Single and Combined Effects of Meropenem, Valproic Acid, and Ketoprofen on Adult Zebrafish Behavior, Oxidative Stress, and Acetylcholinesterase Activity

Abstract

Background: Pharmaceutical compounds frequently co-occur in environmental waters, but studies on their combined effects on animals and humans remain limited. The present study investigated the individual and combined short-term effects of ketoprofen (Kp, a nonsteroidal anti-inflammatory drug inhibiting cyclooxygenase-2), valproic acid (VPA, an anticonvulsant acting as a voltage-gated sodium channel modulator), and meropenem (Mp, a β-lactam antibiotic) at environmentally relevant concentrations on zebrafish behavior, acetylcholinesterase (AChE) activity, and oxidative status. Methods: Adult zebrafish were exposed for 4 days to Kp, VPA, Mp, and their binary and ternary mixtures. Behavioral effects were assessed using 3D novel tank and social behavior tests, while the oxidative stress response was assessed through malondialdehyde (MDA) content, superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities. Results: Zebrafish exposed to Mp showed a notable increase in immobility, whereas those exposed to VPA and Mp + Kp exhibited a significant augmentation of average velocity and counter-clockwise rotations. All treated groups exhibited a notable increase in the time spent near the walls (thigmotaxis), and except for the control and Mp-exposed zebrafish, the other groups mostly stayed in the bottom tank zone (geotaxis). Kp, VPA + Kp, and VPA + Mp + Kp treatments impaired social behavior, with zebrafish displaying less interest in conspecifics. Biochemical analysis demonstrated that both the individual drugs and their combination caused oxidative stress, characterized by decreased GPx activity and increased SOD activity and MDA levels. Moreover, AChE activity was more strongly inhibited in zebrafish exposed to the binary and ternary mixtures than to individual drugs. Conclusions: The results indicate that acute exposure to individual and/or combined pharmaceuticals induces behavioral changes, oxidative damage, and AChE inhibition in zebrafish, highlighting the need to assess the effects of pharmaceutical mixtures for comprehensive ecosystem risks evaluation.

Keywords: behavior; joint effects; oxidative stress; pharmaceuticals; zebrafish.

Figure 1

Behavioral changes in adult zebrafish treated with single and combined pharmaceuticals (Mp = 1 µg/L; VPA = 3 µg/L; Kp = 5 µg/L) for 96 h. Behavioral parameters measured include (a) total distance traveled (cm), (b) mean speed (cm/s), (c) immobility duration (s), (d) time spent near the walls (s), (e) frequency of transitions to the walls, and (f) counter-clockwise rotations. Data are presented as mean ± SD (n = 10 animals per group, with a 1:1 male-to-female sex ratio) and were analyzed using one-way ANOVA followed by Dunnett’s test. Statistically significant differences are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 2

Behavioral changes in adult zebrafish treated with single and combined pharmaceuticals (Mp = 1 µg/L; VPA = 3 µg/L; Kp = 5 µg/L) for 96 h. Behavioral parameters measured include (a) total distance traveled (cm), (b) mean speed (cm/s), (c) immobility duration (s), (d) time spent in the upper half (s), (e) transitions to the upper half, and (f) counter-clockwise rotations. Data are presented as mean ± SD (n = 10 animals per group, with a 1:1 male-to-female sex ratio) and were analyzed using one-way ANOVA followed by Dunnett’s test. Statistically significant differences are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 3

Effects of single and combined pharmaceuticals (Mp = 1 µg/L; VPA = 3 µg/L; Kp = 5 µg/L) acute treatments on adult zebrafish social interaction. Behavioral parameters measured include (a) amount of time that zebrafish spent in the left arm, (b) decision zone, (c) right arm, and (d) middle arm. Data are presented as mean ± SD (n = 10 animals per group, with a 1:1 male-to-female sex ratio) and were analyzed using one-way ANOVA followed by Dunnett’s test. Statistically significant differences are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 4

Biochemical parameters of adult zebrafish after 96 h of treatment with single and combined pharmaceuticals (Mp = 1 µg/L; VPA = 3 µg/L; Kp = 5 µg/L). The following biomarkers were assessed: (a) SOD (U/mg of protein), (b) GPx (U/mg of protein), (c) MDA (nmol/mg of protein), and (d) AChE (nmol ATCh/min/mg of protein). Data are presented as mean ± SD (n = 5) and were analyzed using one-way ANOVA followed by Dunnett’s test. Statistically significant differences are indicated as follows: * p < 0.05, ** p < 0.01, and *** p < 0.001.

Figure 5

Pearson’s correlation analysis between behavioral and biochemical parameters: (a) TSLA and TU, (b) TSNW and TU, (c) TSDZ and TU, (d) AB and TD, (e) GPx and TU, (f) MDA and TU, (g) SOD and MDA, (h) TID and MDA, (i) FTU and AChE. Data points represent individual zebrafish from the following groups: (●) Control, (●) Mp, (●) VPA, (●) Kp, (●) VPA + Mp, (●) VPA + Kp, (●) Mp + Kp, and (●) VPA + Kp + Mp, exposed to environmentally relevant concentrations for 96 h. The solid line represents the linear regression, while the dashed lines show the 95% confidence interval. Abbreviations: TSLA = time spent in the left arm, TSDZ = time spent in the decision zone, TDS = total distance (side view), AV = average velocity (side view), TID = total immobilization duration (side view), TU = time in upper half, FTU = frequency of transitions to upper half, TSNW = time spent near the walls.

Figure 5

Pearson’s correlation analysis between behavioral and biochemical parameters: (a) TSLA and TU, (b) TSNW and TU, (c) TSDZ and TU, (d) AB and TD, (e) GPx and TU, (f) MDA and TU, (g) SOD and MDA, (h) TID and MDA, (i) FTU and AChE. Data points represent individual zebrafish from the following groups: (●) Control, (●) Mp, (●) VPA, (●) Kp, (●) VPA + Mp, (●) VPA + Kp, (●) Mp + Kp, and (●) VPA + Kp + Mp, exposed to environmentally relevant concentrations for 96 h. The solid line represents the linear regression, while the dashed lines show the 95% confidence interval. Abbreviations: TSLA = time spent in the left arm, TSDZ = time spent in the decision zone, TDS = total distance (side view), AV = average velocity (side view), TID = total immobilization duration (side view), TU = time in upper half, FTU = frequency of transitions to upper half, TSNW = time spent near the walls.

Figure 6

Schematic diagram of the experimental setup. (a) 3D novel tank test and (b) social behavior test.