Long-term Cu exposure alters CYP450s activity and induces jejunum injury and apoptosis in broilers.

Copper (Cu) is an essential trace element that plays a crucial role in numerous physiopathological processes related to human and animal health. In the poultry industry, Cu is used to promote growth as a feed supplement, but excessive use can lead to toxicity on animals. Cytochrome P450 enzymes (CYP450s) are a superfamily of proteins that require heme as a cofactor and are essential for the metabolism of xenobiotic compounds. The purpose of this study was to explore the influence of exposure to Cu on CYP450s activity and apoptosis in the jejunum of broilers. Hence, we first simulated the Cu exposure model by feeding chickens diets containing different amounts of Cu. In the present study, histopathological observations have revealed morphological damage to the jejunum. The expression levels of genes and proteins of intestinal barrier markers were prominently downregulated. While the mRNA expression level of the gene associated with CYP450s was significantly increased. Additionally, apoptosis-related genes and proteins (Bak1, Bax, Caspase-9, Caspase-3, and CytC) were also significantly augmented by excessive Cu, while simultaneously decreasing the expression of Bcl-2. It can be concluded that long-term Cu exposure affects CYP450s activity, disrupts intestinal barrier function, and causes apoptosis in broilers that ultimately leads to jejunum damage.

Effect of Copper Exposure on the Cholesterol Metabolism in Broiler Liver.

Copper (Cu) is a kind of widely used dietary supplement in poultry production, and a common environmental pollutant at the same time. Excess Cu exposure has been reported to accumulate in the liver and induce cytotoxicity, but the effect of Cu toxicity on hepatic cholesterol metabolism is still uncertain. Herein, we aimed to reveal the effect of excess Cu on the liver and primary hepatocytes of broilers at various concentrations. We found that 110 mg/kg Cu supplement remarkably increased blood cholesterol levels by detecting serum TC, LDL-C, and HDL-C in the broilers, while there was no significant difference in 220 and 330 mg/kg Cu supplements. In addition, high Cu exposure resulted in severe hepatic steatosis and hepatic cord derangement in the broilers. Oil red O staining of primary hepatocytes showed that Cu treatment caused intracellular neutral lipid accumulation. However, the hepatic TC content indicated a downward trend in both liver tissues and hepatocytes after Cu exposure. Furthermore, the expression of cholesterol metabolism-related indicators (SREBP2, HMGCR, LDLR, and CYP7A1) was notably decreased in the Cu-treated groups. While the expression of the key enzyme of cholesterol esterification (ACAT2) did not change significantly. Taken together, our findings preliminarily revealed excess Cu-induced hepatic cholesterol metabolism dysfunction, providing a deeper understanding of the molecular mechanisms of Cu-induced hepatotoxicity.

Toxicological mechanism of large amount of copper supplementation: Effects on endoplasmic reticulum stress and mitochondria-mediated apoptosis by Nrf2/HO-1 pathway-induced oxidative stress in the porcine myocardium.

Copper (Cu) is an essential micronutrient that is required by all living organisms. However, Cu can also be a potentially toxic metal if excessive dietary supplementation occurs. The current study aimed to investigate the mechanism of Cu toxicity in the cardiomyocytes of large mammal pigs. Here, we used pigs to explore Cu toxicity in the control group (10 mg/kg Cu) and treatment groups (125 mg/kg and 250 mg/kg Cu) for a period of 80 days. Consequently, we identified that large amount intake of Cu led to in oxidative damage, and activation of the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1)-mediated antioxidant pathway, indicating an imbalanced redox status in the myocardium. Furthermore, Cu exposure activated endoplasmic reticulum (ER) stress through upregulating levels of glucose-regulated protein 78 (GRP78), c-Jun N-terminal kinase (JNK), glucose-regulated protein 94 (GRP94), X-box binding protein 1 (XBP1), and C/EBP homologous protein (CHOP). Additionally, mitochondrial fission and fusion homeostasis was disrupted and the copy number of mitochondrial DNA (mtDNA) was reduced under Cu exposure. Furthermore, Cu exposure could induce apoptosis, evidenced by the increased terminal deoxynucleotidyl transferase biotin-d UTP nick end labeling (TUNEL)-positive staining, the upregulated expression levels of Cytoplasm-cytochrome C (Cytc), Bcl-2-associated X protein (Bax), and Cleaved-caspase3, and decreased expression level of B-cell lymphoma-2 (Bcl-2) and Mitochondrial-cytc. In summary, large amount of Cu could trigger Nrf2/HO-1 pathway-mediated oxidative stress, which promotes ER stress and mitochondrial damage pathways, causing apoptosis in cardiomyocytes.

Toxic effects of copper on the jejunum and colon of pigs: mechanisms related to gut barrier dysfunction and inflammation influenced by the gut microbiota.

Copper (Cu) is an essential trace mineral, but its excessive intake can lead to potentially toxic effects on host physiology. The mammalian intestine harbors various microorganisms that are associated with intestinal barrier function and inflammation. In this study, the influences of Cu on barrier function, microbiota, and its metabolites were examined in the jejunum and colon of pigs. Here, we identified that the physical and chemical barrier functions were impaired both in the jejunum and colon, as evidenced by the decreased expression of tight junction proteins (ZO-1, Occludin, Claudin-1, and JAM-1) and mucous secretion-related genes, positive rate of Muc2, and secretion of SIgA and SIgG. Additionally, inflammatory cytokines were overexpressed in the jejunum and colon. Furthermore, Cu might increase the abundances of Mycoplasma, Actinobacillus and unidentified_Enterobacteriaceae in the jejunum, which significantly affected pentose and glucoronate interconversions, histidine metabolism, folate biosynthesis, porphyrin metabolism, and purine metabolism. Meanwhile, the abundances of Lactobacillus and Methanobrevibacter were remarkably decreased and Streptococcus, unidentified_Enterobacteriaceae, and unidentified_Muribaculaceae were significantly increased in the colon, with an evident impact on glycerophospholipid metabolism, retinol metabolism, and steroid hormone biosynthesis. These findings revealed that excess Cu had significant effects on the microbiota and metabolites in the jejunum and colon, which were involved in intestinal barrier dysfunction and inflammation.

Copper induces mitochondria-mediated apoptosis via AMPK-mTOR pathway in hypothalamus of Pigs.

Copper (Cu), one of the heavy metals, is far beyond the carrying capacity of the environment with Cu mining, industrial wastewater discharging and the use of Cu-containing pesticides. Intaking excess Cu can cause toxic effects on liver, kidney, heart, but few studies report Cu toxicity on brain tissue. It is noteworthy that most toxicity tests are based on rodent models, but large mammals chosen as animal models has no reported. To explore the relationship of the Cu toxicity and mitochondria-mediated apoptosis on hypothalamus in pigs, the content of Cu, histomorphology, mitochondrial related indicators, apoptosis, and AMPK-mTOR signaling pathway were detected. Results showed that Cu could accumulate in hypothalamus and lead to mitochondrial dysfunction, evidenced by the decrease of ATP production, activities of respiratory chain complex I-IV, and mitochondrial respiratory function in Cu-treated groups. Additionally, the genes and proteins expression of Bax, Caspase-3, Cytc in treatment group were higher than control group. Furthermore, the protein level of p-AMPK was enhanced significantly and p-mTOR was declined, which manifested that AMPK-mTOR signaling pathway was activated in Cu-treated groups. In conclusion, this study illuminated that the accumulation of Cu could cause mitochondrial dysfunction, induce mitochondria-mediated apoptosis and activate AMPK-mTOR pathway in hypothalamus.

Effects of copper on oxidative stress and autophagy in hypothalamus of broilers.

The purpose of this research was to discuss the effects of copper (Cu)-induced toxicity on oxidative stress and autophagy in hypothalamus of broilers. In this study, 240 one-day-old broilers were randomly divided into 4 groups and the contents of dietary Cu in 4 groups were 11 mg/kg (control group), 110 mg/kg (group I), 220 mg/kg (group II), and 330 mg/kg (group III). The experiment lasted for 49 days and the hypothalamus tissues were collected for histological observation and detection of Cu content. Additionally, the indicators related to oxidative stress in hypothalamus were determined. Moreover, the mRNA expression levels of autophagy-related genes and the protein expression levels of Beclin1, LC3-II/LC3-I, and p62 in hypothalamus were measured. Results showed that the treated groups were observed vacuolar degeneration in hypothalamus compared to control group, and the Cu content in hypothalamus was increased with the increase of dietary Cu. Furthermore, the activities of SOD, CAT, T-AOC were increased in group I and group II and then decreased in group III, and the content of MDA and the mRNA levels of Nrf2, HO-1, SOD-1, CAT, GCLC, GCLM, and GST in treated groups were elevated compared to control group. Moreover, the mRNA expression levels of Beclin1, Atg5, LC3-I, LC3-II and the protein expression levels of Beclin1 and LC3-II/LC3-I up-regulated significantly with the increasing levels of Cu. However, the mRNA expression levels of p62 and mTOR and the protein expression level of p62 down-regulated remarkably. Taken together, our present study evidenced that excessive intake of Cu could induce oxidative stress and autophagy in hypothalamus of broilers.

Liver mitochondrial dysfunction and electron transport chain defect induced by high dietary copper in broilers.

Copper is an important trace mineral in the diet of poultry due to its biological activity. However, limited information is available concerning the effects of high copper on mitochondrial dysfunction. In this study, 72 broilers were used to investigate the effects of high dietary copper on liver mitochondrial dysfunction and electron transport chain defect. Birds were fed with different concentrations [11, 110, 220, and 330 mg of copper/kg dry matter (DM)] of copper from tribasic copper chloride (TBCC). The experiment lasted for 60 d. Liver tissues on d 60 were subjected to histopathological observation. Additionally, liver mitochondrial function was recorded on d 12, 36, and 60. Moreover, a site-specific defect in the electron transport chain in liver mitochondria was also identified by using various chemical inhibitors of mitochondrial respiration. The results showed different degrees of degeneration, mitochondrial swelling, and high-density electrons in hepatocytes. In addition, the respiratory control ratio (RCR) and oxidative phosphorylation rate (OPR) in liver mitochondria increased at first and then decreased in high-dose groups. Moreover, hydrogen peroxide (H2O2) generation velocity in treated groups was higher than that in control group, which were magnified by inhibiting electron transport at Complex IV. The results indicated that high dietary copper could decline liver mitochondrial function in broilers. The presence of a site-specific defect at Complex IV in liver mitochondria may be responsible for liver mitochondrial dysfunction caused by high dietary copper.

Mitochondrial Protein Profile in Mice with Low or Excessive Selenium Diets.

Dietary selenium putatively prevents oxidative damage, whereas excessive selenium may lead to animal disorder. In this study, we investigated the effects of low and excessive levels of dietary selenium on oxidative stress and mitochondrial proteins in mouse liver. Six to eight week old mice were fed a diet with low, excessive, or moderate (control) levels of selenium (sodium selenite). The selenium concentration and oxidative stress-related parameters in hepatic mitochondria were evaluated. Two-dimensional electrophoresis and mass spectrometry were applied to identify the differentially-expressed proteins associated with dietary selenium. The selenium content of the livers in mice with the low selenium diet was significantly lower than that of the control, while that of mice fed excessive levels was significantly higher. In both groups oxidative stress in hepatic mitochondria was found; accompanied by lower superoxide dismutase (SOD) and glutathione peroxidase (GPX) levels and higher malondialdehyde (MDA) content, compared with the control group. Furthermore, ten proteins in the hepatic mitochondria of the selenium-low or -excessive groups with more than two-fold differences in abundance compared with the control group were identified. The differentially-expressed proteins in hepatic mitochondria may be associated with dietary (low or excessive) selenium-induced oxidative stress.

The Protective Roles of Selenium on Hepatic Tissue Ultrastructure and Mitochondrial Antioxidant Capacity in Copper-Overloaded Rats.

The aim of this study was to explore the effects of selenium addition on hepatic tissue ultrastructure and mitochondrial antioxidant capacity in copper-overloaded rats. Eighteen adult male Sprague-Dawley (SD) rats were randomly divided into three groups (n = 6 per group). Each group received 1 mL (intragastrically) of water (control, group I) or water containing copper chloride (CuCl2, 1 mol/L) (group II) or a mixture of CuCl2 (1 mol/L) with sodium selenite (Na2SeO3, 0.05 mol/L) (group III) once daily for 30 days. Histological examination revealed normal hepatocyte structure and no ultrastructural changes in mitochondria in controls. In contrast, group II exhibited severe ultrastructural alterations, fuzzy mitochondrial membranes, irregularly shaped and fragmented rough endoplasmic reticulum (RER), and the high melanin content; group III also exhibited larger amounts of engulfing vesicles (EV) in the cytoplasm. Compared to controls, the CuCl2 treatment lowered (P < 0.05) hepatocyte enzymatic activities and increased lipid peroxidation as indicated by an increase in malondialdehyde (MDA) levels. The effects of CuCl2 were attenuated by simultaneous administration with Na2SeO3. These results indicated that the adverse effects of copper toxicity can be partially attenuated by providing a source of selenium.

Effects of dietary zinc oxide nanoparticles on growth performance and antioxidative status in broilers.

Broilers in four groups were fed a basal diet supplemented with 60 mg/kg zinc oxide (60-ZnO; control), or 20, 60, or 100 mg/kg ZnO nanoparticles (20-, 60-, and 100-nano-ZnO, respectively). Compared with the controls, after 14 days, birds in the 20- and 60-nano-ZnO groups had significantly greater weight gains and better feed conversion ratios. However, the body weight of birds in the 100-nano-ZnO group was dramatically reduced after 28 days. Relative to the control group, the total antioxidant capability (T-AOC) in serum and liver tissue was significantly higher in the 20-nano-ZnO group at all time points and also significantly higher in the 60- and 100-nano-ZnO groups in serum on days 28 and 35 and in liver tissues on days 21 and 28. Compared with the controls, the activity of copper-zinc superoxide dismutase (Cu-Zn-SOD) was significantly greater in the 60- and 100-nano-ZnO groups in serum on days 28 and 35 and in liver tissues after 21 days. Catalase activity in serum samples was significantly higher in the 20- and 60-nano-ZnO groups relative to the control and 100-nano-ZnO birds, but catalase activity in liver tissue was not affected by different nano-ZnO levels. Malondialdehyde content in serum and liver tissues was significantly reduced in the 20-, 60-, and 100-nano-ZnO groups compared with that in the control group at all time points except day 42. Taken together, our data indicate that appropriate concentration of dietary ZnO nanoparticles improves growth performance and antioxidative capabilities in broilers, and 20 mg/kg nano-ZnO is the optimal concentration.

L-arginine prevents reduced expression of endothelial nitric oxide synthase (NOS) in pulmonary arterioles of broilers exposed to cool temperatures.

This study investigated nitric oxide synthase (NOS) expression in the endothelium of pulmonary arterioles of broilers during the development of pulmonary hypertension syndrome (PHS). PHS was triggered by exposing broilers to sub-thermoneutral (cool) temperatures and an additional 1.0% L-arginine was added to the basal diet to evaluate the effects of supplemental L-arginine on nitric oxide (NO) production, endothelial NOS expression, and the incidence of PHS. Cumulative mortality from PHS, right/total ventricle weight ratios (RV/TV), and body weights were recorded. Plasma NO concentration and NOS expression in the endothelium of pulmonary arterioles with an outer diameter ranging from 100 to 200 microm were determined. Birds exposed to cool temperatures had increased pulmonary hypertension and PHS mortality and diminished endothelial NOS expression. Supplemental dietary L-arginine reduced PHS mortality and elicited higher NOS expression within the pulmonary endothelium coincident with elevated NO production. The results demonstrated that broilers developing PHS exhibited diminished NOS expression in the endothelium of their pulmonary arterioles. Supplemental L-arginine prevented the reduced expression of NOS in the pulmonary endothelium, which might contribute to the increased production of NO by the pulmonary vasculature.

L-Arginine inhibiting pulmonary vascular remodelling is associated with promotion of apoptosis in pulmonary arterioles smooth muscle cells in broilers.

OBJECTIVE: Pulmonary vascular remodelling is one of the important pathological bases of broiler pulmonary hypertension syndrome (PHS). Nitric oxide (NO) has been found to inhibit proliferation and to induce apoptosis in pulmonary artery smooth muscle cells (SMC) in mammals with pulmonary hypertension. The present study was conducted to evaluate the effects of NO precursor l-arginine on pulmonary vascular remodelling in broilers with pulmonary hypertension induced by cold exposure and to examine whether NO-induced apoptosis in pulmonary arteriole SMC is involved in the regulatory mechanisms. METHODS: Two hundred and forty mixed-sex commercial broilers were equally assigned to three groups and reared in normal brooding temperatures before day 14. Starting on day 14 continuing until the end of the experiment, the control group was brooded in normal temperatures whereas the other two groups were subjected to low ambient temperatures with or without l-arginine added to the basal diets. Cumulative PHS mortality and body weight were recorded in each group. Right/total ventricle ratio (RV/TV), plasma NO concentration and pulmonary vascular morphological changes were analyzed. TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay was used to detect apoptosis in pulmonary arteriole SMC. RESULT: l-Arginine, in group A, had no effect on body weights under cold temperature condition. Birds kept in group B had increased PHS mortality, RV/TV ratio, vessel wall area/vessel total area ratios (WA/TA) and mean media thickness in pulmonary arterioles (mMTPA) (P<0.05). Percentages of apoptotic SMC in pulmonary arterioles in group B were not altered by cold exposure (P>0.05). Supplemental dietary l-arginine in group A elevated plasma NO level (P<0.05), reduced PHS mortality (P<0.05), attenuated pulmonary vascular remodelling and increased the percentages of apoptotic SMC (P<0.05) when compared with the group B. CONCLUSION: Supplemental l-arginine partially inhibited pulmonary vascular remodelling that occurred secondary to increased pulmonary pressure; NO-induced apoptosis in arteriole SMC might contribute to its regulatory effect on pulmonary vascular structural changes.