
==== Front
Poult Sci
Poult Sci
Poultry Science
0032-5791
1525-3171
Elsevier

S0032-5791(24)00746-6
10.1016/j.psj.2024.104167
104167
IMMUNOLOGY, HEALTH AND DISEASE
Cadmium causes spleen toxicity in chickens by regulating mitochondrial unfolded protein response and nuclear receptors response
Xu Wang-Ye *1
Li Xiao-Wei *1
Sun Jin-Xu *
Malhi Kanwar Kumar *
Li Xue-Nan lixuenan@neau.edu.cn
*†‡§2
Li Jin-Long ⁎†‡
⁎ Department of Clinical Veterinary Medicine, College of Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
† Key Laboratory of the Provincial Education Department of Heilongjiang for Common Animal Disease Prevention and Treatment, Northeast Agricultural University, Harbin, 150030, China
‡ Heilongjiang Key Laboratory for Laboratory Animals and Comparative Medicine, Department of Clinical Veterinary Medicine, Northeast Agricultural University, Harbin, 150030, China
§ Department of Obstetrics & Gynaecology; Li Ka Shing Institute of Health Sciences; School of Biomedical Sciences; and The Chinese University of Hong Kong-Sichuan University Joint Laboratory for Reproductive Medicine, The Chinese University of Hong Kong, Hong Kong, China
2 Corresponding author: lixuenan@neau.edu.cn
1 These authors contributed equally to this study and shared the first authorship.

03 8 2024
11 2024
03 8 2024
103 11 10416715 4 2024
30 7 2024
© 2024 The Authors
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Cadmium (Cd) is a heavy metal that pollutes the environment and threatens human and animal health via the food chain. The spleen is one of the target organs affected by Cd toxicity. However, the mechanism of Cd toxicity is not fully understood. In this study, 80 chicks were allocated into 4 groups (n = 20) and exposed to different doses of CdCl2 (0 mg/kg, 35 mg/kg, 70 mg/kg and 140 mg/kg) for 90 d. The pathological changes in the spleen, mitochondrial dynamics-related factors, cytochrome P450 (CYP450) enzyme system contents, activities, transcription levels, nuclear receptors (NRs) response molecule levels, and mitochondrial unfolded protein-related factors were detected. The findings indicate that exposure to Cd significantly leads to spleen injury. In Cd groups, the total contents of CYP450 and cytochrome b5 (Cyt b5) increased, and the activities of the CYP450 enzyme system (APND, ERND, AH, and NCR) changed. The NRs response was induced, and the gene levels of AHR/CAR and corresponding CYP450 isoforms (CYP1B1, CYP1A5, CYP1A1, CYP2C18, CYP2D6 and CYP3A4) were found altered. The study found that Cd exposure altered the mRNA expression levels of mitochondrial dynamics-related factors, such as OPA1, Fis1, MFF, Mfn1, and Mfn2, breaking mitochondrial fusion and cleavage and ultimately leading to mitochondrial dysfunction. Changes were detected in the gene levels of several mitochondrial unfolded protein response (mtUPR)-related factors, namely (SIRT1, PGC-1α, NRF1, TFAM, SOD2, and HtrA2). Cd also altered the gene levels of mitochondrial function-related factors (VDAC1, Cyt-C, COA6, PRDX3, RAF and SIRT3). It is showed that Cd can initiate the NRs response, influence the homeostasis of the CPY450 enzyme system, trigger the mtUPR, impair mitochondrial function, and ultimately lead to Cd toxicity in the spleen of chickens.

Keywords

cadmium
spleen
CYP450s enzyme system
mtUPR pathway
nuclear receptors (NR) response
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pmcINTRODUCTION

Cadmium (Cd) is a hazardous heavy metal pollutant with various forms, including Cd chloride, Cd oxide, and Cd carbonate. It is commonly utilized in industrial processes like electroplating and nickel melting. However, Cd's nonbiodegradable nature makes it easy to spread into the environment through industrial waste. Furthermore, Cd has high mobility in the ecosystem, making it effortless for plants, including crops, to absorb it (Hu, et al., 2022). Cd accumulates in different organs of the body through the food chain, ultimately causing toxicity that can have teratogenic and carcinogenic effects. As a result, Cd significantly threatens human and animal health (Zhang, et al., 2022). Cd induces pathologic changes in the animal spleens, as the spleen is one of the Cd toxicity target organs. Cd can destroy the pig spleen's structure and function, leading to necroptosis (Xia, et al., 2023). In mice, Cd caused splenomegaly, and the weight of the spleen increased while the weight of the body decreased (Pathak and Khandelwal, 2007; Hao, et al., 2021). In ducks, Cd induces structure damage in the spleen, such as the wall thickness of the sheath artery (Cao, et al., 2016). Cd causes spleen injury in goats by enhancing pro-oxidant enzymes or inhibiting the variety of antioxidant enzymes that increase ROS (Majumder et al., 2019). Cd treatment causes spleen cell autophagy in chickens and changes the icon levels (Chen, et al., 2018). As well known, the spleen is an important immune organ, with hematopoietic and blood storage functions and is involved in metabolism, spleen injury will not only cause the destruction of immune function (Demenesku, et al., 2014; Wang, et al., 2017), but also have an impact on the body's metabolism. However, the mechanism of Cd-induced splenic injury in chicks has not been precisely determined.

The nuclear receptors (NR) can regulate the transcription of genes and are involved in metabolism, determining sex, differentiation, reproductive development, maintaining homeostasis, and sensing cell signals (Li and Wang, 2010). NRs are divided into steroid, nonsteroid, and orphan nuclear receptors (ONR) with no known endogenous ligands (Mazaira, et al., 2019; Frigo, et al., 2021). The other receptors have certain ligands that exist in usual, while ONRs lack specific ligands and are activated by xenobiotics (Sonoda, et al., 2008). Nuclear xenobiotic receptors (NXR) can respond to endogenous compounds and xenobiotics, including some of the ONRs, such as the Pregnane X receptor (PXR) and the constitutive androstane receptor (CAR) (Mackowiak, et al., 2018). The coordination of cytochrome P450 (CYP450) enzymes, coupling enzymes, and drug transporters helps detoxify and eliminate exogenous drugs (Nebert, 1991). CYP450s bind to the endoplasmic reticulum (microsome) membrane or the matrix of the inner mitochondrial membrane (El-Sherbeni and El-Kadi, 2017; Zhang, et al., 2019b), which are important for the detoxification of xenobiotics (Manikandan and Nagini, 2018). When NXRs are activated by exogenous substances, they enter the nucleus and bind to the corresponding ligands, affecting the expression of CYP450 isozymes, initiating the expression of the CYP450s, and regulating the catabolism of exogenous substances (Dvorak and Pavek, 2010). CYP450 is induced by PXR, CAR, and Aryl hydrocarbon receptor (AHR). Cd exposure could activate XRs and change the expression of CYP450s, leading to the production of reactive oxygen species (ROS) in pig hearts (Zhao, et al., 2021). It also could change the CYP450s mRNA levels in pig liver (Wang, et al., 2021). In chicken liver and heart, Cd changed the CYP450s mRNA levels (Cong, et al., 2019; Guo, et al., 2020). As a result, the Cd toxin will activate NRs response and change the CYP450 subfamilies levels (Zhang, et al., 2021a; Lv, et al., 2023). Although those reports have shown that NRs response and CYP450s play an important role in toxic effects induced by xenobiotics, how Cd influences the NRs response and CYP450s levels in the chicken spleen is still to be clarified.

Cd induces ROS production by bonding to proteins to form sulfhydryl groups, (Wang and Fowler, 2008; Valko, et al., 2016) and ROS is recognized as one of the key factors in the induction of the unfolded protein response (mtUPR) pathway (Runkel, et al., 2013; Shao, et al., 2016). In order to defend the excessive ROS, mtUPR-related factors such as Sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) were activated. During mitochondrial experience dysfunction or the accumulation of unfolded proteins, cells utilize a transcriptional response called the mtUPR to promote cell survival and repair defective mitochondria. However, an excessive mtUPR will result in mitochondrial dysfunction (Lin, et al., 2016), causing cell injury, apoptosis or autophagy. Some reports have demonstrated that mtUPR could cause mitochondrial damage in neuronal cells (Cai, et al., 2022) or disrupt mitochondria in skeletal muscle (Ogunbileje, et al., 2016). However, few reports focus on how mtUPR influences Cd-induced spleen injury in chickens.

People pay more and more attention to the environmental pollution of heavy metals like Cd and the effects of their accumulation in animals. The spleen is a vital part of the immune system that enhances the defence function. As mentioned earlier, Cd is a certain toxin that induces injury in the spleen of animals. NRs response and mtUPR play an important role in histiocytic injury, but how the NRs response and mtUPR influence the chicken spleen process remains indefinite. This study discovered that Cd contributed to spleen injury in chickens, changed CYP450 contents and CYP450 enzymatic activities, and activated NRs response and CYP450 subfamilies levels. Cd also changed the mitochondrial dynamics related to molecule levels and induced fusion and division disorder. All the changes were most likely induced by NRs response and mtUPR. Our study provides a new insight into Cd-induced spleen injury.

MATERIALS AND METHODS

Animal Ethics

All methods and procedures of the experiment were performed according to the requirements of the Animal Ethics Committee of Northeast Agricultural University (NEAUEC20190314).

Animal Experiment and Treatment

Eighty male Hy-Line Variety White chicks (aged 1 d, Xianfeng Chicken Farm, China) were divided into 4 groups in different Cd-dose (each group consisted of 20 chicks): Control (Con), 35, 70, and 140 mg/kg groups. All chickens were fed for 90 d by feed (Charoen Pokphand Group, Tianjin) mixed with CdCl2 of different doses: 0, 35, 70, and 140 mg/kg BW/d. The feeding environment was controlled, and temperature and humidity are compatible with the previous report (Li, et al., 2024). The choice of Cd dose was determined based on previous studies (Guan, et al., 2022). After 90 d, all chickens were euthanized by cardiac blood collection. The spleens of all animals were collected under 4°C and then stored at −80°C for the following research. Spleen tissues were fixed in a formaldehyde solution (4%) to observe histopathological changes.

Hematoxylin and Eosin Staining

Ethanol was used to dehydrate the spleen tissues, which were then embedded in paraffin. Afterwards, these sections were stained with hematoxylin and eosin (H&E) to observe the pathological injury of the spleens. An optical microscope scanner (3DHISTECH Ltd., Budapest, Hungary) was used to scan the spleen sections.

Determination of the CYP450 Contents and CYP450 enzymatic Activities in Chicken Spleen

CYP450 and cytochrome b5 (Cyt b5) levels were determined based on a previous study (Xia, et al., 2016). Aminopyrine N-demethylase enzyme (APND) activities and erythromycin N-demethylase (ERND) activities were determined by establishing an HCHO standard curve (Nash, 1953). The activities of aniline-4-hydroxylase (AH) and NADPH-cytochrome c reductase (NCR) were determined according to the previous papers (Williams and Kamin, 1962; Imai, et al., 1966; Koleva, et al., 1999). The results of the contents were expressed in nmol/mg, and the activities were measured in nmol/min/mg.

The Extraction of RNA and Quantitative Real-Time PCR

A 50 mg spleen sample was used to extract the total RNA using RNAout Reagent (Beijing Tiandz, Inc., Beijing, China). The total RNA was determined using a spectrophotometer through detecting the quality and concentration at 260/280 nm. Then, the total RNA was used to produce complementary DNA (cDNA) using a reverse transcriptase kit (TransGen Biotech, China) as described by the manufacturer's product specification. cDNA was used for the qRT-PCR template. Oligo 7.22 software was used to design the primers for quantitative real-time PCR (qRT-PCR), which are listed in Supplementary Table S1 (Supplementary Material). β-actin was used as the internal reference gene, and all the mRNA transcript levels were calculated by the 2−△△CT formula.

Western Blot

The total protein of the spleen tissue was extracted using RIPA lysis buffer (Cat: R0010, Beijing Solarbio Science and Technology Co., Ltd). 12% SDS-PAGE separated the extracted proteins, which were transferred to an NC membrane. The membranes were washed using a TBST solution (3 times, 5 min each) and blocked for 2 h with 5% fat-free milk at 37°C. Washed 3 times with TBST, the membranes were separately incubated with antibodies against β-actin, PCG-1α and SOD2 overnight at 4°C. After being washed by TBST 3 times, the membranes were incubated by a secondary antibody. In the end, the membranes with proteins were imaged by Amersham Imager 600 (GE, American). The antibodies were listed in Supplementary Table S2 (Supplementary Material) purchased from ABclonal Technology Co., Ltd. (Wuhan, Hubei, China) and Beijing Biosynthesis Biotechnology Co., Ltd. (Beijing, China).

Statistical Analysis

Data analysis and statistics were performed by GraphPad Prism 5.0 (GraphPad Software, San Diego, CA). Statistical analysis was performed using a 1-way analysis of variance (ANOVA). All data was described as mean ± SD. A heat map of the different expression genes was drawn using R Programming Language version 3.2. (*) indicate significant differences of Cd groups compared with the control group, setting to *P < 0.05, **P < 0.01 and ***P < 0.001.

RESULTS

Histopathological Analysis of Spleen

The pathological changes under the microscope are illustrated in Figure 1. In the control group, the structure of the perisplenic membrane was intact, and both splenic cords and splenic sinuses were normal. The splenic nodules appeared clear, with a distinct structure of red and white pulp and neatly arranged cells. There were no abnormal findings in the sheath artery wall thickness (Figure 1, Figure 1). In the low-dose Cd group (35 mg/kg), the boundary between red and white pulp was slightly blurred compared to the control group (Figure 1, Figure 1). In the middle-dose Cd group (70 mg/kg), except for the blurred boundary between red and white pulp, there was slight splenic pulp congestion and bleeding, and the lymphocytes arrangement was sparse (Figure 1, Figure 1). In the high-dose group (140 mg/kg), the boundaries between splenic red pulp and white pulp disappeared, splenic pulp congestion and bleeding were more apparent, the structure of splenic nodules could not be recognized, and the wall thickness of the sheath artery was significantly increased compared to the control group (Figure 1, Figure 1).Figure 1 Histopathological analysis of spleen. 10×: (A-D) Different Cd dose groups (0, 35, 70, 140 mg/kg); 40×: (a-d) Different Cd dose groups (0, 35, 70, 140 mg/kg). Yellow arrows indicated sheath artery, and black arrows indicated lymphocyte.

Figure 1

Results of CYP450 Contents and CYP450 Enzymatic Activities

To investigate the impact of Cd on the CYP450 enzyme system, we analyzed the contents and enzymatic activities of CYP450 (Figure 2). In general, after exposure to Cd for 90 d, with the gradual increase of Cd dose, the CYP450 contents (Figure 2A) and Cyt b5 contents (Figure 2B) increased, and so did the ENRD (Figure 2D) and AH (Figure 2E) activities. On the other hand, the APND and NCR activities (Figure 2, Figure 2) declined. Compared with the control, CYP450 content significantly increased in the high-dose group (140 mg/kg) (P < 0.01), and Cyt b5 contents in the middle-dose and high-dose groups also increased (P < 0.05) (Figure 2B). APND activity decreased significantly with increasing Cd compared to the control group (P < 0.001) (Figure 2C). In high-dose groups, there were significant increases in ERND and AH activities (P < 0.05) and a decrease in NCR activity (P < 0.05).Figure 2 Results of CYP450 contents and CYP450 enzymatic activities. (A) CYP450 Content. (B) Cyt b5 Content. (C) APND Activity. (D) ERND Activity. (E) AH Activity. (F) NCR Activity. Significant differences between the control groups and Cd groups are expressed as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

mRNA Expression Levels of NRs and CYP450 Subfamilies Genes

As shown in Figure 3A, mRNA expression levels of AHR in low and high-dose groups significantly decreased (P < 0.001), while the middle-dose group increased (P < 0.01). The mRNA expression levels of CYP1A1, CYP1A5 and CYP1B1 in Cd groups significantly decreased (P < 0.01 or P < 0.001). The mRNA level of CAR in low and middle-dose groups remarkably increased (P < 0.05 or P < 0.001) while decreasing in the high-dose group (P < 0.01). The CYP2C18, CYP2D6 and CYP3A4 mRNA levels showed no marked changes in low-dose groups. In middle-dose groups, the mRNA levels of CYP2C18 and CYP2D6 increased (P < 0.05 or P < 0.01) and decreased subsequently while still higher than or near the levels of the control groups. CYP3A4 mRNA expression levels showed a decreasing trend. With the increase of Cd dose, in the overall trend, the expression levels of mRNA of CYP1A1, CYP1A5, CYP1B1 and CYP3A4 showed a downward trend, while those of CYP2C18 and CYP2D6 showed an upward trend (Figure 3B).Figure 3 mRNA expression levels of NRs and CYP450 subfamilies genes. (A) The transcript levels of AHR, CYP1A1, CYP1A5, CYP1B1 and CAR, CYP2C18, CYP2D6, CYP3A4 in different Cd groups. (B) Heatmap of CYP450 subfamilies gene mRNA levels. Significant differences between the control groups and Cd groups are expressed as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

Effects of Cd on Mitochondrial-Related Molecules in Chicken Spleen

MFF, Fis1 and Mfn2 (Figures 4A, Figure 4, Figure 4) mRNA levels increased significantly in low and middle-dose groups (P < 0.01 or P < 0.001) and decreased to similar levels to the control in high-dose groups. In low-dose groups, OPA1 and Mfn1 (Figure 4, Figure 4) mRNA levels remarkably increased (P < 0.05), while in middle and high-dose groups, they decreased significantly (P < 0.05, P < 0.01 or P < 0.001)Figure 4 Effects of Cd on mitochondrial dynamics related molecules in chicken spleen. (A-E): the relative transcript levels of MFF, Fis1, OPA1, Mfn1 and Mfn2 in different Cd groups. Significant differences between the control groups and Cd groups are expressed as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Effects of Cd on Mitochondrial mtUPR-Related Factors in Chicken Spleen

To determine whether Cd could influence mtUPR, the mRNA levels of mtUPR-related factors were tested (Figure 5A). Compared with the control groups, except SOD2, the levels of all the genes in low-dose groups increased (P < 0.05, P < 0.01, or P < 0.001). The mRNA levels of SIRT1, PGC1-α, NRF1 and TFAM in the middle-dose groups increased (P < 0.05, p < 0.01, or P < 0.001) while the SIRT1, PGC1-α, NRF1 and HtrA2 mRNA levels in high-dose groups decreased (P < 0.01, or P < 0.001). The level of TFAM in the high-dose group decreased, but the decline was nonsignificant (P > 0.05). This current trend of PCG1-α (Figure 5, Figure 5) was also confirmed in protein levels. For SOD2 groups, there were no remarkable changes in mRNA levels among the different groups, but the protein level changes (Figure 5, Figure 5) were significant (P < 0.01 or P < 0.001). The mRNA levels of all genes were increased in low and medium dose groups, and decreased in high dose groups.Figure 5 Relative mRNA expression and protein levels of mitochondrial UPR-related genes affected by Cd in chicken spleens. (A) the mRNA expression levels of SIRT1, PGC-1α, NRF1, TFAM, SOD2 and Htra2 in different Cd groups. (B, D) Protein levels of PGC-1α. (C, E) Relative protein levels of SOD2. Significant differences between the control groups and Cd groups are expressed as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Effects of Cd on Mitochondrial Functional Protein Genes in Chicken Spleen

The mRNA expression levels of VDAC-1 and Cyt-C (Figures 6A and B) showed an increasing trend with the gradual increase of Cd exposure dose, while the levels of COA6 (Figure 6C) showed a decreasing trend. The mRNA levels of PRDX3, RAF and SIRT3 (Figures 6D–F) showed an overall downward trend. There were no remarkable mRNA level changes in low-dose groups of all the mitochondrial functional protein genes (P > 0.05). In the middle-dose and high-dose groups, VDAC-1 and Cyt-C mRNA levels increased (P < 0.01 or P < 0.001), and the mRNA expression levels of COA6 and PRDX3 decreased (P < 0.01 or P < 0.001). SIRT3 mRNA levels in the low-dose group increased a bit and decreased in the middle-dose group, but the changes were not significant, while the decline in the high-dose group was significant (p < 0.05).Figure 6 mRNA expression levels mitochondrial functional protein genes affected by Cd in chicken spleen. (A-F) the relative mRNA expression levels of VDAC1, Cyt-C, COA6, PRDX3, RAF1 and SIRT3 in different Cd groups. Significant differences between the control groups and Cd groups are expressed as follows: *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

DISCUSSION

Cd accumulates in plants due to soil pollution caused by industrial Cd emissions, which eventually leads to Cd accumulation in animals and poses a threat to the health of humans and animals. The spleen, an essential immune organ, is prone to the toxic effects of Cd (Demenesku, et al., 2014). Cd has been reported to disrupt the structure and function of the spleen, causing morphological changes in chicken spleen (Xu, et al., 2017). Reports have shown that Cd can cause splenic cell apoptosis and autophagy, resulting in splenic injury (Qu, et al., 2019; Chu, et al., 2023). In some reports, mtUPR and NRs could induce injury (Ogunbileje, et al., 2016; Zhang, et al., 2019a; Shi, et al., 2023), but how they work in splenic injury has not been fully elucidated. Our study focused on how Cd influenced mtUPR and NRs in the spleen.

Here, we also confirmed that the Cd-induced morphological changes in the spleen. We found that with the increase in Cd dose, the boundary between red and white pulp in the spleen was gradually blurred, and the number of spleen bodies declined. Compared with the control group, the arrangement of lymphocytes in the Cd dose group reduced, the thickness of the sheath artery wall increased, at the same time, pathological changes in the spleen became obvious with the increase of Cd dose exposure. The results indicated that the spleen was the target organ for Cd to exert toxic effects.

Enzymes associated with CYP450 contribute to the metabolism of both endogenous and exogenous substances within the body (Wang, et al., 2014). Cd changed the contents and activities of the CYP450 enzyme system in the liver of chickens (Cong, et al., 2019). AHR activation induces the CYP1 enzyme, which oxidizes AHR ligands, leading to ligand metabolic clearance and detoxification (Schiering, et al., 2017). NXRs are activated by the exogenous toxin aluminum, inducing CYP450s transcription in the chicken spleens (Cao, et al., 2023). Cd induces metabolic disorders in CYP450 enzymes by disrupting NRs response, which reduces the brain's ability to metabolize exogenous substances (Lv, et al., 2023). We also found that the CYP450 contents (total CYP450 and Cyt b5) and the activities of the enzyme system (AH, APND, NCR and ERND) changed (Figure 2). Results of our study showed that the AHR and CYP1 levels decreased. CAR, a xenosensor, regulates CYP2C18, CYP3A4, and CYP2D6 (Chen and Goldstein, 2009; Chang, et al., 2016; Rakateli, et al., 2023). Report has shown that CYP450s could resist DEHP's toxicity on cardiomyocytes. (Zhang, et al., 2019b). In chicken liver, CYP450s could also be changed by toxin (Zou, et al., 2024). In our study, CAR induced changes in CYP450s, which means Cd induced the disruption of CYP450s. The changes in AHR, CAR and the downstream factors levels meant that NRs response is triggered to defend against the toxin of Cd. Mitochondria is the target organ of Cd toxicity damage. Anyway, Cd caused the CYP450 enzymes to be altered and the beginning of detoxification in the chicken spleen through NRs reaction. All the results indicated the toxin of Cd in the spleen cells induced the CYP450 system response to detoxify to decrease the possible damages.

Under normal conditions, mitochondria undergo fusion and division. This process helps maintain the balance of the mitochondrial system and ensures that cells can function properly. However, in the case of heart injury, there is an increase in Fis1 and MFF expression, while Mfn1 and Mfn2 decline (Boovarahan, et al., 2022). Cd destroys cells by inducing excessive mitochondrial fission (Wu, et al., 2022). Cd treatments enhanced Fis1 and MFF mRNA levels and decreased Mfn1, Mfn2, and OPA1 mRNA levels in sheep kidneys (Wu, et al., 2022). In chicken kidneys, Cd destabilized mitochondrial dynamics related factors (OPA1, Mfn1, Mfn2, and MFF) levels (Xu, et al., 2017). In this study, we got similar results, in 35 mg/kg groups, fusion protein OPA1, Mfn1 and Mfn2 increased, and in the middle-dose and high-dose groups decreased. The results suggested that in the low-dose groups, mitochondria maintain normal function through fusion. Still, there may be a limit to this fusion capacity, and once this limit is exceeded, mitochondrial fusion will be reduced. We found that in low and middle-dose groups, MFF and Fis1 increased significantly and declined in high-dose groups. Similar to the fusion results, capacity was likely limited. All the results showed that Cd influenced spleen mitochondrial dynamics by breaking the normal fusion and decomposition balance of mitochondria and disturbing the normal function of mitochondria.

To confirm how mtUPR influenced the splenic injury induced by Cd, mtUPR-related factors were detected. Cd treatment down-regulates PGC-1α and SIRT1 mRNA levels in sheep kidneys and rabbit livers (Zhang, et al., 2021a; Wu, et al., 2022). It has been reported that PGC1-α, TFAM, Nrf1 and Drp1 are significantly upregulated in neurons with brain injury (Fan, et al., 2021). Cd can also induce the interference of mtUPR-related factors (TFAM, Nrf1, and Htra2) (Ge, et al., 2019) in the chicken kidney. The upregulation of Htra2 level and proteasome activity contributes to the degradation of misfolded proteins (Papa and Germain, 2011) and promotes the degradation of misfolded proteins. Cd increases the transcription level of SOD2 in mice liver (Pi, et al., 2015) and expression in testicular tissue (Zhou, et al., 2022) to protect mitochondria. In our study, the levels of mtUPR-related genes were changed by Cd, and the SIRT1/Htra2 mtUPR pathway was activated. The transcript levels of mtUPR molecules increased with increasing doses of Cd, suggesting Cd dose dependence of mtUPR. It was interesting in our study that in the low and middle-dose groups, all the genes increased but decreased in the high-dose group. It seemed that there was not enough potential to respond to the toxin. Compared to the results of the histopathological sections, the spleen damage was at least partially attributable to the activation of mtUPR.

Mitochondrial function-related genes are detected to confirm the effect of Cd. Cd could upregulate the VDAC1, Cyt C and SIRT3 in chicken kidney (Zhang, et al., 2020). Referred to the previous report, the lack of SIRT3 will reduce the level of acetylated PRDX3 and reduce the antioxidant capacity of PRDX3 (Wang, et al., 2020). The decline of SIRT3 and PRDX3 probably indicates the serious disruption of mitochondrial function. It is reported that RAF1 is enhanced by Cd in piglet epididymis tissues (Zhang, et al., 2021b). In our study, COA6 was decreased by Cd, which means the Cd disrupted electron transport in the mitochondria. Cd feeding changed the mitochondrial functional protein gene levels in this study. In the low-dose groups, mitochondria had enough potential to resist Cd toxin because all the levels did not show remarkable changes. However, in high-dose groups, all of the mRNA levels changed abnormally. The mitochondrial damage from the Cd toxin made the VDAC-1 and Cyt-C mRNA levels increase, while COA6, PRDX3, RAF1 and SIRT3 mRNA levels decrease. The increasing and decreasing trends were concentration-dependent. All the results suggested that Cd damaged the transcription level of functional protein of spleen mitochondria, and the normal function of mitochondria was destroyed, causing the dysfunction of mitochondrial function. According to the study, Cd specifically affects the spleen and disrupts the function of the mitochondria's antioxidant system. These mitochondrial functional proteins are closely related to mitochondrial stress and oxidative-antioxidant homeostasis. The alteration of their transcriptional levels led to abnormal mitochondrial function and abnormal accumulation of unfolded or misfolded proteins, ultimately leading to the mtUPR. Although the mtUPR had the function of promoting cellular survival, it was clear that Cd-triggered mitochondrial abnormality had gone beyond the protective capacity of the mtUPR and ultimately destroyed splenocytes.

In this study, we found that Cd entered cells, activated the NRs response, disrupting the expression of the NRs-regulated CYP450 enzyme system, and causing metabolic disorders that ultimately led to splenic injury. On the other hand, Cd-induced mitochondrial dysfunction disrupted the balance of clearance of unfolded and misfolded proteins within mitochondria, which in turn activated the mtUPR response and also led to spleen injury.

CONCLUSION

In conclusion, our study demonstrated that Cd could cause spleen damage by regulating mtUPR and NRs response (Figure 7). Cd could trigger the NRs response to induce CYP450 contents, and CYP450 enzymatic activities changed. This affected mitochondrial fusion and division, especially at the high dose, changed mitochondrial homeostasis, and disrupted the normal function of mitochondria. Mitochondrial unfolded protein and NRs responses contributed to the changes and damages. Our study provides a new mechanism for the spleen damage caused by Cd in chickens.Figure 7 Mechanism diagram of spleen damage induced by Cd in chicken via mtUPR and NRs response-dependent pathway.

Figure 7

DISCLOSURES

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix Supplementary materials

Image, application 1

ACKNOWLEDGMENTS

This study has received assistance from National Natural Science Foundation of China (No. 32172932 and No. 32302934 ), Key Program of Natural Science Foundation of Heilongjiang Province of China (No. ZD2021C003 ), China Agriculture Research System of MOF and MARA (No. CARS-35 ), Distinguished Professor of Longjiang Scholars Support Project (No. T201908), Heilongjiang Touyan Innovation Team Program and “Academic Backbone” Project of Northeast Agricultural University (No. 20YJXG11 ).

Supplementary material associated with this article can be found in the online version at doi:10.1016/j.psj.2024.104167.
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REFERENCES

Boovarahan S.R. Chellappan D.R. Ali N. AlAsmari A.F. Waseem M. Alabdulrahim A.S. Alzahrani Z.A. Kurian G.A. Diabetic hearts exhibit global DNA hypermethylation that alter the mitochondrial functional genes to enhance the sensitivity of the heart to ischemia reperfusion injury Biomedicines 10 2022 3065 36551820
Cai G. Lin F. Wu D. Lin C. Chen H. Wei Y. Weng H. Chen Z. Wu M. Huang E. Ye Z. Ye Q. Rosmarinic acid inhibits mitochondrial damage by alleviating unfolded protein response Front. Pharmacol. 13 2022 859978
Cao C. Liu Y. Yang Z. Ouyang H. Fu Q. Li X. The mechanisms of aluminum-induced immunotoxicity in chicks Poult. Sci. 102 2023 102251
Cao H. Zhang M. Xia B. Xiong J. Zong Y. Hu G. Zhang C. Effects of molybdenum or/and cadmium on mRNA expression levels of inflammatory cytokines and HSPs in duck spleens Biolog. Trace Elem. Res. 170 2016 237 244
Chang J.H. Chen J. Liu L. Messick K. Ly J. Rifampin-mediated induction of tamoxifen metabolism in a humanized PXR-CAR-CYP3A4/3A7-CYP2D6 mouse model Drug Metabol. Disposit. 44 2016 1736 1741
Chen M. Li X. Fan R. Yang J. Jin X. Hamid S. Xu S. Cadmium induces BNIP3-dependent autophagy in chicken spleen by modulating miR-33-AMPK axis Chemosphere 194 2018 396 402 29223809
Chen Y. Goldstein J.A. The transcriptional regulation of the human CYP2C genes Curr. Drug Metabol. 10 2009 567 578
Chu X. Dai X. Pu W. Guo H. Huang G. Huang B. Cui T. Zhang C. Co-exposure to molybdenum and cadmium triggers pyroptosis and autophagy by PI3K/AKT axis in duck spleens Environm. Toxicol. 38 2023 635 644
Cong Y. Chi Q. Teng X. Li S. The protection of selenium against cadmium-induced mitochondrial damage via the cytochrome P450 in the livers of chicken Biolog. Trace Elem. Res. 190 2019 484 492
Demenesku J. Mirkov I. Ninkov M. Popov Aleksandrov A. Zolotarevski L. Kataranovski D. Kataranovski M. Acute cadmium administration to rats exerts both immunosuppressive and proinflammatory effects in spleen Toxicology 326 2014 96 108 25446329
Dvorak Z. Pavek P. Regulation of drug-metabolizing cytochrome P450 enzymes by glucocorticoids Drug Metabol. Rev. 42 2010 621 635
El-Sherbeni A.A. El-Kadi A.O. Microsomal cytochrome P450 as a target for drug discovery and repurposing Drug Metabol. Rev. 49 2017 1 17
Fan H. Ding R. Liu W. Zhang X. Li R. Wei B. Su S. Jin F. Wei C. He X. Li X. Duan C. Heat shock protein 22 modulates NRF1/TFAM-dependent mitochondrial biogenesis and DRP1-sparked mitochondrial apoptosis through AMPK-PGC1α signaling pathway to alleviate the early brain injury of subarachnoid hemorrhage in rats Redox Biol. 40 2021 101856
Frigo D.E. Bondesson M. Williams C. Nuclear receptors: from molecular mechanisms to therapeutics Ess. Biochem. 65 2021 847 856
Ge J. Zhang C. Sun Y.C. Zhang Q. Lv M.W. Guo K. Li J.L. Cadmium exposure triggers mitochondrial dysfunction and oxidative stress in chicken (Gallus gallus) kidney via mitochondrial UPR inhibition and Nrf2-mediated antioxidant defense activation Sci. Total Environm. 689 2019 1160 1171
Guan T.Q. Qiu B.H. Nurmamedov H. Talukder M. Lv M.W. Li J.L. Cadmium-induced splenic lymphocytes anoikis is not mitigated by activating Nrf2-mediated antioxidative defense response J. Inorg. Biochem. 234 2022 111882
Guo K. Ge J. Zhang C. Lv M.W. Zhang Q. Talukder M. Li J.L. `Cadmium induced cardiac inflammation in chicken (Gallus gallus) via modulating cytochrome P450 systems and Nrf2 mediated antioxidant defense Chemosphere 249 2020 125858
Hao R. Jiang Y. Li F. Sun-Waterhouse D. Li D. MiR-182-5p/TLR4/NF-κB axis contributes to the protective effect of caffeic acid phenethyl ester against cadmium-induced spleen toxicity and associated damage in mice Food Chem. Toxicol. 158 2021 112654
Hu J. Chen G. Xu K. Wang J. Cadmium in cereal crops: uptake and transport mechanisms and minimizing strategies J. Agric. Food Chem. 70 2022 5961 5974 35576456
Imai Y. Ito A. Sato R. Evidence for biochemically different types of vesicles in the hepatic microsomal fraction J. Biochem. 60 1966 417 428 4291137
Koleva M. Kastelova A. Staneva-Stoytcheva D. Stoytchev T. Combined effect of propranolol with nifedipine or with diltiazem on rat liver monooxygenase activities Toxicol. Lett. 105 1999 153 161 10221277
Li H. Wang H. Activation of xenobiotic receptors: driving into the nucleus Expert Opinion Drug Metabol. Toxicol. 6 2010 409 426
Li X.W. Li S. Yang Y. Talukder M. Xu X.W. Li C.X. Zhang C. Li X.N. Li J.L. The FAK/occludin/ZO-1 complex is critical for cadmium-induced testicular damage by disruption of the integrity of the blood-testis barrier in chickens J. Hazard. Mat. 470 2024 134126
Lin Y.F. Schulz A.M. Pellegrino M.W. Lu Y. Shaham S. Haynes C.M. Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response Nature 533 2016 416 419 27135930
Lv M.W. Zhang C. Ge J. Sun X.H. Li J.Y. Li J.L. Resveratrol protects against cadmium-induced cerebrum toxicity through modifications of the cytochrome P450 enzyme system in microsomes J. Sci. Food Agric. 103 2023 5883 5892 37115015
Mackowiak B. Hodge J. Stern S. Wang H. The roles of xenobiotic receptors: Beyond chemical disposition Drug Metabol. Dispos. 46 2018 1361 1371
Majumder R. Datta M. Pal P.K. Bhattacharjee B. Bandyopadhyay D. Protective mechanisms of melatonin on caprine spleen injury induced by cadmium (Cd): An in vitro study Melat. Res. 2 2019 57 75
Manikandan P. Nagini S. Cytochrome P450 structure, function and clinical significance: A review Curr. Drug Targ. 19 2018 38 54
Mazaira G.I. Zgajnar N.R. Lotufo C.M. Daneri-Becerra C. Sivils J.C. Soto O.B. Cox M.B. Galigniana M.D. Nuclear receptors: A historical perspective Method. Mol. Biol. (Clifton, N.J.) 1966 2019 1 5
Nash T. The colorimetric estimation of formaldehyde by means of the Hantzsch reaction Biochem. J. 55 1953 416 421 13105648
Nebert D.W. Proposed role of drug-metabolizing enzymes: regulation of steady state levels of the ligands that effect growth, homeostasis, differentiation, and neuroendocrine functions Mol. Endocrinol. (Baltimore, Md.) 5 1991 1203 1214
Ogunbileje J.O. Porter C. Herndon D.N. Chao T. Abdelrahman D.R. Papadimitriou A. Chondronikola M. Zimmers T.A. Reidy P.T. Rasmussen B.B. Sidossis L.S. Hypermetabolism and hypercatabolism of skeletal muscle accompany mitochondrial stress following severe burn trauma Am. J. Physiol. Endocrinol. Metabol. 311 2016 436 448
Papa L. Germain D. Estrogen receptor mediates a distinct mitochondrial unfolded protein response J. Cell Sci. 124 2011 1396 1402 21486948
Pathak N. Khandelwal S. Role of oxidative stress and apoptosis in cadmium induced thymic atrophy and splenomegaly in mice Toxicol. Lett. 169 2007 95 108 17267144
Pi H. Xu S. Reiter R.J. Guo P. Zhang L. Li Y. Li M. Cao Z. Tian L. Xie J. Zhang R. He M. Lu Y. Liu C. Duan W. Yu Z. Zhou Z. SIRT3-SOD2-mROS-dependent autophagy in cadmium-induced hepatotoxicity and salvage by melatonin Autophagy 11 2015 1037 1051 26120888
Qu K.C. Wang Z.Y. Tang K.K. Zhu Y.S. Fan R.F. Trehalose suppresses cadmium-activated Nrf2 signaling pathway to protect against spleen injury Ecotoxicol. Environm. Saf. 181 2019 224 230
Rakateli L. Huchzermeier R. van der Vorst E.P.C. AhR, PXR and CAR: From xenobiotic receptors to metabolic sensors Cells 12 2023 2752 38067179
Runkel E.D. Liu S. Baumeister R. Schulze E. Surveillance-activated defenses block the ROS-induced mitochondrial unfolded protein response PLoS Gen. 9 2013 e1003346
Schiering C. Wincent E. Metidji A. Iseppon A. Li Y. Potocnik A.J. Omenetti S. Henderson C.J. Wolf C.R. Nebert D.W. Stockinger B. Feedback control of AHR signalling regulates intestinal immunity Nature 542 2017 242 245 28146477
Shao L.W. Niu R. Liu Y. Neuropeptide signals cell non-autonomous mitochondrial unfolded protein response Cell Res. 26 2016 1182 1196 27767096
Shi Y.S. Zhao Y. Li X.N. Li M.Z. Li J.L. Xenobiotic-sensing nuclear receptors as targets for phthalates-induced lung injury and antagonism of lycopene Chemosphere 312 2023 137265
Sonoda J. Pei L. Evans R.M. Nuclear receptors: Decoding metabolic disease FEBS Lett. 582 2008 2 9 18023286
Valko M. Jomova K. Rhodes C.J. Kuča K. Musílek K. Redox- and non-redox-metal-induced formation of free radicals and their role in human disease Arch Toxicol. 90 2016 1 37 26343967
Wang B. Yang S. Hu J. Li Y. Multifaceted interaction of the traditional chinese medicinal herb schisandra chinensis with cytochrome P450-mediated drug metabolism in rats J. Ethnopharmacol. 155 2014 1473 1482 25091466
Wang G. Fowler B.A. Roles of biomarkers in evaluating interactions among mixtures of lead, cadmium and arsenic Toxicol. Appl. Pharmacol. 233 2008 92 99 18325558
Wang H. Han Q. Chen Y. Hu G. Xing H. Novel insights into cytochrome P450 enzyme and solute carrier families in cadmium-induced liver injury of pigs Ecotoxicol. Environm. Saf. 211 2021 111910
Wang P. Wang J. Sun Y.J. Yang L. Wu Y.J. Cadmium and chlorpyrifos inhibit cellular immune response in spleen of rats Environm. Toxicol. 32 2017 1927 1936
Wang Z. Sun R. Wang G. Chen Z. Li Y. Zhao Y. Liu D. Zhao H. Zhang F. Yao J. Tian X. SIRT3-mediated deacetylation of PRDX3 alleviates mitochondrial oxidative damage and apoptosis induced by intestinal ischemia/reperfusion injury Redox Biol. 28 2020 101343
Williams C.H. Kamin H. Microsomal triphosphopyridine nucleotide-cytochrome c reductase of liver J. Biol. Chem. 237 1962 587 595 14007123
Wu Y. Yang F. Zhou G. Wang Q. Xing C. Bai H. Yi X. Xiong Z. Yang S. Cao H. Molybdenum and cadmium co-induce mitochondrial quality control disorder via FUNDC1-mediated mitophagy in sheep kidney Front. Vet. Sci. 9 2022 842259
Xia J. Lin J. Zhu S.Y. Du Z.H. Guo J.A. Han Z.X. Li J.L. Zhang Y. Lycopene protects against atrazine-induced hepatotoxicity through modifications of cytochrome P450 enzyme system in microsomes Experim. Toxicol. Pathol. 68 2016 223 231
Xia Y. Zhang Y. Zhang J. Du Y. Wang Y. Xu A. Li S. Cadmium exposure induces necroptosis of porcine spleen via ROS-mediated activation of STAT1/RIPK3 signaling pathway Environm. Mol. Mutagen. 64 2023 382 392
Xu Z. Jin X. Pan T. Liu T. Wan N. Li S. Antagonistic effects of selenium on cadmium-induced apoptosis by restoring the mitochondrial dynamic equilibrium and energy metabolism in chicken spleens Oncotarget 8 2017 52629 52641 28881757
Zhang J. Zhu Y. Yu L. Yang M. Zou X. Yin C. Lin Y. Research advances in cadmium uptake, transport and resistance in rice (Oryza sativa L.) Cells 11 2022 569 35159378
Zhang L. Yang F. Li Y. Cao H. Huang A. Zhuang Y. Zhang C. Hu G. Mao Y. Luo J. Xing C. The protection of selenium against cadmium-induced mitophagy via modulating nuclear xenobiotic receptors response and oxidative stress in the liver of rabbits Environm. Poll. 285 2021 117301
Zhang Q. Zhang C. Ge J. Lv M.W. Talukder M. Guo K. Li Y.H. Li J.L. Ameliorative effects of resveratrol against cadmium-induced nephrotoxicity via modulating nuclear xenobiotic receptor response and PINK1/Parkin-mediated Mitophagy Food Funct. 11 2020 1856 1868 32068207
Zhang Q. Zhao Y. Talukder M. Han Y. Zhang C. Li X.N. Li J.L. Di(2-ethylhexyl) phthalate induced hepatotoxicity in quail (Coturnix japonica) via modulating the mitochondrial unfolded protein response and NRF2 mediated antioxidant defense Sci. Total Environm. 651 2019 885 894
Zhang Y. Li Y. Zhang J. Qi X. Cui Y. Yin K. Lin H. Cadmium induced inflammation and apoptosis of porcine epididymis via activating RAF1/MEK/ERK and NF-κB pathways Toxicol. Appl. Pharmacol. 415 2021 115449
Zhang Y. Shi G. Cai J. Yang J. Zheng Y. Yu D. Liu Q. Gong Y. Zhang Z. Taxifolin alleviates apoptotic injury induced by DEHP exposure through cytochrome P450 homeostasis in chicken cardiomyocytes Ecotoxicol. Environm. Saf. 183 2019 109582
Zhao X. Li X. Wang S. Yang Z. Liu H. Xu S. Cadmium exposure induces mitochondrial pathway apoptosis in swine myocardium through xenobiotic receptors-mediated CYP450s activation J. Inorg. Biochem. 217 2021 111361
Zhou J. Zeng L. Zhang Y. Wang M. Li Y. Jia Y. Wu L. Su P. Cadmium exposure induces pyroptosis in testicular tissue by increasing oxidative stress and activating the AIM2 inflammasome pathway Sci. Total Environm. 847 2022 157500
Zou Y. Zhang S. Yang J. Qin C. Jin B. Liang Z. Yang S. Li L. Long M. Protective effects of astaxanthin on ochratoxin A-induced liver injury: Effects of endoplasmic reticulum stress and mitochondrial fission-fusion balance Toxins 16 2024 68 38393146
