Evolutionary Mode of Metallothioneins Inferred from Cd-MT Genes of Tetrahymena

From Tetrahymena thermophila （strain BF5）, the coding region of Cd-MT gene was cloned and sequenced. and identified as MTT1 isoform. A serial duplicate structure is discovered in its amino acid sequence, which separates the coding region into three parts （Part 1：7-61; Part 2：64-118; Part 3：122-162）. The alignments among them and comparison with the corresponding parts of MT1 isoform suggest that MT1 and MTT1 isoforms both come from the same ancient gene that is homologous to Part 1, and Cd-MTs of Tetrahymena are aroused by such ancient gene duplication. The prediction of secondary structures and the analysis of the disulfide-bonding state of cysteine show that there are a lot of differences between MT1 and MTT1 isoforms, which maybe relate to their function mechanism.

Biochemical and Immunochemical Characterization of Caenorhabditis elegans Metallothioneins I and II Induced by Cadmium

Metallothioneins in Caenorhahditis elegans (CeMT-Ⅰ and Ⅱ) were punned by the combination of gel filtration ion-exchange chromatography.and high-performance liquid chromatography.Amino acid compositions and amino-terminal sequences of CeMT-Ⅰ and Ⅱ were slightly different from those of vertebrate MTs previously reported, although cysteine residue contents were relatively high.Enzyme immunoassay using anti-CeMT-Ⅱ antibody showed the difference of antigenicity to rat MT-Ⅰ and Ⅱ and even to CeMT-Ⅰ. Immunohistochemical staining revealed the existence of CeMT-Ⅱ in the intestine and the eggs, suggesting the role of MT in detoxification and homeostasis of heavv metals. 1990 Academic Press.Inc.

Metallothioneins Induced by Antimony Accumulation in Earthworm Eisenia foetida Using Filter Paper Contact Method

MTs （Eiseniafoetida metallothioneins） have been recently reported as biomarkers for environmental metal contamination, however no study regarding Sb （antimony） is available. This study aimed to explore possibility of MTs as a biomarker of Sb pollution. A contact method of filter paper was used to investigate Sb inducing MTs in Eiseniafoetida. Spectrophotometrical and SDS-PAGE （sodium dodecyl sulphate-polyacrylamide gel electropheresis） analyses were used to determine the nature of the induced MTs samples. Rabbit liver metallothioneins was used as a standard. The results showed that the molecular weight of induced protein was approximately 6-7 kDa. A characteristic absorption peak at 268 nm was observed, which was consistent with the standard. The amounts of Sb-MTs quantified by cadmium hemoglobin saturation method showed a significant positive relationship with increasing Sb exposure.The experiments prove that Sb can induce Sb-MTs in Eiseniafoetida as a biomarker of antimony pollution.

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.

THE INDUCTION AND EXTRACTION OF METALLOTHIONEINS IN ARTEMIA

A small amount of heavy metal binding protein,identified by BioRad Protein Assay,has been iso-lated from the adult brine shrimp,Artemia franciscanus.This protein has an apparent molecular weight be-tween 6000 to 9000 dalton.a UV absorption peak at 260 instead of 280 nm like most proteins;andhas high affinity towards binding with radioactive labeled 109Cd.These characteristics are similarto that of metallothioneins reported for many vertebrate and invertebrate,marine and terrestrialanimals.After the brine shrimp is exposed to a small amount of Cd2+for 24 h,a large amount ofmetallothionein can be isolated,showing the inducibility of this detoxifying protein in the adult Artemiain a short period of time.

Kinetic Studies on Ligand Substitution Reactions of Metallothioneins with DTPA and EDTA

Cd, Zn-thionelns were isolated from the rat liver by improved Winge method and purified further. The composition was determined. The mobilization of Cd ions from the Cd, Zn-metallothioneins was studied with spectrophotometric method. The results show that the reaction of DTP A or EDTA with Cd, Zn-thioneins occurs through three steps. The first step follows pseudo-first order reaction with association mechanism. The formation constants and the dissociation rate constants of the relevant intermediate ternary complexes and apparent rate constants, Kapp were determined. The properties and structure of Cd, Zn-thioneins and ligands with low molecular weights affect the values of Kapp.

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.

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.

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.

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.

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.