Dexrazoxane does not mitigate early vascular toxicity induced by doxorubicin in mice

Abstract

Apart from cardiotoxicity, the chemotherapeutic agent doxorubicin (DOX) provokes acute and long-term vascular toxicity. Dexrazoxane (DEXRA) is an effective drug for treatment of DOX-induced cardiotoxicity, yet it remains currently unknown whether DEXRA prevents vascular toxicity associated with DOX. Accordingly, the present study aimed to evaluate the protective potential of DEXRA against DOX-related vascular toxicity in a previously-established in vivo and ex vivo model of vascular dysfunction induced by 16 hour (h) DOX exposure. Vascular function was evaluated in the thoracic aorta in organ baths, 16h after administration of DOX (4 mg/kg) or DOX with DEXRA (40 mg/kg) to male C57BL6/J mice. In parallel, vascular reactivity was evaluated after ex vivo incubation (16h) of murine aortic segments with DOX (1 μM) or DOX with DEXRA (10 μM). In both in vivo and ex vivo experiments, DOX impaired acetylcholine-stimulated endothelium-dependent vasodilation. In the ex vivo setting, DOX additionally attenuated phenylephrine-elicited vascular smooth muscle cell (VSMC) contraction. Importantly, DEXRA failed to prevent DOX-induced endothelial dysfunction and hypocontraction. Furthermore, RT-qPCR and Western blotting showed that DOX decreased the protein levels of topoisomerase-IIβ (TOP-IIβ), a key target of DEXRA, in the heart, but not in the aorta. Additionally, the effect of N-acetylcysteine (NAC, 10 μM), a reactive oxygen species (ROS) scavenger, was evaluated ex vivo. NAC did not prevent DOX-induced impairment of acetylcholine-stimulated vasodilation. In conclusion, our results show that DEXRA fails to prevent vascular toxicity resulting from 16h DOX treatment. This may relate to DOX provoking vascular toxicity in a ROS- and TOP-IIβ-independent way, at least in the evaluated acute setting. However, it is important to mention that these findings only apply to the acute (16h) treatment period, and further research is warranted to delineate the therapeutic potential of DEXRA against vascular toxicity associated with longer-term repetitive DOX dosing.

Fig 1. Evaluation of vascular reactivity, following short-term in vivo DOX and DEXRA treatment.

DOX impaired ACh-induced vasodilation, irrespective of DEXRA pre-treatment (A). DEANO-induced vasodilation did not differ between the treatment groups (A). In both the absence and presence of L-NAME, PE-induced contraction remained unaffected (B). For A: Repeated measures two-way ANOVA with Dunnett’s multiple comparisons test. For B: One-way ANOVA with Dunnett’s multiple comparisons test per L-NAME condition. n = 7 in each group. For A: *p<0.05 for vehicle vs. DOX group; #p<0.05 for vehicle vs. DOX with DEXRA group. For B: p>0.05 for vehicle vs. DOX group and vehicle vs. DOX with DEXRA group.

Fig 2. Evaluation of vascular reactivity, following short-term ex vivo DOX and DEXRA incubation.

ACh-induced vasodilation was diminished in the DOX and DOX with DEXRA groups (A). DEANO-induced vasodilation was similar between the treatment groups (A). PE-induced contraction with and without L-NAME was lower following DOX exposure (B). For A: Repeated measures two-way ANOVA with Dunnett’s multiple comparisons test. For B: One-way ANOVA with Dunnett’s multiple comparisons test per L-NAME condition. n = 7 in each group. For A: *, **p<0.05, 0.01 for vehicle vs. DOX group; #, ##p<0.05, 0.01 for vehicle vs. DOX with DEXRA group. For B: *,**p<0.05, 0.01 compared to vehicle.

Fig 3. Evaluation of vascular reactivity, following short-term ex vivo DOX and NAC incubation.

DOX diminished ACh-induced vasodilation, irrespective of NAC (A). DEANO-induced vasodilation did not differ between the treatment groups (A). In both the absence and presence of L-NAME, PE-induced contraction was lower in the DOX group, regardless of NAC (B). For A: Repeated measures two-way ANOVA with Dunnett’s multiple comparisons test. For B: One-way ANOVA with Dunnett’s multiple comparisons test per L-NAME condition. n = 8 in vehicle and DOX groups; n = 7 in DOX with NAC group. For A: *p<0.05 for vehicle vs. DOX group; #p<0.05 for vehicle vs. DOX with NAC group; £ 0.05

Fig 4. Assessment of TOP-IIβ expression in the murine thoracic aorta (TA) and heart, following short-term in vivo DOX treatment.

RT-qPCR (A) and WB (B) show that murine aortic tissue expresses TOP-IIβ. DOX does not alter TOP-IIβ expression in the heart and aorta (A). DOX decreased TOP-IIβ protein levels in cardiac, but not aortic, tissue (B). Representative blots for panel B (C). For A & B: Mann-Whitney U test. n = 6 in each group. p>0.05 for vehicle vs. DOX groups in panel A. *p<0.05 for vehicle vs. DOX groups in panel B. For C: “L”, “V” and “D” stand for ladder, vehicle and DOX, respectively.