Abstract
To investigate the long-term effects of dietary zinc oxide nanoparticle (Nano-ZnO, 20–40 nm) on the relative organ weight, liver function, deposition, and absorption of trace minerals in intrauterine growth retardation (IUGR) pigs, piglets were allocated to NBW (6 normal birth weight piglets fed basal diets), IUGR (6 IUGR piglets fed basal diets) and IUGR+NZ (6 IUGR piglets fed basal diets + 600 mg Zn/kg from Nano-ZnO) groups at weaning (21 days of age), which were sampled at 163 days of age. There were no noteworthy changes in the relative weight of organs, hepatic histomorphology, serum alkaline phosphatase, glutamic pyruvic transaminase and glutamic oxalacetic transaminase activities, and Mn, Cu, and Fe concentrations in leg muscle, the liver, the tibia, and feces among the IUGR, NBW, and IUGR+NZ groups (P>0.05), and no intact Nano-ZnO in the jejunum, liver, and muscle was observed, while dietary Nano-ZnO increased the Zn concentrations in the tibia, the liver, serum, and feces (P<0.05) and mRNA expression of metallothionein (MT) 1A, MT2A, solute carrier family 39 member (ZIP) 4, ZIP14, ZIP8, divalent metal transporter 1, solute carrier family 30 member (ZnT) 1, ZnT4 and metal regulatory transcription factor 1, and ZIP8 protein expression in jejunal mucosa (P<0.05). Immunohistochemistry showed that dietary Nano-ZnO increased the relative optical density of ZIP8 (mainly expressed in cells of brush border) and MT2A (mainly expressed in villus lamina propria and gland/crypt) (P<0.05). In conclusion, long-term dietary Nano-ZnO showed no obvious side effects on the development of the major organs, liver function, and metabolism of Cu, Fe, and Mn in IUGR pigs, while it increased the Zn absorption and deposition via enhancing the expression of transporters (MT, ZIP, and ZnT families) in the jejunum, rather than via endocytosis as the form of intact nanoparticles.
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Introduction
The rapid development of nanotechnology has promoted the material science into a new field — Nanomaterial, which size is in the range of 1–100 nm and has various special properties, and shows broad application prospects in many industries [1, 2]. Zinc oxide nanoparticle (Nano-ZnO) is one of the most studied and widely used nanomaterials, because of its easy synthesis and excellent properties such as anti-bacteria, anti-cancer, antioxidant, and hypoglycemic [3, 4]. Currently, Nano-ZnO is widely used in rubber, ceramics, cosmetics, packaging, semiconductor materials, and biomedical fields [5, 6]. Furthermore, as a high-quality zinc source, Nano-ZnO is also very attractive to the livestock industry. Sun et al. [7] reported that dietary 600 mg Zn/kg of Nano-ZnO had the same positive effects on diarrhea, growth performance, intestinal microbiota, and immunity and antioxidant capacity of piglets as dietary 2000 mg Zn/kg of ZnO (pharmacological dose), which was in line with the results reported by Oh et al. [8]. Our previous study found that added Nano-ZnO to the diet could enhance antioxidant capacity and intestinal barrier of broilers [9]. However, the potential harmful effects of Nano-ZnO on the environment and human health have attracted increasing attention, which has caused controversies in the livestock industry. Some side effects of Nano-ZnO on organs (especially for the liver) and cells had been reported in rat, mice and cell models [1, 10, 11], while dietary supplementation of Nano-ZnO (10, 20, and 40 mg/kg) for 42 days has not observed significant adverse effects on broilers [12]. Our previous studies reported that dietary 800 mg Nano-ZnO/kg or 1200 mg Nano-ZnO/kg for 14 days still did not show significant toxicity in weaned piglets [13, 14]. However, the safety assessment of long-term dietary Nano-ZnO supplementation in large animals (such as pigs) has not been reported.
As a pregnancy syndrome in mammals, intrauterine growth retardation (IUGR) impairs fetal growth and development and negatively affects postnatal growth and feed utilization efficiency, bringing a host of economic losses to the global livestock industry every year, especially for the swine industry [15]. Dietary Nano-ZnO could effectively alleviate the IUGR induced intestinal oxidative stress, inflammatory response, and excessive autophagy of pigs [16,17,18,19]. However, it remains unclear about the possible effects of dietary Nano-ZnO on development of organs, trace mineral metabolism, and liver function in IUGR finishing pigs. Therefore, we further investigated the effects of long-term feeding 600 mg Zn/kg from Nano-ZnO on relative weight of organs, liver function, deposition, and absorption of trace minerals (Mn, Fe, Cu, and Zn) in IUGR pigs. These results could provide partial information about the safety assessment of long-term dietary supplementation with Nano-ZnO in large animals.
Materials and Methods
Animal Care
The Nanjing Agricultural University Institutional Animal Care and Use Committee gave its approval to the animal study and associated protocols used in this work (Permit number SYXK-2019-00142) (Jiangsu, China).
Experimental Design and Sample Collection
This experiment is part of a series of researches to study the effects of long-term dietary Nano-ZnO in IUGR pigs [Duroc × (Landrace × Yorkshire)]. Specific experimental design has been described in previous study [19]. Briefly, a total of 18 male neonatal pigs were selected and grouped to NBW (6 normal birth weight piglets fed basal diet), IUGR (6 IUGR piglets fed basal diet), and IUGR+NZ (6 IUGR piglets fed basal diet + 600 mg Zn/kg from Nano-ZnO) groups at 21 days of age (weaning age). The feeding period was from 21 to 163 days of pig’s age. The nutrient levels and composition are presented in Supplementary Table 1. The source and characterization of Nano-ZnO used in our study have been well described in our previous studies [9, 13, 14]. Briefly, the Nano-ZnO was provided by Zhangjiagang Bonded Area Hualu Nanometer Material Co., Ltd. (Jiangsu, China), and the particles appeared nearly spherical geometry with sizes mainly ranging from 20 to 40 nm. All the piglets were individually housed in stainless steel pen with the leaking manure floor at the appropriate temperature with unlimited access to feed and water.
At 163 days of age, pigs were fasted for 12 h, and then blood samples were taken from their precaval vein and centrifuged to collect serum (4 °C, 3000 g, 10 min). The liver (right lobe) and jejunum samples were collected for morphological analysis and immunohistochemical analysis. The jejunum, liver, and muscle samples were collected and fixed in glutaraldehyde solution for transmission electron microscope (TEM) analysis. Jejunal mucosa was scraped quickly and stored. The pancreas, liver, kidney, spleen, and heart were weighed for the relative organ weight analysis (organ weight (g) / body weight (kg)).
Determination of the Serum Parameters
The serum activities of alkaline phosphatase (AKP, Article number: A059-2-2), glutamic pyruvic transaminase (GPT, Article number: C009-1-1), and glutamic oxalacetic transaminase (GOT, Article number: C010-2-1) were determined via kits (Nanjing Jiancheng Institute of Bioengineering, Nanjing, China).
Hepatic Histomorphology
As reported by Dong et al. [20], the fixed liver samples (paraformaldehyde, 24 h) were dehydrated and embedded in paraffin wax and then sliced and dyed with hematoxylin and eosin. All sections were observed and photographed by a microscope with digital camera (Nikon H550L, Tokyo, Japan).
TEM Analysis
The TEM sections of the jejunum, liver, and muscle were made as previously described [19]. Briefly, the fixed-samples (2.5% glutaraldehyde solution for 48 h) were further fixed with 1% osmium tetroxide and dehydrated and embedded in epoxy resin. TEM sections were observed and photographed under a transmission electron microscope (Hitachi H-7650, Tokyo, Japan) to examine whether Nano-ZnO (black particles, 20–40 nm) existed in the jejunum, liver, and muscle of IUGR pig according to the previous studies [21,22,23,24].
Determination of Trace Mineral Concentrations
Determination of trace mineral concentrations (Zn, Mn, Cu, and Fe) in serum, leg muscle, the liver, the tibia, and feces was based on previous studies [13, 25]. Briefly, the serum was diluted triple with deionized water. For the liver, leg muscle, and the tibia, took 1–1.5 g simple into a digestion tube and added the acid mixture (HClO4:HNO3 = 1:4, v:v) to digest, after which diluted with deionized water to 25 mL. For feces, about 1 g dried samples were weighed for carbonization and ashing and then dissolved with hydrochloric acid and set the volume to 25 mL by deionized water. These solutions were measured by inductively coupled plasma optical emission spectrometry (ICP-OES, USA) after the standard curve was prepared.
Acquisition of cDNA and Determination of mRNA Expression
As previously described [16, 19], extracted the jejunal mucosal total RNA, and obtained the cDNA samples, which were then reversed transcription polymerase chain reaction (RT-PCR). The β-actin (internal reference gene) and the 2 -ΔΔCt method [26] were used to determine the relative mRNA expression of trace mineral metabolism-related genes (metallothionein (MT) 1A; MT2A; carrier family 39 member (ZIP) 4; ZIP14; ZIP8; ZIP5; ZIP10; solute carrier family 30 member (ZnT) 1; divalent metal transporter 1 (DMT1); ZnT4; ZnT2; solute carrier family 40 member 1 (FPN1); metal regulatory transcription factor 1 (MTF1); ATPase copper transporting alpha (ATP7A); transferrin receptor 1 (TFR1); solute carrier family 31 member 1 (CTR1); ATPase copper transporting beta (ATP7B)) and endocytosis-related genes (epidermal growth factor receptor pathway substrate 15 (Eps15); dynamin 1 (DNM1); caveolin 1 (Cav1); dynamin 2 (DNM2); caveolin 2 (Cav2)) in jejunal mucosa. Table 1 lists relevant primer information.
Western Blot Examination
Western blot examination of the jejunal mucosa was carried out according to previous studies [16, 27]. The primary antibody of ZIP8 (dilution rate: 1:2,000) was provided by Affinity Biosciences LTD (Jiangsu, China) and β-actin (dilution rate: 1:1,000, internal reference protein) was provided by Cell Signaling Technology (Danvers, Massachusetts, USA).
Immunohistochemical Analysis
Immunohistochemical analysis of the jejunum was determined according to previous description [28]. The antibodies of ZIP8 (dilution rate:1:200) and MT2A (dilution rate:1:150) were purchased from Affinity Biosciences LTD (Jiangsu, China). The sections were observed and photographed via a microscope with digital camera (Nikon H550L, Tokyo, Japan). Brown deposits indicate the positive immunostaining. The relative optical density of sections was analyzed using Image-Pro Plus 6.0 software according to the previous studies [29, 30].
Statistical Analysis
Used SPSS software (ver. 22.0, SPSS Inc., Chicago, USA) to examine the data, and one-way analysis of variance with Tukey’s post hoc test was used to analyze the statistical differences between groups. Statistically significant differences are shown by P values lower than or equal to 0.05.
Results
The Relative Weight of Organs
No noteworthy changes were observed in the relative weight of organs (the heart, liver, spleen, pancreas, and kidney) among the IUGR, NBW and IUGR+NZ groups (Table 2, P>0.05).
Zn Concentrations in Serum, the Liver, Leg Muscle, and the Tibia
Table 3 shows that in comparison to the NBW group, pigs in the IUGR group had no noteworthy differences in the Zn concentrations of the liver, leg muscle, the tibia, and serum (P>0.05). In comparison to the IUGR group, IUGR+NZ group’ pigs showed observably higher Zn concentrations in the tibia, the liver, and serum (P<0.05), but no significant alteration in the Zn concentration of leg muscle was observed (P>0.05).
Hepatic Histomorphology and Serum Parameters
Figure 1 shows that pigs in the IUGR+NZ, NBW, and IUGR groups all showed normal hepatic architecture and cell morphology. In addition, no noteworthy changes in the activities of serum AKP, GPT, and GOT were observed among the IUGR+NZ, NBW, and IUGR groups (Table 4, P>0.05).
Cu, Fe, and Mn Concentrations in Serum, the Liver, Leg Muscle, and the Tibia and Related mRNA Expression in Jejunal Mucosa
Results showed that no noteworthy differences were found in the concentrations of Mn, Cu, and Fe in the liver (Fig. 2A), leg muscle (Fig. 2B), the tibia (Fig. 2C), and serum (Fig. 2D) among the IUGR+NZ, NBW, and IUGR groups (P>0.05). Moreover, no significant alterations were observed in relative mRNA expression of TFR1, FPN1, CRT1, ATP7A, and ATP7B among the IUGR+NZ, IUGR, and NBW groups (Fig. 2E, P>0.05).
Trace Mineral Concentrations in Feces
Table 5 shows that no noteworthy alterations were found in fecal concentrations of Cu, Fe, and Mn among the IUGR+NZ, IUGR, and NBW groups (P>0.05). However, dietary Nano-ZnO markedly increased the Zn concentration in feces in comparison to the IUGR group (P<0.05), while no significant alteration was found in the Zn concentration of the feces between the IUGR and NBW groups (P>0.05).
TEM Analysis and mRNA Expression of Endocytosis-Related Genes in Jejunal Mucosa
Figure 3A exhibits that no intact Nano-ZnO (black particles, 20–40 nm) was observed in the jejunum, liver, and muscle in the IUGR+NZ group. For the relative mRNA expression of endocytosis related genes in jejunal mucosa (Fig. 3B), no noteworthy differences were obtained in the relative mRNA expression of Eps15, DNM1, DNM2, Cav1, and Cav2 among the IUGR+NZ, IUGR, and NBW groups (P>0.05).
Zn Metabolism-Related Gene mRNA and ZIP8 Protein Expression in Jejunal Mucosa
Figure 4A shows that in comparison to the IUGR group, dietary Nano-ZnO markedly up-regulated the mRNA expression of MT1A, MT2A, ZIP4, ZIP8, ZIP14, ZnT1, ZnT4, DMT1, and MTF1 (P<0.05), but did not markedly affect the mRNA expression of ZIP5, ZIP10, and ZnT2 in jejunal mucosa (P>0.05). Dietary Nano-ZnO markedly up-regulated the ZIP8 protein expression in jejunal mucosa in comparison to the IUGR group (Fig. 4B, P<0.05), while no significant alteration was observed in the expression of these selected genes and ZIP8 protein between the IUGR and NBW groups (P>0.05).
Immunohistochemistry
Figure 5 exhibits that ZIP8 was mainly detected in jejunal villous brush border and absorptive cells, and MT2A was mainly detected in jejunal villus lamina propria and gland/crypt. In comparison to the NBW group, no noteworthy differences were observed in the relative optical density of ZIP8 and MT2A in the IUGR group (P>0.05), while dietary Nano-ZnO observably increased the relative optical density of ZIP8 and MT2A in the jejunum in comparison to the IUGR group (P<0.05).
Discussion
The relative weight of organs has long been considered as a sensitive indicator of organ development and is an important part of the toxicology and risk assessment of drugs or additives [31, 32]. In our study, dietary long-term (from 21 to 163 days of age) Nano-ZnO did not markedly alter the relative weights of the kidney, liver, pancreas, spleen, and heart of IUGR pigs, which suggested that dietary Nano-ZnO did not induce intoxication in these organs or impair the development of these organs in IUGR pigs. Similar to our results, previous studies have found that feeding 250 and 500 mg Nano-ZnO/kg for 7 weeks or 35 weeks did not alter the relative weight of organs (the spleen, pancreas, liver, brain, testis, kidney, and heart) of mice [33, 34]. Oh et al. [8] reported that pigs fed with 200 mg Nano-ZnO/kg for 14 days did not affect the relative weight of spleen.
After the Nano-ZnO was absorbed by the gastrointestinal tract, the Zn can be transported to other tissues and organs for deposition or utilization, such as the liver, muscle, and the tibia [35, 36]. In our present study, long-term dietary Nano-ZnO raised the Zn concentrations in the tibia, serum, and liver of IUGR pigs, which indicated that dietary Nano-ZnO might increase the intestinal absorption and transport and enhanced the deposition of Zn in tissues (the liver and tibia). However, these organs can be damaged or intoxicated when Zn deposition exceeds the tolerance range, especially for the liver [10]. The histomorphology and serum activities of GOP, GTP, and AKP are the sensitive and common indicators for evaluating the hepatic damage induced by high Zn [13, 37]. Interestingly, dietary Nano-ZnO showed no impact on the hepatic histomorphology and serum GOP, GTP, and AKP activities of IUGR pigs in our current study, suggesting that long-term (from 21 to 163 days of age) dietary Nano-ZnO did not induce obvious damage in the liver of IUGR finishing pigs, despite the increased Zn deposition in the liver, the tibia, and serum. This may be connected with the high tolerance of Zn in pigs, and the increased Zn did not reach the extent to induce hepatic damage [38]. Similarly, dietary 800 mg Nano-ZnO/kg or 1200 mg Nano-ZnO/kg for 14 days increased the Zn concentrations of plasma, the liver, and the tibia in piglets, but did not observably change the serum lactate dehydrogenase, GOT, and GPT activities [13, 14]. Kiciova et al. [39] also reported that the basal diet with 500, 1000, and 2000 mg/kg Zn from zinc phosphate-based nanoparticles for 10 days did not show obvious damage on the liver of piglets.
It is supposed that Zn, as an all-important element for normal physiological function and metabolism of the organism, might also show antagonistic effects on some other elements, including Cu, Fe, and Mn [40, 41]. Long-term adding excessive Zn into diets might potentially affect the absorption and deposition of Cu, Fe, and Mn [42, 43]. However, in the current study, dietary Nano-ZnO (600 mg Zn/kg) for 142 days did not markedly alter the Mn, Cu, and Fe concentrations of serum, tissues (leg muscle, the liver, and the tibia), and feces in the IUGR pigs, which agrees with our previous study [13]. Moreover, the mRNA expressions of Fe transport-related genes (TFR1 and FPN1) and Cu transport-related genes (CRT1, ATP7A, and ATP7B) in jejunal mucosa were also not observably changed by dietary Nano-ZnO in our current study. These results suggested that Nano-ZnO did not affect the absorption from intestine or deposition in the selected tissues of Cu, Fe, and Mn. Rincker et al. [44] also reported that dietary 2 g Zn/kg from zinc oxide or zinc methionine for 14 days did not affect the Cu and Fe concentrations in serum and Cu, Fe, and Mn concentrations of whole-body in nursery pigs. One potential reason for no antagonistic effects on the metabolism of other minerals (Fe, Mn, and Cu) here might be due to the improved intestinal morphology and enhanced nutrient absorption of pigs by Nano-ZnO supplementation [19].
We further explored the possible mechanisms by which Nano-ZnO supplementation increased Zn deposition in tissues (the liver and tibia) of IUGR pigs. Our results showed that dietary Nano-ZnO up-regulated the mRNA expression of MTF1, MT (MT1A and MT2A), ZnT family (ZnT1 and ZnT4), ZIP family (ZIP4, ZIP8, and ZIP14), and DMT1 and protein expression of ZIP8 in the jejunal mucosa of IUGR pigs. Mammalian Zn transporters largely fall into the ZnT (SLC30) and ZIP (SLC39) families, which mediate Zn efflux from enterocytes and transport extracellular Zn into the cytoplasm, respectively [45, 46]. MT, an intracellular Zn-binding protein in the intestinal mucosa, is induced into expression by excessive amounts of Zn in the diet and has the ability to excrete excess Zn [33, 47, 48]. Moreover, the DMT1 expresses in the intestinal epithelium and mainly transports divalent metal cations [49, 50]. The MTF1 is involved in the regulation of Zn by inducing the expression of MT and ZnT1 [51]. Therefore, these results indicated that dietary Nano-ZnO might promote Zn into the blood through the jejunum and raise Zn deposition in tissues via enhancing the Zn metabolism-related genes (MT, ZIP, and ZnT families) and ZIP8 protein expression in the jejunal mucosa of IUGR pigs. Similarly, Ling et al. [52] and Chen et al. [53] reported that Nano-ZnO incubation up-regulated mRNA expression of Zn transport genes (MT, MTF1, and ZIP and ZnT families) in the intestinal epithelial cells of yellow catfish. Moreover, as reported by Melia et al. [54], our immunohistochemical results revealed that ZIP8 was mainly expressed in cells of jejunal villous brush border, which are important sites for nutrient absorption in the intestine [55], and MT2A was mainly expressed in villus lamina propria and gland/crypt of the jejunum. The lamina propria is rich in blood and lymphatic vessels to facilitate nutrient efflux into blood/lymph [55]. That dietary Nano-ZnO enhanced the ZIP8 and MT2A in our immunohistochemical analysis further proved that Nano-ZnO could promote Zn transport from the intestine into the blood. Therefore, it can be concluded that the increased Zn deposition in serum and tissues of IUGR pigs may, at least partially, be attributed to the enhanced Zn transport from the jejunal lumen via the regulation of MT, ZnT and ZIP families [45].
Due to the special particle size effects, some previous studies supposed that nanoparticles might be absorbed by intestine epithelial endocytosis [56,57,58], which closely mediated by the endocytosis-related genes. For example, the Eps15 promotes formation of coated vesicle through interaction with adapter protein-2 [59]. The DNM1 and DNM2, as GTPase enzymes in mammals, can detach newly formed vesicles from membranes, and Cav1 and Cav2, involved in invagination of lipid raft domain, are major coat proteins of caveolae [60]. However, in our present study, dietary added Nano-ZnO did not markedly affect the mRNA expression of endocytosis-related genes in the jejunal mucosa of IUGR pigs, including Eps15, DNM1, DNM2, Cav1, and Cav2, suggesting that dietary added Nano-ZnO did not affect the formation and isolation of endocytic vesicles in the jejunal cytomembrane of IUGR pigs. Consistently, the intact Nano-ZnOs (black particles, 20–40 nm) in cells of the jejunum, liver, and muscle in IUGR pigs were not observed in the current study, which further confirmed that Nano-ZnO was not absorbed as the form of intact Nano-ZnO by endocytosis in the jejunum of IUGR pigs. However, Chen et al. [53] reported that dietary Nano-ZnO could be absorbed through intestinal clathrin- and Cav1-mediated endocytosis in yellow catfish. The different effects of Nano-ZnO on the intestinal endocytosis might be due to the difference between catfish and pigs. However, the related mechanism still needs more researches in future.
Conclusion
In summary, our results indicated that IUGR finishing pigs showed catch-up growth in the weight of selected organs (the heart, liver, spleen, pancreas, and kidney), and mineral metabolism and long-term dietary 600 mg Zn/kg from Nano-ZnO had no obvious side effects on the development of the organs, liver function, and metabolism of Cu, Fe, and Mn in IUGR pigs. However, the Zn absorption in the jejunum and deposition in the liver and tibia were increased via enhancing the expression of transporters (MT, ZIP, and ZnT families) in the jejunum by dietary Nano-ZnO, rather than via endocytosis as the form of intact nanoparticles.
Data Availability
The data and materials of this study are available from the corresponding author upon reasonable request.
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This study was supported by the National Natural Science Foundation of China (No.31972598).
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Conceived and designed the experiment by Chao Wang and Tian Wang. Experiment preparation and data collection were performed by Binbin Zhou, Jian Li, Shun Chen, Huijuan Liu, Jiaqi Zhang, and Yudan He. Analyzed the data by Binbin Zhou. The first draft of the manuscript was writing by Binbin Zhou. Review and editing the first draft by Chao Wang. All authors have read and agreed to the published version of the manuscript.
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Zhou, B., Li, J., Zhang, J. et al. Effects of Long-Term Dietary Zinc Oxide Nanoparticle on Liver Function, Deposition, and Absorption of Trace Minerals in Intrauterine Growth Retardation Pigs. Biol Trace Elem Res 201, 4746–4757 (2023). https://doi.org/10.1007/s12011-022-03547-2
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DOI: https://doi.org/10.1007/s12011-022-03547-2