• Molecular & Cellular Toxicology
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• 제4권3호
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• pp.183-191
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• 2008

Volatile organic compounds (VOCs) are common constituents of cleaning and degreasing agents, paints, pesticides, personal care products, gasoline and solvents. And VOCs are evaporated at room temperature and most of them exhibit acute and chronic toxicity to human. Benzene is the most widely used prototypical VOC and the toxic mechanisms of them are still unclear. The multi-step process of toxic mechanism can be more fully understood by characterizing gene expression changes induced in cells by toxicants. In this study, DNA microarray was used to monitor the expression levels of genes in HepG2 cells and HL-60 cells exposed to the benzene on IC20 and IC50 dose respectively. In the clustering analysis of gene expression profiles, although clusters of HepG2 and HL-60 cells by benzene were divided differently, expression pattern of many genes observed similarly. We identified 916 up-regulated genes and 1,144 down-regulated genes in HepG2 cells and also 1,002 up-regulated genes and 919 down-regulated genes in HL-60 cells. The gene ontology analysis on genes expressed by benzene in HepG2 and HL-60 cells, respectively, was performed. Thus, we found some principal pathways, such as, focal adhesion, gap junction and signaling pathway in HepG2 cells and toll-like receptor signaling pathway, MAPK signaling pathway, p53 signaling pathway and neuroactive ligand-receptor interaction in HL-60 cells. And we also found 16 up-regulated and 14 down-regulated commonly expressed total 30 genes that belong in the same biological process like inflammatory response, cell cycle arrest, cell migration, transmission of nerve impulse and cell motility in two cell lines. In conclusion, we suggest that this study is meaningful because these genes regarded as strong potential biomarkers of benzene independent of cell type.

• Molecular & Cellular Toxicology
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• 제4권3호
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• pp.177-182
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• 2008

Caveolin proteins are mediators of cell death or the survival of injured cells, and they are inhibitors of various signaling pathways. The expression of caveolin-, which is involved in the protein kinase A (PKA) signaling pathway, was examined in the differentiated mouse vestibular cell line UB/UE-1 after gentamicin ototoxicity. Caveolae in the vestibular hair cell of healthy guinea pigs were observed through an electron microscope. UB/UE-1 cells were cultured at 95% $CO_2$ with 5% $O_2$ at $33^{\circ}C$ for 48 hours and at 95% $CO_2$ with 5% $O_2$ at $39^{\circ}C$ for 24 hours for differentiation. Cells were treated with 1 mM gentamicin, 0.02 mM H89 (PKA inhibitor), and then incubated for 24 hours. Caveolin-1 expression was examined by western blotting and PKA activity by a $PepTag^{(R)}$ assay. Caveolae were observed in the vestibular hair cells of healthy guinea pigs by electron microscopy. Caveolin-1 was expressed spontaneously in differentiated UB/UE-1 cells and increased after gentamicin treatment. PKA was also over-activated by gentamicin treatment. Both gentamicin-induced caveolin-1 expression and PKA over-activation were inhibited by H89. These results indicate that gentamicin-induced caveolin-1 expression is mediated by the PKA signaling pathway. We conclude that caveolae/ caveolin activity, induced via a PKA signaling pathway, may be one of the mechanisms of gentamicin-induced ototoxicity.

• Molecular & Cellular Toxicology
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• 제4권2호
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• pp.144-149
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• 2008

Despite versatile activity (cancericidal, antimicrobial, hypoxia inducible factor (HIF) inhibition, immune deactivation of DNA bis-intercalation agent, echinomycin, its specific mechanism has been elusive. Of these novel mechanisms, we reported that using human colon cancer cells (HT-29), apoptotic machinery induced by echinomycin might be dependent of caspase-3 pathway. Despite a partial enlightenment of prototypic signal path triggered by echinomycin, the role of Bcl-2 in this signaling pathway is unclear. To address this issue, we explored whether or not echinomycin would overcome the anti-apoptotic impact of Bcl-2 in HT-29 cells by the controlled Bcl-2 overexpression. Prior to this proof, we confirmed that echinomycin induces mitochondrial depolarization, then triggering the mitochondrial pathway of apoptosis with an involvement of upstream cas-pases-3. Transiently transfection with inactive Bax-DNA failed to prevent echinomycin-induced apoptosis in HT-29 cells. To dissect the role of Bcl-2 in echinomycin-induced apoptosis, HT-29 cells were transiently transfected with Bcl-2 DNA for overexpression and then treated with echinomycin for 24h. Combined analyses of DNA fragmentation and flow cytometric analysis clearly verified that echinomycin-induced apoptosis was drastically attenuated by Bcl-2 overexpression, whereas a control vector rarely affected echinomycin-induced apoptosis. Collectively, these data verify that Bcl-2 regulates echinomycin-induced apoptosis in HT-29 cells. To my knowledge, this is the first evidence that of diverse, structured minor groove binders (MGB), the prototypic echinomycin might control the apoptotic signaling via Bcl-2-mitochondrial pathway.

• Molecular & Cellular Toxicology
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• 제4권2호
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• pp.164-169
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• 2008

Methylmercury (MeHg) is known to have devastating effects on the mammalian nervous system. In order to characterize the mechanism of MeHg-induced neurotoxicity, we investigated the analysis of transcriptional profiles on human 8k cDNA microarray by treatment of $1.4{\mu}M$ MeHg at 3, 12, 24 and 48h in human neuroblastoma SH-SY5Y cell line. Some of the identified genes by MeHg treatment were significant at early time points (3h), while that of others was at late time points (48h). The early response genes that may represent those involved directly in the MeHg response included pantothenate kinase 3, a kinase (PRKA) anchor protein (yotiao) 9, neurotrophic tyrosine kinase, receptor, type 2 gene, associated with NMDA receptor activity regulation or perturbations of central nervous system homeostasis. Also, when SH-SY5Y cells were subjected to a longer exposure (48h), a relative increase was noted in a gene, glutamine-fructose-6-phosphate transaminase 1, reported that overexpression of this gene may lead to the increased resistance to MeHg. To confirm the alteration of these genes in cultured neurons, we then applied real time-RT PCR with SYBR green. Thus, this result suggests that a neurotoxic effect of the MeHg might be ascribed that MeHg alters neuronal receptor regulation or homeostasis of neuronal cells in the early phase. However, in the late phase, it protects cells from neurotoxic effects of MeHg.

• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.1-4
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• 2008

The 5-HTT gene is a candidate gene for influencing the clinical response to antidepressant treatment. The purpose of this gene study was to determine the relationship between serotonin transporter gene polymorphism at the SLC6A4 and the response to citalopram in a Korean population with major depressive disorder (MDD). Citalopram was administered for 8 weeks to the 80 patients who completed this study. The severity of depression was assessed with the 21-item Hamilton Depression Rating scale, and the 5-HTTLPR genotypes in the patients were determined using the polymerase chain reaction. Our result did not showed significant differences in, allele, and carrier distribution between the normal group and MDD patients. This study suggest that polymorphism of the 5HTT gene was not associated with citalopram response to MDD in the Korean population.

• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.45-51
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• 2008

Volatile Organic Compounds (VOCs) have been shown to cause nervous system disorders through skin contact or respiration, and also cause foul odors even at low densities in most cases. Also, as a compound itself, VOCs are directly harmful to the environment and to the human body, and may participate in photochemical reactions in air to create secondary pollutants. In this study, HL-60 cells were treated with volatile organic compounds, including ethylbenzene and trichloroethylene, at a value of $IC_50$. Then, the in house-prepared Human HazChem arrayer was utilized in order to compare the gene expression between the two VOCs. After hybridization, 8 upregulated genes and 8 downregulated genes were discovered in the HazChem array. The upregulated genes were identified as SG15, TNFSF10, PRNP, ME1, NCOA4, SRXN1, TXNRD1, and XBP1. The downregulated genes were identified as MME, NRF1, PRARBP, CALCA, CRP, BAX, C7 or f40, and FGFR1. Such results were highly correlated with the quantitative RT-PCR results. The majority of the 16 genes were related with the characteristics of VOCs, including respiratory mechanism, apoptosis, and carcinogenesis-associated genes. Our data showed that our human HazChem array can be used to monitor hazardous materials via gene expression profiling.

• Molecular & Cellular Toxicology
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• 제4권2호
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• pp.124-131
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• 2008

The growing problem of obesity is associated with numerous medical conditions. Several studies have reported that activation of thyroid hormone receptor beta $(THR{\beta})$ is involved in lipid metabolism and thermogenesis. To identify the relationship between the $THR{\beta}$ gene and obesity, we genotyped eighty two single nucleotide polymorphisms (SNPs) in the gene using the Affymetrix array chip in 209 overweight/obese and 155 normal subjects in Korean population. Of the eighty two polymorphisms, the seven SNPs exhibited a significant association with overweight/obesity in three alternative models (codominant, dominant, and recessive models; P<0.05 after adjusting for age and sex) were rs826221 (+267878 T>C), rs4858604 (+186399 A>G), rs1158265 (+200152 T>C), rs1868575 (+206031 G>A), rs1700939 (+238467 T>A), rs1505301 (+241933 T>C), and rs1924768 (+126491 T>C). During haplotype analysis using HapAnalyzer software, 2 haplotypes (block 13: TTAT; block 15: CTGC) containing significant polymorphisms (rs1700939 +238467 T>A and rs4858604 +186399 A>G) were detected to be significantly different. The results suggest that the $THR{\beta}$ gene may be associated with overweight/obesity in Korean population.

• Molecular & Cellular Toxicology
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• 제4권2호
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• pp.113-123
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• 2008

Microglia, which is the primary immune effector cells in the central nervous system, constitutes the first line of defense against infection and injury in the brain. The goal of this study was to determine the protective (anti-inflammation) mechanisms of forsythia suspense (FS) on LPS-induced activation of BV-2 microglial cells. The effects of FS on gene expression profiles in activated BV-2 microglial cells were evaluated using microarray analysis. BV-2 microglial cells were cultured in a 100mm dish $(1{\times}10^7/dish)$ for 24hr and then pretreated with $1{\mu}g/mL$ FS or left untreated for 30 min. Next, $1{\mu}g/mL$ LPS was added to the samples and the cells were reincubated at $37^{\circ}C$ for 30 min, 1hr, and 3hr. The gene expression profiles of the BV-2 microglial cells varied depending on the FS. The oligonucleotide microarray analysis revealed that MAPK pathway-related genes such as Mitogen activated protein kinase 1 (Mapk1), RAS protein activator like 2 (Rasal2), and G-protein coupled receptor 12 (Gpr12) and nitric oxide biosynthesis-related genes such as nitric oxide synthase 1 (neuronal) adaptor protein (Nos1ap), and dimethylarginine dimethylaminohydrolase 1 (Ddah1) were down regulated in FS-treated BV-2 microglial cells. FS can affect the MAPK pathway and nitric oxide biosynthesis in BV-2 microglial cells.

• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.5-10
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• 2008

The effect of obesity on the drug-metabolizing enzymes remains an important issue for clinician since obesity is a world wide epidemic problem. However, little is known about the effects of obesity on flavincontaining monooxygenase (FMO) production and activity. We show here for the first time that in vivo FMO activity determined by urinary ranitidine (RA) metabolites ratio in human, was higher in subjects with a high body mass index (BMI, kg/$m^2$, 21.97-30.32) than in those with an intermediate BMI (range 19.38-21.83). Moreover, there was a significant correlation between FMO activity and BMI in 209 subjects. In high fat diet-induced obese mice, we also observed that the hepatic expression of FMO (225% of lean mice) and the activity measured by the RA Noxidation rate ($513{\pm}58.1$ vs. $349{\pm}66.0$ pmol/hr per mg protein) were significantly higher than in lean mice fed a control diet. Unknown factors rather than leptin or insulin appeared to regulate the hepatic FMO production. Thus, FMO activity may be increased in obese or overweight individuals. Moreover, the regulation of FMO activity in subjects with morbid obesity, with or without complications and its clinical implications, should be investigated further.

• Molecular & Cellular Toxicology
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• 제4권1호
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• pp.16-21
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• 2008

Cell death of trophoblast, particularly by abnormal release of physiological nitric oxide (NO) has been known to be a causative factor of pre-eclampsia. In the present study, effects of intracellular calcium increase enhancing the activity of NO synthases (neuronal NO synthase, nNOS in this trophoblast cells) on the cell death were examined in a human placental full-term cell line (HT-1). Furthermore, we analyzed the possible mechanisms underlying the augmentation of $Ca^{++}$-mediated NOS activity mediated by protein kinases like PKC, PKA, or CaM-KII. In experiments for cell toxicity, a calcium ionophore (ionomycin $10{\mu}M$) enhanced cell death confirmed by MTT assay, and increased significantly nNOS activity determined with a hemoglobin oxidation assay. This cell death was partially protected by pre-treatment of 7-nitroindazole (7-NI, $10{\mu}M$ and $100{\mu}M$), a nNOS-specific inhibitor. Additionally, $Ca^{++}$-ionophore -induced increase of nNOS activity also was partially normalized by pre-treatment of specific inhibitors of protein kinases, PKC, PKA or CaM-KII. Therefore, we suggest that an increase of calcium influx, leading to the activation of nNOS activity, which in turn may result in the death of trophoblast cells by involvement of signaling mechanisms of protein kinases.