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 (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 liver, muscle and 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 liver, 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, liver and 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's 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, liver and 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 (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 glands/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 [56–58], which closely mediated by the endocytosis-related genes. For example, the Eps15 promotes formation of coated vesicle through interaction with adapter protein-2 [59]. 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 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.