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Copper induced oxidative stress in porcine small intestinal epithelial (IPEC-J2) cells. Cells were treated with CuSO4, Cu-Gly, and Cu-Pro at the concentrations of 30 and 120 μM for 10 h. (a) Total superoxide dismutase (SOD) activity was assessed using an assay kit. The result was expressed as a percentage of the control. (b) SOD1 protein expression levels were measured by Western blotting. The densitometry of blot images normalized to β-actin levels for each lane. (c) Reactive oxygen species (ROS) was determined by using 2, 7-dichlorofluorescin diacetate (DCFH-DA). (d) Malondialdehyde (MDA) was measured by an acid reactive substance (TBARS) assay. The result was corrected with the total protein content of cells. Data represent mean values ± standard deviation (n = 3). Significant differences between processing groups are represented by different lowercase letters (p < 0.05).
Source publication
Copper (Cu) is widely used in the swine industry to improve the growth performance of pigs. However, high doses of copper will induce cell damage and toxicity. The aim of this study was to evaluate toxicity, bioavailability, and effects on metabolic processes of varying copper sources using porcine intestinal epithelial cells (IPEC-J2) as a model....
Citations
... In a meta-analysis by Prabhu et al. (2016), it was highlighted that organic forms of Se, like selenomethionine (SeMet) and selenoyeast (Se yeast), are more bioavailable compared to selenite; however, for other trace minerals like Zn and Mn the results of the different studies are conflicting (Prabhu et al., 2019;Silva et al., 2019;Meiler et al., 2021). Li et al. (2019) saw different metabolic patterns, including increased expression of the peptide transporter gene PepT1 and decrease in specific divalent mineral transporter gene CTR1 when porcine intestinal epithelial model cells (IPEC-J2) were provided media with increasing levels of different (inorganic vs. proteinates) Cu sources. ...
The aquafeed ingredient inventory is ever changing, from marine to plant based, and recently evolving to incorporate increasing amounts of low trophic, side stream and circular economy based raw materials, each one contributing with variable amounts and qualities of macro- and micronutrients. Meeting the micronutrient requirement of farmed fish for healthy and efficient growth under normal and challenging conditions is of paramount importance. In this study we run a trial based on a 2 × 4 factorial design with three replications for each dietary treatment, where Atlantic salmon smolt were fed one of 8 experimental diets supplemented with either organic or inorganic mineral premixes (copper, iron, manganese, selenium, and zinc) at four dietary inclusion levels. We saw a trend for higher growth rate in the organic mineral groups irrespective of the dietary mineral levels. Mineral digestibility was negatively correlated with increasing mineral supplementation levels for all tested minerals but Se which increased with the increasing supplementation in the inorganic and up to the 2nd inclusion level in the organic mineral groups. Increasing mineral supplementation affected retention efficiency of Zn, Mn, Cu and Fe while mineral source affected only the retention of Se which was higher in the organic mineral groups. Moreover, fish obtained higher EPA and DHA in their body and increased slaughter yield in the organic as compared to the inorganic mineral groups and corroborated that trace mineral inclusion levels play a key role on salmon fillet's technical quality. More effects from different origin and dietary inclusion levels of trace minerals were seen on fillet yield, fillet technical and nutritional quality, bone strength, skin morphology, organ mineralization and midgut transcriptome.
... In a meta-analysis by Prabhu et al. (2016), it was highlighted that organic forms of Se, like selenomethionine (SeMet) and selenoyeast (Se yeast), are more bioavailable compared to selenite; however, for other trace minerals like Zn and Mn the results of the different studies are conflicting (Prabhu et al., 2019;Silva et al., 2019;Meiler et al., 2021). Li et al. (2019) saw different metabolic patterns, including increased expression of the peptide transporter gene PepT1 and decrease in specific divalent mineral transporter gene CTR1 when porcine intestinal epithelial model cells (IPEC-J2) were provided media with increasing levels of different (inorganic vs. proteinates) Cu sources. ...
... The negative correlations between hair Cu with serum MDA and TBA were also observed in our study. The MDA content reflects the severity of body exposure to free radicals, which are crucial biomarkers in the oxidative stress process 30 . In our previous study, the serum MDA was decreased in 20 mg·kg −1 copper diet compared with 6 and 300 mg·kg −1 copper diet, suggesting that dietary 20 mg·kg −1 copper could effectively protect tissues from oxidative damage 8 (Supplementary Table S1). ...
Ionomics-metabolomics association analysis is a novel method to elucidating the potential mechanisms underlying the effects of dietary copper on the overall health parameters of suckling piglets model. Few studies have elucidated the relationship between the changes of ionic and metabolic homeostasis responses to dietary copper level. The growth performance data was obtained from 180 suckling piglets which access to different copper levels: 6 (low copper diet, LC), 20 (control diet, CON), and 300 (high copper diet, HC) mg·kg⁻¹ copper (based on diet, supplementation from CuSO4), and offered ad libitum from d 14 until weaning at 40 d of age. Dietary high level copper (300 mg·kg⁻¹) increased the ADG and ADFI during d 14 to 28 of piglets. Six elements (Mg, Na, K, P, Cu, and Mn) concentrations significantly changes in hair among the three treatment diets. The significant increased concentrations of Na and K, and decreased concentration of Mg and Mn in 300 mg·kg⁻¹ than 20 mg·kg⁻¹ copper diet was observed. In current study, with the increase in copper level from 20 to 300 mg·kg⁻¹ in diet, the correlation between hair Na, K and Cu, Mn, Zn vanish. Hair Na and K were positively correlated with serum total antioxidant capacity (T-AOC) and negatively correlated with tumor necrosis factor-α (TNF-α). The hair Cu was negatively correlated with serum malondialdehyde (MDA), total bile acid (TBA). The fecal Cu was positively correlated with serum growth hormone (GH). The results suggested that the average daily gain (ADG) in 6 mg·kg⁻¹ copper diet and the average daily feed intake (ADFI) in 20 mg·kg⁻¹ copper diet were decreased than 300 mg·kg⁻¹ copper diet during d 14 to 28 and the ADG was decreased in 6 and 20 mg·kg⁻¹ copper diets in d 29 to 40 of piglets. Dietary 20 mg·kg⁻¹ copper maintain ion homeostasis due to increase the number of positive correlations between macroelements-microelements in hair and serum. Significantly changed Na, K, Mg, Mn and Cu concentrations in hair can reflect the adverse effects of dietary 300 mg·kg⁻¹ copper of suckling piglets. We believe our results may benefit people to gain a better understanding of the ion interactions and metabolic homeostasis of heavy metal elements that are critical to human and animal health.
... As a result, toxic substances such as heavy metals enter the water systems of large cities, where they can pose a risk to human health and wildlife (Banfalvi, 2011). For example, copper in amounts exceeding the permissible concentrations can cause stomach and intestinal upset, kidney depression, anemia, and toxic damage to cells throughout the body (Abu Al-Rub et al., 2006;Li et al., 2019;Malhotra et al., 2020). ...
The use of microalgae Chlorella kessleri VKPM A1-11 ARM (RF, NPO Algobiotechnology) for environmental and energy purposes is considered. The results of our study of the use of C. kessleri microalgae biomass as a biosorbent to purify model wastewater from Cu2+ ions under static conditions are presented. Biosorption is a promising technology for the treatment of industrial effluents containing various heavy metal compounds, but the issues of economic benefits of using biosorbents, their environmental safety and the cost of disposal of used sorbents are subject to much discussion. The paper proposes to dispose the used biosorbent formed after wastewater treatment from copper as an additional fuel. The copper concentration in the filtrate was determined by colorimetric analysis with sodium diethyldithiocarbamate. The cleaning efficiency and sorption capacity of the dry mass of C. kessleri were obtained by calculation. The maximum sorption capacity for Cu2+ ions was 4.2 mg/g. The purification efficiency reached 87% at the initial concentration of Cu2+ ions being 97 mg/l. Tests to estimate the specific heat of combustion of C. kessleri biomass and used biosorbents based thereon were carried out by the calorimetric method using a bomb calorimeter. The specific heats of combustion were 22,125 kJ/kg and 21,674 kJ/kg, respectively. A comparison of these values with traditional energy carriers is given. A technological scheme has been developed for a waste-free cycle of using C. kessleri to treat wastewater from industrial enterprises with the production of several valuable resources as end products, such as purified water, energy resources, fertilizers, and recycled metals. The obtained results of our study can be applied in technologies for post-treatment of wastewater from various industrial enterprises using biological non-waste resources.
... Several studies have demonstrated that organic Cu, such as Cu−glycine and Cu−proteinate, has greater bioavailability compared to inorganic Cu; 75,76 for example, Wen et al. 17 reported that organic Cu provides benefits to intestinal microbiota and reduces Cu excretion in pigs compared with inorganic Cu. Li et al. 77 also found that organic Cu is less toxic and has a higher absorption efficiency compared with inorganic Cu. Copper−proteinate is also reported to reduce blood cholesterol and improve health conditions in broilers. ...
The extensive use of high-concentration copper (Cu) in feed additives, fertilizers, pesticides, and nanoparticles (NPs) inevitably causes significant pollution in the ecological environment. This type of chain pollution begins with animal husbandry: first, Cu accumulation in animals poisons them; second, high Cu enters the soil and water sources with the feces and urine to cause toxicity, which may further lead to crop and plant pollution; third, this process ultimately endangers human health through consumption of livestock products, aquatic foods, plants, and even drinking water. High Cu potentially alters the antibiotic resistance of soil and water sources and further aggravates human disease risks. Thus, it is necessary to formulate reasonable Cu emission regulations because the benefits of Cu for livestock and plants cannot be ignored. The present review evaluates the potential hazards and benefits of high Cu in livestock, the environment, the plant industry, and human health. We also discuss aspects related to bacterial and fungal resistance and homeostasis and perspectives on the application of Cu-NPs and microbial high-Cu removal technology to reduce the spread of toxicity risks to humans.
... When cultured, IPEC-J2 and IPEC-1 form a polarized monolayer with tight junctions (TJ), and microvilli located on their apical side [20] and moreover develop extensive metabolic functions. Those features allow measurements of barrier function and nutrient transport [21] as well as investigations on the impact of external challenges as mycotoxins [22], microbial pathogens [10], and their toxins [23,24], heavy metals such as zinc [25] and copper [26]. Gene expression analysis of both cell lines revealed that IPEC-J2 is higher differentiated in morphology e.g., developing a two-fold higher number of microvilli and villin, as compared to IPEC-1, which indicates a more efficient crosslinking between microvilli [21]. ...
Numerous bioactive plant additives have shown various positive effects in pigs and chickens. The demand for feed additives of natural origin has increased rapidly in recent years to support the health of farm animals and thus minimize the need for antibiotics and other drugs. Although only in vivo experiments can fully represent their effect on the organism, the establishment of reliable in vitro methods is becoming increasingly important in the goal of reducing the use of animals in experiments. The use of cell models requires strict control of the experimental conditions so that reliability and reproducibility can be achieved. In particular, the intestinal porcine epithelial cell line IPEC-J2 represents a promising model for the development of new additives. It offers the possibility to investigate antioxidative, antimicrobial, anti- or pro-proliferative and antiviral effects. However, the use of IPEC-J2 is limited due to its purely epithelial origin and some differences in its morphology and functionality compared to the in vivo situation. With regard to chickens, the development of a reliable intestinal epithelial cell model has attracted the attention of researchers in recent years. Although a promising model was presented lately, further studies are needed to enable the standardized use of a chicken cell line for testing phytogenic feed additives. Finally, co-cultivation of the currently available cell lines with other cell lines and the development of organoids will open up further application possibilities. Special emphasis was given to the IPEC-J2 cell model. Therefore, all publications that investigated plant derived compounds in this cell line were considered. The section on chicken cell lines is based on publications describing the development of chicken intestinal epithelial cell models.
... The affinity of Met to Cu(I) is higher than that of Gly [38]. The enhancement of the endocellular generation of the reactive oxygen forms upon the introduction of Cu(Gly) 2 and the development of oxidative stress have been observed in vitro study of toxicity of different copper sources [39]. ...
... The organs, tissues, blood, feces, urine and diets were analyzed for Cu content using inductively coupled plasma-mass spectroscopy (ICP-MS, Agilent) according to methods described by Li et al. (2020). The short-chain fatty acids in colonic digesta were determined by an ion chromatographic analyzer (ICS-3000, Dionex, U.S.) according to the methods described by Han et al. (2019). ...
... The primers used for quantifying selected genes are listed in Appendix Table 2. Firstly, 30 to 40 mg of sample were homogenized at 4 C. The system for RNA extraction, reverse transcription and real-time quantitative PCR was performed according to Li et al. (2020). In addition, b-actin was used as an endogenous control. ...
... PepT1 protein expression was similarly significantly higher for both levels of Cu-pro treatments. Li et al. (2020) also found similar results in IPEC-J2 cells. Two key considerations have been drawn from the research findings here: firstly, that glycine or small peptides carried Cu into the cell by endocytosis; and secondly, Cu-Gly and Cu-Pro underwent dissociation before transport by ASCT2 and PepT1. ...
Copper (Cu) is an essential trace element in the production of swine. This study was conducted to investigate the effect of 3 different sources of Cu on growth performance, Cu metabolism, and intestinal microorganisms of finishing pigs, so as to estimate the bioavailability of the 3 sources for pigs. A total of 42 male finishing pigs (88.74 ± 5.74 kg) were randomly allocated to 7 treatments. The factors were 3 sources (CuSO4, Cu-glycine, Cu-proteinate) and 2 levels (5 and 20 mg/kg) of Cu, plus one negative control treatment (0 mg/kg added Cu level) for the entire 28-d experiment. The average daily gain (ADG) and feed to gain ratio (F:G) both increased when Cu was added. The Cu level in liver, bile, kidney, serum, lung, urine and feces rose (P <0.001) with increasing dietary Cu level regardless of the source. Meanwhile, pigs receiving organic Cu (glycinate or proteinate) retained more Cu and excreted less Cu than those receiving inorganic Cu (CuSO4), which showed that organic forms were more bioavailable. At the transcriptional level, changes in the level and source of dietary Cu resulted in modulation of transporters. In the jejunal mucosa, import transporter high affinity copper uptake protein 1 (CTR1) and export transporter ATPase copper transporting alpha (ATP7A) in supplemental Cu treatments were down-regulated compared to the control. Also, peptide transporter 1 (PepT1) and lanine-serine-cysteine transporter, type-2 (ASCT2) were significantly (P < 0.01) up-regulated in 20 mg/kg Cu-proteinate and Cu-glycinate treatments, respectively. Microbial diversity was lowest in the 20 mg/kg CuSO4 treatment, and the ratio of Firmicutes to Bacteroidetes was higher in added Cu treatments, especially Cu-glycinate treatment. These results indicate that uptake of different Cu forms is facilitated by different transporters and transport mechanisms, and compared with inorganic Cu, organic Cu provides benefits to intestinal microflora and reduces Cu excretion.
... The culture medium was refreshed every two days, and the organoids were passaged every 6-7 days [28,29]. Li et al. treated porcine intestinal epithelial cells (IPEC-J2) with two doses (30 and 120 μmol/L) of CuSO4 [30]; therefore, the porcine organoid was incubated with 10 and 100 μmol/L CuSO4·5H2O (Sangon Biotech, A100330, Shanghai, China) for 72 hours after passage. The organoid activity was determined by organoid budding efficiency, budding crypt depth, and crypts per organoid [31,32]. ...
Copper (Cu) is an essential micronutrient for animals. Many studies have been conducted on the effects of dietary Cu on growth performance, intestinal morphology, and function of piglets. However, the underlying mechanism remains to be explored. Intestinal stem cells (ISC) drive the development and constant renewal of intestinal epithelium. Therefore, we hypothesized that dietary Cu affects piglets’ intestinal development via modulating ISC activity. A total of eighty-five 21-day-old piglets were randomly assigned to five groups, where 25, 50, 75, 100, and 125 mg CuSO4/kg on a dry matter basis were supplemented to the basal diet at phase 1 (day 0 to 21). Increasing the dietary Cu concentration decreased (p < 0.05) villus width but increased (p < 0.001) the number of Ki67-positive cells. At phase 2 (day 22 to 163), the other 45 pigs were offered the same diets. Villus height in the 125 mg/kg Cu group was greater (p < 0.001) than in the other groups. Moreover, the effects of Cu on ISC activity in vitro were tested to explore the underlying mechanism. Compared to the control group, 10 μmol/L CuSO4·5H2O increased (p < 0.001) the organoid budding efficiency, crypt depth, and crypts per organoid. Dietary Cu improved the intestinal morphology of finishing pigs via promoting cell proliferation and modulating ISC activity.
... Cu is a commonplace animal feed additive due to its vital biological significance for animal growth and development. It is an important component of many enzymes and is involved in many important metabolic pathways, including regulating cell respiration, and it is involved, neurotransmitter transmission, anti-stress, anti-oxidation, assimilation of relevant metal ions, and other cellular functions [2][3][4][5][6]. According to the "Safety Standards for Feed Additives" (No. 2625) issued by the Ministry of Agriculture and Rural Affairs of the People's Republic of China, the maximum concentration of Cu in feed is 125 mg/kg for piglets less than 25 kg, while the Cu content in feed for fattening pigs should be in the range of 3 to 6 mg/ kg and cannot exceed 25 mg/kg. ...
... Cu has an important role in the body's normal metabolism [4], and addition of an appropriate amount of Cu to feed can promote animal growth and improve hair color [12]. Many feed manufacturers apparently intend to add an excessive amount of Cu to feed, some exceeding the national feed standard by dozens of times. ...
Background:
Blackened intestines in slaughtered pigs have been commonly observed in China in recent years. However, no cause has been reported.
Objectives:
We attempted to determine whether the blackening of the pig intestine was related to an excess of copper (Cu) in their feed.
Methods:
In this study, we observed and collected porcine intestines in small- and large-scale pig slaughterhouses in Shandong province from May to October 2018. Twelve types of metal ions were detected in the black intestinal samples.
Results:
The Cu level in the intestine samples was mostly higher than the Chinese national limit for food. Further study showed that Cu supplementation in most commercial porcine feed also exceeded the national standard. An animal model (mouse) that could mimic the intestinal blackening in pigs was established. Compared to control mice, Cu accumulated in the liver and intestines of mice fed an excessive Cu level, confirming the excessive Cu in the feed may be considered the major cause of blackened porcine intestines. Microscopic examination revealed that black intestines had many particles containing Cu in the lamina propria of the intestinal mucosa, and the intestinal mucosal epithelial cells showed degeneration and necrosis.
Conclusions:
In conclusion, overuse of Cu in animal feed can lead to animal poisoning and Cu accumulation in animal products. Such overuse not only harms the health of livestock but can also affect public health.
