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 metallothionein and glutathione status in an acute response to cadmium in Mercenaria mercenaria brown cells

Brown cells that are found in the red glands of Mercenaria mercenaria accumulate, detoxify and excrete cadmium. Brown cell involvement in metal detoxification was due in part to endogenous glutathione (GSH) and protein sulfhydryl. Metallothionein (MT) and GSH have been shown to play an important role in metal detoxification in bivalve molluscs. This study showed that the protein sulfhydryl in brown cells of Mercenaria was in fact MT, that brown cell GSH functioned in acute protection against Cd2+ toxicity, that GSH provided the initial defense against Cd2+ toxicity prior to MT induction and that MT variants were unequal in response to Cd2+. During treatment of Mercenaria with 0.5 and 1.0 ppm Cd2+, brown cells were analyzed for MT by capillary electrophoresis and GSH colorimetrically after 0.25, 1, 2, 3, and 4 days. The data indicated that the cadmium-binding protein was MT with an apparent molecular weight of 9 kDa determined by gel filtration or 6 kDa as indicated by capillary electrophoresis. Glutathione appeared to prevail in the brown cell acute response to 0.5 ppm Cd2+, whereas MT appeared to prevail in the acute response to 1.0 ppm Cd2+. Capillary electrophoresis can be used to monitor and quantify MT and its variants in brown cells without need for prior separation of cytosolic components by chromatography. The change in MT-II was greater relative to the change in MT-I in the brown cell acute response to 0.5 ppm Cd2+, whereas the change in MT-1 was greater relative to the change in MT-II in the acute response to 1.0 ppm Cd2+. The variants of brown cell MT appeared to respond differentially to Cd2+ depending upon the Cd2+ treatment concentration.     

Nuclear factor erythroid 2-related factor-2 activity controls 4-hydroxynonenal metabolism and activity in prostate cancer cells

4-Hydroxynonenal (HNE) is an end product of lipoperoxidation with antiproliferative and proapoptotic properties in various tumors. Here we report a greater sensitivity to HNE in PC3 and LNCaP cells compared to DU145 cells. In contrast to PC3 and LNCaP cells, HNE-treated DU145 cells showed a smaller reduction in growth and did not undergo apoptosis. In DU145 cells, HNE did not induce ROS production and DNA damage and generated a lower amount of HNE-protein adducts. DU145 cells had a greater GSH and GST A4 content and GSH/GST-mediated HNE detoxification. Nuclear factor erythroid 2-related factor-2 (Nrf2) is a regulator of the antioxidant response. Nrf2 protein content and nuclear accumulation were higher in DU145 cells compared to PC3 and LNCaP cells, whereas the expression of KEAP1, the main negative regulator of Nrf2 activity, was lower. Inhibition of Nrf2 expression with specific siRNA resulted in a reduction in GST A4 expression and GS-HNE formation, indicating that Nrf2 controls HNE metabolism. In addition, Nrf2 knockdown sensitized DU145 cells to HNE-mediated antiproliferative and proapoptotic activity. In conclusion, we demonstrated that increased Nrf2 activity resulted in a reduction in HNE sensitivity in prostate cancer cells, suggesting a potential mechanism of resistance to pro-oxidant therapy.     

Cadmium-induced elevation of hepatic isometallothionein concentrations in inbred strains of mice

Susceptibility to Cd toxicity differs among inbred strains of mice. For example, C3H/He mice are sensitive to Cd-induced hepatotoxicity while DBA/2 mice are resistant. Metallothionein (MT), which in rodents exists predominantly as two isoproteins (MT-I and MT-II), is an important endogenous protein in the detoxication of Cd. The present investigation examines the possibility that strain-dependent susceptibility to Cd-induced liver injury is mediated by an inherited inability to accumulate a specific isoform of MT in response to Cd exposure. Hepatic concentrations of MT-I and MT-II were measured in C3H/He (Cd-sensitive) and DBA/2 (Cd-resistant) mice at various times after the administration of non-toxic (2.5 mumol Cd/kg) to hepatototoxic (80 mumol Cd/kg) dosages of Cd. The concentration of MT-I and MT-II in these strains was similar 24 h after injection of non-hepatotoxic dosages of Cd (10 mumol Cd/kg or less) as well as 6-12 h after a mildly hepatotoxic dose of Cd (20 mumol Cd/kg). The concentration of total MT in liver of Cd-sensitive mice was greater than that present in resistant mice 24-72 h after 20 mumol Cd/kg injection. The data indicates that susceptibility to Cd-induced hepatotoxicity observed in C3H/He mice is not due to a deficit in the induction of a particular isoform of MT.     

Modulation of Exogenous Glutathione in Ultrastructure and Photosynthetic Performance Against Cd Stress in the Two Barley Genotypes Differing in Cd Tolerance

Greenhouse hydroponic experiments were conducted using Cd-sensitive (Dong 17) and tolerant (Weisuobuzhi) barley genotypes to evaluate genotypic differences in response of photosynthesis and ultrastructure to Cd toxicity in the presence of exogenous glutathione (GSH). Addition of 20 mg L(-1) GSH in 5 μM Cd culture medium (Cd?+?GSH) significantly alleviated Cd-induced growth inhibition and reduced Cd concentration in leaves and roots especially in the sensitive genotype Dong 17. Exogenous GSH greatly ameliorated Cd-induced damages on leaf/root ultrastructure, e.g., compared with Cd alone treatment, chloroplasts in plants treated with Cd?+?GSH become better or in relatively normal shape with well-structured thylakoid membranes and parallel pattern of lamellae and unfolded more starch grains but less osmiophilic plastoglobuli; nuclei of root cells were better formed and chromatin distributed more uniformly in both genotypes, and number of plastids and mitochondria cristae in Dong 17 resumed to control level. The examination of photosynthetic performance revealed GSH dramatically increased net photosynthetic rate (P(n)), stomatal conductance (G(s)), and transpiration rate (T(r)) in the both genotypes and strongly stimulated Cd-induced decrease in the maximal photochemical efficiency (F(v)/F(m)) especially in the sensitive genotype.     

Serum oxidative stress-induced repression of Nrf2 and GSH depletion: a mechanism potentially involved in endothelial dysfunction of young smokers

Background

Although oxidative stress plays a major role in endothelial dysfunction (ED), the role of glutathione (GSH), of nuclear erythroid-related factor 2 (Nrf2) and of related antioxidant genes (ARE) are yet unknown. In this study we combined an in vivo with an in vitro model to assess whether cigarette smoking affects flow-mediated vasodilation (FMD), GSH concentrations and the Nrf2/ARE pathway in human umbilical vein endothelial cells (HUVECs).

Methods and Results

52 healthy subjects (26 non-smokers and 26 heavy smokers) were enrolled in this study. In smokers we demonstrated increased oxidative stress, i.e., reduced concentrations of GSH and increased concentrations of oxidation products of the phospholipid 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphorylcholine (oxPAPC) in serum and in peripheral blood mononuclear cells (PBMC), used as in vivo surrogates of endothelial cells. Moreover we showed impairment of FMD in smokers and a positive correlation with the concentration of GSH in PBMC of all subjects. In HUVECs exposed to smokers'' serum but not to non-smokers'' serum we found that oxidative stress increased, whereas nitric oxide and GSH concentrations decreased; interestingly the expression of Nrf2, of heme oxygenase-1 (HO-1) and of glutamate-cysteine ligase catalytic (GCLC) subunit, the rate-limiting step of synthesis of GSH, was decreased. To test the hypothesis that the increased oxidative stress in smokers may have a causal role in the repression of Nrf2/ARE pathway, we exposed HUVECs to increasing concentrations of oxPAPC and found that at the highest concentration (similar to that found in smokers'' serum) the expression of Nrf2/ARE pathway was reduced. The knockdown of Nrf2 was associated to a significant reduction of HO-1 and GCLC expression induced by oxPAPC in ECs.

Conclusions

In young smokers with ED a novel further consequence of increased oxidative stress is a repression of Nrf2/ARE pathway leading to GSH depletion.     

Different mechanisms for metal-induced adaptation to cadmium in the human lung cell lines A549 and H441

Sensitivity to Cd and Zn as well as the capacity to develop tolerance were characterized in human lung cells A549 and H441. In the A549 cells, a 2-fold lower LC₅₀ was obtained for Cd compared to Zn, whereas H441 cells were similarly sensitive to both metals. H441 cells were twice as resistant to Cd as the A549 cells. Higher HSP70, but not metallothionein (MT) or glutathione (GSH) levels, could contribute to this better resistance. A 1.5- and 2-fold increase in the LC₅₀ for Cd was obtained in the A549 cells pre-exposed to non-cytotoxic concentrations of Cd (20 μM) or Zn (40 μM) for 24 h. On the other hand, only Zn increased H441 cells’ resistance to Cd. Maximum Zn- and Cd-induced tolerances were reached as early as 3 and 12 h, respectively. Increases in MT-IIa and HSP70 messenger RNA levels were higher in A549 cells, but cycloheximide eliminated the induction of tolerance only in the H441 cells. Protein synthesis is a prerequisite for metal-induced tolerance to Cd in the H441 cells but not the A549 cells. Results obtained with l-buthionine sulfoximine revealed that GSH synthesis is not responsible for the acquired tolerance in both cell lines. However, GSH plays a critical role against Cd toxicity, and pro-oxidant conditions sensitized cells to Cd with different impacts on the metal-induced mechanisms of acquired tolerance. GSH and catalase both provide antioxidative protection, but only the stress related to low GSH content, not that resulting from catalase inhibition, may be alleviated with Zn.     

The peroxynitrite donor 3-morpholinosydnonimine activates Nrf2 and the UPR leading to a cytoprotective response in endothelial cells

Endothelial dysfunction is associated with the formation of peroxynitrite, described to be toxic. Recent data also suggests that peroxynitrite is able to activate the protective Nrf2 pathway and/or the unfolded protein response (UPR). The aim of our work was to study the response of human endothelial cells to 3-morpholinosydnonimine (SIN-1), a peroxynitrite donor, and to highlight the possible protective roles of Nrf2 or the UPR pathway in this response.Immortal and primary human umbilical vein endothelial cells were exposed to SIN-1. SIN-1 incubation led to Nrf2 activation and to the overexpression of Nrf2-regulated genes, heme oxygenase-1 (HO-1) and NAD(P)H quinone oxidoreductase 1. We also demonstrated that this defensive response protected cells against cell death induced by serum starvation, by reducing apoptosis (monitored by caspase-3 activity and DNA fragmentation) and favoring autophagosome formation, as evidenced by LC3-II accumulation. Interestingly, we observed an activation of the UPR, with a rapid and significant overexpression of CHOP in serum starved cells stimulated with SIN-1. While siRNA mediated knockdown of CHOP had no effect on DNA fragmentation, the invalidation of Nrf2 or HO-1 by siRNA strongly increased DNA fragmentation, but also reinforced the SIN-1-induced LC3-II accumulation.This study shows that peroxynitrite, at least at sublethal concentrations and within a narrow concentration range, could exert protective effects on endothelial cells by modulating the balance between autophagy and apoptosis, through Nrf2-dependent pathways.     

Binding of inorganic phosphate to the cadmium-induced dimeric form of metallothionein from rabbit liver.

Recently we have demonstrated that the exposure of monomeric Cd7-metallothionein (MT) to Cd(II) ions in potassium phosphate buffer results in the nonoxidative formation of MT dimers containing approximately two additional Cd(II) ions/monomer subunit [Palumaa, P., Mackey, E. and Vasák, M. (1992) Biochemistry 31, 2181-2186]. In this study, we demonstrate that inorganic phosphate participates in the Cd-induced dimerization of MT. In the absence of phosphate, Cd-induced oligomerization of MT still takes place, but a substantially lower apparent yield of the dimeric form and an additional peak of MT tetramers were detected in gel-filtration experiments. Arsenate exhibits a similar effect to that of phosphate, whereas a number of other anions, i.e. F-, NO3-, SO4(2-), ClO4-, BO3-, SCN-, HCOO- and CH3COO- had no effect on Cd-induced oligomerization of MT. Studies on the pH dependence of MT dimerization indicate that the dianionic form of phosphate is involved in this process. Equilibrium-dialysis experiments using potassium [32P]phosphate established binding of two molecules of phosphate to the dimeric MT form with a dissociation constant, Kd, of 23 +/- 3 microM (20 mM Tris/HCl and 0.1 M KCl, pH 8.0 at 25 degrees C), whereas binding of phosphate was not observed with the monomeric Cd7-MT. The noncovalent nature of phosphate binding to the Cd-induced MT dimers has been demonstrated. The presented data provide the first evidence for the binding of a nonmetallic cellular component to MT.     

Combination effects of chronic cadmium exposure and gamma-irradiation on the genotoxicity and cytotoxicity of peripheral blood lymphocytes and bone marrow cells in rats

This work investigated the effects of chronic cadmium (Cd) exposure combined with γ-ray irradiation on the cytotoxicity and genotoxicity of peripheral blood cells and bone marrow cells in rats. Results showed that when the rats were exposed to low dose (LD) Cd of 0.1mg CdCl?/(kgd) for 8 and 12 weeks, the Cd concentration in blood reached to 135-140 μg/L and no toxic effects on peripheral blood lymphocytes, white blood cells (WBC) and granulocyte-monocyte (GM) progenitor cells were observed except polychromatic erythrocytes (PCE) of bone marrow. Moreover, this chronic LD Cd exposure significantly decreased irradiation-induced micronucleus (MN) formation and hypoxanthine-guanine phosphoribosyl transferase (hprt) mutation in lymphocytes and PCE, while the combination of LD Cd exposure and irradiation induced the additive metallothionein (MT) protein expression in bone marrow cells. When the rats were exposed to a high dose (HD) Cd of 0.5mg CdCl/?(kgd) for 8 and 12 weeks, the blood Cd level approached to 458-613 μg/L and an inflammatory response was induced, meanwhile, MN formation and hprt mutation were markedly increased, and the ratio of PCE/NCE (normochromatic erythrocyte) was significantly decreased. Furthermore, when the rats were exposed to HD Cd plus 2 Gy irradiation, additive toxic effects on MN formation, hprt mutation, PCE damage and GM progenitor cell proliferation were observed, while this combination treatment resulted in an obvious reduction of MT protein compared to HD Cd group. In conclusion, chronic exposure to LD Cd induced the adaptive response to irradiation in the genotoxicity of peripheral blood lymphocytes and PCE of bone marrow by the up-regulation of Cd-induced MT protein, but the combination of HD Cd exposure and irradiation generated the additive effects on the cytotoxicity and genotoxicity in peripheral blood lymphocytes and bone marrow cells.     

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.     

Characterization of the human hepatocellular carcinoma (hepg2) cell line as an in vitro model for cadmium toxicity studies

Biochemical indicators and in vitro models, if they mimic in vivo responses, offer potentially sensitive tools for inclusion in toxicity assessment programs. The purpose of this study was to determine whether the HepG2 cell line would mimic known in vivo or in vitro (or both) responses of mammalian systems when confronted with cadmium (Cd2+). Uptake and compartmentalization of Cd2+, metallothionein (MT) compartmentalization, and glutathione (GSH) depletion were examined. In addition, several cytotoxic and stress effects, e.g., viability (neutral red [NR] uptake, 3-[4,5-dimethylthiozole-2-yl]-2,5,-biphenyl tetrazolium bromide [MTT] dye conversion, and live/dead [L/D]), membrane damage (lactate dehydrogenase leakage), metabolic activity (adenosine triphosphate levels), and detoxification capabilities (GSH content, cytochrome P4501A1/2 [EROD (ethoxyresorufin-o-deethylase)] activity, and MT induction), were measured in both naive (no previous exposure) and Cd2+ preexposed cells. Cadmium uptake increased during a 24-h period. Metallothionein induction occurred in response to both Cd2+ and ZnCl2; however, Cd2+ was the more potent inducer. Both Cd2+ and MT were localized primarily in the cytoplasmic compartment. All biochemical responses, except EROD, showed concentration- response relationships, after 24-h exposure to Cd2+ (ranges 0-3 ppm [26.7 microM]). Cadmium effects were reduced in preexposed cells, indicating adaptive tolerance or increased resistance had occurred. Twenty-four-hour LC50, dose causing death of 50% of the test subjects, values were 0.97, 0.69, and 0.80 ppm (8.7, 6.2, and 7.2 microM) for naive cells and 1.45, 1.21, and 1.39 ppm (12.9, 10.7, and 12.3 microM) for preexposed cells based on the NR, MTT, and L/D assays, respectively. These data indicate that this carcinoma cell line is a useful in vitro model for cadmium toxicity studies.