Melatonin mitigates arsenic and fluoride-induced cardiotoxicity in chickens by maintaining mitochondrial homeostasis

Abstract

Co-exposure to geogenic arsenic and fluoride is widely recognized in numerous countries and has been shown to induce severe cardiac injury in both humans and animals. Our previous studies have demonstrated that mitochondria are primary targets of arsenic and fluoride toxicity, as evidenced by alterations in ultrastructure, mitochondrial membrane potential, respiratory chain function, etc. Therefore, targeting mitochondrial homeostasis to develop pharmacological interventions for mitigating arsenic and fluoride-induced cardiotoxicity holds significant promise. Melatonin has emerged as a potent antioxidant and free radical scavenger. In this study, we reveal the substantial therapeutic potential of melatonin in mitigating arsenic and fluoride-induced cardiac injury in chicken models. A total of 72 one-day-old male Hy-Line Brown broilers were randomly allocated into eight groups: Control Group (basal diet), Arsenic Group (36 mg/kg As₂O₃ in diet), Fluoride Group (400 mg/kg NaF in diet), Arsenic + Fluoride Group (36 mg/kg As₂O₃ and 400 mg/kg NaF in diet), Melatonin Group (2.5 mg/kg melatonin in diet), Arsenic + Melatonin Group (36 mg/kg As₂O₃ and 2.5 mg/kg melatonin in diet), Fluoride + Melatonin Group (400 mg/kg NaF and 2.5 mg/kg melatonin in diet), and Arsenic + Fluoride + Melatonin Group (36 mg/kg As₂O₃, 400 mg/kg NaF, and 2.5 mg/kg melatonin in diet). Following a 23-week intervention with arsenic, fluoride, or their combination, the chickens exhibited significant cardiac damage and myocardial fibrosis. This was evidenced by elevated serum levels of lactate dehydrogenase (LDH), aspartic transaminase (AST), and troponin I (cTn-I), as well as marked changes observed in histopathological examinations. Dietary supplementation with melatonin significantly mitigated these cardiac damages induced by arsenic and fluoride. Mechanistically, we identified that melatonin exerted cardioprotective effects by reducing oxidative stress and apoptosis, and by restoring mitochondrial homeostasis through attenuating pathological mitochondrial fission, enhancing mitochondrial fusion, and promoting mitochondrial biogenesis. Collectively, our findings highlight melatonin as a potent cardioprotective agent against arsenic and fluoride-induced heart injury in chickens.

Keywords: Arsenic; Fluoride; Heart; Melatonin; Mitochondrial homeostasis.

Fig. 1

The effects of melatonin on serum levels of LDH, AST, and cTn-I in chickens exposed to arsenic and fluoride individually or in combination (mean ± SEM, n = 5). (A) Serum LDH levels. (B) Serum AST levels. (C) Serum cTn-I levels. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Fig. 2

Histopathological evaluation of myocardial tissues in chickens by H&E staining and Masson’s trichrome staining (n = 5). (A) The results of H&E staining. (B) The results of MASSON staining. The muscle fibers were stained in red, and the collagen fibers appeared in blue. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Fig. 3

The mRNA expression levels of antioxidant genes related to the NRF2 pathway (n = 5). (A) NRF2: Nuclear factor erythroid 2-related factor 2. (B) SOD1: Superoxide dismutase 1. (C) NQO1: NAD(P)H quinone oxidoreductase 1. (D) HO1: Heme Oxygenase-1. (E) CAT: Catalase. (F) GPX1: Glutathione peroxidase 1. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001.

Fig. 4

The protective effect of melatonin treatment on arsenic and fluoride-induced cardiomyocyte apoptosis. (A) The heart sections were stained with TUNEL. (B) Column bar graph illustrating the proportions of TUNEL-positive cells. (C-E) The protein expression levels of the anti-apoptotic protein BCL2 and the pro-apoptotic protein BAX. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control; ## P < 0.05, ## P < 0.01, ### P < 0.001.

Fig. 5

Alterations in the ultrastructure of mitochondria in chicken cardiomyocytes following arsenic, fluoride, and melatonin treatment (n = 5). (A and B) The representative image of cardiomyocyte mitochondria under TEM (A: scale bar = 2 μm; B: scale bar = 1 μm). (C-G) The mitochondrial size, the mitochondrial matrix density, the proportion of disordered mitochondria, and the width of mitochondrial cristae were quantitatively analyzed. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. Control; ^##P < 0.05, ^##P < 0.01, ^###P < 0.001.

Fig. 6

The effects of melatonin treatment on mitochondrial dynamics and biogenesis in chicken hearts exposed to arsenic, fluoride, or their combination. * P < 0.05, *** P < 0.001 vs. Control; ^##P < 0.05, ^###P < 0.001.