A review of cinnabar (HgS) and/or realgar (As4S4)-containing traditional medicines.

ETHNOPHARMOCOLOGICAL RELEVANCE: Herbo-metallic preparations have a long history in the treatment of diseases, and are still used today for refractory diseases, as adjuncts to standard therapy, or for economic reasons in developing countries. AIM OF THE REVIEW: This review uses cinnabar (HgS) and realgar (As4S4) as mineral examples to discuss their occurrence, therapeutic use, pharmacology, toxicity in traditional medicine mixtures, and research perspectives. MATERIALS AND METHODS: A literature search on cinnabar and realgar from PubMed, Chinese pharmacopeia, Google and other sources was carried out. Traditional medicines containing both cinnabar and realgar (An-Gong-Niu-Huang Wan, Hua-Feng-Dan); mainly cinnabar (Zhu-Sha-An-Shen Wan; Zuotai and Dangzuo), and mainly realgar (Huang-Dai Pian; Liu-Shen Wan; Niu-Huang-Jie-Du) are discussed. RESULTS: Both cinnabar and realgar used in traditional medicines are subjected to special preparation procedures to remove impurities. Metals in these traditional medicines are in the sulfide forms which are different from environmental mercurials (HgCl2, MeHg) or arsenicals (NaAsO2, NaH2AsO4). Cinnabar and/or realgar are seldom used alone, but rather as mixtures with herbs and/or animal products in traditional medicines. Advanced technologies are now used to characterize these preparations. The bioaccessibility, absorption, distribution, metabolism and elimination of these herbo-metallic preparations are different from environmental metals. The rationale of including metals in traditional remedies and their interactions with drugs need to be justified. At higher therapeutic doses, balance of the benefits and risks is critical. Surveillance of patients using these herbo-metallic preparations is desired. CONCLUSION: Chemical forms of mercury and arsenic are a major determinant of their disposition, efficacy and toxicity, and the use of total Hg and As alone for risk assessment of metals in traditional medicines is insufficient.

Expression of functional metallothionein isoforms in papillary thyroid cancer.

Metallothionein (MT) isoforms have not been studied in papillary thyroid cancer. We examined how the functional MT1 and MT2 isoforms were expressed in papillary thyroid cancer (KAT5) cells. We demonstrated that KAT5 cells expressed eight functional MT1 and MT2 isoforms induced by cadmium. Elevated calcium and activated ERK1/2 predated MT expression. The inhibition of either calcium or ERK1/2 significantly blocked the isoform expression. The induction of these isoforms accompanied an increased progression of cell cycle from G0/G1 to G2-M. The alternation in cell cycle disappeared when the expression of MT isoforms was blocked by calcium inhibitor or ERK1/2 inhibitor. Collectively, KAT5 cells express eight functional MT1 and MT2 isoforms in a pathway controlled by calcium and ERK1/2. The elevation of the MT isoforms contributes to the decreased G0/G1 but increased G2-M phase. These results reveal a novel pathway for the expression of the functional MT in papillary thyroid cancer.

Polymorphisms in metallothionein-1 and -2 genes associated with the risk of type 2 diabetes mellitus and its complications.

Metallothionein (MT) as a potent antioxidant can affect energy metabolism. The present study was undertaken to investigate the association between MT gene polymorphism and type 2 diabetes mellitus. Using the PCR-based restriction fragment length polymorphism method, seven single nucleotide polymorphisms (SNPs) in MT genes (rs8052394 and rs11076161 in MT1A gene, rs8052334, rs964372, and rs7191779 in MT1B gene, rs708274 in MT1E gene, and rs10636 in MT2A gene) were detected in 851 Chinese people of Han descent (397 diabetes and 454 controls). Several serum measurements were also examined randomly for 43 diabetic patients and 41 controls. The frequency distributions of the G allele in SNP rs8052394 of MT1A gene were significantly associated with the incidence of type 2 diabetes. There was no difference between patients and controls for the rest of six SNPs. Serum levels of interleukin-6 and tumor necrosis factor-alpha were higher, and serum superoxide dismutase activity was significantly lower in the diabetic group than those in the control group. For diabetic patients, serum superoxide dismutase activity was significantly lower in GG or GA carriers than those of AA carriers of rs8052394 SNP. Increased serum levels in diabetic patients were positively associated with rs964372 SNP, and type 2 diabetes with neuropathy was positively associated with rs10636 and rs11076161. These results suggest that multiple SNPs in MT genes are associated with diabetes and its clinical symptoms. Furthermore, MT1A gene in rs8052394 SNP is most likely the predisposition gene locus for diabetes or changes of serum superoxide dismutase activity.

Induction of functional MT1 and MT2 isoforms by calcium in anaplastic thyroid carcinoma cells.

Metallothionein (MT) expression in carcinogenesis of thyrocytes is unknown. We demonstrated that cadmium induced transcription of all functional MT-1 and MT-2 isoforms and promoted the cell cycle from the G1 to the S phase in thyroid cancer cells, which can be suppressed by the ERK inhibitor. Cadmium exposure stimulated intracellular calcium and the phosphorylation of ERK1/2. Therefore, a common pathway initiated by a rapid rise in calcium and followed by calcium-mediated activation of ERK is involved in the transcriptional induction of functional MT1 and MT2 isoforms and in the progression of the cell cycle in thyroid cancer cells exposed to cadmium.

P53 mediated regulation of metallothionein transcription in breast cancer cells.

Recent studies have shown that only breast cancer epithelial cells with intact p53 can induce metallothionein (MT) synthesis after exposure to metals. In this study, the potential role of p53 on regulation of MT was investigated. Results demonstrate that zinc and copper increased metal response elements (MREs) activity and MTF-1 expression in p53 positive MN1 and parental MCF7 cells. However, inactivation of p53 by treatment with pifithrin-alpha or the presence of inactive p53 inhibited MRE-dependent reporter gene expression in response to metals. MTF-1 levels remained unchanged after treatment with zinc in cells with nonfunctional p53. The introduction of wild-type p53 in MDD2 cells, containing nonfunctional p53, enhanced the ability of zinc to increase MRE-dependent reporter gene expression. The cellular level of p21Cip1/WAF1 was increased in MDD2 cells after p53 transfection, confirming the presence of active p53. The treatment of MN1 and parental MCF7 with trichostatin A led to a sixfold increase in the MRE activity in response to zinc. On the contrary, MRE activity remained unaltered in MDD2 cells with inactive p53. The above results demonstrate that activation of p53 is an important factor in metal regulation of MT.

Biological stress response terminology: Integrating the concepts of adaptive response and preconditioning stress within a hormetic dose-response framework.

Many biological subdisciplines that regularly assess dose-response relationships have identified an evolutionarily conserved process in which a low dose of a stressful stimulus activates an adaptive response that increases the resistance of the cell or organism to a moderate to severe level of stress. Due to a lack of frequent interaction among scientists in these many areas, there has emerged a broad range of terms that describe such dose-response relationships. This situation has become problematic because the different terms describe a family of similar biological responses (e.g., adaptive response, preconditioning, hormesis), adversely affecting interdisciplinary communication, and possibly even obscuring generalizable features and central biological concepts. With support from scientists in a broad range of disciplines, this article offers a set of recommendations we believe can achieve greater conceptual harmony in dose-response terminology, as well as better understanding and communication across the broad spectrum of biological disciplines.

Metallothionein expression as prognostic factor for transitional cell carcinoma of bladder.

OBJECTIVES: To determine whether metallothionein (MT) protein expression is associated with clinical outcomes in patients with transitional cell carcinoma (TCC) of the bladder. METHODS: Archival pathologic radical cystectomy and transurethrally resected specimens and medical charts were reviewed for 123 patients with TCC. Patients were divided into groups based on the TNM stage, tumor grade, and MT protein expression in the primary tumor. Survival and disease progression were correlated with MT expression. RESULTS: The mean patient age was 66 years (range 41 to 92). Of the 123 tumors, 21, 13, 18, 24, 17, and 30 were pathologically staged as pTa, pT1, pT2, pT3, pT4, and pTis, respectively; 28, 15, 14, and 66 tumors had a histologic grade of X, 1, 2, and 3, respectively. On univariate analysis, TNM stage and tumor grade predicted survival and progression outcomes. MT expression was detected in 69 (56.9%) of 123 bladder cancer specimens. Greater MT protein expression was associated with worse overall survival, disease-specific survival, disease-free survival, and disease-free progression (P = 0.0004, P = 0.05, P = 0.0008, and P = 0.0005, respectively). CONCLUSIONS: MT protein expression in the primary tumor of TCC specimens appeared to be associated with overall survival, disease-specific survival, disease-free survival, and disease-free progression. This finding requires additional validation using other data sets.

Metallothionein and liver cell regeneration.

Hepatocytes in adults are in a nonproliferative state but they have high capacity to regenerate within few hours after an injury. After partial hepatectomy or chemical injury, hepatocytes undergo a synchronized multistep process consisting of priming/initiation, proliferation, and termination. These distinct steps are essential for restoring the structure and functions of liver. The mechanisms involved in each of these steps of regeneration are well documented from various laboratories and are described in several reviews. We briefly describe these steps and the involvement of various cytokines and growth factors for cell regeneration in this short review. Liver cell regeneration may also involve stem cell proliferation. The regenerating cells require large amounts of zinc within a short time, and this requirement is met by induction of a zinc and copper binding protein, metallothionein (MT), during the priming step, soon after an injury. There are several reports on the transfer of zinc from MT to various metalloenzymes and transcription factors. Genetically modified mouse models have been used to study the involvement of interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in cell regeneration. The use of an MT-knockout mouse has enabled us to investigate the specific role of MT in liver regeneration after partial hepatectomy, chemical injury, and fibrosis. Several studies have suggested a defective liver regeneration after an injury in MT-knockout mice. There is cumulative evidence that indicates an essential role for MT in liver cell regeneration.

Interaction of metallothionein with tumor suppressor p53 protein.

Previous reports have shown that metallothionein (MT) may modulate p53 activity through zinc exchange. However, little is known on a direct interaction between MT and p53 in cells. The results demonstrate an interaction between MT and p53 can occur in vitro. The complex between MT and p53 was observed in breast cancer epithelial cells with both wild and inactive type of p53. Furthermore, it was shown that wt-p53 was preferentially associated with Apo-MT. Our data suggest that co-expression of MT and p53 and their complex formation in tumor cells may be involved in regulation of apoptosis in these cells.

Augmented hepatic injury followed by impaired regeneration in metallothionein-I/II knockout mice after treatment with thioacetamide.

A previous study (Oliver, J.R., Mara, T.W., Cherian, M.G. 2005. Impaired hepatic regeneration in metallothionein-I/II knockout mice after partial hepatectomy. Exp. Biol. Med. 230, 61-67) has shown an impairment of liver regeneration following partial hepatectomy (PH) in metallothionein (MT)-I and MT-II gene knockout (MT-null) mice, thus suggesting a requirement for MT in cellular growth. The present study was undertaken to investigate whether MT may play a similar role in hepatic injury and regeneration after acute treatment with thioacetamide (TAA). Hepatotoxicity of TAA is caused by the generation of oxidative stress. TAA was injected ip to both wild-type (WT) and MT-null mice. Mice were killed at 6, 12, 24, 48, 60, and 72 h after injection of TAA (125 mg/kg) or 48 h after injection of saline (vehicle control), and different parameters of hepatic injury were measured. The levels of hepatic lipid peroxidation were increased at 12 h in both types of mice; however, lipid peroxidation was significantly less in WT mice than MT-null mice at 48 h after injection of TAA. Analysis of hepatic glutathione (GSH) levels after TAA injection showed depletion of GSH at 12 h in WT mice and at 6 h in MT-null mice; however, significantly more GSH was depleted early (6-24 h) in MT-null mice than WT mice. An increase in hepatic iron (Fe) levels was observed in both types of mice after injection of TAA, but Fe levels were significantly higher in MT-null mice than WT mice at 6-60 h. The levels of hepatic copper (Cu) and zinc (Zn) were significantly higher in WT mice than MT-null mice at 6-60 h for Cu, and at 24 h and 60 h for Zn, respectively. Histopathological examination showed hemorrhagic necrosis in the liver of both types of mice at 12-72 h, with hepatic injury being more prominent in MT-null mice than WT mice. The hepatic MT levels were increased in WT mice after injection of TAA, and were highest at 24-72 h. Immunohistochemical staining for MT in WT mice indicated the presence of MT in both nucleus and cytoplasm of hepatocytes at 24-72 h after TAA injection. Cell proliferation, as assessed by immunohistochemical staining for proliferating cell nuclear antigen, was detected mainly in the livers of WT mice at 48-72 h after TAA treatment. Hepatic proliferation index in MT-null mice was very low as compared to WT mice during liver regeneration after injection of TAA. These results show that the liver cells of MT-null mice with no functional MT are unable to regenerate after TAA-induced hepatic injury, demonstrating an important role for MT in cellular regeneration.

Essentiality, toxicology and chelation therapy of zinc and copper.

Both zinc and copper are essential minerals that are required for various cellular functions. Although these metals are essential, they can be toxic at excess amounts, especially in certain genetic disorders. Zinc and copper homeostasis results from a coordinated regulation by different proteins involved in uptake, excretion and intracellular storage/trafficking of these metals. Apart from zinc transporters (ZnT) families and Cu-ATPase, metallothionein is an important storage protein for zinc and copper. Metallothioneins are intracellular polypeptides with a remarkable ability to bind metallic ions. These proteins bind both essential metals indispensable for the organism and also toxic metals (e.g. cadmium or lead). Metallothioneins play a critical role to maintain zinc and copper homeostasis. In this review, we summarize the toxicity of zinc and copper and the potential treatment for zinc or copper toxicity by zinc- or copper-specific chelators as well as strategy to up-regulate metallothionein.

Zinc supplementation decreases hepatic copper accumulation in LEC rat: a model of Wilson's disease.

The effect of dietary zinc (Zn) supplementation on copper (Cu)-induced liver damage was investigated in Long-Evans Cinnamon rats (LEC), a model for Wilson's disease (WD). Four-week-old LEC (N=64) and control Long-Evans (LE) (N=32) female rats were divided into two groups; one group was fed with a Zn-supplemented diet (group I) and the other was given a normal rodent diet (group II). LEC rats were killed at 6, 8, 10, 12, 18, and 20 wk of age; the LE control rats were killed at 6, 12, 18, and 20 wk of age. Cu concentration in the liver was reduced in LEC rats fed the Zn-supplemented diet compared with LEC rats on the normal diet between 6 and 18 wk of age. Metallothionein (MT) concentration in the livers of LEC rats in group I increased between 12 and 20 wk of age, whereas hepatic MT concentration in LEC rats from group II decreased after 12 wk. Hepatocyte apoptosis, as determined by TUNEL, was reduced in Zn-supplemented LEC rats at all ages. Cholangiocellular carcinoma was observed only in LEC rats in group II at wk 20. These results suggest that Zn supplementation can reduce hepatic Cu concentration and delay the onset of clinical and pathological changes of Cu toxicity in LEC rats. Although the actual mechanism of protection is unknown, it could be explained by sequestration of dietary Cu by intestinal MT, induced by high dietary Zn content.

Inhibition of extracellular signal regulated kinase (ERK) leads to apoptosis inducing factor (AIF) mediated apoptosis in epithelial breast cancer cells: the lack of effect of ERK in p53 mediated copper induced apoptosis.

Recent studies have shown that MEK/ERK-mediated signals play a major role in regulation of activity of p53 tumor suppressor protein. In this study, we investigated whether or not there is functional interaction between p53 and MEK/ERK pathways in epithelial breast cancer cells exposed to copper or zinc. We demonstrated that expression of wild-type p53 induced by copper or zinc significantly reduced phosphorylation of extracellular signal regulated kinase (ERK) in epithelial breast cancer MCF7 cells. Mutation or suppression of p53 in MDA-MB231 and MCF7-E6 cells, respectively, resulted in a strong ERK phosphorylation in the presence of metals. Weak ERK phosphorylation in MCF7 cells induced by copper or zinc was linked to mitochondrial disruption and apoptosis. Furthermore, inhibition of ERK through addition of PD98059 stimulated p53 activation in MCF7 cells and also led to upregulation of p53 downstream targets, p21 and Bax, which is a proapototic member of Bcl-2 family triggering mitochondrial pore opening. Moreover, blockage of the MEK/ERK pathway caused a breakdown of the mitochondrial membrane potential accompanied by an elevation in the ROS production. Disruption of p53 expression attenuated the depolarization of the mitochondrial membrane and ROS generation. Furthermore, PD98059 initiated apoptosis inducing factor (AIF) translocation from mitochondria to the nucleus in MCF7 cells; which are depleted in caspase 3. Interestingly, repression of MEK/ERK pathway did not intensify the cell stress caused by metal toxicity. Therefore, these findings demonstrate that MEK/ERK pathway plays an important role in downregulation of p53 and cell survival. Inhibition of ERK can lead to apoptosis via nuclear relocation of AIF. However, metal-induced activation of p53 and mitochondrial depolarization appears to be independent of ERK. Our data suggest that copper induces apoptosis through depolarization of mitochondrial membrane with release of AIF, and this process is MEK/ERK independent.

Impaired hepatic regeneration in metallothionein-I/II knockout mice after partial hepatectomy.

Although the translocation of metallothionein (MT) from cytoplasm to nucleus has been demonstrated in liver during times of high requirement for zinc (fetal development and the neonatal period), the role of MT in cellular growth is not well understood. In this study, a potential role of MT in liver regeneration was investigated in wild type (WT) and MT-I and MT-II gene knockout (MT-null) mice after 35% partial hepatectomy (PH) or sham laparotomy. Hepatic MT levels and proliferation index were measured at 0, 5, 15, 24, 36, 48, and 60 hrs after PH and 48 hrs after sham laparotomy (control). MT levels were increased in WT mice (peak at 24 hrs after PH) and declined to normal levels by 60 hrs after PH. Immunohistochemical staining for MT in WT mice indicated the presence of MT in both nucleus and cytoplasm of hepatocytes at 24 hrs after PH, whereas MT was present mainly in the cytoplasm at 36-60 hrs after PH and 48 hrs after sham laparotomy. Hepatic proliferation index in both WT and MT-null mice, as determined by argyrophilic nucleolar organizing region staining and proliferating cell nuclear antigen immunohistochemical staining, reached a peak at 48 hrs and declined by 60 hrs after PH. Cell proliferation was significantly less in MT-null mice as compared to WT mice during liver regeneration after PH. These results suggest that MT may play a positive role in hepatic regeneration after PH.

Metallothionein induction is not involved in cadmium accumulation in the duodenum of mice and rats fed diets containing high-cadmium rice or sunflower kernels and a marginal supply of zinc, iron, and calcium.

Rats fed diets with cadmium (Cd) concentrations similar to that found in human diets, and nutritionally marginal with respect to iron (Fe), zinc (Zn), and calcium (Ca) retained 10 times more Cd in the duodenum than rats fed adequate mineral diets. In the current study, 2 experiments were performed to determine the role of intestinal metallothionein (MT) in the accumulation of duodenal Cd, and to determine whether endogenous rice grain Cd is as available as Cd exogenously incorporated into the grain. In Expt. 1, wild-type and MT-null mice were fed 40% rice diets containing marginal or adequate amounts of Fe, Zn, and Ca, and 240 mug Cd/kg. Duodenal Cd was 10 times higher in both wild-type and MT-null mice regardless of their mineral status. In Expt. 2, one group of rats was fed 40% rice diets in which Cd was incorporated into the rice during growth and maturation, and another group was fed 40% rice diets in which Cd was incorporated into the rice during cooking. Each group also was fed either marginal or adequate amounts of Zn, Fe, and Ca. After 5 wk, rats were given a single meal labeled with (109)Cd, and the amount of label retained after 7 d was determined by whole-body counting. Rats with marginal mineral status retained 10 times more (109)Cd than those with adequate status; however, there was no difference between rats fed endogenous or exogenous Cd rice. Although duodenal Cd concentration was 8 times higher in the marginally fed rats, MT concentration was unchanged. These 2 experiments indicate that MT induction is not involved in duodenal Cd accumulation in animals with marginal dietary status of Fe, Zn, and Ca. In addition, they support the hypothesis that marginal deficiencies of Fe, Zn, and Ca, commonly found in certain human populations subsisting on rice-based diets, play an important role in increasing the risk of dietary Cd exposure.

Role of tumor necrosis factor-alpha in the development of spontaneous hepatic toxicity in Long-Evans Cinnamon rats.

The objective of this study was to evaluate the potential role of TNF-alpha in the onset of acute hepatitis in the Long-Evans Cinnamon (LEC) rat, an animal model for inherited copper (Cu) toxicosis. In LEC rats, Cu is accumulated in the liver with age, and clinical signs of acute hepatitis were observed as, icterus, reduced body weight, nasal bleeding, dehydration, and reduced food intake at 12 weeks of age. Cellular changes such as apoptosis in the liver were evident in these rats with increasing age. Positive TNF-alpha and TNFR1 immunostainings were observed in hepatocytes and Kupffer cells in LEC rats. Hepatic levels of caspase-3 activity, TNF-alpha mRNA, and protein were also increased in LEC rats from 6 to 12 weeks of age as compared with control Long-Evans (LE) rats. The neutralization of TNF-alpha by passive immunization or the inhibition of caspase activity can block the apoptotic process initiated by TNF-alpha. In this study, we evaluated the effects of passive immunization of LEC rats with weekly administration of anti-rat TNF-alpha on Cu-induced acute hepatitis. This treatment resulted in a reduction of the percentage of apoptotic cells in the liver, decreased activity of caspase-3, and also in down-regulation of the TNF-alpha gene expression. Thus, these results suggest a major role for TNF-alpha on the pathogenesis of Cu-induced acute hepatitis in LEC rats.

Zinc- or cadmium-pre-induced metallothionein protects human central nervous system cells and astrocytes from radiation-induced apoptosis.

We have shown the protection of human central nervous system (CNS) cultures by zinc (Zn) or cadmium (Cd)-pre-induced metallothionein (MT) synthesis from radiation-induced cytotoxicity (lactate dehydrogenase (LDH) release and neuronal dendritic injury). The present study is to further define the types of cell death induced by different dose levels of radiation and investigate the effect of MT induction (by Zn or Cd) on radiation-induced apoptosis in primary human CNS and astrocyte cultures. Apoptosis was detected by fragmented DNA electrophoresis, TUNEL technique, and propidium iodide staining. Expression of MT protein was examined by immunofluorescent staining. Results showed that exposure of primary human CNS cultures to 15 and 30 Gy gamma-radiation predominantly induced apoptotic cell death, while exposure to 60 Gy gamma-radiation predominantly induced necrotic cell death. Normal primary human CNS cultures showed weak MT staining, while primary human CNS cultures exposed to Zn or Cd showed intense MT staining. The induced apoptotic cell death by exposure to 30 Gy gamma-radiation increased to a maximum level at 12 and 24 h, and was reduced significantly by Zn or Cd pre-induced MT. Using primary human astrocytes, the induction of MT protein by Zn or Cd was further confirmed. The enhanced MT expression also afforded a significant protection from 30 Gy gamma-ray-induced apoptosis in the primary human astrocytes. These results suggest that MT protected human CNS cells from apoptosis following ionizing radiation, probably through its antioxidant property.

Metallothioneins in human tumors and potential roles in carcinogenesis.

Metallothioneins (MT) are a group of low-molecular weight, cysteine rich intracellular proteins, which are encoded by a family of genes containing at least 10 functional isoforms in human. The expression and induction of these proteins have been associated with protection against DNA damage, oxidative stress and apoptosis. Moreover, MT may potentially activate certain transcriptional factors by donating zinc. Although MT is a cytosolic protein in resting cells, it can be translocated transiently to the cell nucleus during cell proliferation and differentiation. A number of studies have shown an increased expression of MT in various human tumors of the breast, colon, kidney, liver, lung, nasopharynx, ovary, prostate, salivary gland, testes, thyroid and urinary bladder. However, MT is down-regulated in certain tumors such as hepatocellular carcinoma and liver adenocarcinoma. Hence, the expression of MT is not universal to all human tumors, but may depend on the differentiation status and proliferative index of tumors, along with other tissue factors and gene mutations. In certain tumors such as germ cell carcinoma, the expression of MT is closely related to the tumor grade and proliferative activity. Increased expression of MT has also been observed in less differentiated tumors. Thus, expression of MT may be a potential prognostic marker for certain tumors. There are few reports on the expression of the different isoforms of MT which have been analyzed by specific gene probes. They reveal that certain isoforms are expressed in specific cell types. The factors which can influence MT induction in human tumors are not yet understood. Down-regulation of MT synthesis in hepatic tumors may be related to hypermethylation of the MT-promoter or mutation of other genes such as the p53 tumor suppressor gene. In vitro studies using human cancer cells suggest a possible role for p53 and the estrogen-receptor on the expression and induction of MT in epithelial neoplastic cells. Some of the evidence supporting a role for MT in both intrinsic and acquired drug resistance is critically evaluated in this review. Since chemoresistance in human tumors is a multifactorial phenomenon, it is difficult to conclude that MT is a more crucial factor than others. Therefore, additional experimental data on MT and its isoforms in human tumors, are needed to elucidate the biological functions of MT during tumor progression, along with other tumor markers.

Copper-binding proteins in human erythrocytes: searching for potential biomarkers of copper over-exposure.

The recognition that copper is essential but also potentially toxic to humans has prompted the search for biomarkers of copper excess. The experimental approach followed here involves the isolation and subsequent characterization of copper-binding molecules (CuBP) from human erythrocytes. Incubation (0-60 min) of freshly obtained erythrocytes in the presence of increasing concentrations of copper (10-50 microM; as 64Cu-histidine) led to time- and concentration-dependent uptake of the radioisotope. A near-maximal incorporation was attained after 20 min, with 45-55% of the radioactivity being recovered in 20,000 x g hemolysate supernatants (S-20). 64CuBP from S-20 were separated by size exclusion and metal-affinity chromatography. Most radioactivity loaded into a Sephadex G-75 column was recovered in association with molecules of MMr greater than 60 KDa (largely accounted for by hemoglobin; Hb). Only negligible amounts of radioactive Cu were associated with metallothionein. With further purification, the higher MMr 64Cu-binding fractions were resolved by Sephadex G-200 into two major peaks. The cpm/microg protein ratios of the first peak (high MMr) were proportional to the concentrations of copper presented to the erythrocytes. The second one contained mostly Hb molecules. Proteins from the first peak were concentrated in an affinity chromatography mini-column, suited to trap CuBP. The higher-affinity CuBP were eluted as a single peak which comprised around 60% of the load. An SDS-PAGE analysis of such peak reveals the presence of three bands, of which two are non-hemoglobin Cu-binding proteins. The latter, whose identity remains to be established, had MMr of approximately 30 and 40 KDa, respectively. Preliminary data indicate that the two bands bind 64Cu within a range of concentrations, relevant to those expected to occur during copper over-exposure conditions.

Zinc-metallothionein protects from DNA damage induced by radiation better than glutathione and copper- or cadmium-metallothioneins.

Protection of radiation-induced DNA damage by metallothionein (MT) has been documented, but there is no detailed information about its efficiency compared to other antioxidants or the effect of metals which bind to MT on the protective effect of MT in radiation-induced DNA damage. In this study, we used a cell-free system to investigate the effect of MT with other antioxidants, such as albumin and glutathione and we compared the efficiency of MT bound to different metals on radiation-induced DNA damage. DNA damage was measured by loss in ethidium bromide/DNA fluorescence and increased mobility of DNA on gel electrophoresis. Gamma rays at 30 Gy induced significant DNA damage and zinc-MT showed a significant higher protection from radiation-induced DNA damage than both glutathione and albumin. Metallothionein bound to other metals, such as copper and cadmium, also showed protection of radiation-induced DNA damage, but the protective effect by zinc-MT was the highest. These results suggest that MT, in particular bound to zinc, is a high-capacity antioxidant to protect radiation-induced DNA damage.