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Pancreatic 13-cells are susceptible to oxidative stress, which is related closely to the islet dysfunction. In the present study, using the pancreatic cell lines HIT-T15 and RINm5F as known in vitro models of impaired [beta]-cell function as well as primary rat islet [beta]-cells, we observed a relationship between intracellular glutathione levels and oxidative stress-mediated cell dysfunction. Hydrogen peroxide and 4-hydroxy2-nonenal caused cell death in HIT-T15 and RINm5F cells at lower concentrations compared with non-[beta]-cells, such as HepG2 and NRK49F cells. The extent of the cytotoxicity caused by the model oxidants was inversely correlated well with intracellular glutathione levels in the cell lines used. Treatment of HIT-T15 and RINm5F cells with L-cysteine or L-cystine significantly augmented the glutathione contents, surpassing the effect of N-acetylcysteine, and abrogated 4-hydroxy-2-nonenal-mediated cytotoxicity almost completely. L-Cysteine increased intracellular glutathione levels in primary [beta]-cells as well. Supplementation of L-cysteine to the RINm5F cell culture inhibited 4-hydroxy-2-nonenal-mediated cytosolic translocation of PDX-1, a key transcription factor for [beta]-cell function. Intrinsic transport activities ([V.sub.max]/[K.sub.m]) of the L-cystine/L-glutamate exchanger in HIT-T15 and RINm5F cells were considerably lower than that in NRK-49F cells, although gene expressions of the exchanger were similar in these cells. Results obtained from the present study suggest that the restricted activity of the L-cystine/L-glutamate exchanger controls the levels of intracellular glutathione, thereby making [beta]-cells become susceptible to oxidative stress. system [x.sup.-.sub.c]; glutathione; hydrogen peroxide; reactive oxygen species; PDX-1

HTiNbO5, HTi2NbO7 and HTiTaO5 nanosheet, prepared by exfoliation of protonated cation-exchangeable layered metal oxides, function as strong solid acids for the esterification of acetic acid and hydrolysis of ethyl acetate, acid-catalyzed reactions. The acid catalytic activity of these titanium niobate/tantalate nanosheets analyzed by H1 magic-angle-spinning nuclear magnetic resonance spectroscopy (H1 MAS NMR), revealed the presence of strong Bronsted active sites in the form of bridging hydroxyl groups.

Transcription factor NF-E2-related factor 2 (Nrf2) regulates the induction of antioxidative proteins, including heme oxygenase-1 (HO-1). Nrf2 is sequestered in the cytoplasm by Keap1 under unstimulated conditions but translocates into the nucleus and transactivates the antioxidant responsive element (ARE) upon exposure to oxidative insults. It has recently been demonstrated that in vitro phosphorylation of Nrf2 on Ser40 by protein kinase C (PKC) facilitates the dissociation of Nrf2 from the Keap1 complex (Huang HC, Nguyen T, and Pickett CB. J Biol Chem 277: 42769-42774, 2002). The present study was designed to examine whether PKC is involved in oxidative stress-mediated nuclear translocation of Nrf2 in vivo and, if so, which PKC isoforms are involved. Induction of HO-1 gene expression by phorone, a glutathione depletor, and 4-hydroxy-2,3-nonenal (4-HNE), an end product of lipid peroxidation, was suppressed by a specific PKC inhibitor, Ro-31-8220, at concentrations that inhibit all isoforms in WI-38 cells. The induction of HO-1 was not affected by prolonged exposure of the cells to 12-O-tetradecanoylphorbol-13 acetate (TPA), suggesting that TPA-insensitive atypical PKC (aPKC) isoforms are involved. An immunocomplex kinase assay revealed that phorone and 4-HNE increased aPKCi activity. In COS-7 cells, 4-HNE induced nuclear translocation of the Nrf2-green fluorescent protein (GFP) fusion protein, but not the Nrf2(S40A)-GFP mutant. In the absence of oxidative insults, the Nrf2(S40E)-GFP mutant was distributed in the nucleus. The Nrf2-GFP accumulation in the nucleus was induced by coexpression of aPKCL, but not by a kinase inactive mutant aPKCi(K274W). The activity of an ARE-driven reporter was increased by coexpression of aPKCi, and this effect was eliminated by Ro-31-8220 in HepG2 cells. The reporter activity induced by 4-HNE was inhibited by coexpression of aPKCi(K274W). These results suggest that phosphorylation of Nrf2 Ser40 by aPKC(s) is involved in the nuclear translocation and ARE transactivation of Nrf2 by oxidative stress. heme oxygenase-1; nuclear translocation; phosphorylation; NF-E2-related factor 2

Background: Parkinson's disease (PD) is an age-related progressive neurodegenerative disorder caused by selective loss of dopaminergic neurons from the substantia nigra (SN) to the striatum. The initial factor that triggers neurodegeneration is unknown; however, inflammation has been demonstrated to be significantly involved in the progression of PD. The present study was designed to investigate the role of the pro-inflammatory cytokine interleukin-1 (IL-1) in the activation of microglia and the decline of motor function using IL-1 knockout (KO) mice.Methods: Lipopolysaccharide (LPS) was stereotaxically injected into the SN of mice brains as a single dose or a daily dose for 5 days (5 mg/2 ml/injection, bilaterally). Animal behavior was assessed with the rotarod test at 2 hr and 8, 15 and 22 days after the final LPS injection.Results: LPS treatment induced the activation of microglia, as demonstrated by production of IL-1β and tumor necrosis factor (TNF) α as well as a change in microglial morphology. The number of cells immunoreactive for 4-hydroxynonenal (4HNE) and nitrotyrosine (NT), which are markers for oxidative insults, increased in the SN, and impairment of motor function was observed after the subacute LPS treatment. Cell death and aggregation of α-synuclein were observed 21 and 30 days after the final LPS injection, respectively. Behavioral deficits were observed in wild-type and TNFα KO mice, but IL-1 KO mice behaved normally. Tyrosine hydroxylase (TH) gene expression was attenuated by LPS treatment in wild-type and TNFα KO mice but not in IL-1 KO mice.Conclusions: The subacute injection of LPS into the SN induces PD-like pathogenesis and symptoms in mice that mimic the progressive changes of PD including the aggregation of α-synuclein. LPS-induced dysfunction of motor performance was accompanied by the reduced gene expression of TH. These findings suggest that activation of microglia by LPS causes functional changes such as dopaminergic neuron attenuation in an IL-1-dependent manner, resulting in PD-like behavioral impairment. [ABSTRACT FROM AUTHOR]

Constitutive androstane receptor (CAR) is a nuclear receptor that regulates the transcription of target genes, including CYP2B and 3A. Phenobarbital activates CAR, at least in part, in an AMP-activated protein kinase (AMPK)-dependent manner. However, the precise mechanisms underlying phenobarbital activation of AMPK are still unclear. In the present study, it was demonstrated that phenobarbital administration to mice decreases hepatic miR-122, a liver-enriched microRNA involved in both hepatic differentiation and function. The time-course change in the phenobarbital-mediated down-regulation of miR- 122 was inversely correlated with AMPK activation. Phenobarbital decreased primary miR-122 to approximately 25% of the basal level as early as 1 h and suppressed transactivity of mir-122 promoter in HuH-7 cells, suggesting that the downregulation occurred at the transcriptional level. AMPK activation by metformin or 5-aminoimidazole-4-carboxamide 1-β-Dribonucleoside had no evident effect on miR-122 levels. An inhibitory RNA specific for miR-122 increased activated AMPK and CAR-mediated trancactivation of the phenobarbital-responsive enhancer module in HepG2 cells. Conversely, the reporter activity induced by the ectopic CAR was almost completely suppressed by co-transfection with the miR-122 mimic RNA. GFP-tagged CAR was expressed in the cytoplasm in addition to the nucleus in the majority of HuH-7 cells in which miR-122 was highly expressed. Co-transfection of the mimic or the inhibitor RNA for miR-122 further increased or decreased, respectively, the number of cells that expressed GFP-CAR in the cytoplasm. Taken together, these results suggest that phenobarbital-mediated down-regulation of miR-122 is an early and important event in the AMPK-dependent CAR activation and transactivation of its target genes. [ABSTRACT FROM AUTHOR]

Patients having a generalised rash with severe liver dysfunction associated with exposure to trichloroethylene (TCE) have been reported mainly in Asian countries. However, no case has been reported in Japan since the 1990s. Here, we describe a case of hypersensitivity syndrome (HS) caused by TCE in a 30-year-old Japanese man. The patient developed a rash, fever and liver dysfunction 21 days after he had been exposed to TCE at his workplace. Serum human herpesvirus (HHV)-6 and cytomegalovirus (CMV) DNA were detected 4 and 7 weeks, respectively, after the onset; the IgG antibody titres to HHV-6 and CMV were significantly elevated 6 and 9 weeks, respectively, after the onset. Patch testing was positive for the metabolites of TCE (i.e. trichloroethanol, trichloroacetic acid and chloral hydrate) but not for TCE itself; these results suggest that the TCE metabolites induced this disease. Human leucocyte antigen-B*1301, which has been reported to be strongly associated with TCE-induced HS, was identified in this patient. In addition, the clinical findings, laboratory data and period of virus reactivation after onset were quite similar to those of drug-induced hypersensitivity syndrome (DIHS). We also review TCE-induced HS from the viewpoint of the similarity to DIHS in this article. Copyright © 2010 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Pancreatic β-cells are susceptible to oxidative stress, which is related closely to the islet dysfunction. In the present study, using the pancreatic cell lines HIT-T15 and RINm5F as known in vitro models of impaired β-cell function as well as primary rat islet β-cells, we observed a relationship between intracellular glutathione levels and oxidative stress-mediated cell dysfunction. Hydrogen peroxide and 4-hydroxy2-nonenal caused cell death in HIT-T15 and RINm5F cells at lower concentrations compared with non-β-cells, such as HepG2 and NRK-49F cells. The extent of the cytotoxicity caused by the model oxidants was inversely correlated well with intracellular glutathione levels in the cell lines used. Treatment of HIT-T15 and RINm5F cells with L-cysteine or L-cystine significantly augmented the glutathione contents, surpassing the effect of N-acetylcysteine, and abrogated 4-hydroxy-2-nonenal-mediated cytotoxicity almost completely. L-Cysteine increased intracellular glutathione levels in primary β-cells as well. Supplementation of L-cysteifle to the RINm5F cell culture inhibited 4-hydroxy-2-nonenal-mediated cytosolic translocation of PDXI, a key transcription factor for 13-cell function. Intrinsic transport activities (Vmax/Km) of the L-cystine/L-glutamate exchanger in HIT-T15 and RINm5F cells were considerably lower than that in NRK-49F cells, although gene expressions of the exchanger were similar in these cells. Results obtained from the present study suggest that the restricted activity of the L-cystine/L-glutamate exchanger controls the levels of intracellular glutathione, thereby making β-cells become susceptible to oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: Many in vitro studies have employed cigarette smoke condensates or soluble smoke components to investigate the biological effects of cigarette smoke. However, neither of these methods evaluates the biological effects of fresh whole cigarette smoke. It is most desirable to conduct in vitro biological studies under conditions which accommodate the dynamic physicochemical character of fresh cigarette smoke. Previously we reported the development of a whole smoke exposure system to assess the biological effects of mainstream cigarette smoke. The exposure system design was based on a combination of the sedimentation procedure and the CULTEX cultivation technique, which includes a systemized air/liquid interface methodology and exposes the cells to fresh smoke at every puff. The aim of this study was to adopt the other biological endpoint to our whole smoke exposure system. We focused on heme oxygenase (HO)-1 mRNA gene expression, an enzyme which has recently been shown to be highly responsible for oxidative stress. In the present study, a dose–response relationship between the HO-1 mRNA expression based on the reverse transcription real-time PCR method and total exposure to cigarette smoke was observed. When a Cambridge filter pad was placed between the cigarette and exposure module, to ensure the cells were only exposed to the gas/vapor phase, the latter, as well as the whole smoke, induced HO-1 mRNA dose dependently. For the next step, acetate plain and charcoal filters with the same pressure drop were prepared to assess the potential ability of charcoal filters with regard to the vapor phase performance. The results revealed reduced HO-1 mRNA gene expression when a charcoal filter was used. Direct whole smoke exposure is a significant approach and may reflect the conditions of exposure essentially resulting from direct contact between cells and a dynamic mixture of gaseous and particulate constituents. We were able to adopt a gene expression assay for oxidative stress to the whole smoke exposure system, following the adaptation of cytotoxicity assays. This system, which includes several advantages involving the post-exposure washing of cells, by adding the exchanging medium and assuring the exposure of the particulate phase through the sedimentation method, may have potential for further investigations into the molecular basis of smoking-related lung disease. [Copyright &y& Elsevier]

SB 203580 has been widely used to specifically shut down the p38 MAP kinase-dependent pathway, although it is capable of inducing c-Raf kinase activity in cells. The present study demonstrates that SB 203580 activates members of the ERK cascade, c-Raf, MEK, and ERK, in human monocytic THP-1 cells. The activation of these kinases was sustained for at least 24 h after SB 203580 treatment and was also observed in U937 cells, suggesting that c-Raf efficiently transduces the signal even in the presence of the inhibitor in these cells. However, the expression of ERK cascade–dependent genes, such as c-fos and IL-1β, was extremely limited. Analysis of the cellular distribution of ERK in SB 203580-treated cells indicated that nuclear translocation of phosphorylated ERK was impaired. Also, nuclear translocation of ERK induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was inhibited by SB 239063, which does not associate with c-Raf and is highly selective for p38 MAP kinase. In addition, the forced expression of the dominant negative mutant of p38 MAP kinase suppressed serum responsive element-dependent transactivation induced by TPA. These results suggest that the steady-state level of p38 MAP kinase activity modulates ERK signaling. [ABSTRACT FROM PUBLISHER]

Heme oxygenase-1 (HO-1) is induced under various oxidative stress conditions, such as lipopolysaccharide (LPS) insult. Induction of HO-1 by LPS is reported to be mediated through interleukin-1β (IL-1β), rather than other inflammatory cytokines in the mouse liver. However, we found that IL-1α/β knockout (KO) mice responded well to LPS insult, as did wild-type mice with respect to HO-1 mRNA induction (about 30-fold increase). In contrast, tumor necrosis factor α KO (TNFαKO) mice responded very weakly to LPS in the HO-1 mRNA expression, but not metallothionein mRNA. Recent studies reveal that nitric oxide from Kupffer cells is involved in HO-1 induction in the liver produced by LPS. Therefore, nitrite and nitrate concentrations in the liver were also measured and these parameters did not increase in either IL-1KO or TNFαKO. In addition, the phosphorylation of c-JUN N-terminal kinase (JNK) and p38, but not extracellular signal-regulated kinase, was very low in TNFαKO mice due to LPS administration. All of these findings indicate that TNFα is a major candidate to trigger HO-1 induction in response to LPS stimulation, and that its message is likely transduced through JNK and p38 pathways. [Copyright &y& Elsevier]

Investigates the regulatory role of protein kinase C isozymes in bufalin-induced cell differentiation and apoptosis. Responses of THP-1 and THP-1/TPA cells to bufalin; Protein kinase C isozymes in THP-1 and THP-1/TPA cells; Effect of protein kinase C inhibitors on bufalin-induced cell differentiation and bufalin-induced apoptosis.

Inbred male ACI/N rats were treated with an intermittent carcinogenic regimen of 2-acetylaminofluorene (2-AAF) until the development of hepatomas (40 weeks), and the activation of 2-AAF by liver S-9 and the oxidation of 2-AAF and 2 other drugs by microsomal functions were periodically examined. The S-9 activity increased at the end of 1 feeding cycle (3 weeks 2-AAF diet and 1 week normal diet), reaching a maximum of 400% of controls after 2 or 3 feeding cycles. It then declined, but the elevated S-9 activity (300 to 250% of controls) was sustained until the development of hyper-plastic nodules in the livers. The microsomal oxidation of 2-AAF to N-hydroxy-2-AAF was activated by dietary 2-AAF, but the activity of cytosol (S-105) to produce mutagen from N-hydroxy-2-AAF was not affected. Cytochrome P-450 content and microsomal oxidation of aminopyrine and aniline were gradually decreased below the normal levels by 2-AAF feeding, although microsomal -hydroxylation of aniline was temporarily elevated to about 130% of control at the first or the second feeding cycle. These results indicate that dietary 2-AAF selectively induces microsomal 2-AAF N-hydroxylase which mediates the oxidation of 2-AAF to N-hydroxy-2-AAF, a proximate carcinogenic or mutagenic metabolite. [ABSTRACT FROM PUBLISHER]

Norharman, widely distributed in our environment, is alone not mutagenic to Salmonella typhimurium TA98 and TA100 either with or without S9 mix, but becomes mutagenic to S. Typhimurium TA98 with S9 mix when non-mutagenic aromatic amines like aniline or o- or m-toluidine are added. Thus norharman has been called a ‘co-mutagen’. In the present study we examined whether or not DNA adducts are formed in DNA of S.typhimurium TA98 by treatment with norharman and aromatic amines using 32P-post-labeling analysis under modified adduct intensification conditions. When a sample of norharman (8 mg) and aniline (4 mg) was incubated with 4 ml of overnight culture of S.typhimurium TA98 in the presence of 20 ml S9 mix for 6 h at 37°C, three adduct spots were detected at a total relative adduct labeling (RAL) of 10.8 ± 2.27/108 nucleotides. Under the same conditions, a mixture of norharman (8 mg) and o-toluidine (4 mg) yielded three adduct spots at a RAL of 3.74 ± 1.71/108 nucleotides. With a combination of norharman and m-toluidine, a single adduct spot was seen at a RAL of 0.04 ± 0.01/108 nucleotides. In contrast, norharman with p-toluidine did not produce adduct spots. Furthermore, neither norharman nor the aromatic amines themselves gave any evidence of adducts. Thus DNA adduct formation by norharman with aromatic amines correlates with the co-mutagenic action of norharman in S.typhimurium TA98. [ABSTRACT FROM PUBLISHER]

Oleanolic acid (OA) has been shown to inhibit mouse skin tumor promotion by 12-O-tetradecanoylphorbol-13-acetate (TPA). This study was designed to examine the effect of OA on the TPA-induced expression of the ornithine decarboxylase (ODC) gene as well as other genes. OA inhibited the induction of ODC activity and mRNA level produced by TPA in the skin of female CD-1 mice. Preapplication of OA (10 μmol) to the mouse dorsal skin produced an approximately 50% decrease in TPA (8 nmol)induced epidermal ODC activity, as well as ODC gene expression. These results suggest that OA inhibits TPA-induced ODC mainly at the transcriptional level. In addition to ODC, TPA also stimulated metallothionetn (MT) gene expression in mouse skin. A dose of 2.5 μmol of OA diminished the TPA-induced MT mRNA 50%. Treatment with OA (10 μmol) after TPA (8 nmol) application also inhibited ODC and MT gene expression which suggests that OA does not compete with TPA for its receptor. OA pretreatment also prevented c-fos gene expression. All of these findings suggest that OA diminishes some signal transduction pathways of TPA to suppress target gene expression in mouse skin. This study suggests that OA might be a general inhibitor against TPA-stimulated gene expression in mouse skin. [ABSTRACT FROM PUBLISHER]