Cardiovascular and Respiratory Toxicity of Protamine Sulfate in Zebrafish and Rodent Models

Figures

• 

  Figure 1. Schematic representation of 94 h (a) and 48 h (b) exposure of zebrafish to the full range of protamine sulfate (PS) concentrations, N(ω)–nitro–l–arginine methyl ester (l–NAME), and unfractionated heparin (UFH), alone or together with PS. Abbreviations: hpf, hour post-fertilization.

• 

  Figure 2. Schematic representation of cardiorespiratory parameters observation in rats treated with unfractionated heparin (UFH) and protamine sulfate (PS), and with N(ω)–nitro–l–arginine methyl ester (l–NAME) and PS.

• 

  Figure 3. Survival (a) and hatching (b) rates for zebrafish embryos exposed to protamine sulfate (PS) concentrations or vehicle (VEH) from the 2nd to 96th hour post–fertilization (hpf). Heart rate (c) values of zebrafish embryos and larvae at 48 and 72 hpf. Morphology (d) of zebrafish larvae at 72 hpf. Abbreviations: NSB, non–inflated swim bladder; PE, pericardial edema. Data are shown as medians with ranges. Note: * p < 0.05; ** p < 0.01, *** p < 0.001 vs. VEH within the group, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 17–29 for each concentration.

• 

  Figure 4. Survival (a) and heart (b) rates of zebrafish embryos at 24 h post–fertilization (hpf) exposed for 24 and 48 h to vehicle (VEH) and protamine sulfate (PS) concentrations. Data are shown as medians with ranges. Note: ** p < 0.01, *** p < 0.001 vs. VEH within the group, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 29–30 for VEH group and for PS at concentrations of 1, 5, and 10 µg/mL; n = 3–20 in PS at concentrations for 25 and 50 µg/mL.

• 

  Figure 5. Heart rate values of zebrafish embryos at 24 h post–fertilization exposed for 24 (a) and 48 h (b) to vehicle (VEH), l–NAME (0.5 mM), unfractionated heparin (UFH, 0.5 U/mL), protamine sulfate (PS), PS together with l–NAME (0.5 mM), or PS together with UFH (0.5 U/mL). Data are shown as medians with ranges. Note: ** p < 0.01, *** p < 0.001 vs. VEH, ^ p < 0.05 ^^^ p < 0.001 vs. PS at the appropriate concentration, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 30 for each concentration.

• 

  Figure 6. The effects of protamine sulfate (PS) and its complexes with unfractionated heparin (UFH) on mean blood pressure (MBP) values. The course of MBP was tracked for 60 min after short–duration intravenous infusion (5 min; 2 mL/h) of PS at a dose of 1.5 (a) or 10 mg/kg (b) following intravenous administration of vehicle (VEH, 1 mL/kg) or UFH (150 or 1000 U/kg, 1 mL/kg). Results are shown as means (lines) with SEMs. The MBP values at 0 min of the experiment were 121.4 (86.6–138.0) for VEH, 110.7 (91.1–129.4) for PS 1.5 mg/kg, 134.6 (122.8–152.7) for UFH 150 U/kg + PS 1.5 mg, 109.4 (93.1–127.4) for UFH 150 U/kg, 116.9 (108.2–135.6) for PS 10 mg/kg, 120.3 (93.5–130.8) for UFH 1000 U/kg + PS 10 mg/kg, 107.7 (95.2–126.6) for UFH 1000 U/kg. Note: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. VEH, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 5–8.

• 

  Figure 7. The effects of protamine sulfate (PS) and its complexes with unfractionated heparin (UFH) on heart rate. The course of heart rate was tracked for 60 min after short–duration intravenous infusion (5 min; 2 mL/h) of PS at a dose of 1.5 (a) or 10 mg/kg (b) following intravenous administration of vehicle (VEH, 1 mL/kg) or UFH (150 or 1000 U/kg, 1 mL/kg). Results are shown as means (lines) with SEMs. The heart rate values at 0 min of the experiment were 438 (365–488) for VEH, 407 (364–438) for PS 1.5 mg/kg, 437 (388–458) for UFH 150 U/kg + PS 1.5 mg, 416 (345–476) for UFH 150 U/kg, 424 (378–517) for PS 10 mg/kg, 410 (385–432) for UFH 1000 U/kg + PS 10 mg/kg, 390 (308–442) for UFH 1000 U/kg. Note: * p < 0.05, ** p < 0.01 vs. VEH, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 5–8.

• 

  Figure 8. The effects of protamine sulfate (PS) and its complexes with unfractionated heparin (UFH) on blood oxygen saturation. The course of blood oxygen saturation was tracked for 60 min after short–duration intravenous infusion (5 min; 2 mL/h) of PS at a dose of 1.5 (a) or 10 mg/kg (b) following intravenous administration of vehicle (VEH, 1 mL/kg) or UFH (150 or 1000 U/kg, 1 mL/kg). Results are shown as means (lines) with SEMs. The blood oxygen saturation values at 0 min of the experiment were 82 (72–89) for VEH, 82 (73–86) for PS 1.5 mg/kg, 85 (81–87) for UFH 150 U/kg + PS 1.5 mg, 85 (84–94) for UFH 150 U/kg, 79 (73–86) for PS 10 mg/kg, 79 (72–89) for UFH 1000 U/kg + PS 10 mg/kg, 87 (80–99) for UFH 1000 U/kg. Note: * p < 0.05, ** p < 0.01; *** p < 0.001 vs. VEH, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 5–8.

• 

  Figure 9. The effects of protamine sulfate (PS) and its complexes with unfractionated heparin (UFH) on perfusion of paw vessels. The course of perfusion of paw vessels was tracked for 60 min after short–duration intravenous infusion (5 min; 2 mL/h) of PS at dose of 1.5 (a) or 10 mg/kg (b) following intravenous administration of vehicle (VEH, 1 mL/kg) or UFH (150 or 1000 U/kg, 1 mL/kg). Results are shown as means (lines) with SEMs. The perfusion of paw vessels at 0 min of the experiment were 0.08 (0.04–0.14) for VEH, 0.07 (0.05–0.11) for PS 1.5 mg/kg, 0.07 (0.02–0.13) for UFH 150 U/kg + PS 1.5 mg, 0.06 (0.03–0.1) for UFH 150 U/kg, 0.09 (0.05–0.13) for PS 10 mg/kg, 0.1 (0.06–0.14) for UFH 1000 U/kg + PS 10 mg/kg, 0.08 (0.05–0.13) for UFH 1000 U/kg. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. VEH, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 5–8.

• 

  Figure 10. The effects of protamine sulfate (PS) and its complexes with unfractionated heparin (UFH) on respiratory rate. The course of respiratory rate was tracked for 60 min after short–duration intravenous infusion (5 min; 2 mL/h) of PS at dose of 1.5 (a) or 10 mg/kg (b) following intravenous administration of vehicle (VEH, 1 mL/kg) or UFH (150 or 1000 U/kg, 1 mL/kg). Results are shown as means (lines) with SEMs. The respiratory rate at 0 min of the experiment were 68 (43–88) for VEH, 68 (63–70) for PS 1.5 mg/kg, 76 (65–83) for UFH 150 U/kg + PS 1.5 mg, 64 (50–75) for UFH 150 U/kg, 67 (48–90) for PS 10 mg/kg, 67 (40–95) for UFH 1000 U/kg + PS 10 mg/kg, 52 (41–59) for UFH 1000 U/kg. Note: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. VEH, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 5–8.

• 

  Figure 11. The maximum effects of protamine sulfate (PS) and its complexes with unfractionated heparin (UFH) on peak exhaled CO2. The course of peak exhaled CO2 was tracked for 60 min after short–duration intravenous infusion (5 min; 2 mL/h) of PS at dose of 1.5 (a) or 10 mg/kg (b) following intravenous administration of vehicle (VEH, 1 mL/kg) or UFH (150 or 1000 U/kg, 1 mL/kg). Results are shown as means (lines) with SEMs. The peak exhaled CO2 values at 0 min of the experiment were 4.2 (4.0–4.7) for VEH, 4.1 (4.0–4.2) for PS 1.5 mg/kg, 4.0 (3.7–4.4) for UFH 150 U/kg + PS 1.5 mg, 4.0 (3.5–4.8) for UFH 150 U/kg, 4.2 (3.6–5.0) for PS 10 mg/kg, 4.5 (4.2–5.0) for UFH 1000 U/kg + PS 10 mg/kg, 4.6 (3.9–5.1) for UFH 1000 U/kg. Note: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. VEH, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 5–8.

• 

  Figure 12. The effects of protamine sulfate (PS) alone and together with N(ω)–nitro–l–arginine methyl ester (l–NAME) on mean blood pressure (MBP). The course of MBP was tracked for 60 min after short–duration intravenous infusion (5 min; 2 mL/h) of PS (15 mg/kg) following intravenous administration of vehicle (VEH; 1 mL/kg) or l–NAME (25 mg/kg; 1 mL/kg). Results are shown as means (line) with SEMs (a) and as medians with ranges (b). The MBP values at 0 min of the experiment were 121.4 (86.6–138.0) for VEH, 139.3 (130.4–153.3) for PS 15 mg/kg, 124.9 (107.1–119.8) for PS 10 mg/kg + l–NAME, and 132.5 (118.5–138.9) for l–NAME alone. Note: * p < 0.05, ** p < 0.01 vs. VEH, ^ p < 0.05 vs. PS 15 mg/kg, Kruskal–Wallis ANOVA with Dunn’s post–hoc test, n = 3–5.