Metallothioneins in Biochemistry and Pathology

The following sections are included:

• CONTENTS

• LIST OF CONTRIBUTORS

The structure and chemistry of mammalian metallothioneins (MTs) with divalent (Zn^II, Cd^II) and monovalent (Cu^I) metal ions pertinent to their role in biological systems are discussed. In human, four MT isoforms designated MT-1 through MT-4 are found. The characteristic feature of these cysteine- and metal-rich proteins is the presence of two metal-thiolate clusters located in independent protein domains. The structure of these clusters is highly dynamic, allowing a fast metal exchange and metal transfer to modulate the activity and function of zinc-binding proteins. Despite the fact that the protein thiolates are involved in metal binding, they show a high reactivity toward electrophiles and free radicals, leading to cysteine oxidation and/or modification and metal release. The unusual structural properties of MT-3 are responsible for its neuronal growth inhibitory activity, involvement in trafficking of zinc vesicles in the central nervous system (CNS), and protection against copper-mediated toxicity in Alzheimer's disease. MT-1/MT-2 also play a role in cellular resistance against a number of metal-based drugs.

Studies involving metallothioneins (MTs) are often performed without reference to a primary biological function or a chemical mechanism of the protein, despite the fact that the critical cysteinyl residues in MT, and their neighboring amino acids, have unique characteristics and determine specific functions. Fluorescence methods have provided new insights into how the redox state of the sulfur donors and the interactions with zinc ions are controlled in mammalian MTs. Two zinc/thiolate clusters modulate zinc affinities over several orders of magnitude, thus allowing fine-tuned control over the cellular availability of free zinc ions. In vivo, not all of the sulfur donors of MT are reduced, and less than seven zinc ions are bound. Thus, MT exists in different states depending on zinc availability and redox poise. Dynamic changes in the sulfur redox state and in zinc binding demonstrate a function of mammalian MT in the interconversion of zinc and redox signals. In the cellular signaling network, MT is an integrated part of the following pathway: signal → reactive species → MT → Zn²⁺ → target. MT is translocated within cells, secreted from cells, and taken up by cells. Its function is important for regulation of the physiological processes that depend on zinc availability, and the pathological processes of cell injury induced by oxidative stress or by thiol-reactive compounds.

Although metallothioneins (MTs) were discovered nearly 40 years ago, their functional role has still not been completely clarified. The role of MTs in the central nervous system has in particular become an intense focus of scientific research. Many papers have confirmed the active and peculiar role played by these proteins in neurodegenerative disorders, even if contrasting results are still present. The involvement of MTs in various neurodegenerative diseases (Alzheimer's disease, frontotemporal dementia, Binswanger's disease, Parkinson's disease, amyotrophic lateral sclerosis, and prion protein disease) is herein reported.

Most neurodegenerative diseases are characterized by a prominent inflammatory response (often called neuroinflammation), which consists mainly of elements of the innate immune response. Whether neuroinflammation is a cause or simply a consequence of the neuropathological events seen during neurodegenerative diseases is unclear, but it is undisputed that chronic inflammation normally leads to permanent scarring and tissue damage. In order to understand this complex biological response, it is essential to identify all factors involved in it. There is a clear consensus that a number of cytokines orchestrate the brain inflammatory response, in terms of both inducing and limiting it, but also control the expression of proteins important for coping with the potential tissue damage. One such protein is metallothionein (MT). Results obtained in MT-1/MT-2–null mice and in MT-1–overexpressing mice strongly suggest that these MT isoforms are important antioxidant, anti-inflammatory, and antiapoptotic proteins in the brain. Results in MT-3–null mice show a very different pattern, with no support for MT-1/MT-2-like functions; rather, MT-3 could be involved in neuronal sprouting and survival.

Autism is a complex disorder with both genetic and environmental factors. Metal disturbances have been reported in autistic individuals. In particular, exposure to ethylmercury (EtHg) in the vaccine preservative thimerosal and exposure to methylmercury (MeHg) through fish consumption have been implicated as environmental contributors to the autism spectrum disorder (ASD) phenotype. Metallothioneins (MTs) are small sulfhydryl (–SH)-rich metal-binding proteins that have important functions in metal homeostasis and protection against metalinduced toxicity. MT1 and MT2 have a high affinity for toxic heavy metals and are induced following mercury (Hg) exposure. It has been suggested that altered or dysfunctional MTs could enhance susceptibility to Hg-induced toxicity, and that alterations in Hg metabolism may contribute to the neurodevelopmental phenotypes present in ASD. One proposed treatment of autism is to attempt to restore MT function; however, there is no evidence in the peer-reviewed literature that MT restoration is an effective treatment for autistic symptoms. To date, there is no evidence for the efficacy of MT in ameliorating MeHg- or EtHg-induced neurotoxicity. Currently, there is nothing in the literature that suggests altered MT homeostasis is a contributing factor to the development of autism.

The physiological role of metallothionein (MT) has been a topic of growing interest, particularly with regard to a potential therapeutic application in trauma of the central nervous system (CNS). An increasing number of studies describe the protective, regenerative, and anti-inflammatory properties of MT-I and MT-II isoforms (MT-I/MT-II) in the context of in vitro and animal models, using, for example, MT-I/MT-II null, overexpressing, or injected mice following induced CNS trauma or disease. MT-I/MT-II respond to trauma by upregulation, and may have roles in metal ion homeostasis and free radical scavenging. Notably, a direct action of MT-I/MT-II on neurons has been shown using in vitro models, whereby the application of exogenous MT-I/MT-II directly increases neurite outgrowth of young neurons and regeneration of injured, mature neurons. The expression and putative functions of MT within the injured CNS will be addressed within this chapter, with particular regard to the MT-I/MT-II isoforms that display neuroprotective and regenerative properties. Intriguingly, a further member of the MT family, MT-III, shows high homology to MT-I/MT-II, yet has a contrasting effect on neuron growth and survival in some models.

The metallothionein (MT) family is a class of low-molecular-weight, cysteine-rich proteins (MT-1, MT-2, MT-3, and MT-4) with high affinity for metal ions. Apart from their involvement in metal ion homeostasis and detoxification, protection against oxidative damage, and cell proliferation and apoptosis, they are also implicated in drug and radiotherapy resistance and several aspects of the carcinogenic process. Variable MT expression has been observed in different cancer types, reaching statistically significant correlation with clinicopathological parameters in some cases; nevertheless, MT expression as a marker of prognosis or as a predictor for the response to either chemotherapy or radiotherapy remains unclear. The present review examines the expression of MT in different human tumors in correlation with resistance to radiation therapy or chemotherapy and patients' final outcome. Detailed studies focused on the expression of MT isoforms and isotypes in different tumor types could elucidate the role of this group of proteins in patients' prognosis and resistance to treatment strategies.

Melanoma is one of the most aggressive human neoplasms, and its incidence is still increasing. To estimate the risk of possible progression and overall survival, Breslow tumor thickness and the invasion level (Clark level) are the most established markers for melanomas at the time of primary diagnosis. In the last decades, overexpression of immunohistochemically labeled metallothioneins (MTs) on paraffin-embedded tissues has turned out to be a highly significant prognostic marker in different tumors. We report the results of a large prospective study on melanoma patients in which MT overexpression was a highly significant marker for progression and survival. In contrast to most other markers, MT overexpression was independent from tumor thickness, and was highly specific even in thin (lowrisk) melanoma patients. In high-risk melanoma patients, sentinel lymph node (SLN) biopsy — a surgical technique with predictive value for progression — was performed. The benefit of this procedure for the individual patient's overall survival remains unclear. Our results corroborate the validity of MT overexpression in primary melanoma as a useful prognostic marker: its accuracy is comparable,and to some degree supplementary to, the results of SLN biopsy.

Mammoth efforts have been made in an attempt to understand the etiology of breast neoplasia. Metallothioneins (MTs) are ubiquitous, low-molecular-weight, metal-binding proteins. In addition to their essential roles in metal homeostasis, MTs have been reported to be overexpressed in breast cancers. Generally, MTs are involved in many processes that support tumorigenesis, encompassing the promotion of cell proliferation and metastasis as well as the disruption of apoptotic mechanisms in breast cancers. There is also increasing evidence which shows the association of MT with tumor grade and chemoresistance, supporting the notion that MT could potentially be a biomarker of prognosis. In this chapter, we shall focus on the contributions of individual MT isoforms in relation to breast carcinogenesis and its eventual pathological outcome.

Metallothionein (MT) is an avid metal-binding (metal + thiol/sulphur-binding) protein in the human body. It binds to trace elements like zinc and copper as well as heavy metals like cadmium, and plays an important role in metal detoxi-fication and homeostasis. MT isoforms are expressed differentially in benign and malignant prostate tissue, with increased MT expression noted in higher-Gleason-grade prostate cancer. MT expression in prostate has been shown to be regulated by high Zn concentration and promoter hypermethylation. MT is known to play a role in the resistance to chemotherapeutic agents such as cisplatin and radiation treatment, presumably by trace metal or free radical scavenging. MT expression in the prostate gland is of particular interest because heavy metals such as Zn, which is present at the highest concentration in prostate compared to other human organs, induce MT expression and may be amenable to therapeutic manipulation in order to improve sensitivity to chemotherapy and radiation. MT may prove to be a useful therapeutic target for novel approaches such as local or systemic heavy metal chelation therapy and gene vectors for treating patients with prostate cancer.

There have been some results reported concerning the expression of metallothionein (MT) in colorectal tumors. This paper reviews four aspects: the contribution of MT in colorectal carcinogenesis, the prognostic significance of MT, MT expression in colorectal liver metastases, and the clinical significance of MT as a drug-resistance protein.

Cardiomyopathy, a condition in which the heart muscle does not work properly, can affect children and adults. Unlike heart disease due to heart attack, where there is a problem with adequate blood flow to the heart, cardiomyopathy refers to the disease of heart muscle itself. The common types of cardiomyopathy are dilated cardiomyopathy, hypertrophic cardiomyopathy, restrictive cardiomyopathy, and specific cardiomyopathy. The causes of cardiomyopathy are unknown, and these diseases can be inherited or caused by other diseases or environmental toxicants. Coronary artery disease causes ischemic cardiomyopathy. Alcohol, especially when a person has a poor diet, causes alcoholic cardiomyopathy. Toxicants, including the anticancer drug doxorubicin, cause toxic cardiomyopathy. Chronic diseases such as diabetes cause disease-related cardiomyopathy, for instance, diabetic cardiomyopathy. Although there are distinct mechanisms for these cardiomyopathies with different etiologies, there are also certain common pathogenic mechanisms; exploring these common mechanisms will be of great importance for developing preventive approaches to these cardiomyopathies. Metallothionein as a stress protein (adaptive protein) was found to play a critical role in protecting the heart from a variety of pathogenic and environmental risks. Therefore, this chapter will summarize the protective effects of metallothionein on the development of these cardiomyopathies, focusing on the experimental evidence, possible mechanisms, and potential clinical implications.

Zinc status, inflammation, and genetic determinants are prominent mechanisms in the pathogenesis of atherosclerosis (AT) and its compliances (cardiovascular diseases). In this review, we report the possible impact of zinc on AT development as well as the role played by a significant genetic determinant involved in inflammation, such as interleukin-6 (IL-6). Genetic polymorphism of IL-6 may affect a different inflammatory response as well as zinc turnover, predisposing to AT. Indeed, zinc deficiency is suggested as a risk factor for AT with advancing aging. The increment of dysfunctional proteins involved in zinc homeostasis, i.e. metallothioneins (MT), caused by persistent inflammation and oxidative stress may further contribute to zinc deficiency and consequently to the development of AT. A zinc supplementation may be useful to achieve healthy aging and, as such, to prevent AT, but it is necessary to consider the individual genetic background (especially when referred to IL-6 and MT polymorphisms) for the success of zinc intervention. Therefore, a zinc genomic approach may offer a reasonable hope for understanding the impact of zinc on molecular processes that maintain health and prevent the development of AT.

Metallothioneins (MTs) are cysteine-rich proteins capable of scavenging free radicals and sequestering metal ions. In the liver, these proteins are involved in copper and zinc metabolism, in the chelation of heavy metals, and in protection against oxidative damage. Because of their properties, MTs are involved in many liver diseases, which can be sorted into the following:

1. Metal storage liver diseases. Zinc, which is an important anticopper agent for Wilson's disease, acts by increasing the concentration of MTs in the enterocytes, thereby reducing metal absorption. Copper also accumulates in the liver in cholestatic diseases, in which MTs are reportedly overexpressed and induced by ursodeoxycholic acid (UDCA), the main drug used to treat cholestasis. The role of MTs in hemochromatosis, an iron-accumulating disease, has yet to be established; but in animal models, it has been suggested that zinc, by increasing MT concentration, could exert a beneficial effect.

2. Toxic liver diseases. By sequestering metal ions and scavenging free radicals, MTs protect against damage caused by exogenous toxic substances, such as cadmium and arsenic, and by the toxic effects on hepatocytes of ethanol and at in alcoholic and nonalcoholic liver diseases.

3. Chronic viral hepatitis. By lowering the inflammatory injury, MTs have a protective action against chronic liver damage; a relationship has also been described between MTs and the severity of liver disease and the response to therapy.

4. Hepatocellular carcinoma. MTs are downexpressed and inversely correlated with tumor stage; an inverse correlation has also been reported between MT concentrations and response to platinum chemotherapy.

Given the ability of zinc to strongly induce MT synthesis, zinc supplementation could be useful not only in Wilson's disease, but also in other liver diseases in which MTs exert a protective effect.

The following sections are included:

• Index