From the Cover: Ethylmercury-Induced Oxidative and Endoplasmic Reticulum Stress-Mediated Autophagic Cell Death: Involvement of Autophagosome-Lysosome Fusion Arrest.

Ethylmercury (EtHg) is derived from the degradation of thimerosal, the most widely used organomercury compound. In this study, EtHg-induced toxicity and autophagy in the mouse kidney was observed and then the mechanism of toxicity was explored in vitro in HK-2 cells. Low doses of EtHg induced autophagy without causing any histopathological changes in mouse kidneys. However, mice treated with high doses of EtHg exhibited severe focal tubular cell necrosis of the proximal tubules with autophagy. EtHg dose-dependently increased the production of reactive oxygen species, reduced the mitochondrial membrane potential, activated the unfolded protein response, and increased cytosolic Ca2+ levels in HK-2 cells. Cell death induced by EtHg exposure was caused by autophagy and necrosis. N-acetyl cysteine and 4-phenylbutyric acid attenuated EtHg-induced stress and ameliorated the autophagic response in HK-2 cells. Furthermore, EtHg blocked autophagosome fusion with lysosomes, which was demonstrated via treatment with wortmannin and chloroquine. Low doses of EtHg and rapamycin, which resulted in minimal cytotoxicity, increased the levels of the autophagic SNARE complex STX17 (syntaxin 17)-VAMP8-SNAP29 without altering mRNA levels, but high dose of EtHg was cytotoxic. Inhibition of autophagic flux by chloroquin increased autophagosome formation and necrotic cell death in HK-2 cells. Collectively, our results show that EtHg induces autophagy via oxidative and ER stress and blockade of autophagic flux. Autophagy might play a dual role in EtHg-induced renal toxicity, being both protective following treatment with low doses of EtHg and detrimental following treatment with high doses.

Protective effect of 70% ethanolic extract of Lindera obtusiloba Blume on tert-butyl hydroperoxide-induced oxidative hepatotoxicity in rats.

Lindera obtusiloba Blume, a native plant of East Asia, has traditionally been used as a folk medicine for liver disease. We studied the in vitro antioxidant and in vivo hepatoprotective activities of a 70% ethanolic extract of L. obtusiloba (LOE) containing 62.9% quercitrin and 22.0% afzelin. LOE prevented tert-butyl hydroperoxide (t-BHP)-induced oxidative damage in HepG2 cells. Along with its high antioxidant potency in vitro, our animal study confirmed that pretreatment with LOE (500 or 2000 mg/kg) for 7 days prior to a single dose of t-BHP (i.p.: 0.5 mmol/kg) significantly lowered the serum levels of alanine and aspartate aminotransferases. In addition, glutathione levels were increased in the liver, and lipid peroxidation levels were decreased in a dose-dependent manner. The histopathological examinations of rat livers showed that LOE significantly reduced the incidence of liver lesions induced by t-BHP. Therefore, we concluded that LOE has merit as a potent candidate to protect the liver against oxidative damage.

Establishment of a new Glivec-resistant chronic myeloid leukemia cell line, SNUCML-02, using an in vivo model.

OBJECTIVE: In this study, we report a newly established chronic myeloid leukemia (CML) cell line, SNUCML-02, which is resistant to imatinib and describe its biological characteristics. MATERIALS AND METHODS: Mononuclear cells were obtained from the bone marrow of a CML patient in blast crisis and were cultured in Dulbecco's modified Eagle's medium/F12 containing 20% fetal bovine serum. After 2 months of primary culture, these cells were injected into nonobese diabetic/severe combined immune-deficient mice via tail vein. Eight weeks after injection, mice were sacrificed and ex vivo culture was performed from the bone marrow cells isolated from the mice. The established cell line was named as SNUCML-02 and the biological features were characterized by cytogenetic analysis, fluorescence in situ hybridization, reverse transcriptase polymerase chain reaction, sequencing analysis, cell proliferation assay, and Western blot analysis. RESULTS: Cytogenetic studies using conventional G-banding and fluorescent in situ hybridization of SNUCML-02 demonstrated classical Philadelphia chromosome, (9;22)(q34;q11.2), and other abnormalities, such as add(11)(q23), +19 and +der(9;22). SNUCML-02 has the same BCR-ABL fusion transcript as was seen in K562 cells, but has no mutations in the ABL kinase domain. SNUCML-02 was more resistant to imatinib (STI571, Gleevec, Glivec) than other CML cell lines (K562, Kcl22, and BV173). SNUCML-02 has constitutive activation of extracellular signal-regulated kinase phosphorylation. In addition, interleukin-3 induced c-ABL phosphorylation and constitutively enhanced extracellular signal-regulated kinase phosphorylation was not inhibited by imatinib in SNUCML-02. CONCLUSION: SNUCML-02 is a new established cell line with a relatively high level of resistance to imatinib, which is useful for investigating the pathogenesis of CML progression, and will be useful in developing optimal therapeutic strategies for this ailment.

Molecular characterization and prognostic significance of FLT3 in CML progression.

To characterize the molecular mechanisms involved in the transition from the chronic phase to blast crisis in chronic myelogenous leukemia (CML), gene expression profiles of leukemic cells from patients in the chronic and blast crisis phases were analyzed using an 8.7K cDNA chip and real-time PCR. A transient transfection analysis was conducted to evaluate the role of FLT3, which was significantly upregulated in the blast crisis patients. Abl and c-Kit induction was detected in K562 cells transfected with FLT3 cDNA (K562/FLT3), and Abl and c-Kit levels were reduced in K562/FLT3 cells transfected with FLT3-siRNA (K562/FLT3-siRNA). The induction of FLT3 in CML cells attenuated imatinib-induced apoptosis. The opposite effect was observed in K562/FLT3-siRNA cells. An increased level of cleaved PARP and decreased level of pro-caspase 3 were noted when K562/FLT3-siRNA cells were treated with imatinib. These findings indicate that FLT3 is associated with disease progression, despite imatinib therapy. These results may help in the prediction of disease progression in CML patients and the development of more appropriate therapeutic modalities.

Protective effect of aqueous extract of Perilla frutescens on tert-butyl hydroperoxide-induced oxidative hepatotoxicity in rats.

The leaves of perilla [Perilla frutescens (L.) Britt. var. japonica (Hassk.) Hara] are often used in Asian gourmet food. The object of this study was to evaluate the protective effects of an aqueous extract of perilla leaves on the tert-butyl hydroperoxide (t-BHP)-induced oxidative injury observed in rat livers. The treatment of the hepatocytes with the perilla leaf extract (PLE) significantly reversed the t-BHP-induced cell cytotoxicity and lipid peroxidation. In addition, PLE exhibited ferric-reducing antioxidant power and 2,2-diphenyl-1-picrylhydrazyl free radical scavenging activities. The in vivo study showed that the pretreatment with PLE (1000 or 3000 mg/kg) for 5 days before a single dose of t-BHP (i.p.; 0.2 mmol/kg) significantly lowered the serum levels of aspartate aminotransferase and alanine aminotransferase, reduced the indicators of oxidative stress in the liver, such as the glutathione disulfide content and lipid peroxidation level in a dose-dependent manner, and remarkably increased the activity of hepatic gamma-glutamylcysteine synthetase. Histopathological examination of the rat livers showed that PLE reduced the incidence of liver lesions induced by t-BHP. Based on the results described above, it is suggested that PLE has the potential to protect liver against t-BHP-induced hepatic damage in rats.

Renal cell carcinoma does not express argininosuccinate synthetase and is highly sensitive to arginine deprivation via arginine deiminase.

Recently, pegylated arginine deiminase (ADI; EC 3.5.3.6) has been used to treat the patients with hepatocellular carcinoma or melanoma, in which the level of argininosuccinate synthetase (ASS) activity is low or undetectable. The efficacy of its antitumor activity largely depends on the level of intracellular ASS, which enables tumor cells to recycle citrulline to arginine. Thus, we examined the expression levels of ASS in various cancer cells and found that it is low in renal cell carcinoma (RCC) cells, rendering the cells highly sensitive to arginine deprivation by ADI treatment. Immunohistochemical analysis revealed that in biopsy specimens from RCC patients (n = 98), the expression of ASS is highly demonstrated in the epithelium of normal proximal tubule but not seen in tumor cells. Furthermore, RCC cells treated with ADI showed remarkable growth retardation in a dose dependent manner. ADI also exerted in vivo antiproliferative effect on the allografted renal cell carcinoma (RENCA) tumor cells and prolonged the survival of tumor-bearing mice. Histological examination of the tumors revealed that tumor angiogenesis and vascular endothelial growth factor (VEGF) expression were significantly diminished by ADI administration. Therefore, these findings suggest that arginine deprivation by ADI could provide a beneficial strategy for the treatment of RCC in ways of inhibitions of arginine availability and neovascularization.

Evaluation of toxicological monitoring markers using proteomic analysis in rats exposed to formaldehyde.

Formaldehyde (FA) is known as a low molecule weight organic compound and one of major components that causes sick building syndrome (SBS), and it has been reported that FA has cytotoxic, hemotoxic, immunotoxic, and genotoxic properties. The International Agency for Research on Cancer (IARC) has characterized FA as a carcinogen. In this study, we investigated the effects of FA on rat plasma proteins by using proteomic approach. Rats were exposed to three different concentrations of FA (0, 5, 10 ppm) for 2 weeks at 6 hours/day and 5 days/week in an inhalation chamber. Malondialdehyde (MDA) assay and carbonyl spectrometric assay were conducted to determine lipid peroxidation and protein oxidation levels and Comet assays were used for genotoxicity evaluation. Level of MDA, carbonyl insertion and DNA damage in plasma, livers, and in the lymphocytes of rats exposed to FA were found to be dose dependently increased. Proteomic analysis using three different pI ranges (3.5-5.6, 5.3-6.9, 6-9) and large size two-dimensional gel electrophoresis (2-DE) showed the presence of 3491 protein spots. A total of 32 (19 up- and 13 down-regulated) proteins were identified as biomarkers of FA, all showed dose dependent expressions in the plasma of rats exposed to FA and of these, 27 protein spots were identified by MALDI-TOF/MS. Several differentiated protein groups were found. Proteins involved in apoptosis, transportation, signaling, energy metabolism, and cell structure and motility were found to be up- or down-regulated. Among these, the identities of SNAP 23, apolipoprotein A-1 and E, clusterin, kinesin, and fibrinogen gamma were confirmed by Western blot assay, and apo E was further analyzed by using 2-DE immunoblot assays to determine isoform patterns. Two cytokine including IL4 and INF-gamma were measured in plasma with respect to fibrinogen gamma changes. In summary, cytotoxicity, and genotoxicity assays, namely MDA lipid peroxidation assay, the carbonyl protein oxidation assay, and Comet genotoxic assay showed that these effects increased on increasing FA levels. Proteomic analysis with three different pI ranges and long size 2-DE gel electrophoresis showed that 32 protein spots were up-or down-regulated. Of these 32 proteins, 7 proteins were confirmed by western blot assay. They could be potential biomarkers for human diseases associated with FA exposure.

The antifibrotic effect of TGF-beta1 siRNAs in murine model of liver cirrhosis.

Liver fibrosis results from chronic damage to the liver by chronic hepatitis, alcohol, and toxic agents. A characteristic of liver fibrosis is an accumulation of extracellular matrix (ECM) protein, which distorts the hepatic architecture by forming a fibrous scar, and the subsequent development of regenerating nodules defines cirrhosis. Transforming growth factor (TGF)-beta1, one of the most powerful profibrogenic mediators, plays a major role in the development of liver cirrhosis and regulates ECM gene expression and matrix degradation. This study elucidates the changes of TGF-beta1-mediated signals during liver fibrogenesis by using RNA interference. In this experiment, the TGF-beta1 siRNAs reduced the expression of TGF-beta1 in the livers of CCl(4) injection compared with those of control group, and the expression of type I collagen and alpha-smooth muscle actin was decreased. In conclusion, this study demonstrates that TGF-beta1 siRNAs inhibit TGF-beta1 expression in the murine model of liver cirrhosis and might be a good therapeutic strategy to prevent liver cirrhosis in human.

Comparison of immunnological and genotoxicological parameters in automobile emission inspectors exposed to polycyclic aromatic hydrocarbons.

In this study, we investigated the immunotoxicities of polycyclic aromatic hydrocarbons in 54 automobile emission inspectors and in 84 control subjects, and evaluated associations between immunological and genotoxicological parameters. Specific surface antigens of peripheral lymphocytes, namely, CD3, CD4, CD8, CD19, and CD69 were subjected to measure immune status in automobile emission inspectors and control subjects. T-and B-cells showed no significant differences between automobile emission inspectors and control subjects (p=0.740 and 0.395). In addition, the ratio of T helper cells to T cytotoxic cells was not deferent (p=0.144). However, T-cell activation was found to be significantly higher in automobile emission inspectors (p=0.041), but not B-cell activation. The levels of two cytokines (IL-4 an INF-γ) and four immunoglobulins (IgA, IgE, IgG, and IgM) were also determined in automobile emission inspectors and control subjects. All immunoglobulin types were lower in automobile emission inspectors, but this was significant only for IgG (0.047). In addition, the levels of two cytokines, IL-4 and INF-γ, were also higher in automobile emission inspectors, though this was not significant. DNA damage in mononuclear and polynuclear lymphocytes and in the level of urinary metabolites, 1-OHP and 2-naphthol, were evaluated in automobile emission inspectors and in control subjects and significant differences were found between the two groups. Examinations of urinary metabolites, DNA damage, and immunological parameters, including leukocyte subpopulations, immunoglobulins, and cytokines, showed that the cytokines levels were associated with the levels of two urinary metabolites, 1-OHP and 2-naphthol.

DNA damage in lymphocytes of benzene exposed workers correlates with trans,trans-muconic acids and breath benzene levels.

Benzene causes many kinds of blood disorders in workers employed in many different environments. These diseases include myelodisplastic syndrome and acute and chronic myelocytic leukemia. In the present study, five occupational work places, including six industrial process types, namely, printing, shoe-making, methylene di-aniline (MDA), nitrobenzene, carbomer, and benzene production were selected, and the levels of breath benzene, and trans,trans-muconic acids (t,t-MA) and phenol in urine were evaluated, as well as hematological changes and lymphocyte DNA damage. The concentration of benzene in breath was less than 3 ppm in the workplaces, and benzene exposure was found to be higher in work places where benzene is used, than in those where benzene is produced. At low levels of benzene exposure, urinary t,t-MA correlated strongly with benzene in air. Highest Olive tail moments were found in workers producing carbomer. Levels of breathzone benzene were found to be strongly correlated with Olive tail moment values in the lymphocytes of workers, but not with hematological data in the six workplaces types. In conclusion, the highest benzene exposures found occurred in workers at a company, which utilized benzene in the production of carbomer. In terms of low levels of exposure to benzene, urinary t,t-MA and DNA damage exhibited a strong correlation with breath benzene, but not with hematological data. We conclude that breath benzene, t,t-MA and lymphocytic DNA damage are satisfactory biomonitoring markers with respect to benzene exposure in the workplace.

Benzene increases the ratio of arachidonic acids to docosahexaenoic acids and inhibits the de novo synthesis of ceramide in the rat liver.

The present study investigated the effects of inhalation exposure of benzene at 0, 10, 200 and 600 ppm for 1, 2 and 4 weeks on n-6 and n-3 fatty acids and ceramide levels in the rat liver. No significant difference in the ratio of saturated fatty acid to unsaturated fatty acid was found on increasing benzene exposure levels, but the ratio of saturated fatty acid to unsaturated fatty acid decreased with increasing benzene exposure times, with the exception of the phospholipids of rats exposed to 200 and 600 ppm of benzene. A significant increase in the ratio of arachidonic acid to docosahexaenoic acid was found in the phospholipids of rats exposed to 200 and 600 ppm of benzene for 4 weeks. In our study, no change in the relative amounts of sphingomyelin in phospholipids, due to benzene exposure at 600 ppm for 4 weeks resulted in the lack of sphingomyelin turnover. However, ceramide levels in the livers of rats exposed to 600 ppm of benzene for 4 weeks were significantly reduced upon increasing the benzene concentration. This result shows that the de novo synthesis of ceramide was significantly inhibited at higher levels of benzene and that the ratio of arachidonic acid to docosahexaenoic acid in phospholipids is dose-dependently related to benzene exposure.

Genomic imbalances in Korean hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most prevalent gastrointestinal malignant tumors in Southeast Asia. Thirty-one cirrhotic HCC, 14 noncirrhotic HCC, and 13 metastastic HCC in the Korean population were investigated on microdissected tissues for chromosomal aberrations by degenerate oligonucleotide-primed polymerase chain reaction (PCR) comparative genomic hybridization. A number of prominent sites of genomic imbalances were observed. The gains of 1q, 6p, 7, 8q, 12q, 13q3-q32, 16p, 17q, and 20q and the losses of 1p, 4q, 6q, 8p, 9p, and 13q regions were observed with a similar high frequency in all types. Various chromosomal aberrations were observed preferentially to specific types. Gains of 4p15-pter, 10q24-qter, 18p11-pter, and 19p10-pter and a loss of 11q14-q22 were observed in the cirrhotic HCC, whereas losses of 14q21-q23 and 10q22-q23 were observed in noncirrhotic HCC. In metastatic HCC, gains of 3q25-qter and Xp21-pter and losses of 21q11-qter and Y were observed. The recurrent gains and losses of chromosomal regions identified in this study are consistent with several previous observations and provide possible candidate regions for the involvement of tumorigenesis and progressions of HCC.