Synergistic Effects of Glutamine Deprivation and Metformin in Acute Myeloid Leukemia

Objective The metabolic reprogramming of acute myeloid leukemia(AML)cells is a compensatory adaptation to meet energy requirements for rapid proliferation.This study aimed to examine the synergistic effects of glutamine deprivation and metformin exposure on AML cells.Methods SKM-1 cells(an AML cell line)were subjected to glutamine deprivation and/or treatment with metformin or bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide(BPTES,a glutaminase inhibitor)or cytarabine.Cell viability was detected by Cell Counting Kit-8(CCK-8)assay,and cell apoptosis and reactive oxygen species(ROS)by flow cytometry.Western blotting was conducted to examine the levels of apoptotic proteins,including cleaved caspase-3 and poly(ADP-ribose)polymerase(PARP).Moreover,the human long noncoding RNA(lncRNA)microarray was used to analyze gene expression after glutamine deprivation,and results were confirmed with quantitative RT-PCR(qRT-PCR).The expression of metallothionein 2A(MT2A)was suppressed using siRNA.Cell growth and apoptosis were further detected by CCK-8 assay and flow cytometry,respectively,in cells with MT2A knockdown.Results Glutamine deprivation or treatment with BPTES inhibited cell growth and induced apoptosis in SKM-1 cells.The lncRNA microarray result showed that the expression of MT family genes was significantly upregulated after glutamine deprivation.MT2A knockdown increased apoptosis,while proliferation was not affected in SKM-1 cells.In addition,metformin inhibited cell growth and induced apoptosis in SKM-1 cells.Both glutamine deprivation and metformin enhanced the sensitivity of SKM-1 cells to cytarabine.Furthermore,the combination of glutamine deprivation with metformin exhibited synergistic antileukemia effects on SKM-1 cells.Conclusion Targeting glutamine metabolism in combination with metformin is a promising new therapeutic strategy for AML.

Bioinformatics Analysis of Metallothionein in Betula phatyphylla

[Objective] This study aimed to analyze the bioinformatics information of Betula phatyphylla metallothionein for subsequent investigation of the important func- tions of metallothionein gene. [Method] The physical and chemical properties, trans- membrane domains, hydrophobicity / hydrophilicity, secondary structure, functional domains and functional classification of B. phatyphylla metallothionein were analyzed and predicted using bioinformatics methods and tools. [Result] The relative molecular mass of B. phatyphylla metallothionein is 7.75 kD and isoelectric point is 5.66; there is a strong hydrophobic region in the center of B. phatyphylla metallothionein and hydrophilic regions in both ends; B. phatyphylla metallothionein may be a non-trans- membrane protein and is mainly constituted by random coil without enzymatic activi- ty, suggesting that it belongs to Metallothio_2 protein family. [Conclusion] This study provided reference for further functional research of B. phatyphylla metallothionein gene.

Metallothionein:An overview

Metallothioneins （MTs） were discovered in 1957 by Margoshes and Vallee and identified as low-molecular weight and sulphydryl rich proteins. It is not surprising that most mammalian tissues contain age related basal levels of MTs since they are involved in metalloregulatory processes that include cell growth and multiplication. In an effort to understand the biology of this intriguing tumor, various biomarkers such as oncogenes, p53 tumor suppressor gene, war 1 protein, proliferating cell nuclear antigen, telomerase, microsatellite markers and cytogenetic changes have been examined. One biomarker which has recently shown to be expressed in various human tumors but still less reported in carcinoma is MT. Immunohistochemical detection of MT proteins in cold acetone-fixed paraffin embedded liver sections was performed by the streptavidin-avidin-biotin immunoperoxidase complex method.

p53 negativity, CDC25B positivity, and metallothionein negativity are predictors of a response of esophageal squamous cell carcinoma to chemoradiotherapy

AIM： Esophageal squamous cell carcinoma is generally sensitive to chemoradiotherapy （CRT）, but some cases are not. Using a retrospective analysis, we aimed to identify the predictors of the response by esophageal squamous cell carcinoma to definitive CRT. METHODS： The intensities of expression of p53, Ki67, Bci-2, Bax, olclin D1, VEGF, CDC25B, and metallothionein （MT） were evaluated immunohistochemically in the biopsy specimens obtained before CRT, and the intensities of their expression were tested for correlations with the clinical effects of CRT. RESULTS： The esophageal squamous cell carcinomas with negative p53, positive CDC25B, and negative MT expression were found to be significantly more sensitive to CRT. In addition, p53 positivity and CDC25B positivity respomd well to CRT. CONCLUSION： Esophageal squamous cell carcinomas with negative p53,positive CDC25B, and negative MT expressions respond well to CRT. Even with p53 positivity, if with CDC25B positivity, CRT can be expected. 2005 The WJG Press and Elsevier Inc. All rights reserved

Cadmium-induced neurotoxicity: still much ado

Cadmium（Cd） is a highly toxic heavy metal that accumulates in living system and as such is currently one of the most important occupational and environmental pollutants. Cd reaches into the environment by anthropogenic mobilization and it is absorbed from tobacco consumption or ingestion of contaminated substances. Its extremely long biological half-life（approximately 20-30 years in humans） and low rate of excretion from the body cause cadmium storage predominantly in soft tissues（primarily, liver and kidneys） with a diversity of toxic effects such as nephrotoxicity, hepatotoxicity, endocrine and reproductive toxicities. Moreover, a Cd-dependent neurotoxicity has been also related to neurodegenerative diseases such as Alzheimer＇s and Parkinson＇s diseases, amyotrophic lateral sclerosis, and multiple sclerosis. At the cellular level, Cd affects cell proliferation, differentiation, apoptosis and other cellular activities. Among all these mechanisms, the Cd-dependent interference in DNA repair mechanisms as well as the generation of reactive oxygen species, seem to be the most important causes of its cellular toxicity. Nevertheless, there is still much to find out about its mechanisms of action and ways to reduce health risks. This article gives a brief review of the relevant mechanisms that it would be worth investigating in order to deep inside cadmium toxicity.

p53 antibodies,metallothioneins,and oxidative stress markers in chronic ulcerative colitis with dysplasia

AIM:To investigate the role of p53 antibodies (p53Abs),metallothioneins (MTs) and oxidative stress markers in the early detection of dysplasia in chronic ulcerative colitis (UC).METHODS:The study included 30 UC patients,15 without dysplasia (group Ⅱ) and 15 with dysplasia (group Ⅲ),in addition to 15 healthy volunteers (group Ⅰ,control subjects).The enzyme-linked immunosorbent assay technique was used to measure serum p53Abs and MTs,while advanced oxidation protein products (AOPPs),and reduced glutathione (GSH) levels were measured by spectrophotometric method in all subjects.RESULTS:In group Ⅱ and group Ⅲ compared to group Ⅰ,there were significant increases in serum levels of AOPPs (145.94 ± 29.86 μmol/L and 192.21 ± 46.71 μmol/L vs 128.95 ± 3.06 μmol/L,P < 0.002 and P <0.001,respectively),MTs (8.18 ± 0.35 μg/mL and 9.20 ± 0.58 μg/mL vs 6.12 ± 0.25 μg/mL,P < 0.05 and P < 0.05,respectively),and p53Abs (20.19 ± 3.20 U/mL and 34.66 ± 1.34 U/mL vs 9.42 ± 1.64 U/mL,P < 0.001 and P < 0.001,respectively).There were significantly higher levels of AOPPs (P < 0.05) and p53Abs (P < 0.001) in UC patients with dysplasia compared to those without dysplasia,while MTs showed no significant difference between the 2 groups (P > 0.096).In contrast,GSH levels showed a significant decrease in both patients' groups (1.87 ± 0.02 μmol/mL and 1.37 ± 0.09 μmol/mL vs 2.49 ± 0.10 μmol/mL,P < 0.05 and P < 0.05 in groups Ⅱ and Ⅲ,respectively) compared with group Ⅰ,and the levels were significantly lower in group Ⅲ than group Ⅱ (P < 0.05).There was a positive correlation between AOPPs and both MTs (r=0.678,P < 0.001) and p53Abs (r=0.547,P < 0.001),and also between p53Abs and MTs (r=0.739,P < 0.001).There was a negative correlation between AOPPs and GSH (r =-0.385,P < 0.001),and also between GSH and both MTs (r=-0.662,P < 0.001) and p53Abs (r=-0.923,P < 0.001).CONCLUSION:Oxidative stress and oxidative cellular damage play an important role in the pathogenesis of chronic UC and the associated carcinogenetic process.p53Abs levels could help in early detection of dysplasia in these conditions.

Effect of Maotai liquor on the liver: an experimental study

BACKGROUND: Epidemiology investigation showed that no worker drunk Maotai liquor for nearly 30 years died of hepatic diseases, and no obvious hepatic fibrosis and cirrho- sis were found in 99 workers who had drunk Maotai liquor for a long period by epidemiology investigation and needle biopsy. The same finding was detected in rats that were drunk by Maotai liquor continued for 56 days. This study was to investigate the effects of Maotai liquor on the liver and its mechanism of preventing hepatic fibrosis. METHODS: After ingestion of Maotai for 56 consecutive days, male SD rats were killed for detecting the levels of metallothionein and malondialdehyde (MDA) in liver tis- sues. Rat hepatic stellate cells (HSCs) and human HSCs were cultured in vitro to observe the effect of Maotai on HSCs proliferation and collagen synthesis. After ingestion of Maotai for 14 consecutive weeks, the livers of male SD rats were harvested for pathohistological examination. RESULTS: The level of metallothionein in the liver of Mao- tai-induced rats increased by 22 folds, whereas the levels of hepatic lipid peroxide and MDA was decreased significantly ( P <0.05) in Maotai-induced animals suffering from CCl4. Maotai demonstrated obvious inhibitory effect on prolifera- tion of HSCs and the inhibition was concentration-depen- dent. Gene expression and protein secretion of collagens could also be inhibited by Maotai. In alcoholic group, typi- cal liver cirrhosis was observed. In Maotai group, however, though fatty degeneration of hepatocytes and mild fibrosis of the interstitium were observed, no obvious hepatic fibro- sis and cirrhosis were found. CONCLUSION: It might be an important mechanism of interfering the progress of hepatic fibrosis that Maotai in- creases the level of metallothionein in the liver and inhibits the activation of HSCs and the synthesis of collagen pro- teins.

Protective role of metallothionein in stress-induced gastric ulcer in rats

AIM: To illustrate the pathophysiological role of metallothionein (MT) in gastric ulcer induced by stress. METHODS: Wistar rats underwent water-immersionrestraint (WIR) stress, ZnSO4 (an MT inducer) treatment, WIR+ZnSO4 or WIR+MT, and the ulcer index (UI) was estimated in excised stomach and liver tissues. The mRNA level of gastric MT was determined by semi-quantitative RT-PCR. The MT content in gastric and hepatic tissues was determined by Cd/hemoglobin affinity assay. The lipid peroxidation products malondialdehyde (MDA) and conjugated dienes (CD) were estimated by use of thiobarbituric acid reactive species and ultraviolet spectrophotometry. RESULTS: WIR stress induced severe gastric mucosal lesions in rats. Compared with control rats, stressed rats had increased lipid peroxide content in serum and stomach and liver tissues. MDA content was increased by 34%, 21% and 29% and CD level by 270%, 83% and 28%, respectively. MT content in the stomach and liver was increased by 0.74- and 1.8-fold, and the MT-mRNA level in the stomach was increased by 26%. Pretreatment with ZnSO4 prevented gastric lesion development (the UI was 87% lower than that without pretreatment), and the MDA and CD content in serum and tissues was lower. The MT content in the liver was double in rats that were not pretreated, and the MT mRNA level in the stomach was 35% higher. MT administration 1 h before the WIR stress prevented gastric lesion development (the UI decreased by 47% compared with that in rats not pretreated), and the MDA and CD content in serum and tissues was significantly lower. CONCLUSION: In WIR-stressed rats, the MT level was increased in serum and in stomach and liver tissues. Pre-administration of exogenous MT or pre-induction of endogenous MT can protect the gastric mucosa against stress-induced ulcers and inhibits the formation of stressinduced lipid peroxide. MT could have a gastroprotective effect and might be a new interventive and therapeutic target in stress-induced gastric ulcers.

Generation and analysis of expressed sequence tags from the salt-tolerant eelgrass species, Zostera marina

Zostera marina, a monocotyledonous angiosperm, is one of the most important seagrass species. To inves- tigate the salt-tolerance mechanism and discover salt-tolerant genes in Z. marina, a cDNA library was con- structed. Single-pass sequencing of the 5＇ ends of 4 081 clones yielded 4 002 high quality expressed sequence tags （ESTs）, which were assembled into 241 contigs and 1 673 singletons, representing 1 914 unigenes. The average length of the ESTs was 582 bp, with sizes ranging from 100-1 500 bp. Basic Local Alignment Search Tool （BLASTX） analysis revealed that 1 664 unigenes had significant homology to known genes in the Na- tional Center for Biotechnology Information （NCBI） non-redundant （nr） database （E-value≤5-10）. Among them, the two most abundant genes encoded metallothionein （157 ESTs） and chlorophyll a/b-binding pro- tein （38 ESTs）, accounting for 7.1% and 1.7% of the total ESTs, respectively. Using Kyoto Encyclopedia of Genes and Genomes （KEGG）, 1 462 unigenes were assigned to 1 161 pathways （E-value≤5-10）. A total of 938 unigenes were assigned Gene Ontology （GO） terms based on the GO hierarchy analysis, and InterProScan searches recognized 1 003 InterPro families. Three genes for metallothionein in Z. marina that belonged to Class II was identified. Results of this study will improve understanding of the molecular mechanisms of saline tolerance in Z. marina.

Effects of Mercuric Chloride on Antioxidant System and DNA Integrity of the Crab Charybdis japonica

Mercury （Hg） is one of the commonly encountered heavy metals, which is widespread in inshore sediments of China. In order to investigate the toxicity of Hg on marine invertebrates, we studied the effects of the divalent mercuricion （Hg2＋） （at two final concentrations of 0.0025 and 0.0050mgL-1, prepared with HgC12） on metallothionein （MT） content, DNA integrity （DNA strand breaks） and catalase （CAT） in the gills and hepatopancreas, antioxidant enzyme activities of superoxide dismutase （SOD）, catalase （CAT） and glutathione peroxidase （GPx）, in the hemolymph, gills and hepatopancreas of the portunid crab Chao,bdisjaponica for an experiment period up to 15 d. The results indicated that MT was significantly induced after 3 d, with a positive correlation with Hg2＋ dose and time in the hepatopancreas and a negative correlation with Hg2＋ dose and time in the gills. While CAT in the hemolymph was not detected, it increased in the hepatopancreas during the entire experiment; SOD and GPx in the three tissues were stimulated after 12h, both attained peak value and then reduced during the experimental period. Meanwhile, DNA strand breaks were all induced significantly after 12 h. These results suggested the detoxification strategies against Hg2＋ in three tissues of C. japonica.

Dietary zinc and metallothionein on small intestinal disaccharidases activity in mice

AIM: To examine the effect of increasing dietary zinc (Zn) intake and the lack of metallothionein (MT) expression on activity of small intestinal disaccharidases. METHODS: MT-Ⅰ and Ⅱ knockout (MT-/-) and wild-type (MT+/+) female mice at 3.5 wk of age were randomly fed with a diet containing 2 (2 Zn), 15 (15 Zn) or 50 (50 Zn) mg Zn/kg (n = 8/group/genotype) for 5 wk. Small intestinal segments (duodenum, jejunum and ileum) were collected and either fixed in 10% formalin for histological analysis or snap frozen in liquid nitrogen for sucrase, lactase and maltase activity analyses.RESULTS: Plasma Zn was significantly (P < 0.05) lower (33%) in MT-/- compared with MT+/+ mice fed the 2 Zn diet. Villus height and crypt depth were increased by approximately 15% in MT+/+ mice compared with MT-/- mice. Duodenal disaccharidase activities were significantly higher in MT+/+ compared with MT-/- mice particularly in those fed the 2 Zn diet. For the 50 Zn diet, jejunal sucrase and lactase activities were significantly higher in MT-/- (13 313 ± 2314; 4107 ± 364 μmol glucose/well/min/g tissue, respectively) compared with MT+/+ mice (7054 ± 608; 1818 ± 174). Similarly, ileal lactase activities were higher in MT-/- (1480 ± 192) compared with MT+/+ (629 ± 353) mice particularly those fed the 2 Zn diet. CONCLUSION: Increasing dietary Zn has little effect on disaccharidases activity in MT wild-type mice. The presence of MT may enhance morphological and functional development of the gut.

A cadmium metallothionein gene of ridgetail white prawn Exopalaemon carinicauda(Holthuis,1950) and its expression

Metallothioneins （MTs） are a group of low molecular weight cysteine-rich proteins capable of binding heavy metal ions. A cadmium metallothionein （EcMT-Cd） cDNA with a 189 bp open reading frame （ORF） that encoded a 62 amino acid protein was obtained from Exopalaemon carinicauda. Seventeen cysteines were in the deduced amino acid sequence, and the cysteine （Cys）-rich characteristic was revealed in different metallothioneins in other species. In addition, the deduced amino acid sequence did not contain any aromatic amino acid residues, such as tyrosine （Tyr）, tryptophan （Trp）, and phenylalanine （Phe）. EcMT- Cd mRNA was expressed in all tested tissues （the ovary, muscle, stomach, and hepatopancreas）, and its expression profiles in the hepatopancreas were very different when shrimps were exposed to seawater containing either 50 pmol/L CuSO4 or 2.5 gmol/L CdC12. The expression of EcMT-Cd was significantly up-regulated in shrimp exposed to CuSO4 for 12 h and down-regulated in shrimps exposed to CdC12 for 12 h. After 24 h exposure to both metals, its expression was down-regulated. By contrast, at 48 h the EcMT-Cd was up-regulated in test shrimps exposed to CdC12. The transcript of EcMT-Cd was very low or even absent before the zoea stage, and the expression of EcMT-Cd was detected from mysis larvae-I, then its expression began to rise. In conclusion, a cadmium MT exists in E. carinicauda that is expressed in different tissues and during different developmental stages, and responds to the challenge with heavy metal ions, which provides a clue to understanding the function of cadmium MT

Clinical signification on the expressions of metallothionein in three types cancer of woman

Objective： To study the expressions and significations of metallothionein （MT） in cervical squamous cell cancer （CSC）, bladder transitional cell cancer （BTC） and breast cancer （BC） of woman. Methods： Immunohistochemical method was used to examine the expresses rate of MT in three types of woman cancer tissue. Results： The expressions rates of MT were 54.35% （29146） in BTC, 67.05% （59188） in BC and 57.14% （40/70） in CSC. The positive rate of MT expression was higher in low differentiation group than well differentiation group in BTC and CSC （P 〈 0.05）. Positive of MT in lobular cancer was significance higher than medullary and duct cancers （P 〈 0.05）. Conclusion： The expression of MT is related to differentiation degree, and it is a guidance for clinical choice of chemotherapy project.

Insulin Expression in Rats Exposed to Cadmium

To investigate the effects of cadmium exposure on insulin expression in rats. Methods Eighteen adult SD rats were administered cadmium subcutaneously （0.5, 1.0, and 2.0 mg/kg bw）. The effects on endocrine of pancreas were assessed. The levels of cadmium and zinc in pancreas, blood and urine glucose, serum insulin and urine NAG （N-acyetyl-β-glucosaminidase） were determined. The gene expressions of metallothionein （MT） and insulin were also measured, and the oral glucose tolerance tests （OGTT） were carried out. Results The contents of cadmium in pancreas in cadmium-treated rats were higher than that in the control group, which was associated with slight increase of zinc in pancreas. Cadmium-exposed rats （1.0 and 2.0 mg/kg bw） demonstrated a marked glucose intolerance. But the levels of serum insulin did not change significantly after cadmium administration, and the UNAG had no change in Cd-treated group. The gene expression of insulin decreased in 1.0 and 2.0 mg/kg bw cadmium-exposed groups, compared with the control group. The expression of MT-I was higher in the groups exposed to 1.0 and 2.0 mg/kg bw cadmium while the expression of MT-II was higher in the group exposed to 2.0 mg/kg bw cadmium. Conclusions Cadmium may be accumulated in the pancreas, resulting in the change of the expression of insulin, MT-I and MT-Ⅱ genes. Cadmium can influence the biosynthesis of insulin, but does not induce the release of insulin. The dysfunction of pancreas occurs earlier than that of kidney after administration of cadmium.

Acanthus ilicifolius plant extract prevents DNA alterations in a transplantable Ehrlich ascites carcinoma-bearing murine model

AIM： To investigate the chemopreventive efficacy of the Indian medicinal plant Acanthus ilicifolius L Acanthaceae in a transplantable Ehrlich ascites carcinoma （EAC）- bearing murine model.METHODS： Male Swiss albino mice were divided into four groups： Group A was the untreated normal control; Group B was the EAC control mice group that received serial, intraperitoneal （ip） inoculations of rapidly proliferating 2 × 10^5 viable EAC cells in 0.2 mL of sterile phosphate buffered saline; Group C was the plant extract-treated group that received the aqueous leaf extract （ALE） of the plant at a dose of 2.5 mg/kg body weight by single ip injections, once daily for 10, 20 and 30 consecutive days following tumour inoculation （ALE control）; and Group D was the EAC ＋ ALE- treatment group. The chemopreventive potential of the ALE was evaluated in a murine model by studying various biological parameters and genotoxic markers, such as tumour cell count, mean survival of the animals, haematological indices, hepatocellular histology, immunohistochemical expression of liver metallothionein （MT） protein, sister-chromatid exchanges （SCEs）, and DNA alterations.RESULTS： Treatment of the EAC-bearing mice with the ALE significantly （P 〈 0.001） reduced viable tumour cell count by 68.34% （228.7 × 10^6 ± 0.53） when compared to EAC control mice （72.4 × 10^6 ± 0.49）, and restored body and organ weights almost to the normal values. ALE administration also increased （P 〈 0.001） mean survival of the hosts from 35 ± 3.46 d in EAC control mice to 83 ± 2.69 d in EAC ＋ ALE-treated mice. Haematological indices also showed marked improvement with administration of ALE in EAC-bearing animals. There was a significant increase in RBC count （P 〈 0.001）, hemoglobin percent （P 〈 0.001）, and haematocrit value （P 〈 0.001） from 4.3 ± 0.12, 6.4 ± 0.93, and 17.63 ± 0.72 respectively in EAC control mice to 7.1 ± 0.13, 12.1 ± 0.77, and 30.23 ± 0.57 respectively in EAC ＋ ALE-treated group, along with concurrent decrement （P 〈 0.001） in WBC count from 18.8 ± 0.54 in EAC control to 8.4 ± 0.71 in EAC ＋ ALE. Furthermore, treatment with ALE substantially improved hepatocellular architecture and no noticeable neoplastic lesions or foci of cellular alteration were observed. Daily administration of the ALE was found to limit liver MT expression, an important marker of cell proliferation with concomitant reduction in MT immunoreactivity （62.25 ± 2.58 vs 86.24 ± 5.69, P 〈 0.01）. ALE was also potentially effective in reducing （P 〈 0.001） the frequency of SCEs from 14.94 ± 2.14 in EAC control to 5.12 ± 1.16 in EAC ＋ ALE-treated group. Finally, in comparison to the EAC control, ALE was able to suppress in vivo DNA damage by abating the generations of＇tailed＇ DNA by 53.59% （98.65 ± 2.31 vs 45.06 ± 1.14, P 〈 0.001）, and DNA single-strand breaks （SSBs） by 38.53% （3.14 ± 0.31 vs 1.93 ± 0.23, P 〈 0.01） in EAC-bearing murine liver.CONCLUSION： Our data indicate that, ALE is beneficial in restoring haematological and hepatic histological profiles and in lengthening the survival of the animals against the proliferation of ascites tumour in vivo. Finally, the chemopreventive efficacy of the ALE is manifested in limiting MT expression and in preventing DNA alterations in murine liver. The promising results of this study suggest further investigation into the chemopreventive mechanisms of the medicinal plant A. ilicifolius in vivo and in vitro.

Effect of Feeding Strategies on Molecular Responses of Biotransformation Genes in Crassostrea gigas Exposed to Cadmium

Heavy metals, including cadmium (Cd), are widespread pollutants of great environmental concern because of their ac- cumulation and toxicity. Environmental conditions can affect the accumulation and elimination of Cd in organisms. The aim of this study is to understand the respective responses of metallothioneins (MTs), P-glycoprotein (P-gp), and heat shock protein 70 (hsp70) genes in the mantle and digestive gland of the oyster Crassostrea gigas exposed to 10 g L 1 Cd for 28 days, followed by a depura- tion period of 35 days under different feeding strategies. The MT expression in the digestive gland was higher than in mantle, and was induced in a fluctuating pattern throughout the time of the whole experiment when the oyster was exposed to Cd. The results indicated an enhanced MT activity is required for Cd detoxification. Expression levels of P-gp in the mantle of Cd-exposed oysters fed with algae increased in the first two weeks of the accumulation phase. Then the induction weakened, but the expression still in- creased significantly compared with the control group fed without algae. The P-gp levels in the digestive gland were under-regulated during both the contamination and decontamination periods except in the group fed with Dicrateria inornata at day 0.083. The in- duction of P-gp is possibly related to the important role of pumping Cd out of the cell, while a major implication of P-gp expression inhibition would be a disruption of the protein synthesis. Hsp70 expression exhibited an overall decreasing trend. The highest relative hsp70 inhibition occurred during day 42 to day 63, with approximately 4-fold and 10-fold lower hsp70 levels as compared to the C.gigas feeding with no algae in the mantle and digestive gland, respectively. The induction of hsp70 may be explained by the re- folding of protein in presence of Cd, followed by the inhibition of hsp70 when the protein synthesis was disrupted.

Alterations of organ histopathology and metallolhionein mRNA expression in silver barb,Puntius gonionotus during subchronic cadmium exposure

Common silver barb, Puntius gonionotus, exposed to the nominal concentration of 0.06 mg/L Cd for 60 d, were assessed for histopathological alterations （gills, liver and kidney）, metal accumulation, and metallothionein （MT） mRNA expression. Fish exhibited pathological symptoms such as hypertrophy and hyperplasia of primary and secondary gill lamellae, vacuolization in hepatocytes, and prominent tubular and glomerular damage in the kidney. In addition, kidney accumulated the highest content of cadmium, more than gills and liver. Expression of MT mRNA was increased in both liver and kidney of treated fish. Hepatic MT levels remained high after fish were removed to Cd-free water. In contrast, MT expression in kidney was peaked after 28 d of treatment and drastically dropped when fish were removed to Cd-free water. The high concentrations of Cd in hepatic tissues indicated an accumulation site or permanent damage on this tissue.

Influence of Isoflavones on Cadmium-induced Adverse Effects in Vascular Endothelial Cells (ECV 304)

Objective To study the possible intervention of isoflavones in cytotoxicity induced by cadmium in vascular endothelial cells. Methods An ECV 304 cell line derived from human umbilical vein endothelial cells was adopted. Genistein / daidzein was added prior to or simultaneously with CdCl2, cell viability was determined by MTT assay, and metallothionein mRNA expression was monitored by RT-PCR method. Results Cell viability was higher in isoflavone and CdCl2 co-treated groups than that in CdCl2 treated group, with CdCl2 concentration at 10, 20, 40, and 80 μmol/L, respectively. However this increase was not observed in the group treated with CdCl2 at a concentration of 60 μmol/L. Isoflavones (10-10 mol/L to 10-5 mol/L) were added 24 h before cells were challenged with 80 μmol/L CdCl2 for 24 h or simultaneously with 80 μmol/L CdCl2. Genistein increased cell viability only at 10-5 mol/L, while daidzein caused a dose-dependent increase from 10-10 mol/L to 10-5 mol/L in co-treatment with CdCl2. In pre-treatment, genistein (10-7 to 10-5 mol/L) increased cell viability whereas only 10-5 mol/L of daidzein exerted protection. Apparent protection could be found when the cells were pre-treated with 10-5 mol/L isoflavones for over 12 h, whereas 24 h incubation was required in such a co-treatment, with the exception of daidzein that had a significant protection in only 3 h. Isoflavones (10-6 mol/L) incubated for 3 h to 24 h, increased MT IIA and MT IF mRNA expression, but the induction could not last for more than 24 h. Co-treatment with isoflavones could induce an additional induction of MT IIA mRNA expression in cells exposed to cadmium. However, the additional induction of MT IIA and MT IF mRNA was not seen when pre-treatment was carried out with isoflavones, with the exception of an increase in MT IIA mRNA expression in the daidzein pre-treated group. Conclusion Genistein/daidzein could reverse the cytotoxicity of cadmium either in pre-treatment or in co-treatment. The protection is the strongest in 10-5 mol/L of isoflavones with a dose-dependent pattern. There are differences between genistein and daidzein in their protective effects. Whether the protection of isoflavones is related to their capacity of inducing MT mRNA expression remains to be elucidated.

Role of MetallothioneinlH in Cisplatin Resistance of Non-Small Cell Lung Cancer Cells

Objective： Despite platinum-based adjuvant chemotherapy has improved greatly patients＇ outcomes, drug resistance poses a major impediment to the successful use of such an effective agent. Metallothioneins（MTs） are known to play putative roles in cancer cell proliferation, apoptosis, differentiation, drug resistance and prognosis. The present studiy was to investigte the role of metallethioeinlH（MTIH） in cisplatin resistance of human non-small cell lung cancer（NSCLC） cell lines in vitro or its possible molecular mechanisms. Methods： MTIH mRNA expression in A549 and A549/DDP cells was detected by RT-PCR. A recombinant eukaryotic expression plasmid pcDNA3.1（-）-MT1H was constructed and transfected into A549 cells which express no MTIH. MT1H siRNA was transfected into A549/DDP cells which express MTIH highly. MTIH expression was detected by RT-PCR and Immunoblot. The chemosensitivity to cisplatin was assessed by MTT assay. Apoptosis rate was determined by Tunel and FCM. Bcl-2 and Bax were determined by immunohistochemistry. Results： MT1H mRNA was expressed in A549/DDP but not in A549. After transfection of MT1H, MT1H expression was enhanced and the chemosensitivity to cisplatin was decreased in A549 cells. Inversely, after transfection of MT1H siRNA, MT1H expression was decreased and the chemosensitivity to cisplatin was increased in A549/DDP. The apoptosis rate induced by cisplatin was increased and Bcl-2 was down-regulated but Bax showed little change in A549/DDP cells interferred with MT1H siRNA. Conclusion： MT1H overexpression can promote drug resistance in A549 cells . Down-regulation of MTIH interfered with siRNA can effectively reverses the drug resistance in A549/DDP cells by down-regulating the expression of Bcl-2 and increasing cisplatin induced apoptosis. SiRNA targeting MT1H combined with chemotherapy may be a very promising strategy for treatment of lung cancer.

Metallothionein 1 Isoform Gene Expression Induced by Cadmium in Human Peripheral Blood Lymphocytes

Objective To study the gene expression of metallothionein 1 （MT-1） isoforms in human peripheral blood lymphocytes （HPBLs）. Methods The expression of mRNA representing the seven active MT-I genes was determined in HPBLs by quantitative RT-PCR before and after exposure to cadmium. Results Basal expressions of MT-IX, and MT-1A in HPBLs were similar to expression of housekeeping gene. In contrast, the basal gene expressions of MT- 1 H, IF, 1E, and 1G were a little transcripts in human HPBLs. No signal was detected for MT-1B. There was a sex difference （P〈0.05）. in basal gene expression of MT-1E. The levels of gene expression of MT-1A, 1E, IF, 1G, 1H, and 1X increased, but the level of MT-1B did not increase after exposure to cadmium. Conclusions Gene expressions of MT-1 G, MT-1 H, MT-1 F, and MT-1X in HPBLs can be used as a potential biomarker of cadmium exposure.