Comparative study on the effects of micro- and nano-sized zinc oxide supplementation on zinc-deficient mice

Abstract

Background: Zinc (Zn) is an essential cofactor for physiological homeostasis in the body. Zn oxide (ZnO), an inorganic compound that supplies Zn, exists in various sizes, and its bioavailability may vary depending on the size in vivo. However, comparative studies on the nutritional effects of micro-sized ZnO (M-ZnO) and nano-sized ZnO (N-ZnO) supplementation on Zn deficiency (ZnD) animal models have not been reported.

Objectives: This study investigated the nutritional bioavailability of N-ZnO and M-ZnO particles in dietary-induced ZnD mice.

Methods: Animals were divided into six experimental groups: normal group, ZnD control group, and four ZnO treatment groups (Nano-Low, Nano-High, Micro-Low, and Micro-High). After ZnD induction, N-ZnO or M-ZnO was administered orally every day for 4 weeks.

Results: ZnD-associated clinical signs almost disappeared 7 days after N-ZnO or M-ZnO administration. Serum Zn concentrations were higher in the Nano-High group than in the ZnD and M-ZnO groups on day 7 of ZnO treatment. In the liver and testis, Nano-Low and Nano-High groups showed significantly higher Zn concentrations than the other groups after 14-day treatment. ZnO supplementation increased Mt-1 mRNA expression in the liver and testis and Mt-2 mRNA expression in the liver. Based on hematoxylin-and-eosin staining results, N-ZnO supplementation alleviated histological damage induced by ZnD in the testis and liver.

Conclusions: This study suggested that N-ZnO can be utilized faster than M-ZnO for nutritional restoration at the early stage of ZnD condition and presented Mt-1 as an indicator of Zn status in the serum, liver, and testis.

Keywords: Zinc oxide (ZnO); bioavailability; metallothionein; nanoparticles; zinc deficiency.

Fig. 1. Induction Zn-deficient mice model. (A) Zn content in diets as determined by ICP-MS. Bars represent the means ± SD (n = 6). (B) Zn concentration in the serum of mice during Zn deficiency induction period. The Zn concentration was determined by ICP-MS (n = 4). (C) Clinical signs (alopecia, erect of fur, eye lesion, and tail lesion) of mice with Zn deficiency.

^*Significantly different from basal diet (p < 0.05).

Fig. 2. Effect of M-ZnO and N-ZnO treatment on body weight and food intake of Zn-deficient mice model. (A) Experimental design for treatments of M-ZnO and N-ZnO in Zn-deficient mice. Zn-deficient mice were induced for 4 weeks by feeding a Zn-deficient diet and M-ZnO and N-ZnO oral treatments were performed daily for 4 weeks. (B and C) Changes in body weights and food intake of mice treated with M-ZnO and N-ZnO. Values represent the means ± SD (n = 16). (D) Clinical signs (alopecia, erect of fur, and eye lesion) of mice with ZnD and ZnD with high dose of N-ZnO on 0–21 days.

ZnD, Zn-deficient control group, ZnO, Zn oxide; ML, Micro-Low group (8 mg Zn/kg body weight); MH, Micro-High group (40 mg Zn/kg body weight); NL, Nano-Low group (8 mg Zn/kg body weight); NH, Nano-High group (40 mg Zn/kg body weight); M-ZnO, micro-sized ZnO; N-ZnO, nano-sized ZnO.

Fig. 3. Effect of M-ZnO and N-ZnO treatment on Zn concentration of mice. (A-C) After Zn deficiency induction, low or high doses of M-ZnO or N-ZnO treatments were performed daily for 4 weeks. (A) Zn concentration in the serum of mice after ZnO treatments for 7–28 days (n = 9). (B) Zn concentration in the liver of mice after ZnO treatments for 7–28 days (n = 9). (C) Zn concentration in the testis of mice after ZnO treatments for 7–28 days (n = 6). Values represent the means ± SD.

ZnD, Zn-deficient control group; ML, Micro-Low group (8 mg Zn/kg body weight); MH, Micro-High group (40 mg Zn/kg body weight); NL, Nano-Low group (8 mg Zn/kg body weight); NH, Nano-High group (40 mg Zn/kg body weight); M-ZnO, micro-sized ZnO; N-ZnO, nano-sized ZnO; ZnO, Zn oxide. ^* indicates statistically significant (p < 0.05).

Fig. 4. Histopathology of liver and testis after Zn oxide treatments for 7 and 28 days. Liver and testis tissue samples are stained with hematoxylin and eosin staining. a: Normal group, b: Zn-deficient control group, c: Micro-Low group (8 mg Zn/kg body weight), d: Micro-High group (40 mg Zn/kg body weight), e: Nano-Low group (8 mg Zn/kg body weight), f: Nano-High group (40 mg Zn/kg body weight). Marked degeneration of spermatids and spermatogenic cells (asterisk), widened interstitial spaces (arrow) and vacuolization of seminiferous tubules with epithelial disorganization (arrow head) are noted. Bar size = 5 μm.

ST, spermatids; SG, spermatogenic cells; IS, interstitial space.

Fig. 5. Effect of M-ZnO and N-ZnO treatment on Mt-1 and Mt-2 mRNA expression of mice. (A-D) After Zn deficiency induction, low or high doses of M-ZnO or N-ZnO treatments were performed daily for 4 weeks. (A and B) Relative Mt-1 and Mt-2 mRNA expressions in the liver after ZnO treatments for 7–28 days (n = 3). (C and D) Relative Mt-1 and Mt-2 mRNA expressions in the testis after ZnO treatments for 7–28 days (n = 3). Values represent the means ± SD.

ZnD, Zn-deficient control group; ML, Micro-Low group (8 mg Zn/kg body weight); MH, Micro-High group (40 mg Zn/kg body weight); NL, Nano-Low group (8 mg Zn/kg body weight); NH, Nano-High group (40 mg Zn/kg body weight); M-ZnO, micro-sized ZnO; N-ZnO, nano-sized ZnO; ZnO, Zn oxide. ^* indicates statistically significant (p < 0.05). NS indicates not statistically significant.