Thionein gene expression in Cd++-variants of the CHO cell: correlation of thionein synthesis rates with translatable mRNA levels during induction, deinduction, and superinduction.

The relationship of thionein synthesis rates to translatable cytoplasmic thionein mRNA levels was investigated for the first time in a cultured cell system. Thionein synthesis was induced in Cdr, a cadmium-resistant variant of CHO, by exposure to 2 microM CdCl2. Following a short (1.5 hr) lag, thionein synthesis increases to a rate that is at least 30 times the uninduced rate 7-8 hr after addition of Cd++. This increase is blocked by the coincident addition of a actinomycin D. Cytoplasmic thionein mRNA levels, measured by translation in a modified wheat germ system, increase rapidly following induction to values approximately 25 times uninduced levels within 6-8 hr. The increase in thionein mRNA precede proportionate increases in thionein synthesis by 0.5-1.0 hr. Continued exposure to Cd++ results in a decreased thionein synthesis rate after 8 hr. By 30 hr, the rate is one-half that seen 6-8 hr after induction. Removal of Cd++ after 8 hr results in a rapid decrease in thionein synthesis (t 1/2 approximately 4 hr). Both decreases are inhibited by the addition of actinomycin. In all instances--induction, deinduction, and actinomycin-mediated "super-induction"--translatable thionein mRNA levels and thionein synthesis rates increase, decrease, or are maintained coordinately. The results suggest that thionein synthesis in Cdr is controlled primarily by the level of translatable cytoplasmic thionein mRNA.     

Induction of metallothionein synthesis in cultured cells derived from rabbit kidney

Metallothionein (MT) synthesis in rabbit kidney-derived RK-13 cells was studied. In response to Cd2+, RK-13 cells synthesized proteins closely similar in chromatographic and electrophoretic behaviors to the liver MTs induced in Cd2+-injected rabbit. These proteins were specifically immunoprecipitated by anti-mouse liver MT-II serum. The rate of RK-13 thionein (apoprotein of MT) synthesis rapidly increased after exposure to 1 microgram/ml of Cd2+, and reached the maximum in 7 h. The dose-response curve for the synthesis was biphasic; a sharp increase up to 0.5 microgram/ml and a slower increase at higher concentrations. RK-13 cells retained kidney-specific properties in terms of responsiveness of thionein synthesis to inducers; The MTs were inducible also by Zn2+ and probably by Hg2+, but not by dexamethasone. This system would therefore be a useful model in vitro for studying the regulation of MT synthesis in kidney cells.     

Cell-free synthesis of metallothionein directed by rat liver polyadenylated messenger ribonucleic acid.

Total RNA was isolated from rat liver polyribosomes and fractionated by oligo(dT)-cellulose chromatography to obtain polyadenylated mRNA. The mRNA was translated in a wheat-germ cell-free protein-synthesizing system containing [3H]glycine, [3H]lysine and [3H]serine. Most of the newly synthesized 3H-labelled polypeptides were removed from the cell-free products by precipitation at pH 4.0. 3H-labelled thionein chains, which were soluble at pH 4.0, were purified by activated-thiol-Sepharose 4B chromatography or by gel-filtration chromatography. Polyribosomal thionein mRNA was found to increase by at least 3-fold after parenteral administration and by 20 h thereafter the ratio of thionein mRNA to total mRNA approached that found in controls. Actinomycin D administration in vivo blocked the Zn2+-induced increase in polyribosomal thionein mRNA content. These data strongly suggest that metallothionein is an inducible protein. The mechanism of regulation appears to involve changes in the synthesis de novo of thionein mRNA and hence the pool of thionein mRNA available for translation.     

Control of zinc-thionein synthesis in rat liver.

The rate of [35S]cystine incorporation into hepatic zinc-thionein (a metallothionein) was stimulated, with a maximum of 5-6h, after parenteral administration of 2mg of Zn2+ containing 65Zn. The binding of 65Zn to zinc-thionein was measurable by 2-1/2h and reached a plateau by 18h after the injection. A net increase in the hepatic 65Zn content was observed subsequent to the decrease in the rate of zinc-thionein synthesis. The incorporation of both 65Zn and [35S]cystine into zinc-thionein was inhibited by prior administration of either actinomycin D or cordycepin. A second injection of Zn2+, 20h after the initial injection, yielded a 4.9-fold greater increase in zinc-thionein synthesis compared with that after only one injection; however, this synthesis was also inhibitable by actinomycin D. These data support the concept that hepatic zinc-thionein synthesis responds quickly to changes in Zn2+ status and that Zn2+ is bound subsequent to synthesis of nascent thionein chains. The mechanism of control of zinc-thionein synthesis by Zn2+ appears to involve changes in the amounts of a short-lived, poly(A)-containing RNA whose translation can be derepressed by additional exposure to Zn2+.     

Metallothionein induction in human peripheral blood lymphocytes by heavy metals

Human peripheral blood lymphocytes have the capacity to produce metallothioneins (MTs) as a protective response to cadmium exposure. To define the range of metal species inducing lymphocyte MTs, cellular proteins synthesized after exposure to each of 11 heavy metals were analyzed by gel electrophoresis. Toxic metals such as cadmium, mercury and silver were found to induce thioneins (apoproteins of MTs) at relatively low concentrations (maximum at approximately 10 microM), whereas less toxic metals such as zinc, copper and nickel were inductive at relatively high concentrations (maximum at approximately 200 microM). Tin, lead, iron, cobalt, and manganese did not induce thioneins. The heavy metal specificity of MT induction in the lymphocyte resembles that in the liver, and the regulatory mechanism of MT production seems to be similar in both of these tissues. In the cells exposed to highly toxic metals such as cadmium and mercury, expression of cytotoxicity (represented by decline of cysteine uptake) was remarkable at the metal concentrations higher than those saturating thionein induction, supporting the protective role of MTs against heavy metals.     

Induction of cadmium-thionein in isolated rat liver cells.

The uptake of cadmium by isolated liver cells was linearly related to the cadmium concentration to which the cells were exposed in the medium. Cadmium-treated cells synthesized proteins de novo with the characteristics of cadmium-thionein induced in the liver of cadmium-treated animals. Thionein from liver cells incorporated cadmium and [35S]cysteine, had a Ve/Vo (Sephadex G-50) of 1.8-1.9, and was separated into two subfractions by DEAE-cellulose ion-exchange chromatography. Cycloheximide and actinomycin D when added after a cadmium exposure prevented the synthesis of thionein. However, addition of actinomycin D after synthesis had started only decreased the total amount of thionein synthesized. The concentration of cadmium to which the cells were exposed affected the amount of cadmium-thionein synthesized in 6h. The maximum response occurred when cells were exposed to 0.5 microgram of cadmium/ml; at higher metal concentrations the total amount of cadmium-thionein synthesized declined. The system described in the present paper can be used to study the mode of metal toxicity and the mechanism of cadmium-thionein synthesis.     

Formation and characterization of aurothioneins: Au,Zn,Cd-thionein, Au,Cd-thionein, and (thiomalato-Au)chi-thionein

Three gold-containing thioneins (Au,Zn,Cd-Th, Au,Cd-Th, and (TmSAu)chi Th, where Th = thionein and TmS = thiomalate) have been prepared by the reactions of horse kidney Zn,Cd-thionein with gold thiomalate (AuSTm). When thionein was present in excess, the thiomalate ligand was displaced and the protein chelated the gold in a bidentate fashion. Primarily zinc but also some cadmium was displaced to form Au,Zn,Cd-Th or Au,Cd-Th. Excess AuSTm reacted to form (TmSAu)chi-thionein with monodentate coordination of the protein to each bound gold, retention of the thiomalate, loss of zinc and cadmium, and an increase in the Stokes radius of the product. EXAFS/XANES studies of Au,Zn,Cd-Th and (TmSAu)chi Th established that the oxidation states and coordination environments of gold were Au(I)S2 and that the gold-sulfur bond distances were 229 and 230 pm, respectively. Radioimmunoassay established that the aurothioneins retained their antigenicity to native metallothionein antibodies. Metal exchange reactions with gold were complete within 5-10 min when Zincon or 4-(2-pyridylazo) resorcinol was used to monitor Cd2+ and Zn2+ displacement.     

Nitric oxide protects anterior pituitary cells from cadmium-induced apoptosis

Cadmium (Cd2+) is a potent toxic metal for both plants and animals. Chronic exposure to low doses of Cd2+ results in damage to several organs. We have previously reported that Cd2+ induces apoptosis in anterior pituitary cells by a caspase- and oxidative stress-dependent mechanism. Nitric oxide (NO) synthesis is affected by Cd2+ in several systems. NO has been shown to be either cytoprotective or cytotoxic in many systems. The aim of this study was to evaluate the possible participation of NO in the cytotoxic effect of Cd2+ on rat anterior pituitary cells. Cell viability was evaluated by mitochondrial dehydrogenase activity assay and confirmed by microscopy, studying nuclear morphology. Here we show that DETA NONOate ((Z)-1-[2 (2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate), a long-term NO donor, at concentrations below 0.5 mM, reduces nuclear condensation and fragmentation and reverses the decrease in cellular activity induced by Cd2+. Cd2+, by itself, induced NO synthesis, and inhibition of this synthesis enhanced Cd2+ cytotoxicity. NO also prevented caspase-3 activation and lipidic peroxidation induced by Cd2+. The NO/cGMP pathway does not seem to be involved in the cytoprotective effect of NO. These results indicate that NO has a cytoprotective role in Cd2+ -induced apoptosis, suggesting that endogenous NO could have a physiological role in protecting anterior pituitary cells.     

Characterization and measurement of metallothionein messenger RNA of C57BL mouse liver

Liver poly(A)+RNA of Cd2+-treated C57BL mouse was characterized by cell-free translation, particularly intending to establish a procedure to measure the levels of messenger RNA coding for metallothioneins (MT-mRNA). Intact polysomes were obtained by Mg2+ precipitation from the liver cytoplasm of mice injected with 1 mg Cd2+/kg body wt. Poly(A)+RNA isolated from the polysomes was translated by a wheat germ cell-free system and the [35S]cysteine-labeled translation products were analyzed by sodium dodecyl sulfate (SDS)-15% polyacrylamide gel electrophoresis and fluorography. MTs were identified in the translation products directed by the RNA from the Cd2+-treated mice, but not in the translation products directed by the RNA from untreated mice. Relative incorporation of [35S]cysteine into MTs was determined by densitometrical quantification of the MT bands, and was found to be linear up to a RNA concentration of 150 micrograms/ml in the translation reaction mixture, showing that this system is suitable for the measurement of translatable MT-mRNA levels. Cd2+ stimulated the total levels of cell-free translation (1.4-fold at 20-60 micrograms/ml), not specifically to MT-mRNA. MT-mRNA sedimented at 9S in a sucrose gradient, and its size was comparable with rat and human MT-mRNAs.     

Metallothionein mRNA stability in chicken and mouse cells

Northern blot analysis revealed that metallothionein (MT) mRNAs accumulate after inhibition of protein synthesis with cycloheximide (CHX) in primary cultures of chick embryo hepatocytes and fibroblasts, as well as in an established mouse hepatoma cell line. Inhibition of RNA synthesis with actinomycin D (AMD) led to rapid loss of MT mRNAs in these cells, whereas CHX dramatically retarded the rate of MT mRNA decay (t1/2 greater than 24 h). These results suggest that CHX causes MT mRNA accumulation primarily by increasing stability of MT mRNA. Thus, changes in MT mRNA turn-over rates may play an important role in regulating the accumulation of MT mRNA. The half-lives of MT mRNAs in chicken and mouse cells were determined by oligodeoxyribonucleotide excess solution hybridization with RNA samples extracted after different periods of exposure to AMD. The half-life of chicken MT (cMT) mRNA in uninduced chicken embryo hepatocytes was 3.6 h. Induction of cMT mRNA by pretreatment of these cells with zinc (Zn) prior to exposure to AMD, did not alter the half-life of cMT mRNA significantly. In contrast, cadmium (Cd) induction led to a 2.5-fold increase in the stability of this mRNA. In uninduced chicken embryo fibroblasts, cMT mRNA levels were too low to allow accurate determination of half-life using the methods employed here. However, the half-life of this mRNA in Zn-induced chicken embryo fibroblasts was 6.2 h, whereas it was 9.3 h in Cd-induced cells. Thus, the turn-over rate of cMT mRNA after Cd-induction is very similar in chick embryo fibroblasts and hepatocytes. These data suggest that the accumulation of MT mRNA in chicken cells may reflect, in part, metal-specific effects on MT mRNA stability. The half-lives of mouse MT-I and MT-II (mMT-I and mMT-II) mRNAs in uninduced BNL hepatoma cells were identical (9.2 h), and were not effectively altered after induction by metals (Zn, Cd) or interleukin-1 beta (IL-1 beta). However, mMT mRNAs in pachytene spermatocytes and round spermatids, freshly isolated from the adult testes, were 2.2- to 4.5-fold more stable than in hepatoma cells. These results suggest that cell-type specific accumulation of mMT mRNAs may be regulated, in part, by mRNA stability.     

Interleukin 2 mRNA induction in human lymphocytes: analysis of the synergistic effect of a phorbol ester and phytohemagglutinin

Induction of interleukin 2 (IL2) mRNA synthesis in human tonsillar lymphocytes was studied by quantifying the relative levels of IL2 mRNA in the lymphocytes stimulated under various conditions by the dot hybridization method. A remarkable increase of IL2 mRNA was induced by stimulation with phytohemagglutinin (PHA) in the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The kinetic study revealed that the IL2 mRNA level of the lymphocytes increased from 2 h of culture, reached a maximal level at 12 h, maintained a relatively high level until 48 h and then sharply decreased by 72 h after the stimulation. Inhibition experiments with actinomycin D showed that the increase was due to a transient synthesis of the mRNA after the stimulation, which almost stopped by 12-16 h. DNA synthesis and cell division were not necessary for the induction of IL2 mRNA production but the induction was inhibited by dexamethasone, showing that the production was mainly associated with the G1 phase of the cell cycle. Two-step culture experiments showed that prior exposure of the lymphocytes to TPA for 1 h at 37 degrees C resulted in a remarkable increase of IL2 mRNA on subsequent stimulation with PHA. This suggests that TPA induces certain changes in the biochemical pathway of signal transduction so that the cells can be triggered to express IL2 gene by subsequent stimulation with mitogen.     

镉中毒大鼠睾丸与肝脏金属硫蛋白表达的时相研究

啮齿目动物睾丸对镉毒性较肝脏更敏感.为阐明睾丸的镉毒性分子机制,比较了肝脏与睾丸金属硫蛋白（MT）表达的时相变化.mRNA采用RT-PCR技术分析并用光密度扫描定量;蛋白质定量用ELISA方法.结果显示,睾丸中存在MT,镉中毒后MT1与MT2 mRNA明显升高,但MT没有相应增加;肝脏镉中毒后MTmRNA与MT均明显升高.结果提示：镉虽然能诱导睾丸MTmRNA的转录,但没有促进其MT的合成,这可能是睾丸对镉毒性与致癌作用较肝脏更敏感的重要原因.     

In vivo and ex vivo displacement of zinc from metallothionein by cadmium and by mercury

Divalent cadmium and mercury ions are capable in vitro of displacement of zinc from metallothionein. This process has now been studied in vivo and ex vivo, using the isolated perfused rat liver system, in order to determine if this process can occur in the intact cell. Rats with normal and elevated (via preinduction with zinc) levels of hepatic zinc thionein were studied. Cd(II) completely displaces zinc from normal levels of metallothionein and on a one-to-one basis from elevated levels of metallothionein, both in vivo and ex vivo. Hg(II) displaces zinc from metallothionein (normal or elevated) rather poorly, as compared with Cd(II), in vivo, probably due to the kidneys preference for absorbing this metal. Ex vivo Hg(II) displaces zinc from metallothionein (normal or elevated) on a one-to-one basis, with considerably more mercury being incorporated into the protein than in vivo. The results of double-label ex vivo experiments using metal and [35S]cysteine (+/- cycloheximide) were consistent with the above experiments, indicating that de novo thionein synthesis was not required for short term incorporation of cadmium and mercury into metallothionein. These data are supportive of the hypothesis that cadmium and mercury incorporation into rat hepatic metallothionein during the first few hours after exposure to these metals can occur primarily by displacement of zinc from preexisting zinc thionein by a process which does not require new protein synthesis.     

Identification, cloning and characterisation of a novel copper-metallothionein in tetrahymena pigmentosa. Sequencing of cDNA and expression.

The protist Tetrahymena pigmentosa accumulates large amounts of metal ions, particularly cadmium and copper. This capability is linked to the induction of metallothioneins (MTs), cysteine-rich metal-binding proteins found in protists, plants and animals. The present study focuses on a novel inducible MT-isoform isolated from Tetrahymena after exposure to a non-toxic dose of copper. The cDNA sequence was determined utilising the partial peptide sequence of purified protein. The Cu-MT cDNA encodes 96 amino acids containing 28 cysteine residues (29%) arranged in motifs characteristic of the metal-binding regions of vertebrate and invertebrate MTs. Both the amino acid and nucleotide sequences differ, not only from other animal MTs, but also from the previously characterised Tetrahymena Cd-MT. Both MTs contain the structural pattern GTXXXCKCXXCKC, which may be proposed as a conservative sequence of Tetrahymena MTs. Cu-dependent regulation of MT expression was also investigated by measuring MT-mRNA and MT levels. MT synthesis occurs very quickly and MT contents increase with Cu accumulation. The induction of Cu-MT mRNA is very rapid, with no observable lag period, and is characterised by transient fluctuation, similar to that described for Cd-MT mRNA. The data reported here indicate that, also in the unicellular organism Tetrahymena, two very different MT isoforms, which perform different biological functions, are expressed according to the inducing metal, Cu or Cd.     

Cadmium-resistant Chinese hamster V79 cells with decreased accumulation of cadmium

Cells resistant to 3 x 10(-5) M CdCl2 (Cdr cells) were isolated from cultures of Chinese hamster V79 cells by a procedure that involved stepwise increase in the concentration of Cd2+ and subsequent mass selection. Cdr cells grew as fast as wild-type cells (Cds) in medium without cadmium. Cdr cells were not cross-resistant to other divalent metal ions, such as Hg2+, Ni2+, Pb2+, and Zn2+. Both Cds and Cdr cells induced similar levels of metallothioneins (MT) in response to zinc. Depletion of glutathione (GSH) did not significantly influence the sensitivity of Cdr cells to Cd2+ but markedly enhanced the sensitivity to Cd2+ of Cds cells. Furthermore, the rate of synthesis of GSH after depletion did not differ greatly between sensitive and resistant cells. The rate of uptake of 109Cd2+ by Cdr cells was only 10-15% that by Cds cells. The difference in rates of uptake between Cds and Cdr cells was observed irrespective of the presence or absence of serum in the culture medium. These results indicate that, in this system, resistance to Cd2+ is attributable neither to increased inducibility of MT nor to increases in intracellular levels of GSH, and that only a decrease in the rate of uptake of Cd2+ contributes to the acquisition of resistance to Cd2+. Uptake of Cd2+ by cells was dependent on temperature and the rate of uptake of Cd2+ by Cdr cells was lower at all temperatures examined than the rate of uptake by Cds cells. Cycloheximide did not suppress the uptake of Cd2+, suggesting that uptake does not require synthesis of cell proteins de novo. Preincubation of cells with N-ethylmaleimide suppressed the uptake of Cd2+ to some extent, a result that suggests the involvement of surface SH groups in the uptake of Cd2+ by these cells.     

Measurement of intracellular cadmium with fluorescent dyes. Further evidence for the role of calcium channels in cadmium uptake.

Cellular uptake of Cd2+ has been monitored using intracellularly trapped dyes, Fura 2 and Quin 2, which bind Cd2+ with extremely high affinity, and digital fluorescence imaging has been used to visualize intracellular free Cd2+. The excitation spectrum of the Cd2+ complex of Fura 2 is similar to that of the Ca2+ complex, whereas Cd2+ displaces Ca2+ from Quin 2 and reduces fluorescence. Fluorescence of Fura 2-loaded cells increased when 50 microM extracellular Cd2+ was added and fluorescence of Quin 2-loaded cells decreased. Cd2+ uptake by GH3 pituitary cells, which occurs in part via voltage-sensitive L-type calcium channels, was increased by BAY K8644 and depolarization and decreased by nimodipine. When Fura 2 and Quin 2 were used to measure Cd2+ uptake by glial C6 cells, which have no L-channel activity, high K+ and BAY K8644 did not change the apparent rate of Cd2+ uptake. GH3 and C6 cells were incubated with Cd2+ for 24 h and loaded with Fura 2, and fluorescence was measured before and after addition of tetrakis-(2-pyridylmethyl)ethylenediamine (TPEN), a membrane permeant chelator with extremely high affinity for metals. TPEN had little effect on fluorescence of Fura 2-loaded GH3 and C6 cells not exposed to Cd2+ but decreased fluorescence of cells that had been incubated with 1-10 microM Cd2+. Fluorescence ratio imaging of Fura 2-loaded cells was used to image intracellular free Cd2+ for both GH3 and C6 cells. Cd2+ uptake over 30-180 min could be followed by the increase in 340/380 fluorescence ratio and the increase in fluorescence ratio was reversed within 5 min by TPEN. The results provide further evidence for the importance of voltage-gated calcium channels to Cd2+ uptake of certain cells.     

Induction and degradation of Zn-, Cu- and Cd-thionein in Chang liver cells

Human liver cells (Chang liver) were exposed to 5 micrograms Zn, 2.5 micrograms Cu or 1 microgram Cd/ml in cultured medium. These exogeneous heavy metals were accumulated by the cells and induced de novo synthesis of metallothionein after a 3-h incubation period. The production of Zn-, Cu- or Cd-thionein started in the cells with accumulation of 1 nmol Zn, 0.3 nmol Cu and 0.1 nmol Cd/mg cytosol protein and subsequently the amounts of metal-binding thioneins increased in agreement with the relative amount of metal accumulated in the cytosol over a 24-h period. When cells containing Zn- or Cu-thionein were placed in metal free medium, 70% or 25% of the zinc or copper bound to each original metallothionein was released after 3 h; bound metals decreased to 85% and 65% respectively after 24 h. The disappearance of metal from metallothionein correlated with increases of metal in the medium. On the other hand, 35S-counts incorporated into Zn- and Cu-thionein decreased only to 40% and 15% of the levels in the original metallothionein after 3 h; 35S-counts decreased to 65% and 45%, respectively, after 24 h, indicating that metals bound to metallothionein decreased more quickly than 35S-counts. These results suggest that metals were released from metallothionein and were excreted into the medium. However, 35S- and 109Cd-counts in Cd-thionein changed very little, if at all, in the cells even after a 24-h incubation period. Our data strongly suggest that Zn- and Cu-thionein are degraded in the cells, but that Cd-thionein remains longer than either Zn- or Cu-thionein. When cells containing Zn-thionein were incubated in metal-free medium, Zn-thionein was digested in the cells and peptide fragments ranging about 200-400 daltons were excreted from the cells.     

Effect of 72 Hz pulsed magnetic field exposure on macromolecular synthesis in CCRF-CEM cells.

Cells from the T-lymphoblastoid cell line, CCRF-CEM, have been exposed in vitro to a quasirectangular, asymmetric electromagnetic field pulsed at 72 Hz at 37 degrees for periods of 30 min to 24 h. RNA synthesis, assessed by incorporation of 3H-uridine, increased (relative to control cells) 2-fold after 30 min in exposed cells and achieved its greatest increase of 3.2-fold relative to controls after 2 h exposure. Increased precursor incorporation was observed at all subsequent exposure times up to 24 h. Synthesis of mRNA was similar, but not identical to that observed with total cellular RNA. Additionally, protein synthesis, determined by incorporation of radioactive precursor into acid-precipitable material, was increased 2.8-fold, compared to controls, after 2 h exposure. Longer exposure times resulted in an exponential decrease in precursor incorporation to 1.1-times control levels after 24 h. Using a dye reduction assay, mitochondrial activity was also found to be increased over a 24 h exposure period. No effect of electromagnetic field exposure was found on cellular synthesis of DNA. These data are generally consistent with other reports documenting effects of electromagnetic field exposure on macromolecular synthesis in vitro.     

镉对克氏原螯虾肝胰腺抗氧化系统的影响

采用毒性试验方法,研究了不同浓度Cd2+(1.72、3.44、6.89、13.77和27.55mg·L-1)对克氏原螯虾肝胰腺抗氧化酶(SOD、CAT、GSH-PX)和抗氧化物质(GSH、Vc)的影响.结果表明:超氧化物歧化酶(SOD)活性受低浓度Cd2+诱导和高浓度Cd2+抑制,且受抑制程度与Cd2+浓度呈正相关.过氧化氢酶(CAT)活性总体表现为先激活后下降,且在第3天达最大值,CAT活性对低浓度Cd2+(≤6.89 mg·L-1)暴露敏感,而高浓度Cd2+对其影响较小.谷胱甘肽过氧化物酶(GSH-PX)活性对Cd2+浓度敏感,当Cd2+≤6.89 mg · L-1时,GSH-PX活性先升高后下降,更高浓度下GSH-PX活性在暴露后第1天即表现出抑制作用.还原型谷胱甘肽(GSH)含量在Cd2+≤6.89 mg·L-1时始终被诱导,且均在第1天达最大值,而高浓度Cd2+(≥13.77 mg·L-1)对GSH含量影响不明显.维生素C(Vc)对Cd2+胁迫敏感,各处理组Vc含量在第1天均显著下降,且下降程度与Cd2+浓度呈正相关,之后具有一定的合成恢复能力.抗氧化酶和非酶抗氧化物质在抵御Cd2+胁迫反应中共同发挥作用,且大多表现出明显的时间和剂量效应性.GSH-PX和Vc可以作为Cd2+污染的潜在生物指示物.