6.82 ± 0.21
A Group 9.48 ± 0.28
a
9.21 ± 0.16
a
13.84 ± 0.66
a
B Group 8.41 ± 0.52
a
8.42 ± 0.21
a
13.21 ± 0.08
a
C Group 9.65 ± 0.23
a
7.97 ± 0.28
a
15.72 ± 1.76
a,b,c
Different letters express significative differences (P<0.05).
a, b, c
: A statistically
signicant dierence exists between the mean (±SE) values represented by dierent
letters on the same column. (
P<0.05).
a
: Control Group,
b
: A Group,
c
: B Group
TABLE IV
Superoxide dismutase activity (µmol·mg
–1
protein, mean ± standard
deviation) in 24–96 hours naproxen exposed and controls Gammarus pulex
24 hours 48 hours 96 hours
Control Group 4.83 ± 0.43 5.47 ± 0.62 5.23 ± 0.48
A Group 4.91 ± 0.27
a
3.98 ± 0.33
a
3.44 ± 0.43
a
B Group 4.67 ± 0.23
a
3.41 ± 0.17
a
3.10 ± 0.17
a
C Group 4.11 ± 0.74
a,b,c
3.68 ± 0.21
a
2.54 ± 0.28
a,c
Different letters express significative differences (P<0.05).
a, b, c
: A statistically
signicant dierence exists between the mean (±SE) values represented by dierent
letters on the same column. (
P<0.05).
a
: Control Group,
b
: A Group,
c
: B Group
TABLE III
Catalase levels (units·mg
-1
protein·min
-1
, mean ± standard deviation)
in 24–96 hours naproxen exposed and controls Gammarus pulex
24 hours 48 hours 96 hours
Control Group 4.90 ± 0.24 4.08 ± 0.32 4.64 ± 0.19
A Group 4.87 ± 0.33 4.31 ± 0.23 4.71 ± 0.12
B Group 5.67 ± 0.11
a,b
5.98 ± 0.25
a,b
6.43 ± 0.09
a,b
C Group 8.54 ± 0.34
a,b,c
8.37 ± 0.33
a,b,c
9.76 ± 0.73
a,b,c
Different letters express significative differences (P<0.05).
a, b, c
: A statistically
signicant dierence exists between the mean (±SE) values represented by dierent
letters on the same column. (
P<0.05).
a
: Control Group,
b
: A Group,
c
: B Group
Effects of Naproxen, in Gammarus pulex / Seker ____________________________________________________________________________________
4 of 8
TABLE III indicated the response of catalase, another important
antioxidant enzyme, upon the administration of naproxen to G. pulex.
The data show that there is a remarkable increase (P<0.05) in CAT
activity in the whole body tissue of G. pulex, especially at 48 and
96h after exposure compared to the control group. These changes
were observed at a dose of 6.87 and13.75 mg·L
-1
of naproxen, and it
is remarkable (P<0.05) to note that no deaths were reported among
G. pulex throughout the entire treatment duration at these doses.
observed in G. pulex at this dose throughout the treatment period.
It is the crucial point that notable alterations were not observed in
this parameter at lower doses (P>0.05).
The results presented of GSH in TABLE IV provide additional
evidence supporting the presence of oxidative stress resulting from
exposure to Naproxen. A remarkable decrease in GSH levels was
observed in the whole body tissue of G. pulex in parallel with the
dose increase in all groups compared to the control group. These
ndings strongly suggest the occurrence of oxidative stress in G. pulex
following naproxen exposure. It is noteworthy that no deaths were
Antioxidant enzymes play a vital role in protecting cells by
scavenging and inhibiting the formation and activity of harmful free
radicals. They are essential for preventing tissue damage caused by
chemical compounds. Industrial substances can affect the activities
of these enzymes, either increasing or decreasing them, while some
enzymes remain unaffected [38]. Numerous studies have investigated
the impact of NSAIDs including naproxen on antioxidant enzymes in
aquatic organisms [18, 19, 20, 21, 23, 39, 40, 41, 42]. Naproxen have
been shown to induce oxidative stress and cellular degenerative
processes by increasing of enzymes such as SOD and CAT activities
within tissues during sublethal exposure [20, 43]. Lipid peroxidation
is a process that necessitates the uptake of oxygen (O
2
) and leads
to the generation of superoxide radicals. Exposure of G. pulex to
carvedilol may induce the production of superoxide dismutase and
catalase, potentially resulting in the accumulation of O
2
and H
2
O
2
.
This accumulation of O
2
and H
2
O
2
could contribute to an elevation in
lipid peroxidation levels in G. pulex. The increased lipid peroxidation
observed in the experimental groups might be attributed to enhanced
oxygen uptake and a more pronounced induction of SOD and CAT
activity. Lipid peroxidation is recognized as one of the detrimental
effects caused by reactive oxygen species. During this process,
lipid peroxides readily decompose, leading to the release of highly
reactive carbonyl fragments, including malondialdehyde (MDA) and
4–hydroxynonenal (4–HNE). Tissue levels of MDA or the extent of lipid
peroxidation are commonly assessed through the measurement of
tissue thiobarbituric acid reactive substances (TBARS), which are
indicative of tissue damage caused by free radicals [44]. Several
studies [19, 20, 43, 45, 46] have reported increased MDA activity in
sh and aquatic invertebrates exposed to NSAIDs. The level of MDA
activity exhibited a remarkable increase (P<0.05) in tested whole
body tissue in a time–dependent manner after naproxen treatment
of G. pulex.
Superoxide dismutase (SOD) enzymes play a crucial role in
neutralizing superoxide radicals and can counteract lipid peroxidation
induced by oxidative stress. In the present study, naproxen
administration resulted in the increase in SOD activity. The changes
in SOD activity observed in tissues of aquatic animals after naproxen
and other NSAIDs administration align with the ndings of various
other researchers [23, 47, 48, 49]. Lucero et al. [21] investigated the
_____________________________________________________________________________Revista Cientifica, FCV-LUZ / Vol. XXXIV, rcfcv-e34505
5 of 8
SOD activity in the Hyalella azteca using a naproxen concentration
of 76.6 and 339.2 mg·kg
-1
and reported a increase in SOD content
following naproxen administration in the Daphnia manga [20]. The
present studies’ results are consistent with the ndings of the above
mentioned authors. However, several studies [48, 50] have reported
an decrease in SOD activity in D. magna and Rhamdia quelen when
exposed to NSAIDs such as ibuprofen and diclofenac. For example,
Cikcikoglu Yıldirim et al. [23] reported a remarkable decrease followed
by a decrease in SOD activity in G. pulex exposed to ibuprofen and
propranolol. These discrepancies may be attributed to variations
in animal species and the administration method of NSAIDs in the
respective studies. A notable rise in superoxide dismutase (SOD)
activity was evident at 24 and 48 h (P<0.01). However, no remarkable
differences were observed between the treatment groups and the
control group after 48 h (P>0.05). This pattern aligns with the role of
SOD as the primary defense against reactive oxygen species. SOD
functions by converting superoxide radicals into oxygen molecules
or hydrogen peroxide [51]. The observed increase in catalase (CAT)
activity suggests that SOD activity could possibly increase leading
to higher hydrogen peroxide production, which was subsequently
detoxied by CAT.
This study demonstrated that the average catalase (CAT) activity in
G. pulex exhibited a signicant increase (P<0.05), particularly at the
96
th
h. The increase in CAT activity was measured at 2.59, 11.18, and
2.16% respectively, in the respective experimental groups at the 96
th
h.
Similar remarkable increases in CAT activity were observed by Zivna et
al. [52] in Danio rerio exposed to acetylsalicylic acid, and by Cikcikoglu
Yildirim et al. [53] in G. pulex exposed to various concentrations
of ibuprofen over 24 and 96 h. Costa et al. [54] and Parolini et al.
[18] Pawłowska et al. [55] described an increase in CAT activity in
the R. philippinarum and Ruditapes decussatus, D.polymorpha and
Heterocypris incongruens following administration of NSAIDs such as
diclofenac, ibuprofen and naproxen. However, it could be seen that
there are studies reporting conicting ndings. Nunes et al. [56] found
that diclofenac had no effect on CAT activity in Solea senegalensis.
Similar observations were reported in Cyprinus carpio exposed to
acetylsalicylic acid [52]. In this study, a dose–dependent increase in
CAT activity was observed. In Scrobicularia plana, ibuprofen exposure
produced biochemical alterations (SOD, CAT, lipid peroxidation) in
several organs indicating oxidative stress and damage to lipids [57].
In the current investigation, there was a remarkable decline
observed in the concentration of GSH within the treatment
groups, compared to the control group. This nding substantiates
the occurrence of cellular oxidation and implies an inadequate
functioning of the glutathione system in effectively clearing ROS
generated by naproxen. In results are in line with previous study,
where naproxen administration decreased the level of GSH compared
to the experimental and control groups in Wistar rats [58]. In the
study, naproxen administration resulted in the increase of superoxide
dismutase (SOD) activity, leading to higher levels of H
2
O
2
, which may
have been mitigated by the action of catalase (CAT), which was also
bioactivated. The decrease in GSH content could be attributed to the
elimination of H
2
O
2
by GSH under conditions of stress. Additionally,
there is a strong and significant correlation between MDA and
glutathione levels, indicating that higher MDA levels correspond to
lower glutathione levels. There is a strong and signicant correlation
between MDA and glutathione levels; This indicates that higher MDA
levels correspond to lower glutathione levels [59].
CONCLUSION
The ndings of this study demonstrated the capacity of naproxen
to induce oxidative stress in G. pulex and increase MDA levels even at
extremely low concentrations. This situation; Since it will negatively
affect natural water resources and the creatures living there, it should
be considered as an environmental risk and effective methodologies
should be developed to prevent naproxen from entering the aquatic
environment. Heavy metals, pesticides, organic pollutants, ammonia
and nitrates, and drug residues can cause harmful effects on aquatic
creatures such as G. pulex. Examining the effects of these substances
on G. pulex can provide important data for the health of the ecosystem
and conservation efforts. This study provides a more comprehensive
understanding of how toxic substances affect the physiology of G.
pulex. One possible mechanism of action of these toxic materials
is their ability to modify the characteristics of lipid peroxidation
constituents and the antioxidant status in aquatic invertebrates. The
alterations observed in antioxidant enzymes suggest that an adaptive
stress response may be triggered in organisms exposed to naproxen,
aiming to restore the redox balance and prevent oxidative damage.
Consent for publications
The author read and approved the nal manuscript for publication.
Ethics approval and consent to participate
No human or vertebrate animals were used in the present research.
Availability of data and material
The data that support the ndings of this study are available from
the corresponding author upon reasonable request
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