Saponin Esculeoside A and Aglycon Esculeogenin A from Ripe Tomatoes Inhibit Dendritic Cell Function by Attenuation of Toll-like Receptor 4 Signaling.

Dendritic cells (DCs) can initiate immune response through the presenting antigens to naïve T lymphocytes. Esculeoside A (EsA), a spirosolane glycoside, is reported as a major component in the ripe fruit of tomato. Little is known about the effect of tomato saponin on mice bone marrow-derived DCs. This study revealed that EsA and its aglycon, esculeogenin A (Esg-A), attenuated the phenotypic and functional maturation of murine DCs stimulated by lipopolysaccharide (LPS). We found that EsA/Esg-A down-regulated the expression of major histocompatibility complex type II molecules and costimulatory molecule CD86 after LPS stimulation. It was also determined that EsA-/Esg-A-treated DCs were poor stimulators of allogeneic T-cell proliferation and exhibited impaired interleukin-12 and TNF-α production. Additionally, EsA/Esg-A was able to inhibit TLR4-related and p-NFκB signaling pathways. This study shows new insights into the immunopharmacology of EsA/Esg-A, and represents a novel approach to controlling DCs for therapeutic application.

In Vivo Kinematic Analysis of the Axial Shoulder Rotation in the Standing and Supine Positions Using 3D/2D Registration and Electromyography.

Background There has been no report comparing shoulder kinematics and muscle activities during axial shoulder rotation in different positions. The purpose of this study was to investigate differences in shoulder kinematics and muscle activities during axial shoulder rotation in healthy subjects between standing and supine positions using three-dimensional/two-dimensional (3D/2D) registration techniques and electromyography (EMG). Methods Eleven healthy males agreed to participate in this study. We recorded the fluoroscopy time during active shoulder axial rotation with a 90° elbow flexion in both standing and supine positions, simultaneously recording surface EMG of the infraspinatus, anterior deltoid, posterior deltoid, and biceps brachii. Three-dimensional bone models were created from CT images, and shoulder kinematics were analyzed using 3D/2D registration techniques. Muscle activities were evaluated as a ratio of mean electromyographic values to 5-sec maximum voluntary isometric contractions.  Results Scapular kinematics during axial shoulder rotation in the supine position showed similar patterns with those in the standing position. The scapula was more posteriorly tilted and more downwardly rotated in the supine posture than in standing (P < 0.001 for both). Acromiohumeral distance (AHD) in the supine posture was significantly larger than in standing. Muscle activities showed no significant differences between postures except for biceps (P < 0.001). Discussion Shoulder kinematics and muscle activities during axial rotation were similar in pattern between standing and supine postures, but there were shifts in scapular pose and AHD. The findings of this study suggest that posture may be an important consideration for the prescription of optimal shoulder therapy following surgery or for the treatment of shoulder disorders.

Dihydropyrazine suppresses TLR4-dependent inflammatory responses by blocking MAPK signaling in human hepatoma HepG2 cells.

Dihydropyrazines (DHPs), including 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), are glycation products generated through non-enzymatic reactions in vivo and in food. They are recognized as compounds that are toxic to organisms as they produce radicals. However, our previous study indicated that DHP-3 suppressed Toll-like receptor 4 (TLR4) expression and decreased the phosphorylation of nuclear factor-κB (NF-κB) in lipopolysaccharide (LPS)-treated HepG2 cells. TLR4 signaling is involved in the onset of various inflammatory diseases, and NF-κB and mitogen-activated protein kinase (MAPK) play important roles in TLR4 signaling. Thus, we aimed to elucidate the effects of DHP-3 on MAPK signaling and in turn on the activated TLR4 signaling pathway. In LPS-stimulated HepG2 cells, DHP-3 reduced the phosphorylation of MAPK, extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38. The expression of c-jun, a subunit of activator protein-1, was decreased by DHP-3 treatment. Furthermore, DHP-3-induced suppression of MAPK signaling resulted in a decrease in various inflammatory regulators, such as interleukin-6, CC-chemokine ligand 2, and cyclooxygenase-2. These results suggest that DHP-3 exerts an inhibitory effect on TLR4-dependent inflammatory response by suppressing MAPK signaling.

Molecular mechanism of dihydropyrazine-induced cytotoxicity: the possibility of an independent pathway from the receptor for advanced glycation end products.

Dihydropyrazines (DHPs) are one of glycation products that are non-enzymatically generated in vivo and in food. We had previously revealed that 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), a methyl-substituted DHP, elicited redox imbalance and cytotoxicity in cultured cells. However, the molecular mechanisms underlying DHP-3-induced cytotoxicity remain unclear. To address this issue, we examined the involvement of the receptor for advanced glycation end products (RAGE) in DHP-3-induced cytotoxicity. To evaluate the role of RAGE, we prepared HeLa cells that constitutively expressed RAGE and its deletion mutant, which lacks the cytoplasmic domain (RAGEΔcyto), using an episomal vector. After transfection with the vector, cells were selected following incubation with multiple concentrations of hygromycin to remove non-transfected cells. The expression of RAGE and RAGEΔcyto in the cells was confirmed by immunoblotting. RAGE and RAGEΔcyto were apparently expressed in transfected cells; however, there were no significant differences in DHP-3-induced cytotoxicity between these cells and mock vector-transfected cells. These results suggested that DHP-3 elicits cytotoxicity in a RAGE-independent manner.

Ablation of Selenbp1 Alters Lipid Metabolism via the Pparα Pathway in Mouse Kidney.

Selenium-binding protein 1 (Selenbp1) is a 2,3,7,8-tetrechlorodibenzo-p-dioxin inducible protein whose function is yet to be comprehensively elucidated. As the highly homologous isoform, Selenbp2, is expressed at low levels in the kidney, it is worthwhile comparing wild-type C57BL mice and Selenbp1-deficient mice under dioxin-free conditions. Accordingly, we conducted a mouse metabolomics analysis under non-dioxin-treated conditions. DNA microarray analysis was performed based on observed changes in lipid metabolism-related factors. The results showed fluctuations in the expression of numerous genes. Real-time RT-PCR confirmed the decreased expression levels of the cytochrome P450 4a (Cyp4a) subfamily, known to be involved in fatty acid ω- and ω-1 hydroxylation. Furthermore, peroxisome proliferator-activated receptor-α (Pparα) and retinoid-X-receptor-α (Rxrα), which form a heterodimer with Pparα to promote gene expression, were simultaneously reduced. This indicated that reduced Cyp4a expression was mediated via decreased Pparα and Rxrα. In line with this finding, increased levels of leukotrienes and prostaglandins were detected. Conversely, decreased hydrogen peroxide levels and reduced superoxide dismutase (SOD) activity supported the suppression of the renal expression of Sod1 and Sod2 in Selenbp1-deficient mice. Therefore, we infer that ablation of Selenbp1 elicits oxidative stress caused by increased levels of superoxide anions, which alters lipid metabolism via the Pparα pathway.

Synthesis of Oxygen-Containing Heterocyclic Compounds by Iron-Catalyzed Alkylative Cyclization of Unsaturated Carboxylic Acids and Alcohols.

Iron-catalyzed alkylative cyclization of alkenes bearing oxygen nucleophiles with secondary and tertiary alkyl bromides through carbon-carbon and carbon-oxygen bond formations has been developed. A broad substrate scope is an attractive feature of this synthetic method, providing a variety of potentially bioactive five- and six-membered oxygen-containing heterocycles. The reaction pathway is proposed to involve a radical addition of the in situ-formed alkyl radical to an alkene followed by carbon-oxygen bond-forming intramolecular cyclization.

The effect of dihydropyrazines on lipopolysaccharide-stimulated human hepatoma HepG2 cells via regulating the TLR4-MyD88-mediated NF-κB signaling pathway.

Dihydropyrazines (DHPs), including 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), are glycation products that are spontaneously generated in vivo and ingested via food. DHPs generate various radicals and reactive oxygen species (ROS), which can induce the expression of several antioxidant genes in HepG2 cells. However, detailed information on DHP-response pathways remains elusive. To address this issue, we investigated the effects of DHP-3 on the nuclear factor-κB (NF-κB) pathway, a ROS-sensitive signaling pathway. In lipopolysaccharide-stimulated (LPS-stimulated) HepG2 cells, DHP-3 decreased phosphorylation levels of inhibitor of NF-κB (IκB) and NF-κB p65, and nuclear translocation of NF-κB p65. In addition, DHP-3 reduced the expression of Toll-like receptor 4 (TLR4) and the adaptor protein myeloid differentiation primary response gene 88 (MyD88). Moreover, DHP-3 suppressed the mRNA expression of tumor necrosis factor-alpha (TNFα), and interleukin-1 beta (IL-1ß). Taken together, these results suggest that DHP-3 acts as a negative regulator of the TLR4-MyD88-mediated NF-κB signaling pathway.

Methoxylation of Acyl Fluorides with Tris(2,4,6-trimethoxyphenyl)phosphine via C-OMe Bond Cleavage under Metal-Free Conditions.

Acyl fluorides are subjected to methoxylation with tris(2,4,6-trimethoxyphenyl)phosphine (TMPP) to afford the corresponding methyl esters in good to excellent yields. This transformation is featured by C(sp2)-OMe bond cleavage under metal-free conditions. Unprecedented utilization of TMPP as a methoxylating agent realized the installation of an OMe group into the desired products.

Synthesis of Dinaphtho[2,3-d:2',3'-d']anthra[1,2-b:5,6-b']dithiophene (DNADT) Derivatives: Effect of Alkyl Chains on Transistor Properties.

To investigate organic field-effect transistor (OFET) properties, a new thienoacene-type molecule, 4,14-dihexyldinaphtho[2,3-d:2',3'-d']anthra[1,2-b:5,6-b']dithiophene (C6-DNADT), consisting of π-conjugated nine aromatic rings and two hexyl chains along the longitudinal molecular axis has been successfully synthesized by sequential reactions, including Negishi coupling, epoxidation, and cycloaromatization. The fabricated OFET using thin films of C6-DNADT exhibited p-channel FET properties with field-effect mobilities (µ) of up to 2.6 × 10-2 cm2 V-1 s-1, which is ca. three times lower than that of the parent DNADT molecule (8.5 × 10-2 cm2 V-1 s-1). Although this result implies that the installation of relatively short alkyl chains into the DNADT core is not suitable for transistor application, the origins for the FET performance obtained in this work is fully discussed, based on theoretical calculations and solid-state structure of C6-DNADT by grazing incidence wide-angle X-ray scattering (GIWAXS) and atomic force microscopy (AFM) analyses. The results obtained in this study disclose the effect of alkyl chains introduced onto the molecule on transistor characteristics.

ß-Naphthoflavone, an exogenous ligand of aryl hydrocarbon receptor, disrupts zinc homeostasis in human hepatoma HepG2 cells.

Recent studies have demonstrated a relationship between the disruption of zinc homeostasis and the onset of diseases. However, little is known about the factors that disrupt zinc homeostasis. Here, we investigated the effects of ß-naphthoflavone, an exogenous ligand of aryl hydrocarbon receptor (AHR), on intracellular zinc levels. Human hepatoma HepG2 cells were treated with ß-naphthoflavone for 3 days, and intracellular labile and total zinc levels were assessed through flow cytometry and inductively coupled plasma atom emission spectroscopy, respectively. The mRNA levels of zinc transporters were determined by real-time PCR. Treatment of cells with ß-naphthoflavone induced a decrease in intracellular labile zinc in a dose-dependent manner, with significantly decreased levels observed at 1 µM compared with controls. Additionally, intracellular total zinc levels demonstrated a decreasing trend with 10 µM ß-naphthoflavone. Zinc pyrithione recovered the decrease in intracellular labile zinc levels induced by ß-naphthoflavone, while zinc sulfate had no effect. Moreover, significant decreases in the mRNA levels of zinc transporters ZnT10 and ZIP5 were observed in response to 10 µM ß-naphthoflavone. These results demonstrated that ß-naphthoflavone has the potential to disrupt zinc homeostasis in hepatocytes. Although the underlying mechanism remains to be determined, suppression of zinc transporter transcription through AHR activation may be involved in the ß-naphthoflavone-induced disruption of intracellular zinc levels.

Comparison of glenohumeral joint rotation between asymptomatic subjects and patients with subacromial impingement syndrome using cine-magnetic resonance imaging: a cross-sectional study.

BACKGROUND: The purpose of this study to compare glenohumeral joint motion during active shoulder axial rotation between subacromial impingement syndrome (SIS) shoulders and asymptomatic shoulders using cine-magnetic resonance imaging (cine-MRI). Measurement of glenohumeral joint motion via manual intervention does not assess the usual glenohumeral joint motion, and the glenoid surface cannot be confirmed manually. However, cine-MRI can produce clear images of glenohumeral joint rotation. Therefore, we sought to measure the active ROM of the glenohumeral rotation using cine-MRI. METHODS: Seventy-three shoulders in 42 asymptomatic volunteers and 110 SIS shoulders in 103 consecutive patients were included in this study. We evaluated 36 matched pairs (72 shoulders in total) adjusting for baseline characteristics with propensity score matching method. The patients underwent cine-MRI during axial rotation of the adducted arm. During imaging, participants rotated their shoulder from the maximum internal rotation to the maximum external rotation over the first 10 s and then back to the maximum internal rotation over the subsequent 10 s. We assessed internal/external rotation, and compared the asymptomatic and SIS shoulders in this regard. Evaluation of rotation angles was performed on a series of axial images through the humeral head center. RESULTS: The mean internal rotation angles of the asymptomatic and patient groups were 55° ± 10° and 41° ± 23°, respectively, (P = .002; 95% Confidence Interval [CI], 51-58 vs 33-49); the mean external rotation angles were 47° ± 15° and 21° ± 25°, respectively, (P < .001; CI, 42-52 vs 13-29). CONCLUSIONS: Compared to asymptomatic shoulders, SIS shoulders showed significantly restricted glenohumeral rotation as determined by cine-MRI. Our results suggested that the significant limitation of active glenohumeral rotation might be associated with rotator cuff dysfunction.

Synthesis and Physicochemical Properties of Dibenzo[2,3- d:2',3'- d']anthra[1,2- b:5,6- b']dithiophene (DBADT) and Its Derivatives: Effect of Substituents on Their Molecular Orientation and Transistor Properties.

We have synthesized dibenzo[2,3- d:2',3'- d']anthra[1,2- b:5,6- b']dithiophene (DBADT) and several derivatives bearing alkyl and phenyl groups at various positions. The optical and electrochemical properties of the synthesized compounds were investigated. All the fabricated OFET devices exhibited typical p-type behavior under ambient conditions, and diphenyl-substituted analogue-based OFET devices showed excellent mobility, as high as 0.66 cm2 V-1 s-1. The surface morphology and molecular orientation in thin films were also investigated using atomic force microscopy (AFM) and two-dimensional grazing incidence X-ray diffraction (2D-GIXD). It was found that the substituents and their positions affect the molecular orbitals, molecular orientation, and morphology of the thin films, producing different FET performance.

Nickel-Catalyzed Decarbonylative Alkylation of Aroyl Fluorides Assisted by Lewis-Acidic Organoboranes.

Herein, nickel-catalyzed decarbonylative C-F bond alkylation of aroyl fluorides with organoboron reagents is reported. Aroyl fluorides are more chemically stable than the corresponding aroyl chlorides and can be readily synthesized from the corresponding carboxylic acids. The fluoronickel intermediate formed via oxidative addition interacts with Lewis-acidic trialkylboranes, and the subsequent decarbonylative alkylation proceeds. This new synthetic methodology allows 1,2-bifunctionalization of aromatic carboxylic acids via palladium-catalyzed ortho-C-H arylation. In addition, an unprecedented 1,4-nickel migration on ortho-arylated aroyl fluorides was observed. As a demonstration of the synthetic utility of the present reaction, the sequential 1-alkyl-2-arylation of 3-hydroxy-2-naphthoic acid was accomplished via chemoselective alkylation at a fluorocarbonyl moiety and the subsequent C-O bond arylation at an acetoxy group.

Dioxin-induced increase in leukotriene B4 biosynthesis through the aryl hydrocarbon receptor and its relevance to hepatotoxicity owing to neutrophil infiltration.

Dioxin and related chemicals alter the expression of a number of genes by activating the aryl hydrocarbon receptors (AHR) to produce a variety of disorders including hepatotoxicity. However, it remains largely unknown how these changes in gene expression are linked to toxicity. To address this issue, we initially examined the effect of 2,3,7,8-tetrachrolodibenzo-p-dioxin (TCDD), a most toxic dioxin, on the hepatic and serum metabolome in male pubertal rats and found that TCDD causes many changes in the level of fatty acids, bile acids, amino acids, and their metabolites. Among these findings was the discovery that TCDD increases the content of leukotriene B4 (LTB4), an inducer of inflammation due to the activation of leukocytes, in the liver of rats and mice. Further analyses suggested that an increase in LTB4 comes from a dual mechanism consisting of an induction of arachidonate lipoxygenase-5, a rate-limiting enzyme in LTB4 synthesis, and the down-regulation of LTC4 synthase, an enzyme that converts LTA4 to LTC4. The above changes required AHR activation, because the same was not observed in AHR knock-out rats. In agreement with LTB4 accumulation, TCDD caused the marked infiltration of neutrophils into the liver. However, deleting LTB4 receptors (BLT1) blocked this effect. A TCDD-produced increase in the mRNA expression of inflammatory markers, including tumor-necrosis factor and hepatic damage, was also suppressed in BLT1-null mice. The above observations focusing on metabolomic changes provide novel evidence that TCDD accumulates LTB4 in the liver by an AHR-dependent induction of LTB4 biosynthesis to cause hepatotoxicity through neutrophil activation.

Nrf2-ARE-Dependent Alterations in Zinc Transporter mRNA Expression in HepG2 Cells.

Zinc transporters are solute carrier family members. To date, 10 zinc transporters (ZnTs) and 14 Zrt-, Irt-like proteins (ZIPs) have been identified. ZnTs control intracellular zinc levels by effluxing zinc from the cytoplasm into the extracellular fluid, intracellular vesicles, and organelles; ZIPs also contribute to control intracellular zinc levels with influxing zinc into the cytoplasm. Recently, changes in zinc transporter expression have been observed in some stress-induced diseases, such as Alzheimer's disease and diabetes mellitus. However, little is known regarding the mechanisms that regulate zinc transporter expression. To address this, we have investigated the effect of a well-established stress response pathway, the nuclear factor erythroid 2-related factor 2 (Nrf2)-antioxidant responsive element (ARE) pathway, on zinc transporter mRNA levels. Exposure to 10-4 M tert-butylhydroquinone (t-BHQ), which activates Nrf2-ARE signaling, for 6 h significantly increases ZnT-1, ZnT-3, and ZnT-6 mRNAs levels, and significantly decreases ZnT-10 and ZIP-3 mRNA levels. These changes are not observed with 10-6 M t-BHQ, which does not activate Nrf2-ARE signaling. Furthermore, t-BHQ exposure does not affect metal responsive element transcription, a cis element that is activated in response to intracellular free zinc accumulation. From these results, we believe that the transcription of ZnT-1, ZnT-3, ZnT-6, ZnT-10, and ZIP-3 is influenced by the Nrf2-ARE signal transduction pathway.

3-Hydro-2,2,5,6-tetramethylpyrazine: A novel inducer of zinc transporter-1 in HepG2 human hepatocellular carcinoma cells.

Dihydropyrazine compounds, including 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), are low-molecular-weight glycation products spontaneously generated in vivo and also ingested via food. Our preliminary study using microarray analysis demonstrated that DHP-3 induced zinc transporter-1 (ZnT-1) in HepG2 cells. It is well known that the increase of intracellular zinc is a sensitive stimulating factor for ZnT-1 protein induction; however, there is little information about the induction of ZnT-1 by low-molecular-weight chemical compounds. Here, we attempted to clarify the mechanism of ZnT-1 induction by DHP-3. A significant increase of ZnT-1 mRNA was observed 6h after DHP-3 treatment at concentrations over 0.5mM, and disappeared 24h after exposure. This induction pattern followed that of metal-responsive transcription factor 1 (MTF-1) mRNA, a metalloregulatory protein that serves as a major transcription factor of ZnT-1. Moreover, DHP-3 yielded transcriptional activation of MTF-1 in a luciferase reporter assay. The intracellular zinc content was unaffected by the compound; however, oxidative stress was observed in cells under the same conditions that activated the MTF-1 signaling pathway. These results suggest that DHP-3 is a novel ZnT-1 inducer and acts via activation of the MTF-1 signaling pathway. Additionally, the activation of MTF-1 by this compound likely occurs through oxidative stress.

Identification of dihydropyrazine-glutathione adducts.

Dihydropyrazines (DHPs) are glycation intermediates generated both in vivo and in food. DHPs can lead to the formation of a variety of different radical species, which can lead to DNA damage and enzyme inhibition. In addition, the presence of DHPs can lead to a decrease in cellular glutathione (GSH) levels, and induce the expression of antioxidant genes. In this study, the products resulting from the reaction of DHP with GSH have been analyzed in detail, with some of the products being separated by reversed-phase HPLC. The structures of the isolated DHP-GSH adducts were determined by FAB-MS and NMR analyses. These data suggested that the reaction of DHP with a thiol moiety could be involved in oxidative stress, because an increase in the amount of DHP-GSH adducts would result in a decrease in the cellular GSH levels.

Effect of dihydropyrazines on human hepatoma HepG2 cells: a comparative study using 2,3-dihydro-5,6-dimethylpyrazine and 3-hydro-2,2,5,6-tetramethylpyrazine.

Dihydropyrazines (DHPs) are glycation products that are nonenzymatically generated in vivo and in food. In this study, we compared the effects of 2,3-dihydro-5,6-dimethylpyrazine (DHP-1), a low toxicity DHP, and 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3), a high toxicity DHP on the redox indices in HepG2 cells. An apparent increase in intracellular hydrogen peroxide concentration was observed at 24 hr after 1 mM DHP-3 treatment. In addition, DHP-3 exposure significantly increased the mRNA levels of heme oxygenase-1 (HO-1) and glutamate cysteine ligase catalytic subunit (GCLC), which are stress-responsive genes, at 6 hr (HO-1 and GCLC), 12 hr (HO-1 and GCLC) and 24 hr (GCLC) after exposure. These indices, with the exception of the increase in GCLC mRNA after a 6 hr exposure, were not affected by treatment with 1 mM DHP-1. HO-1, GCLC, and nuclear factor erythroid 2-related factor 2 (Nrf2) protein levels also increased at 6 hr (Nrf2), 12 hr (Nrf2, HO-1 and GCLC) and 24 hr (GCLC) after DHP-3 treatment. The increase in HO-1 and Nrf2 protein levels were observed with lower concentration (0.5 mM) of DHP-3, and in agreement with this, antioxidant responsive element-luciferase reporter activity was significantly increased with exposure to at least 0.5 mM DHP-3. These results support our previous report establishing that oxidative stress is in part involved in the effects of DHP on mammalian cells. Additionally, our results suggest that the cell response to DHP-3 exposure was exerted via the activation of the Nrf2-ARE signal pathway.

Selenium-binding protein 1: its physiological function, dependence on aryl hydrocarbon receptors, and role in wasting syndrome by 2,3,7,8-tetrachlorodibenzo-p-dioxin.

BACKGROUND: Selenium-binding protein 1 (Selenbp1) is suggested to play a role in tumor suppression, and may be involved in the toxicity produced by dioxin, an activator of aryl hydrocarbon receptors (AhR). However, the mechanism or likelihood is largely unknown because of the limited information available about the physiological role of Selenbp1. METHODS: To address this issue, we generated Selenbp1-null [Selenbp1 (-/-)] mice, and examined the toxic effect of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in this mouse model. RESULTS: Selenbp1 (-/-) mice exhibited only a few differences from wild-type mice in their apparent phenotypes. However, a DNA microarray experiment showed that many genes including Notch1 and Cdk1, which are known to be enhanced in ovarian carcinoma, are also increased in the ovaries of Selenbp1 (-/-) mice. Based on the different responses to TCDD between C57BL/6J and DBA/2J strains of mice, the expression of Selenbp1 is suggested to be under the control of AhR. However, wasting syndrome by TCDD occurred equally in Selenbp1 (-/-) and (+/+) mice. CONCLUSIONS: The above pieces of evidence suggest that 1) Selenbp1 suppresses the expression of tumor-promoting genes although a reduction in Selenbp1 alone is not very serious as far as the animals are concerned; and 2) Selenbp1 induction by TCDD is neither a pre-requisite for toxicity nor a protective response for combating TCDD toxicity. GENERAL SIGNIFICANCE: Selenbp1 (-/-) mice exhibit little difference in their apparent phenotype and responsiveness to dioxin compared with the wild-type. This may be due to the compensation of Selenbp1 function by a closely-related protein, Selenbp2.

Possible involvement of glutathione balance disruption in dihydropyrazine-induced cytotoxicity on human hepatoma HepG2 cells.

Dihydropyrazines (DHPs), formed by nonenzymatic glycation, are known to exert various effects in vitro and in vivo, such as generation of radical species, DNA strand breakage, enzyme inhibition, and inhibition of bacterial growth. However, their effects on mammalian cells remain elusive. To address this issue, we investigated the effects of a range of DHP concentrations on human hepatoma HepG2 cells using 2,3-dihydro-5,6-dimethylpyrazine (DHP-1), 2,3-dihydro-2,5,6-trimethylpyrazine (DHP-2), and 3-hydro-2,2,5,6-tetramethylpyrazine (DHP-3) as model compounds. All of the tested compounds exerted cytotoxic activity against HepG2 cells in the range of 10 µM-1 mM, and significantly so at the highest concentration. DHP-3 was the most effective drug, and it also caused a significant decrease in the ratio of intracellular reduced and oxidized glutathione (GSH/GSSG). In addition, the cytotoxic effect of DHP-3, but not DHP-1 and DHP-2, was enhanced by the inhibition of GSH biosynthesis using 100 µM l-buthionine-(S,R)-sulfoximine (BSO). From these results, it is suggested that the mechanisms of cytotoxicity exerted by DHP-3 are distinct from those exerted DHP-1 and DHP-2. In addition, it is possible that the disruption of intracellular glutathione balance induced by DHP-3 is related to its effect on HepG2 cells.