Evaluation of DNA integrity and mitochondrial membrane potential in catfish Pseudoplatystoma magdaleniatum, induced by exposure 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 fish. 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 catfish Pseudoplatystoma magdaleniatum. For this study, the fish 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 flow 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 sufficient 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, specific, and reliable technique, compared to traditional methods.


Introduction
Pharmaceuticals are all prescription or over-the-counter drugs, as well as veterinary therapeutic drugs, for the treatment of both human and animal diseases. On the other hand, personal care products (PCP) are used to improve the quality of daily life. In recent years, concern has increased about the presence of PPCPs (Pharmaceutical and Personal Care Products) in the different compartments of the aquatic environment, such as water, sediments, and biota, in concentrations that are capable of generating harmful effects for both aquatic animals and humans; becoming a major concern, as PPCPs has tended to increase in human and veterinary medicine, leading to a steady release to the environment (Petrie et al. 2014;Aristizabal-Ciro et al. 2017;Ebele et al. 2017;Pico et al. 2019).
Contamination with PPCPs in water can occur by different routes: the rst is the absorption of PPCPs by the body after use, followed by excretion and release into the sewage system or septic tank (Dey et al. 2019). After incomplete wastewater treatment, these waters can be used as biosolids, applied as fertilizers on agricultural lands, in addition to leaching processes, PPCPs can reach groundwater (Kim et al. 2009;Petrie et al. 2014;Dey et al. 2019;White et al. 2019); these PPCPs can also reach freshwater through runoff from the soil treated with these biosolids. The second way is through its manufacture since the wastewater from these production factories goes directly to the WWTP. In addition, PPCPs can be discharged through shower, bath, sink, and swimming wastes (Archer et al. 2017;Ebele et al. 2017). 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-steroidal anti-in ammatory drug (NSAID), this drug reversibly inhibits the synthesis of prostanoids (prostaglandins, prostacyclins, and thromboxanes) (González-Mira et al. 2016), by non-selectively 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).
Among aquatic organisms, sh is an excellent tool for the analysis of possible genotoxic alterations, due to their ability to metabolize, concentrate and store water-borne pollutants (Ternjej et al. 2010). 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 (Mojica et al. 2016;Herrera-Cruz et al. 2019). This sh inhabits the Magdalena and 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 food security as a source of food (Friedrich-Ebert-Stiftung and Foro Nacional Ambiental 2015). In addition, cat sh, being at the top of the food chain, indirectly contributes to the control and regulation of the aquatic system where it lives.
The Magdalena basin, made up of the Cauca and Magdalena rivers, the main rivers of Colombia, is the main recipient of domestic wastewater, water contaminated with pesticides used in crops, illegal gold extraction, and discharges from industries such as oil re ning and tanneries (Tejeda-Benítez et al. 2018). The investigations in the Magdalena 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 rivers is wastewater from the main cities Bogotá, Medellín, Cali, and Barranquilla (Galvis and Mojica 2007;Tejeda-Benitez et al. 2016). In the main cities of the country, some investigations have 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).
Ibuprofen is considered a ubiquitous pollutant as it has been found in tributaries and e uents from wastewater treatment plants, surface waters, drinking water, sludge, and hospital e uents; Ibuprofen concentrations have been reported worldwide in the range of 0.001 and 75.8 µg / L (Gutiérrez-Noya et al. 2020) Therefore, it is possible that ibuprofen is present in the Magdalena river basin, causing alterations in the cat sh that inhabit these areas and being one of the possible causes of its decrease in the basin. The determination of possible genotoxic alterations, such as mitochondrial membrane potential (ΔΨM) and DNA integrity can be determined by blood cells, which are constantly exposed to reactive oxygen species and provide a relatively non-invasive source for the biomonitoring, also the sampling is done relatively easily and the sh can be returned to the water after collection without having to sacri ce them to carry out the study (Ternjej et al. 2010). The analyzes can be carried out using ow cytometry, since it is a fast and reliable technique to quantify and characterize some cell populations, allowing the evaluation of immunopathological processes in sh (Alzamora-Gonzales et al. 2015).
The ΔΨM is generated by proton pumps (Complexes I, III, and IV), it is an indispensable component in the energy storage process during oxidative phosphorylation. The ΔΨM together with the proton gradient forms the transmembrane potential of hydrogen ions that are used to produce ATP, resulting from the redox transformations associated with the activity of the Krebs cycle. A prolonged decrease or increase in normal levels of ΔΨm can induce a loss of cell viability and be a cause of various alterations (Zorova et al. 2018).
Another parameter that can be measured by ow cytometry is the integrity of the DNA. The determination of both parameters is possible by the use of different stains; DiOC6 (3,3'-dihexyloxacarbocyanine iodide), is a uorescent dye, its function is to stain the endoplasmic reticulum of a cell, membranes of vesicles, and mitochondria, the union is possible through the hydrophilic groups of the dye (Rieger et al. 2011). On the other hand, propidium iodide (PI) is used to determine if cells are viable, underwent apoptosis or necrosis, through differences in the integrity of the plasma membrane and permeability. This dye is one of the most used since it is inexpensive, stable, and is a good indicator since it can exclude living cells. PI binds to double-stranded DNA by intercalation between base pairs. The ability of PI to enter a cell depends on the permeability of the membrane. In late apoptotic and necrotic cells, plasma and nuclear integrity decrease, allowing PI to pass through the membranes, interspersing with nucleic acids (Özgen et al. 2000;Rieger et al. 2011).
Given that ibuprofen is one of the most consumed drugs in the world ((Ngo and Bajaj 2020) and so far there is no information on the possible alterations in mitochondrial membrane potential and DNA integrity caused by ibuprofen in Neotropical sh such as Pseudoplatystoma magdaleniatum, it is necessary to determine if ibuprofen at different doses (0, 25, 50 µg / L) exposed for four months to P. magdaleniatum produces genotoxic alterations, determined by ow cytometry, is one of the possible causes of its decline.

Fish Husbandry
The striped cat sh Pseudoplatystoma magdaleniatum (Siluriformes: Pimelodidae) presents sexual dimorphism, reproductive migrations with temporality for spawning ( All the sh were caught in the Cauca River. Sexually mature striped cat sh were used. For the experimentation, six individuals were arranged per experimental unit, separated into males and females 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. The experimental units were divided into 3 control groups 0, 25, and 50 µg/L. Due to photochemical degradation and sh uptake of ibuprofen, ibuprofen (1335508 USP, Rockville, USA) concentrations were readjusted in each tank.
Concentrations ranged from 17.8-27.9 µg/L (25 µg/L) to 41.7-52.2 µg/L (50 µg/L), and the control group always remained below the limit of quanti cation, Table 1. 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. Week 17 0.304 ± 0.001 0.413 ± 0.121 0.376 ± 0.026 LOQ = limit of quanti cation (0.500 µg/L).
The sh were fed with live feed Astyanax sp (ad libitum), grown at the Fish Culture Research Institute. As described in Table 2, the quality of the water in the tanks increased; Parameters related to water quality and optimal conditions for sh maintenance were measured weekly, a portable multiparameter (HACH, Sension + MM15, USA) was used to determine dissolved oxygen, temperature, pH and saturation percentage of oxygen.
Ammonia nitrogen and ammonia were measured using a kit (API, USA). Values are expressed as mean ± SD.
SD standard deviation.

Experimental design
The sh caught for the experimentation are sh of sexual maturity size. 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, in separate tanks, the sh were divided into three groups according to their exposure: 0 control, 25 and 50 µg/L, the control group (0 µg/L). 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 before being analyzed (maximum time 24 hours). Ibuprofen was quanti ed using an ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry (

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. Whole blood was used for all analyzes, as blood is an excellent source of DNA. The analyzes are carried out with white blood cells (leukocytes) and not red blood cells (erythrocytes) since they lack a nucleus.

Determination of Mitochondrial Membrane Potential (ΔΨM)
For the determination of ΔΨM in the blood samples, 3,3'-dihexyloxacarbocyanine iodide (DiOC 6 , Molecular Probes by Life Technologies, Thermo Fisher Scienti c) was used, this analysis was evaluated with an adapted version of the protocol described by Zamzami et al. (1996). 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 polystyrene tube was used to deposit the DiOC6, a phosphate buffer (PBS) at a nal concentration of 800 nM and 10 µL of blood. Subsequently, to stain the cells and simultaneously assess their viability, a nal concentration of 1 mg / mL of propidium iodide (PI, Thermo Fisher Scienti c) was added.
Samples were incubated for 30 minutes and ΔΨM was measured by ow cytometry (LSRFortessa, BD Biosciences). Subsequently, it was separated in three ways according to the intensity of DIOC6 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 forms of separation. The temporal analysis was carried out in the high uptake and intermediate uptake cells of DIOC 6 , Fig. 1. The mitochondrial membrane potential was determined at the levels, low, medium, and high ΔΨM.
Flow cytometry data were analyzed using FlowJo software version 10.6.2 (Tree Star Inc., Ashland, Oregon, United States).

Analysis of Flow Cytometry
The determination of mitochondrial membrane damage was performed by excluding aggregates by selecting the cell population of interest after contrasting the size (FSH) and granularity (SSC) to select unique events. After performing this exclusion, DiOC 6 -positive cells were compared with propidium iodide (PI)-negative cells, differentiating between the populations of erythrocytes and leukocytes, Fig. 2.
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. 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
Studies carried out in sh have shown that the ibuprofen present in the waters can cause an increase in the activity of glutathione-S-transferase 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 blood cells 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.
An analysis of ΔΨM was performed, as an indicator of cell viability. This analyzed parameter re ects the pumping of hydrogen through the inner membrane in the processes of electron transport and oxidative phosphorylation. 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. Table 3 shows the results for a two-way analysis of variance (ANOVA), where it is observed that there is no statistically signi cant difference in the PI + samples between the different treatments, nor in the comparisons concerning the four months of exposure. For the ΔΨM there is no statistically signi cant difference between the 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. It is di cult to make comparisons with other research since there is not much literature on this type of analysis in tropical sh and it could be inferred that this drug does not produce loss of mitochondrial function in the concentrations in which the experiment is carried out.  Table 4 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 0.76 (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 5 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. Comparing these results with other studies where DNA integrity is determined by comet assay, ibuprofen alterations were evident in monocytic cells of Hoplias malabaricus, with exposure of 10-1000 ng / mL (Ribas et al. 2014); in Rhamdia quelen with ibuprofen exposure of 66. 40 ng/L, where a statistically signi cant DNA loss was obtained after 5 and 28 days of exposure of 22.74-34.32% (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 the present study carried out by ow cytometry, it is di cult to make comparisons with other analysis techniques, however, it can be pointed out that hypodiploid cells were not observed, nor was there a loss of linearity that could be interpreted as coming from DNA. It is probable that at the concentrations at which the experimentation was carried out, this drug does not cause DNA damage, but because it is an endocrine disruptor, characterized by not following dose-response patterns, it is possible that at lower exposure concentrations it may cause obvious alterations. Therefore, in this study, they could not be demonstrated.

Conclusions
This study is one of the rst to analyze alterations in mitochondrial membrane potential and DNA integrity by ow cytometry in Neotropical sh. The results for the ΔΨM showed a statistically signi cant difference for the mean ΔΨM and the low ΔΨM, but without differences between the ΔΨM after four months of exposure, indicating that at these concentrations and exposure times there is no loss of function. mitochondrial, caused by ibuprofen. On the other hand, there were no alterations in the integrity of the DNA, the percentages of DNA without fragmentation were higher than 90% in all sexes, levels, and exposure times. It is concluded that the cat sh Pseudoplatystoma magdaleniatum exposed to ibuprofen at concentrations of 25, 50 µg/L for four months, does not produce alterations in the mitochondrial membrane potential or the integrity of the DNA. 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 were one of the causes of the decline in this species in the Cauca and Magdalena River Basin of Colombia.

Declarations
Ethics approval and consent to participate: These species were caught with the authorizations required by

Consent for publication: Not applicable
Availability of data and materials: The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.
Competing interests: The authors declare that they have no con ict of interest.
Funding: This study was funded by the Ministry of Science, Technology, and Innovation of Colombia (grant number 111569944244).
Authors' contributions: Sara E. Gallego R, performed activities related to Methodology, Formal Analysis, Investigation, Writing -Original Draft. Gustavo A. Peñuela was involved in the Funding Acquisition, Resources, Conceptualization, Supervision, and Project Administration. All authors contributed to writing the manuscript. Figure 1 Cell staining with DIOC6 and PI, and separation according to the intensity of DIOC6 (low, medium, and high ΔΨM).