• Journal of Life Science
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• v.24 no.9
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• pp.1012-1018
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• 2014

Sasa quelpaertensis Nakai (Korean name, Jeju-Joritdae) is one of the most abundant plants on Mt. Halla, Jeju Island, and it has long been used in traditional medicines. Recent studies have reported it as possessing various beneficial functions, including anti-inflammatory, anti-diabetic, anti-hypertension, anti-gastritis, anti-oxidant, and anti-cancer effects. However, the molecular mechanisms of its anti-cancer activity have not been clearly elucidated. In this study, we investigated the anti-cancer effects and mechanism of S. quelpaertensis on human colon cancer HT-29 cells. Cell growth inhibition by S. quelpaertensis was determined by MTT assay. Apoptosis was performed by DNA fragmentation, flow cytometry with propidium iodide staining (PI), and reverse transcription-polymerase chain reaction (RT-PCR) to confirm the anti-apoptotic factors, such as inhibitor of apoptosis (IAP) family members. $NO^{\bullet}$ production was determined by Griess assay. S. quelpaertensis treatment resulted in the time- and dose-dependent inhibition of the cell viability of HT-29 cells by inducing apoptosis, as evidenced by the accumulation of the sub-G1 cell population stained by PI, as well as the ladder-like DNA fragmentation in a dose-dependent manner. S. quelpaertensis-inducing apoptosis was accompanied by the induction of S cell cycle arrests, increasing $NO^{\bullet}$ concentrations, and the down-regulation of IAPs, including X-chromosome-linked IAP (XIAP), cellular IAP-1 (cIAP-1), cIAP-2, and survivin. Taken together, these findings have important implications for future clinical developments of S. quelpaertensis in colon cancer treatment.

• Molecular & Cellular Toxicology
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• v.3 no.3
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• pp.177-184
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• 2007

As a possible feasibility of the extrapolation between in vivo and in vitro systems, we investigated the global gene expression from both mouse liver and mouse hepatic cell line treated with hepatotoxic chemical, acetaminophen (APAP), and compared between in vivo and in vitro genomic profiles. For in vivo study, mice were orally treated with APAP and sacrificed at 6 and 24 h. For in vitro study, APAP were administered to a mouse hepatic cell line, BNL CL.2 and sampling was carried out at 6 and 24 h. Hepatotoxicity was assessed by analyzing hepatic enzymes and histopathological examination (in vivo) or lactate dehydrogenase (LDH) assay and morphological examination (in vitro). Global gene expression was assessed using microarray. In high dose APAPtreated group, there was centrilobular necrosis (in vivo) and cellular toxicity with the elevation of LDH (in vitro) at 24 h. Statistical analysis of global gene expression identified that there were similar numbers of altered genes found between in vivo and in vitro at each time points. Pathway analysis identified glutathione metabolism pathway as common pathways for hepatotoxicty caused by APAP. Our results suggest it may be feasible to develop toxicogenomics biomarkers or profiles by comparing in vivo and in vitro genomic profiles specific to this hepatotoxic chemical for application to prediction of liver toxicity.

• Toxicological Research
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• v.8 no.2
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• pp.343-357
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• 1992

Tumor necrosis factor-${\alpha}$(TNF), a polypeptide hormone secreted primarily by activated macrophages, was originally identified on the basis of its ability to cause hemorrhagic necrosis and tumor regression in vivo. Subsequently, TNF has been shown to be an important component of the host responses to infection and cancer and may mediate the wasting syndrome known as cachexia. These systemic actions of TNF are reflected in its diverse effects on target cells in vitro. TNF initiates its diverse cellular actions by binding to specific cell surface receptors. Although TNF receptors have been identified on most of animal cells, regulation of these receptors and the mechanisms which transduce TNF receptor binding into cellular responses are not well understood. Therefore, in the present study, the mechanisms how TNF receptors are being regulated and how TNF receptor binding is being transduced into cellular responses were investigated in rat liver plasma membranes (PM) and ME-180 human cervical carcinoma cell lines. $^{125}I$-TNF bound to high ($K_d=1.51{\pm}0.35nM$)affinity receptors in rat liver PM. Solubilization of PM with 1% Triton X-100 increased both high affinity (from $0.33{\pm}0.04\;to\;1.67{\pm}0.05$ pmoles/mg protein) and low affinity (from $1.92{\pm}0.16\;to\;7.57{\pm}0.50$ pmoles/mg protein) TNF binding without affecting the affinities for TNF, suggesting the presence of a large latent pool of TNF receptors. Affinity labeling of receptors whether from PM or solubilized PM resulted in cross-linking of $^{125}I$-TNF into $M_r$ 130 kDa, 90 kDa and 66kDa complexes. Thus, the properties of the latent TNF receptors were similar to those initially accessible to TNF. To determine if exposure of latent receptors is regulated by TNF, $^{125}I$-TNF binding to control and TNF-pretreated membranes were assayed. Specific binding was increased by pretreatment with TNF (P<0.05), demonstrating that hepatic PM contains latent TNF receptors whose exposure is promoted by TNF. Homologous up-regulation of TNF receptors may, in part, be responsible for sustained hepatic responsiveness during chronic exposure to TNF. As a next step, the post-receptor events induced by TNF were examined. Although the signal transduction pathways for TNF have not been delineated clearly, the actions of many other hormones are mediated by the reversible phosphorylation of specific enzymes or target proteins. The present study demonstrated that TNF induces phosphorylation of 28 kDa protein (p28). Two dimensional soidum dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) resolved the 28kDa phosphoprotein into two isoforms having pIs of 6.2 and 6.1. The pIs and relative molecular weight of p28 were consistent with those of a previously characterized mRNA cap binding protein. mRNA cap binding proteins are a class of translation initiation factors that recognize the 7-methylguanosine cap structure found on the 5' end of eukaryotic mRNAs. In vitro, these proteins are defined by their specific elution from affinity columns composed of 7-methylguanosine 5'-triphosphate($m^7$GTP)-Sepharose. Affinity purification of mRNA cap binding proteins from control and TNF treated ME-180 cells proved that TNF rapidly stimulates phosphorylation of an mRNA cap binding protein. Phosphorylation occurred in several cell types that are important in vitro models of TNF action. The mRNA cap binding protein phosphorylated in response to TNF treatment was purifice, sequenced, and identified as the proto-oncogene product eukaryotic initiation factor-4E(eIF-4E). These data show that phosphorylation of a key component of the cellular translational machinery is a common early event in the diverse cellular actions of TNF.

• Molecular & Cellular Toxicology
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• v.4 no.1
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• pp.31-44
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• 2008

The forced swim test (FST) is the most widely used model for assessing potential antidepressant activity. Although it has been shown that lateral septum is involved with the FST-related behavior, it is not clear whether antidepressant treatments could alter the FST-induced gene expression profile in the lateral septum. In the present study, the gene expression profiles in response to FST and reboxetine pretreatment were observed in the lateral septum of rats. Reboxetine is known as a most selective serotonin norepinephrine reuptake inhibitor. In addition, we compared the changes in gene expression profile between reboxetine response and nonresponse groups, which were determined by counting FST-related behavior. After FST, lateral septum from controls and reboxetine pretreated group were dissected and gene expression profiles were assessed using an Affymetrix microarray system containing 15,923 genes. Various genes with different functions were changed in reboxetine response group compared with reboxetine nonresponse group, In particular, pleiotrophin, orexin receptor 2, serotonin 2A receptor, neuropeptide Y5 receptor and thyroid hormone receptor $\beta$ were decreased in reboxetine response group, but Lim motif-containing protein kinase 1 (Limk1) and histone deacetylase 1 (HDAC1) were increased. Although further studies are required for direct roles of these genes in reboxetine response, the microarray may provide tools to find out potential target genes and signaling pathways in antidepressant response.

• Molecular & Cellular Toxicology
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• v.4 no.2
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• pp.157-163
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• 2008

Formation of ${\beta}$-amyloid $(A{\beta})$ fibrils has been identified as one of the major characteristics of Alzheimer's disease (AD). Inhibition of $A{\beta}$ fibril formation in the CNS would be attractive therapeutic targets for the treatment of AD. Several small compounds that inhibit amyloid formation or amyloid neurotoxicity in vitro have been known. Citrate has surfactant function effect because of its molecular structure having high anionic charge density, in addition to the well-known antibacterial and antioxidant properties. Therefore, we hypothesized that citrate might have the inhibitory effect against $A{\beta}$ fibril formation in vitro and have the protective effect against $A{\beta}$-induced neurotoxicity in PC12 cells. We examined the effect of citrate against the formation of $A{\beta}$ fibrils by measuring the intensity of fluorescence in thioflavin-T (Th-T) assay of between $A{\beta}_{25-35}$ groups treated with citrate and the control with $A{\beta}_{25-35}$ alone. The neuroprotective effect of citrate against $A{\beta}$-induced toxicity in PC12 cells was investigated using the WST-1 assay. Fluorescence spectroscopy showed that citrate inhibited dose-dependently the formation of $A{\beta}$ fibrils from ${\beta}$-amyloid peptides. The inhibition percentages of $A{\beta}$ fibril formation by citrate (1, 2.5, and 5 mM) were 31%, 60%, and 68% at 7 days, respectively in thioflavin-T (Th-T) assay. WST-1 assay revealed that the toxic effect of $A{\beta}_{25-35}$ was reduced, in a dose-dependent manner to citrate. The percentages of neuroprotection by citrate (1, 2.5, and 5 mM) against $A{\beta}-induced$ toxicity were 19%, 31 %, and 34%, respectively. We report that citrate inhibits the formation of $A{\beta}$ fibrils in vitro and has neuroprotective effect against $A{\beta}$-induced toxicity in PC12 cells. Neuroprotective effects of citrate against $A{\beta}$ might be, to some extent, attributable to its inhibition of $A{\beta}$ fibril formation. Although the mechanism of anti-amyloidogenic activity is not clear, the possible mechanism is that citrate might have two effects, salting-in and surfactant effects. These results suggest that citrate could be of potential therapeutic value in Alzheimer's disease.

• Molecular & Cellular Toxicology
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• v.5 no.2
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• pp.126-132
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• 2009

The usage and types of chemicals are advancing, specializing, large-scaled increasing, and new chemical exposed workers are concerning to occupational disease. The generation of reactive oxygen in the body from carcinogen, mutation and DNA damage in cancer is protected by natural antioxidants (phytochemicals) with antimutagenic effect. There were many reports of ginsenoside Rb$_1$, Rg$_1$ grievances of the genetic mutation to suppress the effect confirm the genetic toxicity test with chromosomal aberration test and the Comet (SCGE) assay confirmed the suppression effect occurring chromosomal DNA damage. We had wanted to evaluate the compatibility and sensitivity between the chromosomal aberration (CA) test and the Comet assay. We used the CA test and Comet assay to evaluate the anti-genotoxicity of ginsenoside Rb$_1$ and Rg$_1$, in CHO-K1 (Chinese hamster ovary fibroblast) cell in vitro, composed negative control (solvent), positive control (benzo[a]pyrene), test group (carcinogen+variety concentration of ginsenoside) group. The positive control was benzo[a]pyrene (50 $\mu$M), well-known carcinogen, and the negative control was the 1 % DMSO solvent. The test group was a variety concentration of ginsenoside Rb$_1$, Rg$_1$ with 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10%. In chromo-somal aberration test, we measured the number of cells with abnormally structured chromosome. In Comet assay, the Olive tail moment (OTM) and Tail length (TL) values were measured. The ratio of cell proliferation was increased 8.3% in 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1%, 10% Rb$_1$ treated groups, and increased 10.4% in 10$^{-10}$%, 10$^{-8}$%, 10$^{-6}$%, 10$^{-4}$%, 10$^{-2}$%, 1% Rg$_1$ treated groups. In the CA test, the number of chromosomal aberration was decreased all the Rb$_1$ and Rg$_1$ treated groups. In the Comet assay, the OTM values were decreased in all the Rb$_1$ and Rg$_1$ treated groups. To evaluate the compatibility between CA and Comet assay, we compared the reducing ratio of chromosomal abnormalities with its OTM values, it was identified the antimutagenicity of ginsenoside, but it was more sensitive the CA test than the Comet assay. Ginsenoside Rb$_1$ and Rg$_1$ significantly decrease the number of cells with chromosomal aberration, and decrease the extent of DNA migration. Therefore, ginsenoside Rb$_1$, Rg$_1$ are thought as an antioxidant phytochemicals to protect mutagenicity. The in vitro Comet assay seems to be less sensitive than the in vitro chromosomal aberration test.

• Molecular & Cellular Toxicology
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• v.3 no.2
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• pp.90-97
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• 2007

8-Hydroxyquinoline is used as antibacterial agent and antioxidant based on its function inducing the chelation of ferrous ion present in host resulting in production of chelated complex. This complex being transported to cell membrane of bacteria and fungi exerts antibacterial and antifungal action. In this study, we have carried out in vitro genetic toxicity tests and microarray analysis to understand the underlying mechanisms and the mode of action of toxicity of 8-hydroxyquinoline. TA1535 and TA98 cells were treated with 8-hydroxyquinoline to test its toxicity by basic genetic toxicity test, Ames and two new in vitro micronucleus and COMET assays were applied using CHO cells and L5178Y cells, respectively. In addition, microarray analysis of differentially expressed genes in L5178Y cells in response to 8-hydroxyquinoline were analyzed using Affymatrix genechip. The result of Ames test was that 8-hydroxyquinoline treatment increased the mutations in base substitution strain TA1535 and likewise, 8-hydroxyquinoline also increased mutations in frame shift TA98. 8-Hydroxyquinoline increased micronuclei in CHO cells and DNA damage in L5178Y. 8-Hdroxyquinoline resulted in positive response in all three tests showing its ability to induce not only mutation but also DNA damage. 783 Genes were initially selected as differentially expressed genes in response to 8-hydroxyquinoline by microarray analysis and 34 genes among them were over 4 times of log fold changed. These 34 genes could be candidate biomarkers of genetic toxic action of 8-hydroxyquinoline related to induction of mutation and/or induction of micronuclei and DNA damage. Further confirmation of these candidate markers related to their biological function will be useful to understand the detailed mode of action of 8-hydroxyquinoline.

• Molecular & Cellular Toxicology
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• v.3 no.2
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• pp.107-112
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• 2007

o-Nitrotoluene is used to synthesize artificial dyes and raw materials of urethane resin. In this study, we have carried out in vitro genetic toxicity tests and microarray analysis to understand the underlying mechanisms and the mode of action of toxicity of onitrotoluene. TA1535 and TA98 cells were treated with o-nitrotoluene to test its toxicity by basic genetic toxicity test. Ames and two new in vitro micronucleus and COMET assays were applied using CHO cells and L5178Y cells, respectively. In addition, microarray analysis of differentially expressed genes in L5178Y cells in response to o-nitrotoluene was analyzed using Affymatrix genechip. The result of Ames test was that o-nitrotoluene treatment did not increase the mutations both in base substitution strain TA1535 and in frame shift TA98. o-Nitrotoluene has not increased micronuclei in CHO cells. But onitrotoluene increased DNA damage in L5178Y cell. Two-hundred two genes were initially selected as differentially expressed genes in response to o-nitrotoluene by microarray analysis and forty four genes among them were over 2 times of log fold changed. These forty four genes could be candidate biomarkers of genetic toxic action of o-nitrotoluene related to induction of mutation and/or induction of micronuclei and DNA damage. Further confirmation of these candidate markers related to the DNA damage will be useful to understand the detailed mechanism of action of o-nitrotoluene.

• Molecules and Cells
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• v.28 no.6
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• pp.529-536
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• 2009

Genome sequencing of the pig is being accelerated because of its importance as an evolutionary and biomedical model animal as well as a major livestock animal. However, information on expressed porcine genes is insufficient to allow annotation and use of the genomic information. A series of expressed sequence tags of 5' ends of five full-length enriched cDNA libraries (SUSFLECKs) were functionally characterized. SUSFLECKs were constructed from porcine abdominal fat, induced fat cells, loin muscle, liver, and pituitary gland, and were composed of non-normalized and normalized libraries. A total of 55,658 ESTs that were sequenced once from the 5′ ends of clones were produced and assembled into 17,684 unique sequences with 7,736 contigs and 9,948 singletons. In Gene Ontology analysis, two significant biological process leaf nodes were found: gluconeogenesis and translation elongation. In functional domain analysis based on the Pfam database, the beta transducin repeat domain of WD40 protein was the most frequently occurring domain. Twelve genes, including SLC25A6, EEF1G, EEF1A1, COX1, ACTA1, SLA, and ANXA2, were significantly more abundant in fat tissues than in loin muscle, liver, and pituitary gland in the SUSFLECKs. These characteristics of SUSFLECKs determined by EST analysis can provide important insight to discover the functional pathways in gene networks and to expand our understanding of energy metabolism in the pig.

• Molecular & Cellular Toxicology
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• v.4 no.4
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• pp.318-322
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• 2008

Obesity is an increasing worldwide health problem that is strongly related to the imbalance of food intake and energy metabolism. It was well-known that several substances in the hypothalamus regulate food intake and energy metabolism. We planned an integrative study to elucidate the mechanism of the development of obesity. Firstly, to find candidate genes with the marvelous effect, the different expression in the hypothalamus between ob/ob and 48-h fasting mice was investigated by using DNA microarray technology. As a result, we found 3 genes [peroxisome proliferator activated receptor, gamma, coactivator 1 alpha (Ppargc1a), calmodulin 1 (Calm1), and complexin 2 (Cplx2)] showing the different hypothalamic expression between ob/ob and 48-h fasting mice. Secondly, a genetic approach on PPARGC1A gene was performed, because PPARGC1A acts as a transcriptional coactivator and a metabolic regulator. Two hundred forty three obese female patients with body mass index (BMI)${\geq}$25 and 285 control female subjects with BMI 18 to<23 were recruited according to the Classification of Korean Society for the Study of Obesity. Among the coding single nucleotide polymorphisms (cSNPs) of PPARGC1A, 2 missense SNPs (rs8192678, Gly482Ser; rs3736265, Thr612Met) and 1 synonymous SNP (rs3755863, Thr528Thr) were selected, and analyzed by PCR-RFLP and pyrosequencing. For the analysis of genetic data, chi-square ($X^2$) test and EH program were used. The rs8192678 was significantly associated with obese women (P<0.0006; odds ratio, 1.5327; 95% confidence interval, 1.2006-1.9568). Haplotypes also showed significant association with obese women ($X^2$=33.28, P<0.0008). These results suggest that PPARGC1A might be related to the development of obesity.