Reversal of Doxorubicin-Induced Bone Loss and Mineralization by Supplementation of Resveratrol and MitoTEMPO in the Early Development of Sparus aurata

Abstract

Doxorubicin is a widely used chemotherapeutic drug known to induce bone loss. The mechanism behind doxorubicin-mediated bone loss is unclear, but oxidative stress has been suggested as a potential cause. Antioxidants that can counteract the toxic effect of doxorubicin on the bone would be helpful for the prevention of secondary osteoporosis. We used resveratrol, a natural antioxidant, and MitoTEMPO, a mitochondria-targeted antioxidant, to counteract doxorubicin-induced bone loss and mineralization on Sparus aurata larvae. Doxorubicin supplemented Microdiets increased bone deformities, decreased mineralization, and lipid peroxidation, whereas Resveratrol and MitoTEMPO supplemented microdiets improved mineralization, decreased bone deformities, and reversed the effects of doxorubicin in vivo and in vitro, using osteoblastic VSa13 cells. Partial Least-Squares Discriminant Analysis highlighted differences between groups on the distribution of skeletal anomalies and mineralization of skeleton elements. Calcium and Phosphorus content was negatively affected in the doxorubicin supplemented group. Doxorubicin reduced the mRNA expression of antioxidant genes, including catalase, glutathione peroxidase 1, superoxide dismutase 1, and hsp90 suggesting that ROS are central for Doxorubicin-induced bone loss. The mRNA expression of antioxidant genes was significantly increased on resveratrol alone or combined treatment. The length of intestinal villi was increased in response to antioxidants and reduced on doxorubicin. Antioxidant supplements effectively prevent bone deformities and mineralization defects, increase antioxidant response and reverse doxorubicin-induced effects on bone anomalies, mineralization, and oxidative stress. A combined treatment of doxorubicin and antioxidants was beneficial in fish larvae and showed the potential for use in preventing Doxorubicin-induced bone impairment.

Keywords: MitoTEMPO; bone deformities; doxorubicin; mineralization; oxidative stress; resveratrol.

Figure 1

Survival and growth parameters of gilthead seabream larvae at the end of the trial. Survival of seabream (A), total length (B), dry weight (C). One-way ANOVA, Tukey’s multiple comparisons test, **—p ≤ 0.01. Acronyms: resveratrol (RES), doxorubicin (DOX), MitoTEMPO (MT), doxorubicin + resveratrol (DOX+RES) and doxorubicin + MitoTEMPO (DOX+MT).

Figure 2

Histology of gut of gilthead seabream supplemented with antioxidants and pro-oxidants microdiets at 45 DAH (days after hatched). Stained with hematoxylin and eosin (10×) initial-initial larvae of 20 dah (A). Length of villus measured on ImageJ (B). One-way ANOVA, Tukey’s multiple comparisons test,*—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, and ****—p ≤ 0.0001. Acronyms: resveratrol (RES), doxorubicin (DOX), MitoTEMPO (MT), doxorubicin + resveratrol (DOX+RES) and doxorubicin + MitoTEMPO (DOX+MT).

Figure 3

Incidence of skeletal deformities. Partial Least-Squares Discriminant Analysis (PLS-DA score) between the antioxidants and pro-oxidants groups (A), heatmap of distribution of skeletal deformities (B), incidence of Deformities (C), deformities charge (D), number of affected areas (E), severity of malformation (number of affected areas) (F). One-way ANOVA, Tukey’s multiple comparisons test, *—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, and ****—p ≤ 0.0001. Acronyms: resveratrol (RES), doxorubicin (DOX), MitoTEMPO (MT), doxorubicin + resveratrol (DOX+RES) and doxorubicin + MitoTEMPO (DOX+MT).

Figure 4

Distribution of skeletal deformities. Incidence of deformities in between the antioxidant and pro-oxidant supplemented groups. Head region (A), cephalic region (B), prehaemal region (C), caudal region (D), caudal fin (E), haemal or pre-caudal region (F), anal fin (F), dorsal fin (G), and heat map of the specific deformities between the antioxidant and pro-oxidant supplemented groups (H). Two-way ANOVA, Tukey’s multiple comparisons test, *—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, and ****—p ≤ 0.0001. Acronyms: resveratrol (RES), doxorubicin (DOX), MitoTEMPO (MT), doxorubicin + resveratrol (DOX+RES), doxorubicin + MitoTEMPO (DOX+MT), Cephalic vertebrae (CeV), Pre-haemal vertebrae (PHV), Haemal vertebrae (HV), Caudal vertebrae (CV), Anal fin (AF), Dorsal fin (DF), and Caudal fin (CF).

Figure 5

Mineralization of the skeletal elements. Partial Least-Squares Discriminant Analysis (PLS-DA score) between the antioxidants and pro-oxidants groups on the mineralization of the skeleton elements (A), Mineralized vertebrae of seabream fed with Microdiets (B), and Extracellular matrix mineralization assay of Vsa13 cells (C). One-way ANOVA, Tukey’s multiple comparisons test, *—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, and ****—p ≤ 0.0001. Acronyms: Alizarin red-S (AR-S), Growth medium (GM), Osteogenic medium (OM), resveratrol (RES), doxorubicin (DOX), MitoTEMPO (MT), doxorubicin + resveratrol (DOX+RES), and doxorubicin + MitoTEMPO (DOX+MT).

Figure 6

Developmental status and mineralization patterns of gilthead seabream fed different microdiets. Percentage of mineralized, mineralizing, cartilaginous, unmineralized and absent; cranial vertebrae neural arch (A), prehaemal vertebrae neural arches and ribs (B), haemal vertebrae neural (C), modified haemal arch and caudal vertebrae neural arches (D), caudal fin vertebrae (CFV), urostyle and hypural (E), and caudal fin rays (F). Heat map of mineralization pattern of skeleton element between the antioxidant and pro-oxidant supplemented groups (G). Graph: percentage +/− SEM. Acronyms: resveratrol (RES), doxorubicin (DOX), MitoTEMPO (MT), doxorubicin + resveratrol (DOX+RES) and Doxorubicin + MitoTEMPO (DOX+MT), Cephalic vertebrae neural arches (CVN), Pre-haemal vertebrae neural arches (PVN), Pre-haemal vertebrae ribs (PVR), Haemal vertebrae neural arches (HVN), Haemal vertebrae haemal arches (HVH), Caudal vertebrae Modified haemal arches (MHA), Caudal fin vertebrae neural arches (CFVN), Caudal fin vertebrae hypural (CFVH), Caudal fin vertebrae urostyle (CFVU), and Caudal fin rays (Rays).

Figure 7

Mineral analysis of gilthead seabream fed with microdiets. Calcium (A), phosphorus (B), calcium/phosphorus ratio (C). One-way ANOVA, Tukey’s multiple comparisons test, *—p ≤ 0.05, **—p ≤ 0.01. Acronyms: resveratrol (RES), doxorubicin (DOX), MitoTEMPO (MT), doxorubicin + resveratrol (DOX+RES), and doxorubicin + MitoTEMPO (DOX+MT).

Figure 8

Oxidative stress and antioxidant status on antioxidant and pro-oxidant treatment. Lipid peroxidation of the gilthead seabream larvae feed with antioxidant and pro-oxidant diets, initial- initial larvae of 20 days (A). mRNA expression of expression of antioxidants genes on VSa13 cells treated with Antioxidants and Pro-oxidants (Resveratrol, MitoTEMPO, Hydrogen peroxide and Doxorubicin); Catalase (cat) (B), superoxide dismutase 1 (sod1) (C), glutathione peroxidase 1 (gpx1) (D), heat shock protein 90 kDA alpha 1 (hsp90) (E) and osteopontin (spp1) (F). One-way ANOVA, Tukey’s multiple comparisons test, *—p ≤ 0.05, **—p ≤ 0.01, ***—p ≤ 0.001, and ****—p ≤ 0.0001. Acronyms: Resveratrol (RES), Doxorubicin (DOX), MitoTEMPO (MT), doxorubicin + resveratrol (DOX+RES), doxorubicin + MitoTEMPO (DOX+MT).