Cadmium is a bioaccumulative toxic heavy metal element that can accumulate in reproductive organs and cause reproductive system disorders [1, 25]. In the present study, we observed that Cd could dose-dependently deposited in the magnum tissue and a positive correlation occurred between the accumulation of Cd in the magnum and Cd content in egg whites. Previous researches have indicated that environmental Cd exposure was usually associated with chickens’ fallopian tube damage and poor egg quality, including poor eggshell and egg white quality [26]. The oviductal magnum epithelial cells of laying hens synthesize and secrete a large amount of egg white proteins in daily cycles, including OVM, OVAL, LYZ, ORM1 and AVD, among which OVM plays a key role in the gel properties of egg white and determines the albumen height and Haugh unit of egg white [23, 27, 28]. We assessed the impacts of dietary Cd on the expressions of major genes involved in egg white protein synthesis in oviductal magnum of laying hens. Results indicated that the expression of OVM gene was up-regulated in 15 mg/kg Cd group, but the expressions of OVM, OVAL, LYZ and ORM1 genes were obviously down-regulated when layers treated with 60 mg/kg Cd. Ovalbumin is the most abundant protein in egg white, accounting for more than 50% of the total egg protein [29]. Lysozyme is a well-known antibacterial protein, which accounts for about 3.4% of the total egg white proteins [30]. In contrast, we found that 60mg/kg Cd exposure obviously increased the expression of AVD gene, which is also a critical egg white antimicrobial protein with a strong ability to bind biotin [31]. However, the increased concentration of egg white avidin may adversely affect the innate immunity of newborn chicks [32]. Therefore, these results indicated that Cd could accumulate in the oviductal magnum of laying hens, which was closely related to the Cd content in egg white. Besides, Cd affects the synthesis of egg white protein by disturbing the expression of egg white-related protein genes in the oviductal magnum, but the specific mechanism needs to be further explored.
Cadmium has been known as a reproductive toxicant, and the induction of oxidative stress is widely considered as one of the major mechanisms by which Cd exerts reproductive toxicity [33]. Cd is implicated in the increase of ROS and the induction of oxidative stress through indirect mechanisms, that is, by affecting the activity of ROS scavengers or depleting GSH [34, 35]. Cd exposure can lead to oxidative damage and apoptosis of hens’ shell-gland, kidney, ovary and hepatocytes [1, 2, 3, 4]. In the present study, the concentration of Cd exposure decreased the activities of CAT and T-SOD and the level of GSH, and increased the content of MDA in the magnum tissue. These specific biomarkers are considered to be closely related to oxidative stress responded to Cd exposure [9]. A major function of Nrf2 that has been extensively studied is its role in resistance to oxidative stress [36]. Previous studies have indicated that the Nrf2-knockout mice are more susceptible to chemical toxicity and disease conditions related to oxidative pathology [37, 38]. Nrf2 is considered to be the “master regulator” of the antioxidant reaction, which protects cells from the toxicity of free radical by modulating the expression of genes encoding antioxidants through interacting with the antioxidant response elements (ARE) [39, 40]. Many studies have shown that Nrf2 signaling pathway can attenuate the toxicity of multiple organs induced by Cd, including in the kidney, testicular, liver, etc. [7, 41, 42]. In this study, the transcription or protein expression level of Nrf2 were obviously downregulated in 30 and 60 mg/kg Cd treatment groups. Meanwhile, the expressions of Nrf2 target genes NQO1, HO-1, GCLC and GCLM also showed similar trends. Keap1, as a key endogenous repressor of Nrf2, can control the stability and accumulation of Nrf2 [43, 44]. In this study, the rapid up-regulation of Keap1 protein expression level in the 60 mg/kg Cd group further confirmed the inhibitory effect of Cd on the Nrf2 signaling pathway. Besides, the expressions of Nrf2-activited genes SOD1, SOD2 and SOD3 genes were down-regulated significantly in the 60 mg/kg Cd treatment group. These results suggested that 60 mg/kg Cd exposure induced oxidative stress through inhibiting Keap1-Nrf2-ARE signaling pathway and the activities of antioxidant enzymes in the magnum tissues of laying hens.
Inflammation is a protective response of organism to injury. Many studies have indicated that Cd exposure impaired innate immune parameters, triggered ROS generation and inflammation [1, 3]. Consistently, the current study found that the relatively high-dose Cd exposure upregulated the expressions of TNFα, IL-1β and NF-κB genes obviously in the magnum of laying hens. IL-1 and TNFα represent the typically pro-inflammatory cytokines that are released rapidly in response to tissue damage or infection [45, 46], which have also been identified as the downstream targets of NF-κB [47]. Studies demonstrated that Nrf2 not only participates in the regulation of oxidative/xenobiotic stress response, but also suppresses the inflammatory response. The decrease in Nrf2 with subsequent increase in oxidative stress markers led to the activation of TGF-β and NF-kB [48]. These results suggested that Cd exposure triggered an inflammatory response in the magnum tissue, and this effect may be mediated by activating NF-κB via inhibiting the Nrf2/HO-1 signaling pathway.
Studies suggest that mitochondrial dysfunction may contribute to reproductive toxicity in response to Cd [49]. Mitochondrial structural damage and mitochondria-mediated apoptosis and autophagy have been reported in Cd-treated chicken liver and kidney [7, 50]. For the genes involved in mitochondria dysfunction, the present study indicated that Cd exposure down-regulated the expressions of Mfn1 and Mfn2, and up-regulated the mRNA level of MFF in the oviductal magnum of laying hens. The formation of excess ROS depends heavily on dysfunctional mitochondria, and it is important to note that mitochondrial ROS formation can result in the opening of the mitochondrial permeability transition pore (mPTP) [51]. During conditions of excess ROS and Ca2+ overload, opening the mPTP helps to maintain Ca2+ levels in the mitochondrial matrix [52]. However, in dysregulated mPTP, matrix metabolites are released, which is accompanied by depolarization of the mitochondrial membrane potential (MMP) and inhibition of oxidative phosphorylation, ultimately leading to mitochondrial damage [53, 54]. In the present study, we found that dietary Cd exposure upregulated the mRNA levels of mPTP-related proteins, including GRP75, VDAC1 and MCU obviously. Known as a master regulator of mitochondrial biogenesis, PGC-1α plays a role in energy metabolism and mitochondria homeostasis [55]. A reduction in PGC-1α expression appears to active PINK1/Parkin-mediated mitophagy [56]. Studies have shown that the PINK1/Parkin pathway is the primary mechanism involved in mitophagy, which occurs when the balance of mitochondrial division and fusion is upset by Cd [57]. Accordingly, we found that exposure to Cd results in a decrease in PGC1α gene level in magnum while expression of PINK1, Parkin, Bnip3, LC3I and LC3II increases. These results suggested that, after exposure to Cd, the expression of mitochondrial fusion factors was limited, whereas the expression of mitochondrial fission factors was enhanced, which caused an imbalance in mitochondrial dynamics. Mitophagy is triggered by mitochondrial damage mediated by PINK1/Parkin.
In previous studies, ER stress (ERS) was found to induce mitophagy. Autophagy may be thought of as a cyto-protective response to an overload of unfolded or misfolded proteins during ERS [58]. Gao et al. (2020) demonstrated that blocking activation of the ERS response prevented the mitophagy process initiated by plumbum [59]. In the present study, we found that the expressions of ATF4, ATF6, IRE1α, CHOP, GRP78 and GRP94 were increased in the group of 60 mg/kg Cd, indicating that Cd can induce ER stress by activating ATF4, IRE1 and ATF6 signaling pathways in the magnum of laying hens. Several regulatory components link the ERS, unfolded protein response (UPR) and mitochondrial function, of which MAMs play a central role. ATF4 was reported to control expression of the ubiquitin ligase Parkin, a crucial regulator of mitochondria function and dynamics [60]. Phosphorylated MFN2 is a receptor of Parkin, which interacts with Parkin to promote the ubiquitination of mitochondrial proteins [61]. Xin et al. (2019) indicated that knockdown of Mfn2 led to aggravation of the PERK/ATF4 pathway [62]. Lin et al. (2021) demonstrated that excessive ROS accumulation is a critical activator of ERS, leading to mitochondrial Ca2+ accumulation via IP3R–GRP75–VDAC1 complex, and consequently programmed necrosis [63]. These results suggest that Cd exposure can promote mitophagy by inducing ER stress and disrupting the MAMs coupling.