Gene expression profile by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the liver of wild-type (AhR+/+) and aryl hydrocarbon receptor-deficient (AhR-/-) mice.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is one of the most toxic environmental pollutants that cause various biological effects on mammals. The purpose of our study was to identify the genes involved in hepatotoxicity and hepatocarcinogenesis caused by TCDD. C57BL/6 (AhR+/+, wild type) and B6.129-AhR/J (AhR-/-, knock out) mice were injected i.p. with a single treatment of TCDD at the dose of 100 microg/kg body weight. Relative liver weight was significantly increased at 72 hr after TCDD treatment without an apparent histopathological change in AhR+/+ mice (p<0.05). TCDD treatment also significantly increased activity of serum alanine aminotransferase in AhR-/- mice (p<0.05). The liver was analyzed for gene expression profiles 72 hr later. As compared with AhR-/- mice, the expression of 51 genes (>3-fold) was changed in AhR+/+ mice; 28 genes were induced, while 23 genes were repressed. Most of the genes were associated with chemotaxis, inflammation, carcinogenesis, acute-phase response, immune responses, cell metabolism, cell proliferation, signal transduction, and tumor suppression. This study suggests that the microarray analysis of genes in the liver of AhR+/+ and AhR-/- mice may help to clarify the mechanism of AhR-mediated hepatotoxicity and hepatocarcinogenesis by TCDD.

Protective effect of diallyl disulfide on oxidative stress-injured neuronally differentiated PC12 cells.

The effects of diallyl disulfide (DADS), a garlic-derived compound, on the viability of neuronal cells and cell signals, including phosphatidylinositol 3-kinase (PI3K)/Akt, glycogen synthase kinase-3 (GSK-3), cytochrome c, caspase-3, and poly(ADP-ribose) polymerase (PARP), were investigated in PC12 cells neuronally differentiated by nerve growth factor. To evaluate the toxicity of DADS itself, nPC12 cells were treated with several concentrations of DADS, and 3,(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and trypan blue stain revealed that the viability was not affected by low concentration of DADS, up to 20 microM, but it was decreased at higher than this concentration. The levels of free radicals and membrane lipid peroxidation were significantly increased in nPC12 cells when treated with more than 50 microM DADS, and treatment of PC12 cells with 100 microM DADS killed the cells by inhibiting PI3K/Akt and by promoting activation of GSK-3 and caspase-3, release of cytochrome c, and cleavage of PARP. To evaluate the protective effects of low concentration of DADS on oxidative stress-injured nPC12 cells, the viability of the cells (pretreated with DADS for 2 h vs. not pretreated) was evaluated 24 h after exposure to 100 microM H2O2 for 30 min. Compared to the cells treated with 100 microM H2O2 only, pretreatment of the cells with 20 microM DADS before exposure to 100 microM H2O2 increased the viability and induced activation of PI3K and Akt, inactivation of GSK-3, and inhibition of cytochrome c release, caspase-3 activation, and PARP cleavage. These results indicate that low concentration of DADS has neuroprotective effects by activating PI3K/Akt and by inhibiting GSK-3 activation, cytochrome c release, caspase-3 activation, and PARP cleavage, whereas high concentration is rather cytotoxic. Therefore, some specific optimum concentration of DADS may be a new potential therapeutic strategy for oxidative stress injured in vitro model of neurodegenerative diseases.

Epigallocatechin gallate prevents oxidative-stress-induced death of mutant Cu/Zn-superoxide dismutase (G93A) motoneuron cells by alteration of cell survival and death signals.

This study was undertaken to evaluate the effect of the G93A mutation in the human Cu/Zn-superoxide dismutase gene (hSOD1) on the phosphatidylinositol-3-kinase (PI3K)/Akt and glycogen synthase kinase-3 (GSK-3) pathway in motoneuron, and to determine the role of epigallocatechin gallate (EGCG) on oxidative stress-injured motoneurons. The viability of G93A mutant cells was less than that of wild-type cells, and the activation of PI3K and the phosphorylation of Akt and GSK-3 in G93A mutant cells decreased compared with wild-type hSOD1 4.1 cells. In the experiment to evaluate the effect of oxidative stress and/or EGCG on these motoneurons, after exposure to 400 microM H2O2, the MTT assay revealed greatly reduced viability of G93A mutant cells compared with wild-type cells, and pre-treatment of these cells with EGCG before H2O2 exposure increased the viability of both cell lines. Western blot analysis showed that the G93A mutation and oxidative stress decreased survival signals including PI3K/Akt but increased death signals including GSK-3; however, pre-treatment with EGCG increased survival signals but decreased death signals. These results suggest that PI3K/Akt and GSK-3 activities are altered in G93A mutant cells and EGCG-induced activation of PI3K/Akt and inhibition of GSK-3 could be a new potential therapeutic strategy for ALS associated with oxidative injury.

Phosphatidylinositol-3 kinase/Akt and GSK-3 mediated cytoprotective effect of epigallocatechin gallate on oxidative stress-injured neuronal-differentiated N18D3 cells.

Epigallocatechin gallate (EGCG) is one of most famous compounds of green tea. EGCG suppresses apoptosis induced by oxidative radical stress through several mechanisms. This study was designed to investigate whether EGCG plays a cytoprotective role by activating phosphatidylinositol-3 kinase (PI3K)/Akt-dependent anti-apoptotic pathway and inhibiting glycogen synthase kinase-3 (GSK-3) activity in oxidative stressed N18D3 neural cells. N18D3 cells, mouse neuroblastoma X dorsal root ganglion hybrid cell line, were pre-treated with EGCG or z-VAD-fmk, non-selective caspase inhibitor used as a control substance, for 2 h. The N18D3 cells were then exposed to low concentration of H(2)O(2) (100 microM) for 30 min, and further incubated for 24 h. MTT (3,[4,5-dimethylthiazol]-2-yl) assay and trypan blue staining were used to identify cell viability. Immunoreactivity (IR) of PI3K, Akt, and GSK-3 beta were measured by Western blotting. MTT assay and trypan blue staining showed that EGCG and z-VAD-fmk significantly increased cell viability, and IR of PI3K, phospho-Akt and phospho-GSK-3 beta was significantly increased in the cells treated with EGCG, but not in z-VAD-fmk treated. These results imply that EGCG has neuroprotective effect by increasing PI3K/Akt-dependent anti-apoptotic signals.

Inhibitory effects of ochratoxin A on nerve growth factor-induced neurite extension through downregulation of p38 MAP kinase and AP-1 activation in cultured pheochromocytoma cells.

Ochratoxin A (OTA) induces microcephaly in animals and in vitro cultured whole embryos. Inhibition of neuronal cell differentiation was proposed as underlying mechanisms responsible for OTA-induced microcephaly. Previously it was found that OTA inhibited differentiation of cultured rat embryonic midbrain cells into neurons. In this study, the influence of OTA on differentiation in PC-12 cells, a widely accepted model cells for study of neuronal differentiation was examined. Cell differentiation was assessed by measurement of neurite extension and quantified by the number of neurites extended. OTA decreased serum and nerve growth factor (NGF)-induced neurite extension in a concentration-dependent manner. Since MAP kinase and transcription factors have been implicated in cell differentiation of neuronal cells, and our previous study demonstrated that p38 MAP kinase and AP-1 are activated during PC 12 cell differentiation, the effect of OTA on NGF-induced p38 MAP kinase and transcription factor activation was examined. Co-treatment of OTA with NGF resulted in inhibition of NGF-induced p38 MAP kinase and AP-1 activation. Moreover, SB203580, a specific inhibitor of p38 MAP kinase blocked p38 MAP kinase and AP-1 activation accompanied by further inhibition of neurite extension. The present study shows that OTA inhibited cell differentiation of PC-12 cells, and this inhibitory effect may be related to inhibition of the activation of the p38 MAP kinase in conjunction with transcription factors AP-1. This finding suggests that the inhibitory effect on neuronal cell differentiation by OTA might be a mechanism responsible for OTA-induced microcephaly.

Epigallocatechin gallate protects nerve growth factor differentiated PC12 cells from oxidative-radical-stress-induced apoptosis through its effect on phosphoinositide 3-kinase/Akt and glycogen synthase kinase-3.

The effects of epigallocatechin gallate (EGCG) on the phosphoinositide 3-kinase (PI3K)/Akt and glycogen synthase kinase-3 (GSK-3) pathway during oxidative-stress-induced injury were studied using H2O2-treated PC12 cells, which were differentiated by nerve growth factor (NGF). Following 100 microM H2O2 exposure, the viability of differentiated PC12 cells (EGCG or z-VAD-fmk pretreated vs. not pretreated) was evaluated the number of viable cell with Trypan blue and 3,4,5-dimethylthiazol-2-yl (MTT). Additionally, expression of cytochrome c, caspase-3, poly(ADP-ribose) polymerase (PARP), PI3K/Akt and GSK-3 was examined using Western blot analyses. EGCG or z-VAD-fmk-pretreated PC12 cells showed an increase of viability compared to untreated PC12 cells, and pretreatment of PC12 cells with either agent induced a dose-dependent inhibition of caspase-3 activation and PARP cleavage. However, inhibition of cytochrome c release was only detected in EGCG-pretreated cells. Upon examination of the PI3K/Akt and GSK-3 upstream pathway, Western blots of EGCG pretreated cells showed decreased immunoreactivity (IR) of Akt and GSK-3 and increased IR of p85a PI3K, phosphorylated Akt and phosphorylated GSK-3. In contrast, no changes were seen in z-VAD-fmk-pretreated cells. These results show that EGCG affects the PI3K/Akt, GSK-3 pathway as well as downstream signaling, including the cytochrome c and caspase-3 pathways. Therefore, it is suggested that EGCG-mediated activation of PI3K/Akt and inhibition of GSK-3 could be a new potential therapeutic strategy for neurodegenerative diseases associated with oxidative injury.

Inhibitory effect of glycolic acid on ultraviolet B-induced c-fos expression, AP-1 activation and p53-p21 response in a human keratinocyte cell line.

Glycolic acid, an alpha-hydroxy acid derived from fruit and milk sugars, has been commonly used as a cosmetic ingredient since it was known to have photo-protective and anti-inflammatory effects, and anti-oxidant effect in UV-irradiated skin. However, little has been known about the functional role of glycolic acid on UV-induced skin tumorigenesis. We previously found that glycolic acid inhibited UV-induced skin tumor development in hairless mouse. In this study we investigated anti-tumor promoting mechanism of glycolic acid on the UV-induced skin tumor development. The ability of glycolic acid to inhibit the UVB-induced cytotoxicity, apoptosis and expression of apoptosis-regulatory genes (p53 and p21) was examined. We also investigated whether glycolic acid could inhibit UVB-induced alternation of cell cycle, c-fos expression and activation of transcription factor AP-1 in cultured immortalized human keratinocyte HaCaT cells. Glycolic acid treatment attenuated the UVB-induced cell cytotoxicity as well as apoptosis. Glycolic acid also inhibited the UVB-induced expression of c-fos and the activation of transcription factor AP-1, and inhibited mRNA levels of apoptosis-regulatory gene (p53 and p21). These results suggest that glycolic acid may exert the inhibitory effect on the UVB-induced skin tumor development by blocking the UVB-induced of apoptosis and cytotoxicity through inhibition of c-fos expression and activation of AP-1 in addition to the inhibition of p53-p2l response pathway.

Inhibitory effect of peroxisome proliferator-activated receptor gamma agonist on ochratoxin A-induced cytotoxicity and activation of transcription factors in cultured rat embryonic midbrain cells.

The effects of 15-deoxy-delta12,14-prostaglandin J2 (15-deoxy PGJ2) on ochratoxin A (OTA)-induced neurotoxicity and on the activation of transcription factors activator protein-1 (AP-1) and nuclear factor-kappa B (NF-kappaB) were investigated in cultured rat embryonic midbrain cells. Twelve-day rat embryo midbrain cells were cultured for 48 h. OTA (0.5 or 1 microg/ml) and/or 1.5-deoxy PGJ2 (0.5 microM) were then added for 48 h. Cell number and neurite outgrowth were determined to assess the neurotoxicity of OTA. AP-1 and NF-kappaB activation was determined by gel mobility shift assay after 3 h of exposure to OTA and/or 15-deoxy PGI2. OTA caused concentration-dependent reductions in neurite outgrowth and cell number, and induced AP-1 and NF-kappaB activation. Cotreatment with 15-deoxy PGJ2 (0..5 microM) blocked OTA-induced decrease in neurite outgrowth and cell number and inhibited AP-1 and NF-kappaB activation. 15-Deoxy PGJ2 (0.5 microM) caused the expression of peroxisome proliferator-activated receptor-gamma (PPAR-gamma) in the cells. Results show that 1.5-deoxy PGJ2 blocked OTA-induced neurotoxicity by inhibiting AP-1 and NF-kappaB activation in cultured rat embryonic midbrain cells.