Metabolomics Reveals the Effects of Copper on Mitochondria-mediated Apoptosis in Kidney of Chicken (Gallus Gallus)

Background: Copper (Cu), a common feed additive in diets for animals, is effective in improving growth performance and feed e�ciency. However, excessive intake of Cu can cause toxic effects. Kidney is the main target organ of Cu, but the relationship between Cu-induced nephrotoxicity and its metabolic process remains unclear. Results: For deeply investigating the nephrotoxicity induced by Cu, a total of 240 broiler chicks were fed with different contents of Cu (11, 110, 220, and 330 mg/kg Cu) for 49 d in this study (60 chicks per group). The results of TUNEL staining showed that Cu could induce apoptosis in kidney with increasing of TUNEL-positive cells. Additionally, a total of 62 differential metabolites were detected by liquid chromatography-mass spectrometry (LC-MS), and mainly enriched in the metabolic pathways including ribo�avin metabolism, glutathione metabolism, sphingolipid metabolism, and glycerophospholipid metabolism, which were closely related to mitochondrial metabolism. Meanwhile, the decrease of mitochondrial membrane potential, impairment of mitochondrial respiratory function, and the increase of mitochondrial membrane permeability indicated that renal mitochondria were damaged by excess Cu. Furthermore, the increase of mRNA and protein levels of Drp1, Bax, Bak-1, CytC, Caspase-3/cleaved Caspase-3 and the decrease of mRNA and protein levels of OPA1, Mfn1, Mfn2, and Bcl-2 con�rmed that Cu could induce mitochondria-mediated apoptosis in kidney. Conclusions: These results highlighted that mitochondrial metabolism could be considered as an important factor in in�uencing Cu toxicity, which for further demonstrating that Cu could induce mitochondria-mediated apoptosis in kidney of broilers.


Background
As a necessary trace element for poultry, copper (Cu) plays a positive effect in mitochondrial respiratory, hematopoiesis, and anti-oxidative function in organisms [1,2].However, excess Cu can lead to toxicity by disturbing osmotic regulation and causing metabolic disturbance [3,4,5].Due to the positive and negative effects of Cu on animal health, the content of Cu in feed has been raised concern.In practice, adding Cu to feedstuffs for poultry can improve growth performance and feed e ciency.Nevertheless, long-term excessive intake of Cu can cause toxic effects in animals and humans due to the enrichment of the food chain.Especially for mitochondria, excess Cu can disrupt redox balance by impairing the respiratory enzyme complexes, which leads to reactive oxygen species (ROS) formation, catalyzes Fenton/Haber-Weiss reactions and thus makes damaged to antioxidant defense system [6,7,8].Previous studies have demonstrated that large amounts of Cu were found in various animals, including poultry [9,10].Additionally, the renal toxicity induced by Cu has been widely concerned since the kidney is the target organ of Cu [11].Indeed, numbers of studies pointed out that Cu could cause nephrotoxicity via reactive oxygen production, energy de ciency, and mitochondrial dysfunction [11,12].
It is universally acknowledged that oxidative damage and mitochondrial toxicity are associated with apoptosis.Apoptosis, an active and gene-control cell death, is highly regulated within metabolic process.It is characterized by chromatin condensation, DNA cleavage, and the formation of apoptotic bodies [13].
The mechanism of apoptosis is mostly complex and mainly divided to endogenous and exogenous pathways.The endogenous pathway, known as mitochondria-mediated pathway, can be activated whereby stimuli in the extracellular matrix, which induce permeation of the outer mitochondrial membrane (OMM) and collapse of mitochondrial membrane potential [14].In this process, numbers of signal molecules such as Bax (pro-apoptotic) or Bcl-2(anti-apoptotic) can be recruited to OMM, which result in the decrease of the mitochondrial membrane potential (MMP), and cytochrome C (CytC) was released to the cytoplasm.CytC can promote the formation of apoptosome as a death factor, which activates Caspase-3 by stimulating enzymatic reactions and leads to apoptosis [15].At present, the nephrotoxicity of Cu has attracted widespread attention, and apoptosis is considered as one of the underlying mechanisms of Cu poisoning.
It is reported that mitochondrial morphology is closed related to apoptosis [16].Actually, the maintenance of mitochondrial morphology depends on its fusion and ssion in normal physiological state.The dynamic balance in mitochondria is crucial for maintaining cellular homeostasis.When mitochondrial dynamics is in imbalance, the metabolic processes such as energy generation, immunity, and apoptosis will be affected [17].Recent studies have pointed out that numbers of components related to ssion and fusion machinery implicated in mitochondrial shape changes including dynamin-related protein 1 (Drp1) and mitochondrial fusion proteins (Mfn1, Mfn2, and OPA1) are indispensable for apoptosis [18,19].In addition, some of ndings revealed that excessive mitochondrial ssion could trigger the increasing open degree of mitochondria permeability transition pore (MPTP) and promote the release of CytC from mitochondria, leading to mitochondrial-mediated apoptosis [18,20].
Cellular metabolism and the pathways of cell death are regarded as the important factors in regulating cell fate.When the cell homeostasis is disrupted, the metabolites should be changed due to cell function affected.If metabolic energy is not enough to satisfy the requirements of biosynthesis and homeostasis maintenance, cells will cease to function normally and trend to apoptosis eventually.Thus, the detection of metabolite change has certain signi cance for re ecting the physiological state of the body.In recent years, metabolomics techniques are widely used in toxicology and disease diagnosis.Metabolomics is the method for quantitative analysis of the overall variation of metabolic substances in a certain period, which can rsthand re ect the nal status of biosystems, and it can reveal the relationship between metabolites and physiological or pathological changes [21].Various studies have showed that the metabolites play a key role in phenotypic regulation and biological synthesis by interacting with genes and proteins [22,23].Indeed, the changes of intracellular metabolites may reveal the metabolic pathway during the process of apoptosis [24].In present research, liquid chromatography-mass spectrometry (LC-MS) was used to detect the changes in metabolites in kidney, and mitochondria dynamics and apoptosis was explored, which further explore the role of mitochondria-mediated apoptosis on Cu-induced nephrotoxicity from the point of metabolomics.

Animals and treatment
A total of 240 newborn chicks (1 day of age, weighting 43 ± 0.32 g) were divided into 4 groups at random (including one control group and three treated groups, 60 broiler chicks per group, half male and half female).The ingredient and nutritional levels of the basic feed were followed in Table S1, and Cu sulfate was selected as Cu sources.Broilers were fed with the basal ration containing 11 mg•kg − 1 Cu in control group, and the Cu levels in three treated groups were as follow: 110 mg•kg − 1 Cu, 220 mg•kg − 1 Cu, and 330 mg•kg − 1 Cu, respectively, which was referred to the previous studies [3,6,25].All the broilers were given ad libitum access to water and diet.On the day 49 of the treatment, the serum was collected for blood biochemistry test, and the kidneys were collected from broilers after euthanasia with nembutal.The procedures were authorized by the Ethics Committee of South China Agricultural University (Permission number: NO. 2017A087).

Histological observation
Tissues were xed in 4% paraformaldehyde for 24 h.The steps of making pathological section and hematoxylin eosin (H&E) staining were referred to our previous studies [3,6].The renal tubular damage score was obtained according to the previous studies [26,27].

TUNEL staining assay
Kidney tissues were immobilized in 4% paraformaldehyde and para n-embedded tissue slices were prepared referring to the standard procedure.TUNEL assay was executed by TUNEL test kit (Beyotime, China) based on the guidance note.Sections were treated with proteinase K and hydrogen peroxide.Then, the sections were incubated with biotin-dUTP solution at 37℃ at dark for 60 min.After being washed with phosphate buffer solution (PBS), the Streptavidin-HRP detected solution was used to incubate 30 min at room temperature and then incubated with diaminobenzidine (DAB).Hematoxylin was used for nuclear labeling.The positive rate of TUNEL staining was measured by Image J.

Ultrastructural observation
The preparation of ultrathin sections and the acquisition of electron microscope photos were performed by the Test Center of South China Agricultural University.The speci c methods could be found in supplementary materials.

ROS detection
ROS was detected by uorescent probe DCFH-DA by ow cytometry according to the guidance note (Beyotime, China).Brie y, the kidney tissues were digested into individual cells and DCFH-DA was used to incubate for 20 min at 37℃.Fluorescence intensity was analyzed by ow cytometry.

Metabolomics analysis
Page 6/23 50 mg of tissues in control group and 330 mg/kg Cu group were taken for metabolite extraction.The method of sample extraction and the analytical equipment platform for LC-MS was from Waters, and the parameter setting was also referred to Luan et al. [28].The original data were processing by employing for baseline ltration, peak extraction, integration, time correction, peak calibration, and normalization.The resultant data were processed into SIMCA-14.1 software (Umetrics, Sweden) and imported to principle component analysis (PCA) and orthogonal partial least squared discriminant (OPLS-DA).The variable importance in the projection (VIP) > 1 and P < 0.05 were identi ed as the potential metabolic markers.The metabolites were identi ed by comparing with HMDB online databases and the metabolic pathways were analyzed by KEGG pathway database and MetaboAnalyst 4.0.

Measurement of mitochondrial oxygen consumption
The extraction of renal mitochondria was performed referring to previous study [29].The mitochondrial oxygen consumption was detected by Oxygraph-2k instrument (O2k, Oroboros instruments, Austria) at 37 ± 0.001°C.After protein quanti cation, the same amounts of mitochondria were added to the respiration chamber containing 2 mL of respiration medium (The recipe was seen in supplementary materials).

Determination of MMP and MPTP
MMP was detected by JC-1 assay (Thermo Fisher Scienti c, USA) following the guidance note.Brie y, the kidney tissues were digested into individual cells and washed with PBS for three times.JC-1 was used to incubate for 20 min at 37℃.After washing, cells were analyzed under the emission wavelength of 590 nm and 525 nm by ow cytometry.
MPTP was determined by MPTP Assay Kit with Calcein AM uorescence probe based on the guidance note (Beyotime, China).Brie y, the kidney tissues were digested into individual cells and washed in PBS for three times.Cells were incubated with Calcein AM staining solution in a dark room for 30 min at 37℃.Then, Calcein AM staining solution was removed and the cells were resuspended in DMEM at 37℃ for 30 min in the dark.After washing, uorescence intensity was analyzed by ow cytometry.
GAPDH was used as the reference gene and the mRNA expression was calculated according to 2 −△△CT method.

Western blot analysis
The total proteins were extracted with radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China).Then, the extracted protein contents were determined by BCA assay (Beyotime, China).10 mg of protein was separated by electrophoresis, transferred to membranes, and blocked.Incubation of primary and secondary antibodies was proceeded as previously described [3].After washing with TBST, ECL solution (Millipore, U.S.A.) was used to visualize the blot.The protein expression was analyzed by Image J.

Evaluation of renal injury induced by Cu
In this study, the body weight of broilers was signi cantly increased in 220 mg/kg Cu group and remarkably decreased in 330 mg/kg Cu compared to control group (Fig. S2).Furthermore, the Cu content in serum and kidney and the biochemical indicators (BUN, Crea, and UA) were increased with increased intake of Cu (Table S3-4).The kidney coe cient was obtained to analysis the pathological effects induced by Cu.As presented in Fig. 1D, the kidney coe cient was elevated after treating 220 mg/kg and 330 mg/kg Cu in comparison with control group, which might indicate that pathological swelling could be induced by Cu.In order to verify the pathological damage in kidney induced by Cu, H&E staining was executed to observe the changes of histological morphology.As shown in Fig. 1A, the vacuoles degenerations in renal tubular epithelial cells could be found in all treated group in comparison with the control group, especially in the 330 mg/kg Cu group (white arrows).Additionally, renal tubular injury was evaluated with the scores of renal tubular damaged, which showed that the score of tubular injury was signi cantly increased with elevated Cu (P < 0.01 or P < 0.001) (Fig. 1E).For exploring whether ROS were the source of kidney injury, ROS levels were evaluated and the results suggested that the ROS production was increased remarkably with the elevated Cu (P < 0.01 or P < 0.001) (Fig. 1C&G).
TUNEL staining is the classical method to detect apoptotic cells with DNA fragmentations, and the apoptotic cells were stained with brown.As demonstrated in Fig. 1B, the brown-strained cells (black arrows) in kidney were elevated remarkably with the increase of Cu level, the color depth and areas were also increased.Quantitative analysis of TUNEL-positive cells also showed the elevated positive rate with the increased Cu (P < 0.001) (Fig. 1F).

Effects of Cu on the change of metabolites in kidney
In order to investigate the relevancy between metabolic process and apoptosis, the kidney metabolites in the 330 mg/kg Cu group and control group were analyzed by metabolomics.The non-targeted metabolomics data were collected in the modes of positive and negative ion by LC-MS.PCA and OPLS-DA were used to pattern discriminant analysis.The clustering of QC plots in PCA indicates that the analytical method was high repeatability and low variability (Fig. 2A-B).Additionally, the plots representing the 330 mg/kg Cu group were signi cantly separated from those of control group in both positive and negative modes, which indicated a signi cant change of the metabolites and a success of modeling (Fig. 2).
The differential metabolic substances were chosen on the basis of VIP > 1, P < 0.05.There were 62 differential metabolites were shown in Fig. 3A&B (heatmap and volcano plot) and Table.S5&6 (including positive and negative ion).Among of above, 30 metabolites were elevated and 32 metabolites were reduced in the 330 mg/kg Cu group in comparison with control group.

Cu induced mitochondrial damage in kidney
In this study, mitochondrial morphology was observed by TEM.As shown in Fig. 4A, the nuclei were normal without shrinkage in control group, and mitochondrial membrane structures were intact.Similarly, no obvious pathological changes were observed in the 110 mg/kg Cu group.However, the nuclear deformation and shrinkage was found in the 220 mg/kg Cu group, and mitochondrial cristae partly disintegrated (red arrows).What's notable was that mitochondrial vacuolar degeneration, break and disappearance of the mitochondrial cristae could be seen in the 330 mg/kg Cu group.
Next, the effect of Cu on mitochondrial respiratory function is measured by real-time OCR using Oxygraph-2k instrument.Schematic diagram was shown in Fig. S1.The respiration without adding any reagents is routine respiration.State 4 is the respiration in presence of speci c substrates.State 3 is the respiration with saturated substrate and ADP.RCR was obtained from the rate between state 3 and state 4 respiration.After the addition of oligomycin, the oxygen consumption was decreased, which represented the oxygen consumption exploited for ATP production and known as ATP-linked respiration.Subsequently, mitochondrial uncoupler CCCP was added to maximize mitochondria respiratory for obtaining the Max.Furthermore, SRC could be calculated by the difference between MAX and State 4 respiration.As presented in Fig. 4B, the routine respiration, RCR, MAX, and ATP-linked respiration were decreased remarkably with the increasing Cu level (P < 0.05, P < 0.01 or P < 0.001).SRC was showed a down trend with the increase Cu level, but the difference was not signi cant (P > 0.05).
Flow cytometry was used to detect the MMP and MPTP, and the results were shown in Fig. 4C-D.JC-1 is widely used as a uorescent probe to detect MMP.JC-1 exists as monomers at low MMP and emits green uorescence after laser excitation.However, JC-1 can emit red uorescence in the form of aggregates at high MMP.Therefore, the transition from red to green uorescence can re ect a decrease of MMP.As shown in Fig. 4C, the decreased trend of MMP could be detected via the transformation from red to green uorescence, and the proportion of cells with low MMP were signi cantly increased with the raising Cu level, which showed dosage effect (P < 0.001).Additionally, the effects of Cu on MPTP in kidney were presented in Fig. 4D.The opening degree of mitochondria membrane pore was inversely proportional to the uorescence density of Calcein AM.The results showed that the mean uorescence density of Calcein AM was decreased with the elevated Cu, and the remarkable change was shown in 330 mg/kg Cu group in comparison with control (P < 0.01).

Cu induced mitochondrial dynamics disorder in kidney
As exhibited in Fig. 5A, the mRNA level of Drp1 was enhanced dramatically when treated with 220 mg/kg and 330 mg/kg Cu compared with control group (P < 0.01 or P < 0.001).However, the mRNA expression levels of OPA1, Mfn1 and Mfn2 were decreased with the elevated Cu level.The expressions mitochondrial dynamic-related proteins were shown in Fig. 5B and the quantitative result were exhibited in Fig. 5C&D.
The protein expression levels were showed the similar trend of mRNA expression.As shown, the protein expression level of Drp1 up-regulated and OPA, Mfn1, and Mfn2 down-regulated with the elevated Cu level.

Cu induced mitochondria-mediated apoptosis in kidney
Mitochondria-mediated apoptosis is controlled by multitudinous signal factors.Thus, the mRNA and protein expression of Bak-1, Bax, Bcl-2, CytC, cleaved Caspase-3 and the mRNA expression of p53 were detected and the results were seen in Fig. 6.The mRNA and protein expression levels of Bak-1, Bax, CytC, and cleaved Caspase-3 were enhanced with the elevated Cu, and the remarkable difference was exhibited in 220 mg/kg Cu and 330 mg/kg Cu group in comparison with control group (P < 0.05, P < 0.01 or P < 0.001).Additionally, the mRNA expression of p53 in all treated groups were signi cantly enhanced compared to control group (P < 0.05, P < 0.01 or P < 0.001).Unlike the others, the mRNA and protein expression levels of Bcl-2 were memorably reduced in 330 mg/kg Cu compared with control group (P < 0.05).

Discussion
Cu is an essential nutrient in poultry, which is usually prescribed to increase feed e ciency and improve growth performance.However, Cu can be toxic to poultry when ingested overdose.Kidney is considered as the storage organ for Cu, so it is of important signi cance to explore the nephrotoxicity induced by Cu.Previous study had found that Cu could accumulate in kidney and cause pathological damage [11], and the elevated ROS levels and injury scores also con rmed the renal damage could be caused by Cu in present study.In addition, many studies indicated that Cu could induce apoptosis, but the metabolic changes and metabolic processes involved in Cu-induced apoptosis were poor studied.In this study, metabolomic techniques were used to detect Cu-induced metabolite differences and metabolic pathways, which may re ect the effects of mitochondria-dependent apoptosis in kidney induced by Cu.
In this study, TUNEL staining assay was used to detect Cu-induced apoptotic cells in kidney.The obtained results of TUNEL staining showed that the apoptotic cells were signi cantly increased with the elevated Cu level.Meanwhile, TEM also showed that the karyopyknosis was found in kidney with the increased intake level of Cu.These results con rmed that apoptosis can be induced by Cu in kidney, and the doseeffect was shown.Therefore, the samples in the control group and the 330 mg/kg Cu group were selected for metabolomics analysis to reveal the metabolic pathway of Cu-induced apoptosis in the kidney.
The results of metabolomics showed that a total of 62 differential metabolites were associated with Cuinduced nephrotoxicity in kidney, which mainly affected ribo avin metabolism, glutathione metabolism, sphingolipid metabolism, and glycerophospholipid metabolism.Ribo avin metabolism is closely linked to mitochondrial energy metabolism.In this metabolic pathway, avin adenine dinucleotide and avin mononucleotide are the two important redox cofactors in mitochondrial respiratory chain, which are vital for energy production.Some studies pointed out that avoprotein might be involved in superoxide production, which revealed that ribo avin metabolism might play a part in apoptosis via oxidative stress [31,32].Additionally, glutathione metabolism is an essential metabolic pathway for cellular growth and homeostasis.It is reported that the low level of glutathione can lead to high level of mitochondrial ROS and cause the oxidative damage to proteins, lipids, and DNA [33].Moreover, sphingolipid metabolism, a complex network composed of interconnected metabolites with ceramide as the central hub, which can cooperate with Bax and Bak to increase mitochondrial metabolic e ciency.The role of sphingolipid metabolism during apoptosis has been extensively explored [34,35].As the hub in sphingolipid metabolism, the increased level of ceramide can respond to the pro-apoptotic signal, inducing the key factors in the apoptosis cascade.Meanwhile, mitochondrial fusion/ ssion and apoptosis are always accompanied by changes in the glycerophospholipid composition (including phosphatidylethanolamine, phosphatidylcholine, lysophosphatidylcholine and so on) on the out surface of cell membranes or organelles membranes, which is extremely related to glycerophospholipid metabolism pathway [36].In this study, 330 mg/kg Cu could increase the level of avin mononucleotide to change the level of ribo avin metabolism, which might affect glycerophospholipid metabolism pathway (decreased levels of phosphatidylcholine, lysophosphatidylcholine and increased level of phosphatidylethanolamine) and glutathione metabolism pathway (decreased levels of oxidized glutathione and glutathione) via pentose and glucuronate interconversion and TCA cycle.Additionally, Cu-induced signi cantly increased levels of ceramide (d18:1/24:1(15Z)) and 3'-O-sulfogalactosylceramide resulted in the change of sphingolipid metabolism pathway also be related to the change of glycerophospholipid metabolism and glutathione metabolism.The metabolomics results suggested that the abnormal activities of ribo avin metabolism, glutathione metabolism, sphingolipid metabolism, and glycerophospholipid metabolism were probably related to Cu-induced apoptosis in kidney.
Interestingly, the differences metabolites and metabolic pathways caused by Cu exposure in present study were closely related to mitochondrial dysfunction.Thus, the mitochondria-mediated apoptosis induced by Cu in kidney was investigated.Firstly, mitochondrial morphology and function were evaluated.The results of TEM showed mitochondrial cristae fracture and vacuolization in Cu treatment groups.
Besides that, the respiratory function detection of the extracted mitochondria also re ected the toxic effect of Cu on renal mitochondria.In this study, the OCR was increased immediately after adding substrates, which con rmed that extracted mitochondria were active.Among the relevant indicators obtained, RCR can well re ect the mitochondrial function and status.A high level of RCR suggest the high coupled effect between ATP production and electron transport in respiratory chain.Meanwhile, ATP-linked respiration represents the oxygen consumption linked to ADP phosphorylation.MAX is depended on the activity of the complex enzymes.Additionally, the SRC is considered as the capacity of the mitochondria to respond to the energy requirement, which is a measure of cell's tness [30].Our study found that the routine respiration, RCR, MAX, and ATP-linked respiration were decreased signi cantly with the increasing level of Cu, and SRC was also shown a down trend.These indicated that Cu could weaken the ATP synthesis, electron transport e ciency, and energy storage capacity in renal mitochondria.Moreover, the results of ow cytometry showed that Cu could lead to the increase of mitochondrial low membrane potential cells, and the degreed of mitochondrial membrane permeability was enhanced.These suggested that Cu could cause mitochondrial dysfunction and structural impairment in kidney.
In this study, the mitochondrial dynamics was evaluated.Mitochondrial dynamics is a process that can be de ned as spatiotemporal changes in mitochondrial structure, number and location within the cell.Fission and fusion in mitochondria are required for homeostasis and mitochondrial health especially when cells suffer from various stimuli and conditions [19].Mitochondrial ssion is a process in which daughter mitochondria are produced from the mother mitochondria leading to improve mitochondrial network ssion.In addition, the separation of dysfunctional mitochondrial section requires Drp1 to be recruited into OMM leading to membrane depolarization.However, excess ssion within mitochondria can impair metabolic function and derange mitochondrial dynamics [37].In contrast to ssion, mitochondrial fusion is a collaborative process that two originally different mitochondria physically merge into one.It also includes the fusion of OMM and the inner mitochondrial membrane (IMM).The fusion of OMM is regulated by Mfn1 and Mfn2, and fusion of the IMM is controlled by OPA1 [38].In the present study, the mRNA and protein levels of Drp1 were increased markedly with the increased contents of Cu.However, the mRNA and protein levels of mitochondrial fusion-related protein including OPA1, Mfn1, and Mfn2 were decreased signi cantly with the Cu contents elevated.These suggest that excess Cu could cause the excessive mitochondrial ssion, which leading to mitochondrial homeostasis disequilibrium.
Mitochondria can be considered as a key factor in programmed cell death, and many studies have found that the mitochondrial-mediated apoptosis can be induced by types of toxic factors [39,40].Upon most occasions, mitochondria-mediated apoptosis is controlled by Bcl-2 family which consists of both proapoptotic factors (Bax/Bak) and anti-apoptotic factor Bcl-2.Once apoptotic signals are received, the proapoptotic proteins will transfer to the mitochondrial membrane and Bcl-2 protein will down-regulated, which lead to permeabilize the OMM, MMP decreasing, and release of intermembrane pro-apoptotic substances, including CytC.And then, Caspase-3 stops contact with peripheral cells, reassembles the cytoskeleton, discontinues DNA duplication, destroys DNA, disrupts the nuclear structure, and disintegrates the cells into apoptotic bodies [14].Additionally, p53 can exert a crucial part in regulating cell-cycle arrest, DNA double-stand break and mitochondria-dependent apoptosis in response to various stresses [41].In present study, the decrease of MMP was accompanied by an increase of MPTP in kidney induced by Cu.Furthermore, the mRNA and protein expression levels of Bax, Bak-1, CytC, and cleaved Caspase-3 and the mRNA expression level of p53 were enhanced with the increased Cu level, but the mRNA and protein levels of Bcl-2 were down-regulated.This nding was further con rmed that excessive intake of Cu could cause mitochondria-mediated apoptosis in kidney.

Conclusion
In this study, metabolomics was used for the rst time to evaluate mitochondria-mediated apoptosis caused by Cu in kidney.The mechanisms of Cu-induced apoptosis involve the metabolic pathways including ribo avin metabolism, glutathione metabolism, sphingolipid metabolism, and glycerophospholipid metabolism.Furthermore, excess Cu could induce mitochondria-mediated apoptosis in kidney, which might be closely related to above metabolic pathways.Thus, metabolomics, the most closely re ecting the phenotype, was effective in investigating the toxic mechanisms induced by Cu exposure in kidney of broiler chickens.

Abbreviations
mitochondrial membrane LC-MS: Liquid chromatography-mass spectrometry MAX: Maximal respiration MMP: Mitochondrial membrane potential MPTP: Mitochondria permeability transition pore

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