Evaluation of mitochondrial membrane potential and DNA integrity in Catsh Pseudoplatystoma magdaleniatum, when exposed for prolonged times to different concentrations of ibuprofen.

There are few studies to date that determine the effects of ibuprofen on mitochondrial membrane potential (ΔΨM) and DNA integrity in neotropical sh. The objective of this study is to determine if four months’ exposure to ibuprofen in different concentrations (25 and 50 µg/L) produces effects on ΔΨM and alters the integrity of DNA in striped catsh Pseudoplatystoma magdaleniatum. For this study, the sh were placed in tanks with water at constant concentrations of 0 (control), 25, and 50 µg/L of ibuprofen for four months. Subsequently, blood samples were taken for analysis of ΔΨM and DNA integrity, using a ow cytometer LSRFortessa BD Biosciences. After four months of exposure to ibuprofen at different concentrations, the results showed no increase in Low ΔΨM, indicating that there are no alterations in the mitochondrial membrane potential. On the other hand, the percentages of DNA damage were below 0.39, which indicates that there were no alterations in DNA integrity. It is possible that under the conditions in which this study was conducted (ibuprofen levels, exposure time), they are not sucient to demonstrate the effects caused by this drug. Higher ibuprofen levels and/or longer exposures may be required to determine alteration in ΔΨM and DNA integrity. Flow cytometric analysis for these types of samples is a fast, specic, and reliable technique, compared to traditional methods.


Introduction
The function of pharmaceuticals is as medicines or to improve the quality of daily life (Giang et al. 2018;Aguilar-Romero et al. 2020). However, their presence in aquatic environments and their possible harmful effects on aquatic organisms have caused concern in recent years (Liu et al. 2020). The main entry routes for pharmaceuticals to aquatic environments are wastewater treatment plants due to their incomplete disposal (Montagner and Jardim 2011;Giang et al. 2018;Meijide et al. 2018;García-Cambero et al. 2019;Aguilar-Romero et al. 2020), and to a lesser extent, the combination of untreated rivers and runoff waters (González-Mira et al. 2016), aquaculture, and pharmaceutical manufacturing sites (Liu et al. 2020). Conventional wastewater treatment plants are not designed to eliminate most pharmaceutical products (Tiedeken et al. 2017), most of them are also persistent due to their continuous diffusion in the aquatic environment, which can remain dissolved in the water column or accumulate in sediments (González-Mira et al. 2016). Most of these compounds and their metabolites are biologically active (Sathishkumar et al. 2020) and can cause alterations in aquatic organisms exposed for long periods, causing endocrine alterations, genotoxicity, carcinogenicity, and fetal malformations, among others (Giang et al. 2019;Liu et al. 2020) Ibuprofen is a non-selective non-steroidal anti-in ammatory drug (NSAID), this drug reversibly inhibits the synthesis of prostanoids (prostaglandins, prostacyclins, and thromboxanes) (González-Mira et al. 2016), by nonselectively inhibiting cyclooxygenase 1 and 2 enzymes and blocking the synthesis of prostaglandins and thromboxanes (Motov et al. 2020); Ibuprofen interferes with the cyclooxygenase pathway, decreasing the catalysis of prostaglandin biosynthesis from arachidonic acid (Parolini 2020). This drug can cause alterations in reproduction and development (Xia et al. 2017), oxidative stress, hematological changes, and DNA damage in sh (Mathias et al. 2018).
The striped cat sh Pseudoplatystoma magdaleniatum is an endemic species and the second most commercially important in the Colombian shery. However, it is among the critically endangered species (Mojica et al. 2012), mainly due to habitat degradation, embalming of rivers, over shing, deforestation, and organic and inorganic contamination ( Cauca basins, the main rivers of Colombia; historically, these basins have presented unsolved environmental problems, derived from deforestation, erosion, and contamination by solid and liquid waste (Galvis and Mojica 2007;Noreña-Ramirez et al. 2012;Zapata et al. 2015;Tejeda-Benítez et al. 2018). This threatens both the economy of the communities where the cat sh live, where it is a main source of work, income, and their food security as a source of food (Friedrich-Ebert-Stiftung and Foro Nacional Ambiental 2015).
The Magdalena river basin is the main recipient of domestic wastewater, as well as contaminated water derived from pesticides used in crops, illegal gold extraction, and some industries such as oil re ning, tanneries (Tejeda-Benítez et al. 2018). The investigations carried out in the Magdalena river basin have been carried out mainly in the analysis of heavy metals, leaving aside the investigations for the determination of some pollutants such as pharmaceutical products in the Magdalena and Cauca rivers (Noreña-Ramirez et al. 2012;Tejeda-Benitez et al. 2016;Tejeda-Benítez et al. 2018). The main source of contamination in the Magdalena and Cauca basins is wastewater from the main cities Bogotá, Medellín, Cali, and Barranquilla (Galvis and Mojica 2007;Tejeda-Benitez et al. 2016). In these cities, some research has been carried out on the content of PPCPs in the waters, nding that the greatest contribution of pharmaceutical products is non-steroidal anti-in ammatory drugs, anticonvulsants, and antibiotics (Gracia-Lor et al. 2012;Hernández et al. 2015;Aristizabal-Ciro et al. 2017;Bedoya-Ríos et al. 2018;Arias 2019;Pemberthy et al. 2020).
Several studies have detected concentrations of ibuprofen in tributaries and e uents of wastewater treatment plants, surface waters, drinking water, sludge, and hospital e uents. Ibuprofen concentrations have been reported around the world in the range of 0.001 and 75.8 µg / L. (Gutiérrez-Noya et al. 2020). Ibuprofen may be present in the Magdalena river basin, causing alterations in the cat sh that inhabit these areas and possibly being one of the causes of their decrease in the basin. The determination of the possible alterations in the mitochondrial membrane potential (ΔΨM) and the integrity of the DNA can be determined by ow cytometry, since it is a fast and reliable technique to quantify and characterize some cell populations, allowing the evaluation of processes immunopathological in sh (Alzamora-Gonzales et al. 2015).
The ΔΨM regulates the synthesis of adenosine-tri-phosphate (ATP), the production of reactive oxygen species (ROS), the sequestration of calcium in the mitochondria, the import of mitochondrial proteins, and the dynamics of the mitochondrial (Luna-Ortiz et al. 2013;Zorova et al. 2018;Restrepo et al. 2019). ΔΨM is important for many mitochondrial processes and is related to mitochondrial and cellular health (Allauca et al. 2019).
Because ibuprofen is one of the most widely consumed drugs in the world (Ngo and Bajaj 2020), it is possible that it is present in the main rivers of Colombia and is one of the possible causes of the decline of striped cat sh P. magdaleniatum in the last four decades. For this, a controlled experimental study was carried out for four months with different doses (0, 25, 50 µg/L) of ibuprofen in males and females of P. magdaleniatum, determining the alterations in the mitochondrial membrane potential and the integrity of the DNA, by ow cytometry.

Fish Husbandry
The striped cat sh Pseudoplatystoma magdaleniatum (Siluriformes: Pimelodidae) is manifested through sexual dimorphism, reproductive migrations with temporality for spawning (Arce et al. 2014), and not possessing scales. The cat sh is nocturnal, feeds on sh, some arthropods, and seeds (Santamaría Merchán 2013).
All the sh were caught in the Cauca River, Colombia. Sexually mature striped cat sh, P. magdaleniatum were used per experimental tank, in which they were divided into males and females (three male and three female cat sh were distributed in individual tanks) with average weights and lengths of 1.86 ± 0.49 kg and 61.14 ± 4.76 cm for males and 2.07 ± 0.64 kg and 63.86 ± 6.01 cm for females. All experimentation was carried out at the Fish Culture Research Institute of the University of Córdoba (CINPIC) located in Montería, in the department of Córdoba. The sh were acclimatized for two months in tanks of 3250 m 3 , with a 12/12 photoperiod throughout the year.
The sh were fed with live feed Astyanax sp (fed on demand), grown in sh farming. To guarantee the quality of the water in the ponds, Table 1

Experimental design
The sh caught for the experimentation are sh of sexual maturity size, however, the breeding season waited; the experiment lasted four continuous months, the time necessary for this species to develop its gametes (Palacio 2009;Arce et al. 2014). The experimentation was carried out with three independent experiments, the sh were divided into three groups according to their exposure: 0 control, 25 and 50 µg/L, the control group (0 µg/L) always remained below the limit of detection. Due to the photochemical degradation and absorption of ibuprofen by the sh, 50% of the water in the tanks was replaced weekly and the ibuprofen concentrations were readjusted in each tank. Water samples were taken in amber glass containers, kept at 4-6°C for 24 hours.
Ibuprofen was quanti ed using an ultra-performance liquid chromatography-tandem mass spectrometer

Blood Sampling
Blood samples were taken 14 days after the addition of ibuprofen and after four months. Blood samples were collected by a direct puncture in the tail vein with the help of a vacutainer; the tubes in which the samples were collected contained EDTA K2 anticoagulants. Blood samples were taken for each treatment and all samples were processed separately. Blood was drawn from each sh and kept for 1 hour at 4-6 ° C and in the absence of ultraviolet light until reaching the laboratory for analysis.

Determination of Mitochondrial Membrane Potential (ΔΨM)
For the determination of ΔΨM in the blood samples, 3,3'-dihexyloxacarbocyanin iodide (DiOC 6 , Molecular Probes by Life Technologies, Thermo Fisher Scienti c) was used. This uorescent dye is used for mitochondrial staining under the in uence of the permeability transition (Rojas et al. 2000;Rieger et al. 2011). For analysis, a tube was used to add the DIOC 6 in phosphate buffer (PBS) to a nal concentration of 800 nM, then 10 µL of blood was added. Subsequently, to stain the cells to simultaneously assess their viability, 1 µg /mL of propidium iodide (PI, Thermo Fisher Scienti c) was added to each tube. The samples were incubated for 30 minutes and the ΔΨM was measured by ow cytometry (LSRFortessa, BD Biosciences). Subsequently separated in three ways according to the intensity of the DIOC 6 in the Mo o XDP using a 70 µm nozzle, at a frequency of 100 thousand Hz, with a minimum e ciency of 98 for each of the three separations ways. The temporality analyzes had been made on the high uptake and intermediate uptake cells of DIOC 6 , Fig. 1.
The ΔΨM of the blood was assessed with an adapted version of the protocol described by Zamzami et al. (1996). A polystyrene tube was used to deposit 3.3′-dihexyloxacarbocyanine iodide (DiOC6, Molecular Probes) in PBS at a nal concentration of 80 nM and 7-aminoactinomycin D (7-AAD, Molecular Probes) at a nal concentration of 2 µg/ml. The compounds were pipetted, and then, 10 µl of blood were added, the nal volume of the reaction was 300 µl. Subsequently, to stain the cells to simultaneously evaluate their viability, a nal concentration of 1 mg/ml of propidium iodide (PI) (Molecular Probes, USA) was added. The samples were incubated for 30 min, and ΔΨM was measured using ow cytometry (LSRFortessa™, BD Biosciences). The samples were excited using a 488 nm solid phase laser, and uorescence from DiOC6 and 7-AAD has detected at 530/30 nm and 630/30 nm, respectively. The ow cytometry data were analyzed using FlowJo version 7.6.2 (FlowJo, LLC, USA) software. For the determination of DNA integrity, an exclusion of aggregates was also performed by selecting the cell population of interest after contrasting the size (FSH) and granularity (SSC), to select the unique events. Once the unique events were selected, employing PI-A and PI-W, the single cells were selected to determine the DNA integrity employing the histogram, Fig. 3.

Statistical Analysis
Statistical analysis was performed using Statgraphics Centurion XVII (StatPoint Inc., USA). The evaluation of the normality of the continuous variables was performed via the Shapiro-Wilk test. Nonparametric statistics were applied to those variables that were not normally distributed. An analysis of variance (ANOVA) was used to evaluate the existence of signi cant differences between ΔΨM and DNA integrity. If this gave a statistically signi cant difference, a post-ANOVA by the least signi cant difference test (LSD-Fisher) was used. Statistical differences for PI + and ΔΨM were analyzed by two-way analysis of variance (ANOVA) with exposure time, concentration, and "time x concentration" interaction as variables. For all statistical analyzes, the signi cance criterion was established at p < 0.05.

Results And Discussion
Various studies have been carried out in sh on the alterations that ibuprofen present in the waters can cause.
Finding increased glutathione-S-transferase activity in the kidney, reduced glutathione peroxidase activity, decreased white blood cell count, causing nephrotoxicity and immunosuppressive effect (Mathias et al. 2018), increased cardiac output in embryos, decreased cell density (Zhang et al. 2019), and a signi cant reduction in the hatching rate (Xia et al. 2017). So far, no publications have been found on the possible alterations on the integrity of the DNA in P. magdaleniatum caused by the presence of ibuprofen in the waters.
For this study, blood samples of striped cat sh, Pseudoplatystoma magdaleniatum, collected from sh exposed to concentrations of 25 µg/L and 50 µg/L of ibuprofen for four consecutive months, and control sh without exposure, were analyzed. Environmental concentrations were used, as in some of the studies mentioned above.
An analysis of ΔΨM was performed, as an indicator of cell viability. This re ects the hydrogen pumping through the internal membrane in the electron transport and oxidative phosphorylation processes. These processes are necessary for the production of ATP, which means that mitochondrial dysfunction is closely related to an alteration in the membrane potential that would cause a decrease in the production of ATP (Padmini and Usha Rani 2011).
We analyzed the variations between the ΔΨM and cells with damage to the cell membrane, positive for PI. High ΔΨM, however, comparing the ΔΨM between the treatments and after exposure, the Medium ΔΨM presents statistically signi cant differences (p < 0.05) with an increase in the percentage after four months of treatment, as well as a decrease in the percentages of Low ΔΨM after four months of exposure to ibuprofen. Despite this difference between Medium and Low ΔΨM, there was no decrease in ΔΨM after four months of treatment with ibuprofen, therefore, this drug can be indicated at concentrations of 25 and 50 µg/L, for four months, not produces loss of mitochondrial function (Blanco and López-Armada 2005).  Table 3 shows the analysis of ΔΨM, the median uorescence intensity, determined by sex and ibuprofen concentration in the different analysis times. For females exposed to 25 µg/L of ibuprofen and presenting ratios 0.75 and 076 (High ΔΨM and Medium ΔΨM, respectively), it is indicated that at the time of the assay their leukocytes had a less mitochondrial function at time zero. However, females exposed to 50 µg/L and presenting a ratio of 1.63 had mitochondrial hyperactivity at time zero. Both events are due to a type of stress that can be interpreted respectively as depolarization and hyperpolarization. Meanwhile, the analyses performed after four months of exposure to this ibuprofen show, for males exposed to a concentration of 50 µg/L with ratios of 0.68 and 0. 70 (High ΔΨM and Medium ΔΨM, respectively), that 32% and 30% of their leukocytes present lower DIOC 6 uptake than the control at the time of the test. For those that have a ratio above 1 (High ΔΨM), there are 12, 28, and 34% of leukocytes with some hyperpolarization and, as these do not exceed 1.5, this may be due to the uctuations of the test. High ΔΨM: samples with high mitochondrial membrane potential.
Medium ΔΨM: samples with medium mitochondrial membrane potential.
Low ΔΨM: samples with low mitochondrial membrane potential.
Different lowercase letters in the columns indicate statistically signi cant differences (p < 0.05).
So far, no comparable results have been found where the analysis for ΔΨM in blood samples is performed by ow cytometry. However, the determination of ΔΨM by ow cytometry is suggested as a biomarker due to its higher speci city and quick quantitative assessment of the possible risk of exposure to this type of pharmaceutical (Padmini and Usha Rani 2011). Table 4 shows the integrity of the DNA, the results for time zero and after four months of exposure to ibuprofen, do not present statistically signi cant difference between the different treatments, and no effect on DNA is evidenced, since, after four months of exposure to ibuprofen, the average for males and females is 92.30% for 2n, which indicates that the vast majority do not present fragmentation in nonfragmented DNA and only 0.32% have some type of DNA damage.  (Rocco et al. 2010); and in Oreochromis niloticus, where exposure to 300 ng/L ibuprofen caused genotoxic effects in both acute (48 h) and subchronic (10 days) exposure (Ragugnetti et al. 2011). For this study, no hypodiploid cells were evident, nor was there any loss of linearity that could be interpreted as being from DNA. It is probable that, at the concentrations at which the analysis was performed, this pharmaceutical will not cause DNA damage.

Conclusions
This study is one of the rst to analyze alterations in the mitochondrial membrane potential and DNA integrity using ow cytometry. Cat sh Pseudoplatystoma magdaleniatum were exposed to different concentrations (0, 25, and 50 µg/L) of ibuprofen for four months. The results for the ΔΨM showed a statistically signi cant difference for the medium and Low ΔΨM, but without an increase in the Low ΔΨM, which indicates at these concentration levels and exposure time, there is no loss of mitochondrial function, caused by ibuprofen. There were no alterations in the integrity of the DNA, the percentages of DNA without fragmentation were higher than 90% in all sexes, connections, and exposure times. More research is needed at different levels of ibuprofen concentration, and longer exposure times; since under the conditions of this study, it was not possible to demonstrate the effects caused by ibuprofen on ΔΨM and DNA integrity.

Declarations
Funding: This study was funded by the Ministry of Science, Technology, and Innovation of Colombia (grant number 111569944244).
Con ict of Interest: The authors declare that they have no con ict of interest.

Availability statement
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.