Origin, Function, and Fate of Metallothionein in Human Blood.

Toxic heavy metals, toxic organic compounds, reactive oxygen species (ROS), infections, and temperature are well-known metallothionein (MT) inducers in human blood. The current review aims to summarize synthesis, function, and fate of human blood MT in response to the known MT inducers. Part of the MTs that are synthesized in different organs such as the liver, kidney, and spleen is transported and stored in different blood cells and in plasma. Cells of the circulatory system also synthesize MT. From the circulation, MT returns to the kidney where the metal-bound MTs are degraded to release the metal ion that in turn induces MT expression therein. The blood MTs play important roles in metal detoxification, transportation, and storage. By neutralizing ROS, MTs protect blood cells from oxidative stress-induced cytotoxicity and genotoxicity. Arguably, MTs are also involved in immune suppression. Given the permeating distribution of blood MT throughout the body as well as its diverse role in the protection against harmful environmental factors and in metal homeostasis, MT could be better recognized as a major public health protein.

Zinc Content in Cord Blood Is Associated with Maternal Age and Parity.

At childbirth (parturition), zinc (Zn) homeostasis in cord blood (CB) can be affected by a number of factors: Zn in maternal blood, parturition related stress as well as metallothionein (MT). Both Zn and stress are known inducers of MT which is primarily involved in Zn homeostasis. This study analyzed Zn concentration [Zn], in CB components and MT-2A transcription in CB mononuclear cells (MNC) in relation to primiparous and multiparous childbirth. [Zn] in CB (n = 47) plasma, erythrocytes, and MNCs were measured by atomic absorption spectrophotometry (λ = 213.9 nm). The MT-2A transcription in CB-MNC was quantified using real-time PCR. Significant correlations (Pearson r) were found between: plasma-[Zn] and erythrocyte-[Zn] (p = 0.002); [Zn] and MT-2A messenger RNA (mRNA) (p = 0.000) in CB-MNC. Student's t tests showed higher levels of MT-2A mRNA and MNC-[Zn] in CB of older (≥25 years) compared to younger mothers (≤24 years) (p = 0.043 and p = 0.016, respectively). Significantly higher [Zn] was found in CB plasma (p = 0.017) and MNC (p = 0.041) of older primiparous compared to the younger primiparous and older multiparous mothers respectively. MT-2A mRNA in CB-MNC was significantly lower in CB of younger primiparous mothers compared to their older counterparts (p = 0.001). Path analysis showed that MNC-[Zn] (ß = 0.83; p = 0.000) had a greater influence on MT-2A mRNA expression, compared to parity (ß = -0.14; p = 0.033). Higher [Zn] in CB of primiparous mothers could be linked to higher stress during parturition, however, might be beneficial for the growth and development of the child. Together MNC-[Zn] and parity contributed ~70 % of the MT-2A transcription in CB-MNC.

Arsenic Induction of Metallothionein and Metallothionein Induction Against Arsenic Cytotoxicity.

Human exposure to arsenic (As) can lead to oxidative stress that can become evident in organs such as the skin, liver, kidneys and lungs. Several intracellular antioxidant defense mechanisms including glutathione (GSH) and metallothionein (MT) have been shown to minimize As cytotoxicity. The current review summarizes the involvement of MT as an intracellular defense mechanism against As cytotoxicity, mostly in blood. Zinc (Zn) and selenium (Se) supplements are also proposed as a possible remediation of As cytotoxicity. In vivo and in vitro studies on As toxicity were reviewed to summarize cytotoxic mechanisms of As. Intracellular antioxidant defense mechanisms of MT are linked in relation to As cytotoxicity. Arsenic uses a different route, compared to major metal MT inducers such as Zn, to enter/exit blood cells. A number of in vivo and in vitro studies showed that upregulated MT biosynthesis in blood components are related to toxic levels of As. Despite the cysteine residues in MT that aid to bind As, MT is not the preferred binding protein for As. Nonetheless, intracellular oxidative stress due to As toxicity can be minimized, if not eliminated, by MT. Thus MT induction by essential metals such as Zn and Se supplementation could be beneficial to fight against As toxicity.

UDP-dependent glycosyltransferases involved in the biosynthesis of steviol glycosides.

A short-term experiment was designed to measure the transcript levels of downstream genes contributing to the biosynthesis of steviol glycosides. Stevia rebaudiana plants were subjected to long- and short-day conditions for different time intervals. Samples from both lower and upper leaves were collected. Using quantitative real-time polymerase chain reaction, the transcript levels of three UDP-dependent glycosyltransferases, UGT85C2, UGT74G1 and UGT76G1, were studied. The results were compared with the steviol glycoside contents measured in the leaves, which were quantified by reversed phase HPLC. In the same daylength condition, steviol glycoside concentration and the transcript levels of the three UGT genes were higher in upper leaves than in lower leaves. Steviol glycosides accumulated more in plants under short-day conditions. Under these conditions, a highly significant correlation was found between UGT85C2 transcription and total steviol glycoside accumulation in the upper leaves. This suggests that the glycosylation of steviol to form steviolmonoside is the rate-limiting step in the glycosylation pathway of steviol glycosides. In these upper leaves, a relatively high accumulation of rebaudioside A compared to stevioside was also observed, however, without correlation with the transcription of UGT76G1.

Metallothionein in human immunomagnetically selected CD34(+) haematopoietic progenitor cells.

Human umbilical CD34(+) immature haematopoietic cells were rapidly and efficiently obtained from light density MNC (mononuclear cells) by MACS (magnetic cell sorting). An ex vivo expanded population of CD34(+) was cultured in serum-free medium supplemented with cytokines FL (flt3 ligand), SCF (stem cell factor) and TPO (thrombopoietin) in order to obtain a sufficient number of CD34(+) cells. CD34(+) cells expanded from cord blood for 7 days were demonstrated to increase in the absolute number of CD34(+) cells by 5.12 ± 2.47-fold (mean ± S.D., n = 3). Flow cytometric analysis demonstrated that the percentage of CD34 antigen expression after expansion of the culture was 97.81 ± 1.07%, whereas it was 69.39 ± 10.37% in none-expanded CD34(+) cells (mean ± S.D., n = 3), thus defining a system that allowed extensive amplification accompanied by no maturation. MTs (metallothioneins), low molecular weight, cysteine-rich metal-binding proteins, exhibit various functions, including metal detoxification and homoeostasis. We here examined the expression pattern of functional members of the MT gene family in immature CD34(+) cells and compared it with more mature CD34(-) cells in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 medium, with or without different zinc supplements for 24 h. Relative quantitative expression of MT isogenes in the mature CD34(-) cells was higher than in the immature CD34(+) cells. IHC (immunohistochemical staining) revealed an increased MT protein biosynthesis in CD34(-) cells, greater than in CD34(+) cells. Therefore, the role of MT in differentiation of human haematopoietic progenitor cells from human cord blood is reported for the first time.

Role of metallothionein in differentiation of leukemia cells.

Metallothioneins (MTs), a group of small, cystein-rich proteins, possess various functions, including metal detoxification and homeostasis. We here report new findings on the participation of MT in cellular differentiation processes. MT isogene transcription was significantly increased in phorbol-12-myristate-13-acetate (PMA) incubated leukemic DAMI cells, which supports its role in cellular differentiation. To further address this possibility, we constructed one stable MT-2A overexpressing DAMI cell line. Increase of cell size, intracellular granulation and megakaryocytic specific antigen expression such as CD41 and CD42, and arresting cell proliferation have validated the role of MT in differentiation in this cell line.

Protective effect of metallothionein on oxidative stress-induced DNA damage.

Metallothioneins (MTs) are a family of low molecular weight proteins with a high cysteine and metal ion content. They are found in most cells and tissues and can be induced by a number of substances, including various forms of oxidative stress. MTs play a central role in essential trace element homeostasis and in metal detoxification. Because of their peculiar structure, characterized by a large content of thiol groups, MTs also act as a potent antioxidant by protecting against various injuries resulting from reactive oxygen (ROS) or nitrogen species (RNS). In this review, the involvement of MT in the protection of DNA against oxidative stress is discussed.

Differential quantitative zinc-induced expression of human metallothionein isogenes in haematopoietic precursor cell lines.

The expression pattern of functional members of the metallothionein (MT) gene family was studied in the haematopoietic precursor cell lines, K562, DAMI, MEG-01, and ELF-153 in order to strengthen the proposed function of MT in differentiation. Cells were cultured in RPMI 1640 with 10% (v/v) foetal calf serum, with or without different zinc supplements. Expression of MT isogenes was analysed by quantitative real-time PCR (RT-PCR) using mRNA extracted from cultured cells. The more mature K562, DAMI, and MEG-01 cell lines exhibited transcription of all MT isogenes, except MT-3 and MT-4. Relative quantitative expression of MT isogenes in the mature cell lines such as K562, DAMI, and MEG-01 was higher than in the immature ELF-153 cell line. Immunohistochemical staining (IHC) reveals an increased MT protein biosynthesis in more mature cell lines such as K562, DAMI and MEG-01 greater than in the immature ELF-153 cell line. Real-time PCR and immunohistochemical staining for investigating the effect of phorbol ester and hemin (haematopoietic differentiation stimuli) on expression of MT isogenes in K562 cells reveals that phorbol ester induces increased MT transcription and biosynthesis. Therefore, to our knowledge, the role of MT in differentiation in human haematopoietic precursor cell lines is here reported for the first time.

Serum-induced expression of metallothionein isoforms in K-562 cells.

The metallothionein (MT) expression was studied in the hematopoietic precursor cell line K-562, after serum deprivation and reconstitution of the cells in medium with 10% (v/v) FCS. Serum deprivation for 72 h markedly downregulated the MT mRNA expression, only the isoforms most abundant in normal K-562 cells were clearly detectable. Within 1-1.5 h after serum supplementation however, a definite induction of MT mRNA was noticed, and all isoforms were induced. Forty-eight hours after serum stimulation, the MT mRNA expression of all isoforms decreased again. Also MT protein levels increased twofold 24 h after serum stimulation. These results suggest that MT has a function in the re-entry of resting cells into the cell cycle, this function however could not be assigned to a specific MT isoform. The induction of MT after serum stimulation was independent of protein synthesis, but dependent on phosphorylation.

Pressure- and temperature-induced unfolding and aggregation of recombinant human interferon-gamma: a Fourier transform infrared spectroscopy study.

The effect of hydrostatic pressure on the secondary structure of recombinant human interferon-gamma (rhIFN-gamma) and its biologically inactive truncated form rhIFN-Delta C15 has been studied using Fourier-transform IR (FTIR) spectroscopy. In situ observation of the pressure-induced changes using the diamond anvil cell shows that the alpha-helical structure is mainly transformed into disordered structure at high pressure. Increasing pressure also induces the formation of a gel. Addition of 0.5 M MgCl(2) significantly reduces the pressure stability. Releasing the pressure below 300 MPa results in the formation of intermolecular antiparallel beta-sheets, which is seldom observed. This suggests that the intermolecular beta-sheet of rhIFN-gamma is stabilized by electrostatic interactions that are disrupted at high pressure. For comparison we also studied the effect of temperature. Temperature-induced changes reflect extensive transformation of alpha-helical structure into intermolecular antiparallel beta-sheet, as is usually observed for most proteins.

Recent developments in quantification methods for metallothionein.

The metallothioneins (MT), a family of proteins with relatively low molecular weight (6-7 kDa), are characterised by the intrinsic presence of 20 cysteinyl groups in their structure, which confers unique metal binding properties to the molecule. Since MT are involved in biological roles, quantification of MT remains an important task. To date, a large number of determination methods have been developed. In this paper recent developments, from 1995 to the present, in methodology employed in quantification studies of total MT and MT polymorphism are described. Different fields were taken into consideration, such as (i) separation techniques and hyphenated systems, (ii) electrochemical methods, (iii) immunological methods and (iv) quantification of MT mRNA. The data presented are based on our own and published results. A brief overview of the use of metallothionein as a biomarker is included as a relevant example of the importance of MT quantification. Finally, general problems associated with determination and evaluation of obtained results within the above four topics are mentioned.