Toxicity Assessment of an Anti-Cancer Drug of p-Toluene Sulfonamide in Zebrafish Larvae Based on Cardiovascular and Locomotion Activities

Abstract

p-Toluene sulfonamide (p-TSA), a small molecular drug with antineoplastic activity is widely gaining interest from researchers because of its pharmacological activities. In this study, we explored the potential cardio and neural toxicity of p-TSA in sublethal concentrations by using zebrafish as an in vivo animal model. Based on the acute toxicity assay, the 96hr LC₅₀ was estimated as 204.3 ppm, suggesting the overall toxicity of p-TSA is relatively low in zebrafish larvae. For the cardiotoxicity test, we found that p-TSA caused only a minor alteration in treated larvae after no overall significant alterations were observed in cardiac rhythm and cardiac physiology parameters, as supported by the results from expression level measurements of several cardiac development marker genes. On the other hand, we found that acute p-TSA exposure significantly increased the larval locomotion activity during the photomotor test while prolonged exposure (4 days) reduced the locomotor startle reflex activities in zebrafish. In addition, a higher respiratory rate and blood flow velocity was also observed in the acutely treated fish groups compared to the untreated group. Finally, by molecular docking, we found that p-TSA has a moderate binding affinity to skeletal muscle myosin II subfragment 1 (S1), ATPase activity, actin- and Ca²⁺-stimulated myosin S1 ATPase, and v-type proton ATPase. These binding interactions between p-TSA and proteins offer insights into the potential molecular mechanism of action of p-TSA on observed altered responses toward photo and vibration stimuli and minor altered vascular performance in the zebrafish larvae.

Keywords: cardiotoxicity; larva; neurotoxicity; p-TSA; zebrafish.

Figure A1

Interaction of p-TSA against (A–L) zebrafish endogenous target proteins. (i) Two-dimensional (2D) interaction diagram, (ii) ribbon structure, and (iii) hydrophobic surface perspective of the protein.

Figure A1

Interaction of p-TSA against (A–L) zebrafish endogenous target proteins. (i) Two-dimensional (2D) interaction diagram, (ii) ribbon structure, and (iii) hydrophobic surface perspective of the protein.

Figure A1

Interaction of p-TSA against (A–L) zebrafish endogenous target proteins. (i) Two-dimensional (2D) interaction diagram, (ii) ribbon structure, and (iii) hydrophobic surface perspective of the protein.

Figure A1

Interaction of p-TSA against (A–L) zebrafish endogenous target proteins. (i) Two-dimensional (2D) interaction diagram, (ii) ribbon structure, and (iii) hydrophobic surface perspective of the protein.

Figure A2

Comparison of ~128 hpf zebrafish larval morphology of one represented larva after ~24 h exposure of (A) 0 (control), (B) 10 ppm, (C) 50 ppm, and (D) 100 ppm of p-toluene sulfonamide in lateral view (scale bar = 0.5 mm). Quantification of (E) body and (F) eye axis lengths of the fishes from each group (n = 30). The data were analyzed using one-way ANOVA test, followed by Dunnett’s multiple comparisons test, and presented as mean with SD.

Figure 1

The mortality rate of zebrafish larvae after 96 h of exposure to p-Toluene Sulfonamide (p-TSA) to determine the median lethal dose (LC₅₀).

Figure 2

Cardiac physiology parameter endpoints ((A) Stroke volume, (B) heart rate of atrium, (C) ejection fraction, (D) cardiac output, (E) heart rate of ventricle, and (F) shortening fraction) of zebrafish larvae at 72 hpf after 24 h incubation in 10, 50, and 100 ppm of p-Toluene Sulfonamide (p-TSA). The statistical difference was calculated using Ordinary One-Way ANOVA with Dunnet multiple comparison test. The data are expressed as mean with SD (n = 29; * p < 0.05).

Figure 3

Cardiac rhythm parameter endpoints ((A) Atrium-ventricle beat interval, (B) sd1 of atrium chamber, (C) sd2 of ventricle chamber, (D) ventricle-atrium beat interval, (E) sd2 of atrium chamber, and (F) sd1 of ventricle chamber)of zebrafish larvae at 72 hpf after 24 h incubation in 10, 50, and 100 ppm of p-Toluene Sulfonamide (p-TSA). The statistical difference was calculated using Ordinary One-Way ANOVA with Dunnet multiple comparison test. The data are expressed as mean with SD (n = 29; * p < 0.05).

Figure 4

Vascular performance parameter endpoints ((A) Maximum blood flow velocity and (B) average blood flow velocity) of zebrafish larvae after 24 h incubation in 10, 50, and 100 ppm of p-Toluene Sulfonamide (p-TSA). The statistical difference was calculated using Ordinary One-Way ANOVA with Dunnet multiple comparison test. The data are shown as mean with SD (n = 30, except 50 ppm group (n = 29); * p < 0.05, *** p < 0.001).

Figure 5

(A) Total distance traveled per minute by 128 hpf zebrafish larvae after 1-day exposure of 0 (control), 10 ppm, 50 ppm, and 100 ppm of p-Toluene Sulfonamide (p-TSA) during both light and dark cycles. The data were analyzed by a two-way ANOVA test with Geisser-Greenhouse correction, continued with Dunnett’s multiple comparisons test. (B,C) Comparison of total distance traveled by the larvae in light and dark cycles, respectively. The data were analyzed using the Kruskal–Wallis test, followed by Dunn’s multiple comparisons test. All data are expressed in median with 95% CI (n = 135 for control and 10 ppm groups, n = 134 for 50 ppm group, n = 136 for 100 ppm group; **** p < 0.0001).

Figure 6

(A) Total distance traveled per second by 192 hpf zebrafish larvae after 4-day exposure of 0 (control), 10, 50, and 100 ppm of p-Toluene Sulfonamide (p-TSA) during the vibrational startle response assay. The data were analyzed using a two-way ANOVA test with Geisser-Greenhouse correction, followed by Dunnett’s multiple comparisons test. (B) A comparison of the total distance traveled by the tested zebrafish larvae during the occurrence of the tapping stimuli. The data were analyzed using the Kruskal–Wallis test, continued with Dunn’s multiple comparisons test. All data are expressed in median with 95% CI (n = 135 for control and 10 ppm groups, n = 122 for 50 ppm group, n = 90 for 100 ppm group; **** p < 0.0001).

Figure 7

(A) Oxygen consumption level per minute by 96 hpf zebrafish larvae after 1-day exposure of 0 (control), 10, 50, and 100 ppm of p-Toluene Sulfonamide (p-TSA) during the respiratory rate assay. The data were analyzed using a two-way ANOVA test with Geisser-Greenhouse correction, followed by Dunnett’s multiple comparisons test. (B) Comparison of total oxygen consumption of the tested zebrafish larvae. The data were analyzed using one-way ANOVA, continued with Dunnett’s multiple comparisons test. All data expressed in the median with 95% CI (n = 69; *** p < 0.001, **** p < 0.0001).

Figure 8

The expression pattern of cardiovascular development-related genes ((A) amhc, (B) myh6, (C) vegfaa, (D) vmhc, (E) gata1, (F) gata4, (G) hbbe1, (H) hbbe2, (I) hbae1, and (J) tbx5) in 5 dpf zebrafish larvae after 1 day exposure of p-Toluene Sulfonamide (p-TSA). The data were analyzed by using one-way ANOVA test, followed with Dunnett’s multiple comparisons test, and are presented as mean with SD (n = 3 groups with a total of 210 zebrafish larvae; * p < 0.05,** p < 0.01).

Figure 8

The expression pattern of cardiovascular development-related genes ((A) amhc, (B) myh6, (C) vegfaa, (D) vmhc, (E) gata1, (F) gata4, (G) hbbe1, (H) hbbe2, (I) hbae1, and (J) tbx5) in 5 dpf zebrafish larvae after 1 day exposure of p-Toluene Sulfonamide (p-TSA). The data were analyzed by using one-way ANOVA test, followed with Dunnett’s multiple comparisons test, and are presented as mean with SD (n = 3 groups with a total of 210 zebrafish larvae; * p < 0.05,** p < 0.01).

Figure 9

Summary of the present study demonstrated various alterations that occurred in zebrafish larvae after p-Toluene Sulfonamide (p-TSA) exposure (↑: upregulated, ↓: downregulated, -: no significant change).