Copper homeostasis dysregulation promoting cell damage and the association with liver diseases

Abstract

Copper plays an important role in many metabolic activities in the human body. Copper level in the human body is in a state of dynamic equilibrium. Recent research on copper metabolism has revealed that copper dyshomeostasis can cause cell damage and induce or aggravate some diseases by affecting oxidative stress, proteasome, cuprotosis, and angiogenesis. The liver plays a central role in copper metabolism in the human body. Research conducted in recent years has unraveled the relationship between copper homeostasis and liver diseases. In this paper, we review the available evidence of the mechanism by which copper dyshomeostasis promotes cell damage and the development of liver diseases, and identify the future research priorities.

Introduction

Copper (Cu) is an essential micronutrient in the human body and an important component of many key metabolic enzymes, ^[1] including copper-zinc superoxide dismutase (SOD1), cytochrome C oxidase (CCO), and lysyl oxidase (LOX). Because copper exists in the two forms of Cu (I)/Cu (II), it is a crucial cofactor in the body's redox system. In March 2022, researchers discovered a new form of copper-dependent cell death, i.e., cuproptosis, ^[2] which further underlines the importance of copper homeostasis in the human body. Copper levels in the human body are in dynamic equilibrium, and both inherited and acquired causes of copper dyshomeostasis can lead to or exacerbate certain diseases ^[3] such as Menkes disease, Wilson's disease (WD), and cancer. Recent studies have shown that copper dyshomeostasis can promote cell damage through a number of pathways and is closely related to the occurrence and development of a variety of liver diseases. ^[4,5] In this paper, we summarize the findings of recent studies and provide a reference for the diagnosis and treatment of liver diseases in the future.

Copper Homeostasis Regulation

In humans, the dietary Cu is primarily taken up at the duodenum. Copper absorbed by copper transporter 1 (CTR1) and divalent metal transporter 1 (DMT1) of enterocyte is delivered to the chaperones or sequestered by metallothionein (MT) and glutathione (GSH). The majority of Cu is transported to the liver via blood circulation. Copper homeostasis is strictly regulated from different dimensions, the early literature has depicted the detailed regulatory mechanisms. ^[6] In the body, the liver is the central organ of copper metabolism. Copper homeostasis of hepatocytes rely on duodenal absorption and bile excretion. In Figure 1, a schematic diagram of copper metabolism at cellular and molecular levels in humans is shown.

Figure 1.

Copper homeostasis in liver depends on the balance between absorption of copper in the duodenum and its excretion in the bile. (1) After being reduced by Cyt B or other reductases, Cu (I) is transported into the duodenum enterocytes by the non-specific DMT1 and the specific CTR1. Chaperones inside the cell transport copper to different cellular organelles or bind MT to store Cu. ATP7A either pumps copper into the TGN to assist cuproenzyme production or exports copper out of the cell. The Cu (I) in blood is oxidized Cu (II), which is then predominantly bound to albumin and α2-macroglobulin in the portal circulation and delivered to the liver. (2) After being reduced by STEAP, Cu (I) is transported into hepatocytes by CTR1 and distributed by chaperones. ATOX1 carries copper to ATP7B, which then transports copper into the TGN for integration into cuproenzymes (i.e., CP) and release by hepatocytes. It is likely that GSH and MT bind Cu for intracellular copper stores. CCS delivers Cu to SOD1. Cox17 carries Cu to CCO in the mitochondria. Excess copper is also transported across the canalicular membrane into bile for excretion by ATP7B. ATOX1: Antioxidant 1 copper chaperone; CCO: Cytochrome C oxidase; CCS: Copper chaperone for superoxide dismutase; Cox17: Cytochrome C oxidase assembly protein 17; CP: Ceruloplasmin; CU ⁺: Copper (I); CU2 ⁺: Copper (II); Cyt B: Cytochrome B; DMT1: Divalent metal transporter 1; SOD1: Copper-zinc superoxide dismutase; STEAP: Six trans membrane epithelial antigen of the prostate; TGN: Trans-Golgi network; CTR1: Copper transporter 1; MT: Metallothionein; GSH: Glutathione.

In terms of copper absorption, studies have illustrated that the abundance of CTR1, the high-affinity Cu transporter, is negatively regulated by the cellular copper concentration. When copper deficiency, CTR1 is up-regulated in enterocytes and hepatocytes, which increases the cellular uptake of copper. At the same time, the liver will induce copper conservation for reducing copper losses. In response to Cu excess, CTR1 of enterocyte and hepatocyte adducted and metallothionein transcription increased to bind the excess Cu for reducing Cu toxicity. However, Lin et al^[7] reported that DMT1 apparently transports copper intracellularly even when in excess, which explains copper is continuously absorbed by virtually all tissues and triggers pathological changes in WD. The copper incorporated by hepatocytes is secreted out of hepatocytes through different ATP7B-dependent manners: under physiological Cu conditions, hepatic ATP7B located in the trans-Golgi network and assemble Cu (I) to ceruloplasmin (CP), which is targeted to other tissues (such as the brain and placenta) through the circulation system; in respond to copper excess, ATP7B translocate to either the apical surface or lysosome, where copper exports via bile. The copper complexed with bile salts cannot be recycled by the intestine and eliminated with feces. Overall, there was no significant feedback relationship between the liver and the gut.

Copper Dyshomeostasis Promotes Cell Damage

Copper and oxidative stress

Reactive oxygen species (ROS) are by-products of cellular respiration and aerobic metabolism under physiological conditions. ^[8] Low and moderate levels of ROS are known to play an important role in cellular signaling in the body; however, high concentration of ROS can cause structural and functional damage at the cellular level, increase the risk of carcinogenesis, and induce cell death by oxidizing intracellular structures. ^[9] Under physiological conditions, the intracellular copper is in a dynamic equilibrium state. Copper overload or deficiency can induce oxidative stress in the cells.

Intracellular copper overload leads to oxidative stress

Intracellular copper overload induces the production of ROS by Fenton and Haber–Weiss reactions. Copper can reduce its intracellular content through the consumption of reduced GSH, resulting in increased sensitivity of cells to harmful stimulation. ^[10] Studies have shown that intracellular copper overload induces oxidative stress, leading to changes in mitochondrial membrane permeability, DNA damage, and increased expression of pro-apoptotic proteins, such as B-cell lymphoma-2 associated X protein (BAX), caspase-3, 9, and then induce apoptosis mediated by mitochondria. ^[11] Shao et al^[12] showed that an increase in intracellular copper can induce apoptosis through the accumulation of ROS and the activation of mitochondrial fission-related DRP1 protein, which promoted the accumulation of P53 in mitochondria.

Intracellular copper deficiency leads to oxidative stress

SOD converts intracellular superoxides, such as O ^2- into molecular oxygen and hydrogen peroxide, and reduces the accumulation of oxygen free radicals in the body. The activity of SOD1 is closely related to that of copper. ^[13] Copper deficiency limits the activity of several copper-dependent antioxidant enzymes, including catalase and glutathione peroxidase, and intracellular ROS accumulation induces oxidative stress. ^[14,15] SOD1 mutation is an important genetic cause of amyotrophic lateral sclerosis (ALS). Studies have demonstrated that copper deficiency in neurons leads to cell damage and death by increasing the misfolding of mutated SOD1 and altering its hydrophobicity. In addition, oral administration of copper complexes was shown to significantly reduce the number of motor neuron death in ALS mice.

Copper and proteasomes

The human proteasome, named 26S, consists of a 20S core and two 19S regulatory particles.Tumor cells exhibit increased activity of proteasomes, which promotes tumor growth by degrading cyclin-dependent kinase inhibitors (i.e., P21 and P27), tumor suppressors (i.e., P53), apoptosis-inducing factor (AIF), and apoptosis regulatory factor (i.e., BAX). ^[16,17] Gałczyńska et al^[16] demonstrated that proteasome inhibitors induce apoptosis by promoting the entry of cytochrome C (CytC) into the cytoplasmic matrix, binding, and activation of apoptotic protease-activating factor 1 (APAF-1) and caspase 9 precursors. In addition, proteasome inhibition can also trigger apoptosis through the accumulation of unfolded and misfolded proteins. Studies have shown that elevated intracellular copper inhibits the activity of the ubiquitin-proteasome system (UPS), which mainly exists in the form of 26S, which is conducive to the role of UPS and the timely decomposition of abnormal proteins in the cell. Livnat-Levanon et al^[18] found that acute oxidative stress within cells can lead to the rapid breakdown of the 26S proteasome into intact 20S core particles and 19S regulatory particles to reduce proteasome activity in HeLa cells.

Cuproptosis

Cuproptosis is a newly discovered form of cell death triggered by the targeted accumulation of copper in mitochondria. Excess copper in mitochondria binds to and induces the aggregation of lipoylated proteins, which is the key to the occurrence of cuproptosis. Cuproptosis has been shown to be closely related to the disorder of copper metabolism, mitochondrial respiration, and accumulation of lipoylated proteins. The mechanism of cuproptosis is briefly described below in the following three aspects.

Dysregulation of copper homeostasis

Dietary copper is absorbed by the small intestine and transported into the bloodstream by copper transporters. The liver plays a central role in copper metabolism in the body. ^[3,19] Intracellular copper levels are strictly regulated under physiological conditions. Studies have shown that copper ion carriers can overcome the mechanism of maintaining intracellular copper dynamic equilibrium. ^[2] Intracellular copper levels were found to increase after treatment with copper ionophore (i.e., elesclomol). Excessive copper lipoylated protein induces lipoylated protein oligomerization, leading to the occurrence of cuprotosis. In contrast, copper chelator, ferredoxins 1 (FDX-1; a direct binder of copper carrier elesclomol), and lipoyl synthase ( LIAS) gene knockout were shown to reduce the risk of cuprotosis.

Lipoylated protein aggregation

Aggregation of lipoylated protein is the key to cuprotosis. Protein lipoylation is a highly conserved post-translational lysine modification. Four key enzymes are known to occur only in the tricarboxylic acid cycle: dihydrolipoamide branched chain transacylase E2, glycine cleavage system protein H, dihydrolipoamide S-succinyltransferase, and pyruvate dehydrogenase complex acyltransferase dihydrolipoamide transacetylase (DLAT). Copper overload leads to oligomerization of lipoylated proteins, decrease in Fe-S cluster protein level, increase in heat shock protein 70 (HSP70) abundance, and toxic stress of protein, leading to cuproptosis. Genome-wide CRISPR-Cas9 loss-of-function screening identified FDX1 and LIAS genes as regulatory factors of protein lipoylation. Moreover, knockout of related coding genes or inhibition of the above two proteins was found to inhibit the occurrence of cuproptosis. ^[2]

Mitochondrial aerobic respiration regulates cuproptosis

Mitochondrial aerobic respiration plays an important role in the regulation of cuproptosis. ^[2] Compared with cells dependent on glycolysis, cells dependent on mitochondrial aerobic respiration have nearly 1000-fold greater sensitivity to copper ion carriers. Under hypoxic conditions (1% O₂), hypoxia-inducible factor-1 (HIF-1) plays a key regulatory role in the corresponding physiological changes in the organism, ^[20] and prolyl hydroxylase (PHD) is a rate-limiting enzyme of HIF-1 degradation reaction. Cuproptosis rate was not changed in cells cultured under normoxic conditions (21% O₂) treated with PHD inhibitor. In a study by Tsvetkov et al^[2], inhibition of mitochondrial aerobic respiration significantly inhibited the occurrence of cuprotosis, confirming the important role of mitochondrial aerobic respiration in the regulation of cuprotosis.

Copper and angiogenesis

Human angiogenesis is mediated by vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), interleukin 1, 6, 8 (IL-1, 6, 8), and other small molecules. ^[21] Studies have shown that copper can stimulate angiogenesis by directly binding to angiopoietin (ANG) or by binding to HIF-1 to activate the aforementioned angiogenesis-promoting small molecules. In addition, copper can promote angiogenesis by promoting endothelial proliferation and migration, and degradation of the extracellular matrix. ^[22] Decrease in intracellular copper can lead to cell damage and death by inhibiting angiogenesis and cutting off the nutrient supply of tissues.

Copper and apoptosis, autophagy, ferroptosis

X-linked inhibitor of apoptosis protein (XIAP) is a type of apoptosis inhibitor protein; its domains, BIR2 and BIR3, bind to and inhibit the activities of caspase 3, 7, and 9, and then regulate apoptosis. ^[23] Studies have shown a strong binding affinity of XIAP with copper. Binding with copper induces a reversible change in the conformation of XIAP, which impairs its ability to inhibit apoptosis. The structure of copper-XIAP complex is not stable which renders it liable to ubiquitinated proteasome degradation, decreasing the threshold of cell apoptosis. ^[10] In addition to regulating apoptosis, XIAP plays an important role in intracellular copper metabolism. Copper metabolism MURR1 domain1 (COMMD1) is closely related to the subcellular localization of copper-transporting P-type adenosine triphosphatase (ATP7B) in hepatocytes. XIAP regulates cellular copper levels by mediating ubiquitinated proteasome degradation of COMMD1, ^[24] Copper overload can reduce intracellular XIAP levels, leading to increased levels of COMMD1, promoting copper outflow from cells. ^[10,25]

Autophagy plays a key role in regulating multiple liver functions and maintaining hepatocyte homeostasis under physiological conditions, ^[26] whereas excessive autophagy leads to autophagic cell death. ^[27] Copper overload promotes the formation of autophagosomes by activating unc51-like autophagy activating kinase 1,2 (ULK1,2) dependent signal transduction pathway to induce autophagy. ^[28,29,30] Intracellular copper overload leads to apoptosis mainly through oxidative stress and mitochondrial injury. Yang et al^[31] found that intracellular copper overload produces large amounts of ROS that promote transcription and translation of autophagy-related genes; enhanced levels of autophagy in turn can suppress copper-induced mitochondrial dysfunction and apoptosis. In a study, the expression level of autophagy-related genes in ATP7B knockout cells was significantly up-regulated after copper treatment. ^[32] Polishchuk et al^[33] similarly found that excessive copper in the hepatocytes of WD patients and ATP7B-deficient mice promoted the increase of intracellular autophagy through Cu-mTORC1-TFEB signal transduction; in addition, the use of autophagy inhibitor Spautin-1 significantly accelerated the occurrence of apoptosis [Figure 2].

Figure 2.

Schematic illustration of the mechanism by which copper deficiency or overload promotes cell damage. Copper deficiency: (1) Copper directly binds ANG to stimulate angiogenesis and binds HIF-1 to activate IL-1, 6, 8, VEGF, and bFGF, Copper deficiency inhibits angiogenesis. (2) By generating ROS and inducing oxidative stress, SOD1 activity suppression activates apoptotic proteins. Copper overload: (1) By generating ROS, oxidative stress is induced, which increases the levels of APAF-1 and procaspase 9, subsequently activating caspase 3, 7, that further leads to the induction of apoptosis. (2) Proteasome inhibition decreases the expression of anti-apoptotic proteins (e.g., Bcl2) and increases the accumulation of P53 and cyclin-dependent kinase inhibitors P21 and P27, which activates the caspase cascade and results in cell cycle arrest in G1 phase. (3) The ROS-mTOR pathway triggers autophagy, which in turn suppresses copper-induced mitochondrial malfunction and apoptosis, and promotes cellular ferroptosis by reducing GPX4 and ferritin such as FTH-1 to increase the free iron in the cytosol. (4) Copper overload encourages the production of CP and GSH synthesis, which inhibits ferroptosis. (5) Copper-XIAP inhibits XIAP activities and enhances XIAP degradation to promote apoptosis. (6) Lipoylated protein aggregation and decreased Fe-S cluster proteins cause cuproptosis. ANG: Angiopoietin; CytC:cytochrome C; APAF-1: Apoptotic protease-activating factor 1; BAX: B-cell lymphoma-2 associated X protein; Bcl2: B-cell lymphoma-2; bFGF: Basic fibroblast growth factor; CP: Ceruloplasmin; Cu: Copper; DLAT: Pyruvate dehydrogenase complex acyltransferase dihydrolipoamide transacetylase; GPX4: Glutathione peroxidase 4; GSH: Glutathione; HIF-1: Hypoxia-inducible factor-1; ROS: Reactive oxygen species; SOD1: Copper-zinc superoxide dismutase; ULK1: unc51-like autophagy activating kinase 1; VEGF: Vascular endothelial growth factor; XIAP: X-linked inhibitor of apoptosis protein; mTOR: Mammalian target of rapamycin; IL: Interleukin.

Increased intracellular ferritin expression inhibits the occurrence of ferroptosis. Hou et al^[34] found that autophagy promotes cellular ferroptosis by increasing intracellular free iron levels by promoting intracellular ferritin degradation. Guo et al^[35] found that copper overload in mouse sperm cells inhibits the occurrence of apoptosis by inducing autophagy through the ROS-dependent AMPK-mTOR pathway. Conversely, it promotes cellular ferroptosis by reducing GPX4 and ferritin such as FTH-1. However, other studies have shown that copper overload in hepatocellular carcinoma (HCC) cells on the one hand promotes CP synthesis, which leads to a decrease in intracellular iron levels, and on the other hand, promotes intracellular GSH synthesis through Cu-HIF1α-SLC7A11 (a key protein for GSH synthesis) pathway, thereby inhibiting the occurrence of intracellular ferroptosis. ^[36,37]

Copper-mediated oxidative stress injury is a major pathological finding in patients with WD. ^[38] Mitochondrial structural and functional damage plays an important role in the development of WD, and significantly elevated mitochondrial copper levels can be found in patients with early stage WD. Zischka et al^[39] found that in the early stage of WD model mice, as the level of copper in the liver increased, the inner and outer membranes of the mitochondria of the liver cells cross-linked, which caused the gradual structural damage of the mitochondria, and this preceded the oxidative stress damage. There is an intracellular mechanism that eliminates damaged mitochondria through mitophagy to ensure the presence of the necessary number of functional mitochondria to meet the needs of the cell. ^[40] Increase in intracellular copper level and mitochondrial damage can lead to the occurrence of selective autophagy of mitochondria, which can reduce the toxicity of copper and inhibit the occurrence of apoptosis mediated by mitochondria. Cuproptosis is characterized by increased liver copper levels, accumulations of lipoylated proteins, and regulations of aerobic respiration. Mitochondria are at the core of cuprotosis, and selective autophagy of mitochondria may inhibit the occurrence of cuprotosis. Further studies are required to unravel the relationship between autophagy and cuprotosis.

Role of Copper Homeostasis Imbalance in Occurrence and Development of Liver Diseases

Copper and Wilson's disease

WD is an autosomal recessive genetic disorder caused by mutations of the ATP7B resulting in copper accumulation in the liver and other tissues, ^[4] leading to a series of lesions.

WD patients mainly present with liver and nervous system symptoms. Recent studies have found that excessive liver copper in WD patients can cause liver lesions by inhibiting nuclear receptor function and leading to mitochondrial dysfunction. Nuclear receptors (NRS) are ligand-dependent transcription factors involved in the regulation of cellular gene expression. Abnormally elevated copper in WD patients affects cellular lipid metabolism by inhibiting NRS. Liver X receptor (LXR) is one of the major targets of early copper action in WD. In the study by Hamilton et al^[41], treatment of ATP7B-deficient mice with LXR agonist T0901317 alone resulted in significant reductions in liver fibrosis and inflammatory cytokines, and improvement in liver function and histology. Previously, it was believed that abnormally elevated liver copper in WD patients mainly induced oxidative stress through the Fenton reaction, leading to mitochondrial dysfunction. However, studies conducted in recent years have found that mitochondrial copper increase about 200 times higher than the normal level before the occurrence of liver copper-mediated oxidative stress. Copper can directly cause mitochondrial inner and outer membrane crosslinking, mitochondrial membrane potential changes, reduced ATP production, and inhibition of mitochondrial DNA replication, ^[38,39,42] resulting in mitochondrial structure and function disorders, leading to apoptosis.

Earlier diagnosis and initiation of copper chelation therapy can significantly improve the prognosis of WD patients and prevent the occurrence of neurological symptoms. However, early diagnosis of WD with the existing diagnostic methods is typically challenging. Therefore, the development of new diagnostic methods and diagnostic biomarkers is a key imperative. Recent studies have found that blood concentrations of relative exchangeable copper ^[43] and ATP7B peptide ^[44] have high sensitivity and specificity in the diagnosis of WD, and the discovery of these markers has important implications. However, the practical application needs to be further verified.

Oral administration of copper chelator and zinc is the first-line treatment for WD. The adverse effects of long-term oral administration of copper chelator and zinc and the worsening of neurological symptoms are still a challenge in the treatment of WD. Studies on novel hepatocyte-and mitochondrial-directed copper chelators are expected to ameliorate this dilemma. Targeted molecular therapies to restore the localization and function of ATP7B and gene therapy using the adeno-associated virus vector to transduce ATP7B cDNA into defective cells are also under active development. ^[4,42] Copper can promote tumorigenesis and progression through multiple pathways, ^[5] but there is evidence that the incidence of liver malignancy in patients with WD is much lower than in those with other etiologies. ^[45] This may be attributable to long-term copper-chelating therapy in WD patients, masking the "natural" distribution of liver copper in patients with the disease, thereby reducing the incidence of liver malignancy in WD patients. ^[45] There is also evidence that excessive intake of copper can inhibit carcinogenesis induced by chemical toxicants in rats. However, long-term oral administration of copper chelator may lead to iron accumulation and subsequent damage to hepatocytes, which may be one of the causes of HCC in WD patients. In conclusion, HCC is a rare complication in WD patients, and elucidation of this association is helpful for the early diagnosis and management of liver malignancies in WD patients.

Copper and non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) is a group of diseases characterized by the accumulation of fat in the liver, including non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH). NAFLD can eventually lead to cirrhosis and HCC. According to epidemiological surveys, the global prevalence of NAFLD is close to 30%, and it is now the most common chronic liver disease in the world. ^[46] With the increasing recognition of NAFLD in recent years, it is considered the hepatic manifestation of the metabolic syndrome (Mets). In early 2020, an international expert consensus on a new definition of metabolic-associated fatty liver disease (MAFLD) recommended the replacement of the term NAFLD. ^[47] There are similarities and differences between the diagnostic criteria for NAFLD and MAFLD. ^[48] While most patients can be diagnosed with both MAFLD and NAFLD, there are still some patients with NAFLD alone or MAFLD alone. In other words, not all cases of NAFLD can be directly classified as MAFLD and vice versa. As NAFLD has been used in previous literature, this paper will continue to use NAFLD as the name of the disease.

At present, the pathogenesis of NAFLD has not been fully elucidated, but studies have shown a close association of copper dyshomeostasis with NAFLD pathogenesis. Experiments also indicated low hepatic copper in NAFLD animal models. ^[49] Compared to control subjects, both adult and pediatric patients with NAFLD showed copper deficiency in the serum, liver, and hair. ^[50] Tosco et al^[51]conducted a genetic analysis of copper-deficient rats and found that copper deficiency down-regulates β-oxidation of free fatty acids in mitochondria and peroxisome. In addition, copper deficiency leads to impaired mitochondrial function and morphological changes, limiting mitochondrial fatty acid β-oxidation and promoting hepatocyte steatosis. ^[52]

Some studies suggested insufficient copper intake may be associated with NAFLD. ^[53] Fructose can reduce copper absorption by inhibiting the expression of duodenal copper transporter 1 (CTR1). Song et al^[54] found that the levels of copper in serum and liver were decreased in high-fructose-fed mice. Moreover, low copper significantly inhibited the expression of fatty acid β-oxidation-rate-limiting enzyme carnitine palmitoyltransferase I (CPT-I) and up-regulated the expression of fatty acid synthase (FAS), the key enzyme of fatty acid synthesis. The results showed that the increase in free fatty acids and triglyceride synthesis in liver cells induced steatosis and damage in mice. Additionally, an experimental study in rats by Tallino et al^[55] has demonstrated that the combination of low-Cu and high-sucrose diet upregulates the expression and transcription of the genes associated with liver inflammation, fibrogenesis, Adenosine triphosphate (ATP) citrate lyase, and FAS, which induces liver steatosis and promotes lipid peroxidation and liver damage without severe steatosis or obesity. Previous studies have shown that copper deficiency is associated with oxidative stress, liver inflammation, and insulin resistance, which play a key role in NAFLD pathogenesis. ^[55,56,57] In the study by Aigner et al^[53], liver biopsy revealed increased liver iron content in NAFLD patient since copper deficiency decreases hepatic iron export via decreasing the activity of CP and the expression of liver ferroportin. Moreover, iron overload promotes the progression of NAFLD by inducing oxidative stress, insulin resistance, and liver inflammation.

However, many studies have contradicted the findings. Studies have shown sex-based differences in the epidemiology, development, and treatment of some liver diseases. ^[58] In a recent study, the association between blood copper and NAFLD increased with the degree of NAFLD severity only in males. ^[50] The underlying reason may potentially involve the protective role of estrogens ^[59] and the sex-based differences in copper intake. ^[50] Stättermayer et al^[60] found a negative association between liver copper and hepatic steatosis only in patients without Mets. The result is in contrast to the finding of Lan et al^[50], and the discrepancy may be attributed to the fact that the authors did not take into account sex in the experiment. In addition, Aigner et al^[53] reported that NASH patients had lower hepatic copper content compared to NAFLD patients, which suggested that lower liver copper concentration is correlated with NAFLD/NASH progression. However, some previous studies have shown inconsistent relation between copper concentration and the severity of NAFLD. The reason for this difference is not yet known. ^[61,62]

Oxidative stress plays a key role in the pathogenesis of NAFLD, and anti-oxidation is also a direction for future treatment of NAFLD. In recent years, many natural compounds with anti-oxidation effects have been shown to protect against NAFLD. In addition to reducing hepatic steatosis and oxidative stress, most of these compounds have the function of chelating copper. ^[15] These contradictory results suggest that there is a more complex relationship between copper and NAFLD. Thus, further research is required for in-depth characterization of the role of copper in the occurrence and development of NAFLD.

Copper and hepatocellular carcinoma

HCC is the most common malignant tumor of the liver and the fourth leading cause of cancer-related deaths worldwide. ^[63] Studies have documented increased serum and liver copper levels in patients with HCC. ^[5,64] This phenomenon may be closely related to the down-regulation of ATP7A or macropinocytosis in HCC cells. ^[65] Davis et al^[66] showed that HCC cells rely on the copper transport of CTR1 to achieve tumor cell proliferation and metastasis, whereas knockout of the CTR1 gene can significantly inhibit tumor cell proliferation and metastasis. Studies have shown that copper can promote tumor formation through a variety of mechanisms, ^[5,67] such as induction of oxidative stress, loss of original function of the oncogene P53, promotion of angiogenesis and cell metastasis, and increased expression of cyclin D1 gene. The relationship between copper and HCC has not been fully elucidated. However, researchers have shown that treatment of HCC cells with copper enhances their ability to proliferate and migrate. This effect may be related to the up-regulation of proliferating cell nuclear antigen (PCNA), cyclin D1, and β-catenin, and the down-regulation of E-cadherin. Myelocytomatosis (MYC) is a key factor regulating cell growth and is overexpressed in most HCCs. ^[68] Porcu et al^[62] found that elevated extracellular copper promotes MYC expression in HepaRG cells. Elevated MYC binds to the MYC-associated factor X (MAX) to form the MYC/MAX complex, which binds to the CTR1 gene promoter to induce its transcription. This was found to promote the proliferation and migration of tumor cells by increasing the level of intracellular copper.

LOX family is a copper-dependent LOX, and LOX expression level is significantly elevated in human HCC tissues. Studies have shown that the LOX family promotes angiogenesis through TGF-β-mediated p38MAPK-VEGF signal transduction, cross-linking extracellular matrix collagen with each other through the H1F-1/LOX signal transduction pathway, and promoting HCC cell growth and metastasis. ^[69] In a study, LOXL-2 levels in HCC tissues showed a negative correlation with overall survival (OS) and disease-specific survival of HCC patients. Tetrathiomolybdate (TTM) has been shown to inhibit cell proliferation and bone destruction in head and neck squamous cell carcinoma by affecting LOX activity, but its application in HCC needs further study in the future. ^[70]

There are 10 members of the copper metabolism MURR1 domain (COMMD) protein family (COMMD1 –COMMD10), which plays a key role in copper metabolism. COMMD1 is closely associated with the subcellular localization of ATP7B in hepatocytes, and COMMD1 deficiency causes defects in cellular ATP7B transport from cytosolic vesicles to the cytosolic membrane, and impaired hepatic copper excretion results in abnormally elevated intrahepatic copper levels. ^[71] These phenomena have also been observed in HCC cells. ^[72] Yang et al^[36] showed that COMMOD10 influences the development of radio-resistance and ferroptosis in HCC cells by altering the intracellular Cu/Fe balance and HIF-1/CP pathway. Therefore, the copper chelator TEPA can be used as a potential radiosensitizer to combat radioresistance in HCC patients.

As with most tumor diseases, surgical resection of the lesion, organ transplantation, and local tumor ablation are effective treatments for early localized tumors, leading to a marked improvement in patient survival postoperatively. ^[63] However, most patients with HCC have advanced disease at the time of diagnosis, and the existing therapies such as transarterial embolization (TAE)/transarterial chemoembolization (TACE), systemic therapy (such as sorafenib), and immunotherapy offer limited survival benefit and are often followed by post-treatment recurrence. Moreover, the high cost of molecular targeted therapies against potential gene mutations in HCC is a key barrier to their use. In this context, aberrant copper homeostasis in HCC cells provides a new direction for the diagnosis and treatment of HCC. ^[66]

In the study by Yoshii et al, ^[73] trientine (a copper chelator) was shown to significantly inhibit the growth and angiogenesis of HCC by inhibiting endothelial cell proliferation and inducing apoptosis. The HIF-1α level in HCC cells was increased under hypoxia by upregulating the expression of key glycolysis enzymes, including glucose transporter 1, pyruvate kinase M, and lactate dehydrogenase A. Enhanced glycolytic metabolism produces corresponding ATP to meet the energy required for rapid growth and proliferation of HCC cells. Lactate can promote tumor angiogenesis through the lactate/NF-κB/IL-8 axis, which is also associated with rapid tumor recurrence after TAE/TACE. In 2020, Davis et al^[66] confirmed that copper is closely related to the metabolic flexibility of HCC cells under hypoxic conditions. TTM chelation of intracellular copper in HCC can inhibit glycolysis metabolism, and significantly reduce the proliferative activity of HCC. However, the efficacy of TTM combined with TAE/TACE in the treatment of HCC needs further study. The liver has a detoxification function, which is responsible for the absorption, metabolism, and elimination of metal-containing compounds. Studies have shown that copper complexes can induce cell death by promoting DNA double-strand breaks. In the study by Rezaei et al, ^[74] Cu (II) complex was found to induce apoptosis in HEPG2 cells by up-regulating the expression of tumor suppressor gene P53 and apoptosis genes ( BAX and Caspase- 8) and down-regulating the expression of anti-apoptosis gene BCL-2. Some novel copper complexes have also been developed for the photodynamic therapy (PDT) of HCC. ^[75]

A prospective case–control study conducted in the Guangdong province of China found that serum copper levels in patients with HCC were significantly associated with liver cancer-specific survival and OS. Their findings suggested that serum copper level may be an independent predictor of survival of HCC patients. ^[76] In contrast to primary HCC, copper uptake was found to be increased in metastatic HCC cells. Therefore, ⁶⁴CuCl₂-PET/CT can be used for non-invasive evaluation of the intracranial and other extrahepatic metastases of HCC located in the low-uptake area of physiological copper. It can also be used to evaluate the patients before liver transplantation. This is of much significance in improving the prognosis of patients with metastatic HCC. In addition, radioactive copper isotopes can also be used as radiopharmaceuticals in the treatment of extrahepatic metastasis of HCC. ^[77]

The Future and the Outlook

Research conducted in recent years has unraveled the molecular mechanism of copper metabolism and its role in human diseases. The recent discovery of cuprotosis has provided new research directions and ideas for diseases related to the dysregulation of copper homeostasis. It is evident that copper is now an important target in the diagnosis and treatment of related diseases. Copper chelators and supplements are also widely used in diseases caused by copper dyshomeostasis. The relationship between copper dyshomeostasis and liver diseases has been well confirmed, which provides a new direction for the diagnosis and treatment of liver diseases such as WD, NAFLD, and HCC. Additionally, there is still some evidence that alcohol intake ^[78] and hepatitis C infection ^[79] may interact with copper dyshomeostasis in humans. However, much was hitherto less well studied especially about the mechanisms behind these pathological changes and clinical symptoms. We propose that future work should focus on exhibiting potentially novel mechanisms and effects on the pathology and physiology of diseases including WD, NAFLD, and HCC due to copper dyshomeostasis, and exploring the diagnostic and therapeutic potential of copper with them. Other fields of investigation actively explore the relationship between copper and other liver diseases, such as alcoholic liver disease, autoimmune liver disease, viral hepatitis, metabolic liver disease, and etc.

Funding

This work was sponsored by the National Natural Science Foundation of China (Nos. 81972265, 82170602), the National Natural Science Foundation of Jilin Province (No. 20200201324JC), the Project for Middle-aged and Young Excellent Technological Innovation Talents of Jilin Province (No. 20220508079RC) and the Project for Health Talents of Jilin Province (No. JLSWSRCZX 2021-079).

Conflicts of interest

None.