Different expression of sodium-iodide importer (NIS) between lactating breast and thyroid tissues may be due to structural difference of thyroid-stimulating hormone receptor (TSHR).

OBJECTIVE: Thyroid-stimulating hormone (TSH) binds TSH receptor (TSHR) on thyroid cell membranes, which will lead activation of cyclic adenosine 3',5'-monophosphate/protein kinase A signaling pathway. Through this pathway, TSHR regulates the expression of sodium-iodide symporter (NIS) to complete iodine intake. In recent studies, it is found that TSHR is widely expressed in a variety of extra-thyroidal tissues. TSHR expressions as well as distribution in normal mammary gland tissues have not been reported. The physiological mechanism of the TSHR in the extra-thyroidal tissues has also been controversial. METHODS: In this study, immunohistochemistry and immunofluorescence were used to characterize the expression distribution of TSHR protein in lactating breast. DNA sequence of TSHR cDNA from mice lactating breast was determined and then compared with TSHR cDNA from mice thyroidal tissue. RESULTS: A 173 amino acid (AA) fragment deletion was found in the extra-cellular domain of lactating breast TSHR. The expression levels of NIS mRNA were compared between two tissues, and the level of NIS mRNA in lactating breasts was lower than the one in thyroidal tissues. CONCLUSION: The lower expression of NIS in lactating breast may be due to the 173 AA deletion in the TSHR resulting the lower binding of TSH to the TSHR. For the first time, this finding may explain the reason of the lower NIS expression in lactating breast.

Development of SCAR Markers and an SYBR Green Assay to Detect Puccinia striiformis f. sp. tritici in Infected Wheat Leaves.

Stripe rust, caused by the pathogenic fungus Puccinia striiformis f. sp. tritici, is an important disease of wheat worldwide. A rapid and reliable detection of the pathogen in latent infected wheat leaves is useful for accurate and early forecast of outbreaks and timely application of fungicides for managing the disease. Using the previously reported primer pair Bt2a/Bt2b, a 362-bp amplicon was obtained from P. striiformis f. sp. tritici and a 486-bp amplicon was obtained from both P. triticina (the leaf rust pathogen) and P. graminis f. sp. tritici (the stem rust pathogen). Based on the sequence of the 362-bp fragment, two pairs of sequence characterized amplified region (SCAR) primers were designed. PSTF117/PSTR363 produced a 274-bp amplicon and TF114/TR323 produced a 180-bp amplicon from P. striiformis f. sp. tritici, whereas they did not produce any amplicon from P. triticina, P. graminis f. sp. tritici, or any other wheat-infecting fungi. The detection limit of PSTF117/PSTR363 was 1 pg/µl and TF114/TR323 was 100 fg/µl. Both SCAR markers could be detected in wheat leaves 9 h post inoculation. An SYBR Green RT-PCR method was also developed to detect P. striiformis f. sp. tritici in infected leaves with the detection limit of 1.0 fg DNA from asymptomatic leaf samples of 6 h after inoculation. These methods should be useful for rapid diagnosis and accurate detection of P. striiformis f. sp. tritici in infected wheat leaves for timely control of the disease.

[Clinical analysis of efficacy and quality of life of segmental bowel resection for bowel endometriosis].

OBJECTIVE: To evaluate the efficacy and quality of life of segmental bowel resection for bowel endometriosis. METHODS: Totally 62 symptomatic patients with bowel endometriosis undergoing segmental bowel resection were recruited. A visual analogue scale(VAS)and the 36-item short form health survey(SF-36)questionnaire were administered before and at least 1 year after surgery, respectively. Pregnancy rates were also recorded. RESULTS: Sixty-two patients in total underwent follow-up ranging from 12 to 74 months. All patients complained of obvious pain symptoms, including dysmenorrhea, dyspareunia, pain on defecation and chronic pelvic pain. The relief of dysmenorrhea(2.9 ± 2.2 versus 7.5 ± 2.9), dyspareunia(0.7 ± 0.5 versus 4.3 ± 2.2)and pain on defecation(1.6 ± 0.7 versus 7.3 ± 1.9)after surgery was statistically significant(all P<0.01). The scores for all 8 domains of the SF-36 questionnaire were significant improved after segmental bowel resection(all P<0.01). The complication rate was 45%(28/62), including 18 cases of urinary retention, 4 rectovaginal fistulas, 2 cases of vaginal dehiscence, and 1 case each of thrombogenesis, pelvic abscess and general peritonitis. All of the patients with complications recovered well throughout follow-up. The postoperative pregnancy rate of the previous infertile patients was 6/10. Among the 6 gestational cases, 2 had labour, 2 underwent caesarean sections, one had a spontaneous natural abortion, and one underwent uterine curettage. CONCLUSION: Segmental bowel resection could significantly relieve pain and improve quality of life for patients with bowel endometriosis.

Signaling pathways from membrane lipid rafts to JNK1 activation in reactive nitrogen species-induced non-apoptotic cell death.

At present, the signaling pathways controlling reactive nitrogen species (RNS)-induced non-apoptotic cell death are relatively less understood. In this work, various RNS donors are found to induce caspase-independent non-apoptotic cell death in mouse embryonic fibroblasts (MEF). In search of the molecular mechanisms, we first established the role of c-Jun N-terminal kinase (JNK) in RNS-induced non-apoptotic cell death. RNS readily activate JNK, and the jnk1-/- MEF are resistant to RNS-induced cell death. Moreover, the reconstitution of JNK1 effectively restores the sensitivity to RNS. Next, we identified tumor necrosis factor receptor-associated factor 2 (TRAF2) and apoptosis signal-regulating kinase 1 (ASK1) as the essential upstream molecules for RNS-induced JNK activation and cell death. RNS fail to activate JNK and induce cell death in traf2-/- MEF; and reconstitution of TRAF2 effectively restores the responsiveness of traf2-/- MEF to RNS. Moreover, RNS-induced ASK1 activation is impaired in traf2-/- cells and overexpression of a mutant ASK1 protein suppresses RNS-induced cell death in wild-type MEF cells. Last, we explored the signaling events upstream of TRAF2 and found that translocation of TRAF2 and JNK1 onto membrane lipid rafts is required for RNS-mediated JNK1 activation and cell death. Taken together, data from our study reveal a novel signaling pathway regulating RNS-induced JNK1 activation and non-apoptotic cell death.

Mutation of BCL-6 gene in normal B cells by the process of somatic hypermutation of Ig genes.

Immunoglobulin (Ig) genes are hypermutated in B lymphocytes that are the precursors to memory B cells. The mutations are linked to transcription initiation, but non-Ig promoters are permissible for the mutation process; thus, other genes expressed in mutating B cells may also be subject to somatic hypermutation. Significant mutations were not observed in c-MYC, S14, or alpha-fetoprotein (AFP) genes, but BCL-6 was highly mutated in a large proportion of memory B cells of normal individuals. The mutation pattern was similar to that of Ig genes.

c-Jun N-terminal kinase mediates hydrogen peroxide-induced cell death via sustained poly(ADP-ribose) polymerase-1 activation.

Reactive oxygen species (ROS) have been closely associated with both apoptotic and non-apoptotic/necrotic cell death. Our previous study has illustrated that c-Jun-N-terminal kinase 1 (JNK1) is the main executor in hydrogen peroxide (H(2)O(2))-induced nonapoptotic cell death. The main objective of this study is to further elucidate the molecular mechanisms downstream of JNK1 in H(2)O(2)-induced cell death. In this study, poly(ADP-ribose) polymerase-1 (PARP-1), a key DNA repair protein, was readily activated by H(2)O(2) and inhibition of PARP-1 activation by either a pharmacological or genetic approach offered significant protection against H(2)O(2)-induced cell death. More importantly, H(2)O(2)-mediated PARP-1 activation is subject to regulation by JNK1. Suppression of JNK1 activation by a chemical inhibitor or genetic deletion markedly suppressed the late-phase PARP-1 activation induced by H(2)O(2), suggesting that JNK1 contributes to the sustained activation of PARP-1. Such findings were supported by the temporal pattern of nuclear translocation of activated JNK and a direct protein-protein interaction between JNK1 and PARP-1 in H(2)O(2)-treated cells. Finally, in vitro kinase assay suggests that PARP-1 may serve as the direct phosphorylation target for JNK1. Taken together, data from our study reveal a novel underlying mechanism in H(2)O(2)-induced nonapoptotic cell death: JNK1 promotes a sustained PARP-1 activation via nuclear translocation, protein-protein interaction and PARP-1 phosphorylation.

LncRNA HCP5 promotes the development of cervical cancer by regulating MACC1 via suppression of microRNA-15a.

OBJECTIVE: To explore the role of long non-coding RNA (lncRNA) HCP5 in the development of cervical cancer and its underlying mechanism. PATIENTS AND METHODS: Expression levels of HCP5, MACC1 and microRNA-15a in cervical cancer tissues and paracancerous tissues were detected. The relationship between HCP5 expression and prognosis of patients with cervical cancer was analyzed by Kaplan-Meier. Cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay after altering expressions of HCP5 and microRNA-15a by plasmids transfection. The binding condition of HCP5, MACC1 and microRNA-15a was evaluated by luciferase reporter gene assay. The regulatory effect of microRNA-15a on MACC1 expression was determined by Western blot. RESULTS: HCP5 and MACC1 were overexpressed in cervical cancer tissues than those of paracancerous tissues. The survival rate of patients with cervical cancer was negatively correlated to HCP5 expression, but positively correlated to microRNA-15a expression. Luciferase reporter gene assay showed that microRNA-15a was directly bound to HCP5 and MACC1. Besides, overexpression of microRNA-15a could remarkably inhibited MACC1 expression. In vitro experiments showed that HCP5 promoted proliferation of cervical cancer cells, which was reversed by microRNA-15a knockdown. CONCLUSIONS: Overexpressed HCP5 promoted the development of cervical cancer through increasing MACC1 expression by microRNA-15a adsorption.

Research on the effect and mechanism of the CXCR-4-overexpressing BMSCs combined with SDF-1α for the cure of acute SCI in rats.

OBJECTIVE: To evaluate the effect and mechanism of bone marrow stem cells (BMSCs) modified with CXCR-4 gene combined with stromal derived factor-1α (SDF-1α) in the treatment of acute spinal cord injury (SCI) in rats. MATERIALS AND METHODS: CXCR-4 gene was transfected by a virus. Spinal cord injury rats were randomly divided into four groups: control group, SDF-1α group, CXCR-4/BMSC group and combined group. The motor function was evaluated with Blood Brain Barrier (BBB) score and the RNA expression of CXCR-4 were measured by PCR. Apoptosis of spinal cord was measured by TUNEL kit (Hu Bei, China). The protein level of Bcl-2 and Bax were measured by Western-blot. The BBB scores, mRNA CXCR-4 expression, and apoptosis rate were compared between four groups at 1d, 3d, 7d, 14d, 21d after the operation. RESULTS: The exercise ability in combined group restored in early and late periods of SCI. The apoptosis rates in the combined group are less than other three groups; the difference was statistically significant (p < 0.05). Bcl-2 in combined group is higher than the other 3 groups and Bax is less than the other 3 groups, the difference is statistically significant (p < 0.05). CONCLUSIONS: The neurological function of rats with a spinal cord can be improved by BMSCs modified with CXCR-4 combined with SDF-1α. The main mechanism may improve the expression of SDF-1α and decrease the apoptosis of the spinal cord.

Drug Target Identification Using an iTRAQ-Based Quantitative Chemical Proteomics Approach-Based on a Target Profiling Study of Andrographolide.

Identifying the cellular binding targets of drugs and other bioactive small molecules is a crucial step for understanding their molecular mechanisms of action as well as potential off-target effects. The field of chemical proteomics is an emerging discipline in chemical biology using synthetic chemistry and high-throughput detection techniques to study small molecule-protein interactions. In this chapter, we describe a quantitative chemical proteomics protocol combining bioorthogonal click chemistry and quantitation by isobaric tags for relative and absolute quantification (iTRAQ) to identify the specific binding targets of drugs and bioactive small molecules such as natural products. A modified drug probe with a click chemistry-enabling addition is synthesized and used in live cell treatments where it undergoes covalent interactions with its cognate cellular targets. The probes are then ligated to biotin through click chemistry and enriched with avidin beads, followed by iTRAQ labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis for protein identification and relative quantitation discriminating specific targets from nonspecific binding proteins. The presented protocol has been used to successfully profile prominent drugs and natural products including andrographolide, aspirin, curcumin, etc., and can be a powerful tool to study the molecular mechanisms of bioactive small molecules.

Proteomic Profiling of De Novo Protein Synthesis in Starvation-Induced Autophagy Using Bioorthogonal Noncanonical Amino Acid Tagging.

Autophagy is an intracellular degradation process activated by stress factors such as nutrient starvation to maintain cellular homeostasis. There is emerging evidence demonstrating that de novo protein synthesis is involved in the autophagic process. However, up-to-date characterizing of these de novo proteins is technically difficult. In this chapter, we describe a novel method to identify newly synthesized proteins during starvation-mediated autophagy by bioorthogonal noncanonical amino acid tagging (BONCAT), in conjunction with isobaric tagging for relative and absolute quantification (iTRAQ)-based quantitative proteomics. l-azidohomoalanine (AHA) is an analog of methionine, and it can be readily incorporated into the newly synthesized proteins. The AHA-containing proteins can be enriched with avidin beads after a "click" reaction between alkyne-bearing biotin and the azide moiety of AHA. The enriched proteins are then subjected to iTRAQ™ labeling for protein identification and quantification using liquid chromatography-tandem mass spectrometry (LC-MS/MS). By using this technique, we have successfully profiled more than 700 proteins that are synthesized during starvation-induced autophagy. We believe that this approach is effective in identification of newly synthesized proteins in the process of autophagy and provides useful insights to the molecular mechanisms and biological functions of autophagy.

Knowledge and beliefs on corneal donation in Singapore adults.

AIM: To assess the knowledge and willingness of Singapore adults towards corneal donation. METHODS: The study population consists of a cluster random sample of the population living in Bedok North (an area in the eastern part of Singapore). The study population comprised residents aged 21-65 years living in 675 randomly sampled housing units. The participation rate was 65.9% (544/825). All participants were interviewed face to face with a questionnaire formulated according to the modified Horton and Horton model. Knowledge, values, attitudes, and spiritual beliefs of participants were assessed to evaluate their willingness to donate their corneas. RESULTS: 67.0% of participants were willing to donate their corneas. Ethnicity (Chinese) and religion (Christians, Hindus, or those with no religion) were associated with increased willingness to donate corneas. Greater knowledge and increased altruistic values were also associated with increased willingness to donate corneas. CONCLUSION: A proportion of participants were willing to donate their corneas. Awareness of corneal donation is high but specific knowledge should be further increased among adults.

The inactivation of the XP-C gene does not affect somatic hypermutation or class switch recombination of immunoglobulin genes.

The mechanism of somatic hypermutation of immunoglobulin genes is not known, but appears to be linked to transcription and perhaps DNA repair. In order to determine if global DNA repair or the repair of the nontranscribed DNA strand is required for somatic mutation, we have analysed mice whose XP-C gene was inactivated by homologous recombination. Our study shows that hypermutation occurs in XP-C knockout mice with a normal frequency, suggesting that the XP-C gene product is not required for somatic hypermutation. Furthermore, we found that Ig gene switch recombination also is normal in these mice.

Immunoglobulin transgenes as targets for somatic hypermutation.

This review describes studies on somatic hypermutation of immunoglobulin genes that were started in the mid-80s in collaboration with Ralph Brinster. Almost all of the experiments were carried out using Ig transgenes as targets for the somatic mutation mechanism. Ig transgenes can be very good targets of somatic mutation, despite many different transgene integration sites. Thus, the required cis-acting elements must be present within the approximately 10 kb of the transgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region in unmanipulated sequences. Several Ig gene enhancers are permissive for somatic mutation and they do not have to be associated with the Ig promoter they normally interact with. However, the mutation process does seem to be specific for Ig genes. No mutations were found in several housekeeping genes isolated from cells that had very high levels of somatic hypermutation of their Ig genes. This suggests that the Ig enhancers provide the lg gene specificity. An exception is the Bcl-6 gene, encoding a transcription factor, which was found to be mutated in normal human memory B cells. When the transcriptional promoter that is located upstream of the variable region is duplicated upstream of the constant region, this region is mutated as well. This suggests a transcription coupled model in which a mutator factor associates with the RNA polymerase at the initiation of transcription, travels with the polymerase during elongation, and causes mutations during polymerase pausing. Our recent data with an artificial substrate for somatic mutation suggest that the mutations are increased by increased stability of the secondary structures in the nascent RNA, and the specific nucleotides that are mutated are due to preferences of a mutator factor.

Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B1.

Accumulating evidence demonstrates that oxidative damage is one of the underlying mechanisms to the cytotoxicity and carcinogenicity of AFB1. The main objective of this study is to show that AFB1 increases reactive oxygen species (ROS) formation in hepatocytes. The ROS level was detected using a fluorescence probe, 2',7'-dichlorofluorescin diacetate (DCFH-DA), which could be converted to highly fluorescent dichlorofluorescein (DCF) with the presence of intracellular ROS. It was found that AFB1 exposure significantly enhanced DCF fluorescence formation in cultured rat hepatocytes. A dose-response of AFB1 was also observed within the range of 10 nM to 1000 nM. Catalase (CAT) was able to completely prevent the increase of DCF fluorescence in AFB1-treated cells in a dose-dependent manner (from 500 to 2000 U/ml). Moreover, the significant inhibitory effects of desferrioxamine (DFO) and dimethyl sulfoxide (DMSO) on DCF fluorescence formation were also observed in both control and AFB1-treated hepatocytes. Therefore, results from the present study provide in vitro evidence indicating the generation of ROS in cultured rat hepatocytes caused by AFB1 exposure. It is postulated that the metabolic process of AFB1 by cytochrome P450 might be the possible source of the elevated ROS level in AFB1-treated hepatocytes. The enhanced level of ROS may be responsible for the oxidative damage caused by AFB1, which may ultimately contribute to the cytotoxic and carcinogenic effects of AFB1.

Superoxide radical-initiated apoptotic signalling pathway in selenite-treated HepG(2) cells: mitochondria serve as the main target.

The exact role of superoxide radicals (O(2)(*)(-)) in apoptosis is still a matter of debate. The main objective of the present study is to evaluate the apoptotic signalling pathway initiated by O(2)(*)(-). The reductive reaction of sodium selenite with glutathione was used as the intracellular O(2)(*)(-)-generating system. When cells were exposed to 5 to 25 microM selenite, a temporal pattern of apoptotic events was observed following the elevation of O(2)(*)(-), in which cytochrome c release and mitochondrial depolarization preceded caspase-3 activation and DNA fragmentation. The simultaneous treatment with N-acetylcysteine and 4-hydroxy-2,2,6, 6-tetramethylpiperidine-N-oxyl markedly reduced O(2)(*)(-) level and suppressed the mitochondrial changes and the downstream apoptotic events. Moreover, pretreatment with cyclosporin A plus trifluoperazine, two mitochondrial permeability transition (MPT) inhibitors, was capable of attenuating O(2)(*)(-)-mediated cytochrome c release and mitochondrial depolarization, and subsequently inhibiting apoptosis. Thus, the present results provide convincing evidence that O(2)(*)(-) generated from the reductive reaction of selenite with GSH is capable of triggering a mitochondria-dependent apoptotic pathway. Such knowledge may not only help to obtain a better understanding of the apoptotic effect of selenite per se, but of the role of O(2)(*)(-) in initiation and execution of apoptosis.

Salvia miltiorrhiza inhibits cell growth and induces apoptosis in human hepatoma HepG(2) cells.

Salvia miltiorrhiza (SM) is a traditional Chinese herbal medicine, commonly used to treat liver diseases in China for centuries. Several earlier studies have indicated that SM exhibits anti-tumor properties, but its mechanism remains to be elucidated. In this study, we evaluated the molecular mechanism of SM in a human hepatoma cell line, HepG(2). Our results show that SM exerted clear cytotoxic effects, and strongly inhibited the proliferation of HepG(2) cells. It was also observed that SM treatment caused apoptotic cell death as evaluated by: (a), morphological changes by using acridine orange/ethidium bromide staining; (b), DNA fragmentation by TdT-mediated dUTP nick end labeling (TUNEL); and (c), sub-G(1) cell analysis. Furthermore, depletion of intracellular glutathione (GSH) and reduction of mitochondrial membrane potential were found to be involved in the initiation of apoptosis by SM.

Protective effect of ebselen against hydrogen peroxide-induced cytotoxicity and DNA damage in HepG2 cells.

The protective effect of ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), a selenoorganic compound, against hydrogen peroxide (H2O2)-induced cytotoxicity and DNA damage was investigated in a human hepatoma cell line, HepG2. The inhibitory effect of H2O2 on cell growth was determined using the tetrazolium dye colorimetric test (MTT test), and the cytotoxicity and lipid peroxidation were estimated by lactate dehydrogenase (LDH) leakage and malondialdehyde (MDA) formation, respectively. DNA damage was detected using single cell gel electrophoresis (comet assay), and intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA). The results showed that H2O2 suppressed the growth of HepG2 cells and the addition of ebselen significantly reduced the suppression. Furthermore, ebselen also displayed a dose-dependent reduction of LDH leakage and MDA formation in H2O2-treated cells. The results also demonstrate that ebselen was able to reduce the ROS formation and DNA damaging effect caused by H2O2 in a dose-dependent manner. These findings suggest that ebselen has a strong protective ability against the cytotoxicity and DNA damaging effect caused by reactive oxygen species.

Risk assessment of nickel carcinogenicity and occupational lung cancer.

Recent progress in risk assessment of nickel carcinogenicity and its correlation with occupational lung cancer in nickel-exposed workers is reviewed. Epidemiological investigations provide reliable data indicating the close relation between nickel exposure and high lung cancer risk, especially in nickel refineries. The nickel species-specific effects and the dose-response relationship between nickel exposure and lung cancer are among the main questions that are explored extensively. It is also suggested that some confounding factors such as cigarette smoking cannot be neglected. The determination of nickel concentration in lung tissue may be conducive to estimating the nickel exposure level, but it is uncertain whether the high nickel content in lung tissue indicates high lung cancer risk in nickel-exposed workers. Immunologic studies suggest that the suppressive effect of nickel on NK cell activity and interferon production may also be involved in the mechanisms of nickel carcinogenesis. As a potential mutagen, nickel can cause chromosome damage both in vitro and in vivo; and on a molecular basis, nickel is found to induce DNA damage (DNA strandbreaks and crosslinks, infidelity of DNA replication, inhibition of DNA repair, and the helical transition of B-DNA to Z-DNA) by binding of nickel ions to DNA and nuclear proteins. The discovery of oncogene promises both a challenge and an opportunity for nickel carcinogenesis research. It can be predicted that, with the rapid development of molecular biology and oncology, new approaches will be established for both understanding and controlling nickel-induced occupational lung cancer.

Microcystic cyanobacteria extract induces cytoskeletal disruption and intracellular glutathione alteration in hepatocytes.

Microcystins are a group of highly liver-specific toxins, although their exact mechanisms of action remain unclear. We examined the effects of microcystic cyanobacteria extract (MCE) collected from a contaminated water source on the organization of cellular microtubules (MTs) and microfilaments (MFs) in hepatocytes. We also investigated the effects on lactate dehydrogenase (LDH) leakage and intracellular glutathione (GSH). Primary cultured rat hepatocytes exposed to MCE (equivalent to 125 microg/mL lyophilized algae cells) showed a characteristic disruption of MTs and MFs in a time-dependent manner. Under these conditions, MCE caused aggregation of MTs and MFs and a severe loss of MTs in some cells. Moreover, MCE-induced cytoskeletal alterations preceded the LDH leakage. On the other hand, the treatment of cells with MCE led to a dose-dependent increase of intracellular GSH. However, time-course study showed a biphasic change of intracellular GSH levels with a significant increase in the initial stage followed by a decrease after prolonged treatment. Furthermore, pretreatment with N-acetylcystein (NAC), a GSH precursor, significantly enhanced the intracellular GSH level and decreased the MCE-induced cytotoxicity as well as cytoskeleton changes. In contrast, buthionine-(S, R)-sulfoximine, a specific GSH synthesis inhibitor, increased the cell susceptibility to MCE-induced cytotoxicity by depleting the intracellular GSH level. These findings suggest that intracellular GSH plays an important role in MCE-induced cytotoxicity and cytoskeleton changes in primary cultured rat hepatocytes. Increasing intracellular GSH levels protect cells from MCE-induced cytotoxicity and cytoskeleton changes.

Cadmium-induced oxidative cellular damage in human fetal lung fibroblasts (MRC-5 cells).

Epidemiological evidence suggests that cadmium (Cd) exposure causes pulmonary damage such as emphysema and lung cancer. However, relatively little is known about the mechanisms involved in Cd pulmonary toxicity. In the present study, the effects of Cd exposure on human fetal lung fibroblasts (MRC-5 cells) were evaluated by determination of lipid peroxidation, intra-cellular production of reactive oxygen species (ROS), and changes of mitochondrial membrane potential. A time- and dose-dependent increase of both lactate dehydrogenase leakage and malondialdehyde formation was observed in Cd-treated cells. A close correlation between these two events suggests that lipid peroxidation may be one of the main pathways causing its cytotoxicity. It was also noted that Cd-induced cell injury and lipid peroxidation were inhibited by catalase and superoxide dismutase, two antioxidant enzymes. By using the fluorescent probe 2',7'-dichlorofluorescin diacetate, a significant increase of ROS production in Cd-treated MRC-5 cells was detected. The inhibition of dichlorofluorescein fluorescence by catalase, not superoxide dismutase, suggests that hydrogen peroxide is the main ROS involved. Moreover, the significant dose-dependent changes of mitochondrial membrane potential in Cd-treated MRC-5 cells, demonstrated by increased fluorescence of rhodamine 123 examined using a laser-scanning confocal microscope, also indicate the involvement of mitochondrial damage in Cd cytotoxicity. These findings provide in vitro evidence that Cd causes oxidative cellular damage in human fetal lung fibroblasts, which may be closely associated with the pulmonary toxicity of Cd.