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Both lipid accumulation and oxidative stress are major pathologic contributors to the development of hepatic steatosis. Treatment with molybdate reduces hepatic levels of lipids in diabetic rats. Potential activities of molybdate as an antioxidant have also been demonstrated in various animal models. In the present study, we evaluated the effects of sodium molybdate dihydrate (SM) on hepatic steatosis and associated disturbances in a widely used mouse model of the metabolic disease. Male C57Bl/6 mice at 10 weeks of age were fed a diet deficient in methionine and choline (MCD) and bottled water containing SM for four weeks. The SM treatment markedly attenuated MCD-induced accumulation of lipids, mainly triglycerides, in the liver. Lipid catabolic autophagic pathways were activated by SM in the MCD-fed mouse livers, as evidenced by a decreased level of p62 expression. MCD-induced oxidative damage, such as lipid and protein oxidation, was also alleviated by SM in the liver. However, the level of MCD-induced hepatocellular damage was not affected by SM. Taken together, these findings suggest that molybdate can be used in the treatment and prevention of hepatic steatosis without inducing adverse effects in the liver. To the best of our knowledge, this is the first experimental study to investigate the effects of molybdate in non-alcoholic fatty liver disease, and also the first that demonstrates molybdate-induced autophagy.

// Seungwoo Lee 1 , Soohee Kim 1 , Seungwoo Hwang 2 , Nathan J. Cherrington 3 and Doug-Young Ryu 1 1 BK21 Plus Program for Creative Veterinary Science Research, Research Institute for Veterinary Science and College of Veterinary Medicine, Seoul National University, Gwanak-gu, Seoul 08826, Republic of Korea 2 Korean Bioinformation Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Republic of Korea 3 College of Pharmacy, University of Arizona, Tucson, AZ 85721, U.S.A. Correspondence to: Doug-Young Ryu, email: dyryu@snu.ac.kr Keywords: nonalcoholic fatty liver disease, endoplasmic reticulum stress, apoptosis, autophagy Received: November 25, 2016 Accepted: June 04, 2017 Published: June 28, 2017 ABSTRACT Nonalcoholic fatty liver disease (NAFLD) is categorized into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) and has emerged as a risk factor for more critical clinical conditions. However, the underlying mechanisms of NAFLD pathogenesis are not fully understood. In this study, expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis and autophagy were analyzed in normal, NAFL and NASH human livers by western blotting. Levels of some ER stress-transducing transcription factors, including cleaved activating transcription factor 6, were higher in NASH than in the normal tissues. However, the expression of a majority of the ER chaperones and foldases analyzed, including glucose-regulated protein 78 and ER protein 44, was lower in NASH than in the normal tissues. Levels of apoptosis markers, such as cleaved poly (ADP-ribose) polymerase, were also lower in NASH tissues, in which expression of some B-cell lymphoma-2 family proteins was up- or down-regulated compared to the normal tissues. The level of the autophagy substrate p62 was not different in NASH and normal tissues, although some autophagy regulators were up- or down-regulated in the NASH tissues compared to the normal tissues. Levels of most of the proteins analyzed in NAFL tissues were either similar to those in one of the other two types, NASH and normal, or were somewhere in between. Together, these findings suggest that regulation of certain important tissues processes involved in protein quality control and cell survival were broadly compromised in the NAFLD tissues.

(Received: October 27, 2015; Revised: December 16, 2015; Accepted: December 24, 2015)Abstract : Exercise is one of the most common stressors in horses. Although various physiological parameters suchas cortisol respond to exercise, there is no reliable parameter for the measurement of exercise-induced stress insport horses. This study was performed to discover a new biomarker with high sensitivity for exercise-induced stress.The expression of fos mRNA was increased more than 10-fold in horse blood samples collected after an hour ofexercise, as compared with before the exercise. The plasma cortisol levels were also increased after the exercise,but only by about two-fold. The fos mRNA levels were well-correlated with plasma cortisol concentrations. Thesefindings suggest that fos mRNA expression in blood may be useful for the measurement of exercise-induced stressin horses.Keywords : biomarker, exercise, fos, horse, stress

Arsenic is an environmental pollutant, and its liver toxicity has long been recognized. The effect of arsenic on liver protein expression was analyzed using a proteomic approach in monkeys. Monkeys were orally administered sodium arsenite (SA) for 28 days. As shown by 2D-PAGE in combination with MS, the expression levels of 16 proteins were quantitatively changed in SA-treated monkey livers compared to control-treated monkey livers. Specifically, the levels of two proteins, mortalin and tubulin beta chain, were increased, and 14 were decreased, including plastin-3, cystathionine-beta-synthase, selenium-binding protein 1, annexin A6, alpha-enolase, phosphoenolpyruvate carboxykinase-M, erlin-2, and arginase-1. In view of their functional roles, differential expression of these proteins may contribute to arsenic-induced liver toxicity, including cell death and carcinogenesis. Among the 16 identified proteins, four were selected for validation by Western blot and immunohistochemistry. Additional Western blot analyses indicated arsenic-induced dysregulation of oxidative stress related, genotoxicity-related, and glucose metabolism related proteins in livers from SA-treated animals. Many changes in the abundance of toxicity-related proteins were also demonstrated in SA-treated human hepatoma cells. These data on the arsenic-induced regulation of proteins with critical roles may help elucidate the specific mechanisms underlying arsenic-induced liver toxicity.

In this study, we investigated global gene expression in primary rat hepatocytes treated with three active hepatocarcinogens (prednisolone, dehydroepiandrosterone and monocrotaline), three inactive hepatocarcinogens (hydrocortisone, glycyrrhetinic acid and lithocholic acid) and two unclassified chemicals (griseofulvin (possibly active) and prednisone (possibly inactive)). At 48 h after treatment with these eight chemicals, cells were harvested for RNA extraction. Global gene expression analyses were conducted using oligonucleotide microarrays to detect genes whose expression was altered. Differentially expressed genes (DEG) analysis, principal component analysis (PCA), gene set enrichment analysis (GSEA) and KEGG pathway analysis were also conducted. Seven-hundred and sixty genes whose expression was altered >1.2-fold (P

To clarify the role of stem cells in hepatocarcino- genesis, the expression of epithelial cell adhesion molecule (EpCAM) and proliferating cell nuclear antigen (PCNA) was investigated in mouse hepatic tumors and embryonic cell lineages. Ten ICR mice were treated with diethylnitrosamine (DEN) at 14 days of age and sacrificed at 36 weeks subsequent to DEN treatment to obtain the hepatic tumors. Mouse embry- onic stem cells, hepatic progenitor cells and hepatocyte‑like cells, representing 0, 22 and 40 days of differentiation, respec- tively, were treated in vitro with DEN at four doses (0, 1, 5 and 15 mM; G1, G2, G3 and G4, respectively) for 24 h and RNA was isolated. A total of 71 hepatic tumors were obtained from the DEN-treated mice. EpCAM expression was increased mainly in hepatic tumor cells, although it was also detected in the surrounding visually normal cells. Double staining showed that EpCAM and PCNA were co-expressed in numerous tumor cells. In vitro, EpCAM expression was significantly different for G4 at day 0 (P

Silver nanoparticles (AgNPs) have emerged as an important class of nanomaterials for a wide range of industrial and medical applications. However, the unique properties of AgNPs could potentially lead to unexpected hazards to both human health and the well being of the environment. Possible mechanisms of AgNP-induced toxicity include the stimulation of oxidative stress, genotoxicity and apoptosis. In this study, a number of previous studies are therefore summarized that demonstrate oxidative stress-, genotoxicity- and apoptosis-related changes brought about by AgNPs in cultured cells and animal tissues. The physicochemical properties of AgNPs that are involved in encouraging such changes are also discussed.

Genomic analysis in the local lymph node assays (LLNAs) is useful for assessing skin sensitization of chemicals and providing insights into mechanisms of sensitization. In this study, we collected 1406 genes from previous microarray findings, validated changes in their expression by RT-PCR analysis in local lymph nodes draining skin exposed to different sensitizers, and interpreted their biological function through pathway-based genomic analysis, in which 468 genes were identified as being in the KEGG pathway database. The top-ranked functions (P

－ While it’s been shown that arsenic impairs male reproductive function, it remains unclear whether the mechanism involves an effect on testosterone (T) production. We examined plasma T and luteinizing hormone (LH) levels in mice given water containing either 20 or 40 mg/L sodium arsenite (SA). The plasma T levels were lower in SA-treated mice than in controls and correlated well with testicular T levels within individuals. However, SA treatment did not significantly affect plasma LH levels. The ratio of plasma T to LH was reduced by the treatment with 40 mg/L SA. These results suggest arsenic-induced defect in testicular testosterone production in mice.Keywords: Arsenic, Testis, Testosterone, Mouse INTRODUCTION The individual testosterone (T) levels vary markedly, which has been previously observed in mice (Bartke et al ., 1973; Bartke and Dalterio, 1975; Klomberg et al ., 2002). Plasma T levels were ranged from 0.24 to 37.7 ng/ml (n=76), and from 0.19 to 60.1 ng/ml (n=73) in adult male mice (Klomberg

Exercise capacity is a valuable trait in horses, and it has been used as a horse selection criterion. Although exercise affects molecular homeostasis and adaptation in horses, the mechanisms underlying these effects are not fully described. This study was carried out to identify changes in the blood profiles of microRNAs (miRNAs) and mRNAs induced by exercise in horse leukocytes. Total RNAs isolated from the peripheral blood leukocytes of four Warmblood horses before and after exercise were subjected to next-generation sequencing (NGS) and microarray analyses to determine the miRNA and mRNA expression profiles, respectively. The expressions of 6 miRNAs, including 4 known and 2 novel miRNAs, were altered by exercise. The predicted target genes of the differentially expressed miRNAs identified by NGS were matched to the exercise-induced mRNAs determined by microarray analysis. Five genes (LOC100050849, LOC100054517, KHDRBS3, LOC100053996, and LOC100062720) from the microarray analysis were matched to the predicted target genes of the 6 miRNAs. The subset of mRNAs and miRNAs affected by exercise in peripheral blood leukocytes may be useful in elucidating the molecular mechanisms of exercise-associated physiology in horses.

Cytotoxicity induced by silver nanoparticles (AgNPs) and the role that oxidative stress plays in this process were demonstrated in human hepatoma cells. Toxicity induced by silver (Ag(+)) ions was studied in parallel using AgNO(3) as the Ag(+) ion source. Using cation exchange treatment, we confirmed that the AgNP solution contained a negligible amount of free Ag(+) ions. Metal-responsive metallothionein 1b (MT1b) mRNA expression was not induced in AgNP-treated cells, while it was induced in AgNO(3)-treated cells. These results indicate that AgNP-treated cells have limited exposure to Ag(+) ions, despite the potential release of Ag(+) ions from AgNPs in cell culture. AgNPs agglomerated in the cytoplasm and nuclei of treated cells, and induced intracellular oxidative stress. AgNPs exhibited cytotoxicity with a potency comparable to that of Ag(+) ions in in vitro cytotoxicity assays. However, the toxicity of AgNPs was prevented by use of the antioxidant N-acetylcysteine, and AgNP-induced DNA damage was also prevented by N-acetylcysteine. AgNO(3) treatment induced oxidative stress-related glutathione peroxidase 1 (GPx1) and catalase expression to a greater extent than AgNP exposure, but treatment with AgNO(3) and AgNPs induced comparable superoxide dismutase 1 (SOD1) expression levels. Our findings suggest that AgNP cytotoxicity is primarily the result of oxidative stress and is independent of the toxicity of Ag(+) ions.

Background It has been suggested that the altered iron metabolism in liver tumors, characterized by the iron-deficient phenotype, is of importance for tumor growth. Aim This study was performed to elucidate the mechanisms underlying iron deficiency in liver tumors by examining how the liver tumor development affects the expression of iron metabolism-related genes. Methods Iron metabolism reference values were analyzed in the sera of diethylnitrosamine-induced hepatocellular adenoma-bearing mice. Expression of iron metabolism-related genes was analyzed in adenomas and surrounding non-tumor tissues, and a subgroup of adenoma-bearing mice loaded with iron 72 h before sacrifice. Results Iron content of the adenoma tissues was 2.0–2.5-fold lower compared to surrounding and age-matched control tissues. There was no significant difference in serum iron levels between the adenoma-bearing and control mice, while the adenoma-bearing mice exhibited a 2.4-fold lower level of serum transferrin saturation. Expression of iron metabolism-related genes was dysregulated in the adenomas. Iron loading affected protein expression similarly in the adenomas and surrounding tissues suggesting that iron-responsive regulation of the proteins was not impaired. However, the mRNA expression for ceruloplasmin and divalent metal transporter 1 (DMT1) IRE(+) in the adenomas was altered independently of iron status, and the dysregulation may contribute to diminished iron content. Conclusion These findings suggest that diethylnitrosamine-induced liver adenoma-bearing mice have abnormal iron metabolism and that dysregulation of iron metabolism-related genes contributes to iron deficiency in the adenomas.

Apx toxins have been identified as important virulence factors of Actinobacillus pleuropneumoniae, the etiologic agent of porcine pleuropneumonia. In some A. pleuropneumoniae serotypes, Apx toxins are secreted by the cell membrane proteins encoded by apxIIIB and apxIIID genes. In an effort to develop a live vaccine strain against A. pleuropneumoniae, we inactivated the apxIIIB and apxIIID genes in A. pleuropneumoniae 1536, a serotype 2 strain, resulting in the DeltaapxIIIB/DapxIIID mutant strain (1536DeltaBDeltaD). Immunization of pigs with live 1536DeltaBDeltaD A. pleuropneumoniae conferred protection against homologous challenge with wild-type A. pleuropneumoniae 1536. Thus, impaired Apx toxin secretion may decrease the virulence of A. pleuropneumoniae and may be an effective strategy for the development of a live-attenuated A. pleuropneumoniae vaccine.

This research was supported by a research grant(07141KFDA682) from Korea Food and Drug Administration in 2007.

As the applications of industrial nanoparticles are being developed, the concerns on the environmental health are increasing. Cytotoxicities of titanium dioxide nanoparticles of different concentrations (5, 10, 20 and 40 microg/ml) were evaluated in this study using a cultured human bronchial epithelial cell line, BEAS-2B. Exposure of the cultured cells to nanoparticles led to cell death, reactive oxygen species (ROS) increase, reduced glutathione (GSH) decrease, and the induction of oxidative stress-related genes such as heme oxygenase-1, thioredoxin reductase, glutathione-S-transferase, catalase, and a hypoxia inducible gene. The ROS increase by titanium dioxide nanoparticles triggered the activation of cytosolic caspase-3 and chromatin condensation, which means that titanium dioxide nanoparticles exert cytotoxicity by an apoptotic process. Furthermore, the expressions of inflammation-related genes such as interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-8 (IL-8), TNF-a, and C-X-C motif ligand 2 (CXCL2) were also elevated. The induction of IL-8 by titanium dioxide nanoparticles was inhibited by the pre-treatment with SB203580 and PD98059, which means that the IL-8 was induced through p38 mitogen-activated protein kinase (MAPK) pathway and/or extracellular signal (ERK) pathway. Uptake of the nanoparticles into the cultured cells was observed and titanium dioxide nanoparticles seemed to penetrate into the cytoplasm and locate in the peri-region of the nucleus as aggregated particles, which may induce direct interactions between the particles and cellular molecules, to cause adverse biological responses.

There has been some concern that certain non-sensitizing irritants may yield false positive results in the murine local lymph node assay (LLNA). This study compared gene expression profiles in lymph nodes draining skin following exposure to sensitizers and irritants, to identify gene transcripts that could distinguish sensitizers from irritants. After treating CBA/N mouse ears for 3 days with the sensitizers 1-chloro-2,4-dinitrobenzene, 2-phenyl-4-ethoxymethylene-5-oxazolone, or toluene-2,4-diisocyanate or the non-sensitizing irritants croton oil or nonanoic acid, auricular lymph nodes and ear tissues were excised. Sensitizer-induced changes in parameters such as ear thickness, lymph node weight, and cell count also occurred in irritant-treated mouse tissues. However, gene transcripts such as Ifi27, Il12rb1, Ifng, and Zbp1, which are related to T-cell activation, were shown by gene expression microarrays and real-time RT-PCR analyses to be up-regulated in auricular lymph nodes by sensitizers exclusively. These findings suggest that gene expression analysis may enable distinction between sensitizing chemicals and non-sensitizing irritants.

In transgenic zebrafish (Danio rerio), green fluorescent protein (GFP) is a promising marker for environmental pollutants. In using GFP, one of the obstacles which we faced was how to compare toxicity among different toxicants or among a specific toxicant in different model species with the intensity of GFP expression. Using a fluorescence detection method, we first validated our method for estimating the amount of GFP fluorescence present in transgenic fish, which we used as an indicator of developmental toxicity caused by the well-known toxicant, arsenite. To this end, we developed mosaic transgenic zebrafish with the human heat shock response element (HSE) fused to the enhanced GFP (EGFP) reporter gene to indicate exposure to arsenite. We confirmed that EGFP expression sites correlate with gross morphological disruption caused by arsenite exposure. Arsenite (300.0 microM) caused stronger EGFP fluorescence intensity and quantity than 50.0 microM and 10.0 microM arsenite in our transgenic zebrafish. Furthermore, arsenite-induced apoptosis was demonstrated by TUNEL assay. Apoptosis was inhibited by the antioxidant, N-acetyl-cystein (NAC) in this transgenic zebrafish. The distribution of TUNEL-positive cells in embryonic tissues was correlated with the sites of arsenite toxicity and EGFP expression. The EGFP values quantified using the standard curve equation from the known GFP quantity were consistent with the arsenite-induced EGFP expression pattern and arsenite concentration, indicating that this technique can be a reliable and applicable measurement. In conclusion, we propose that fluorescence-based EGFP quantification in transgenic fish containing the hsp70 promoter-EGFP reporter-gene construct is a useful indicator of development toxicity caused by arsenite.

This study evaluated oxidative stress-induced apoptosis as a possible mechanism of arsenite toxicity in zebrafish liver cell line (ZFL cells). The heat shock protein 70 (HSP70), a chaperone protein, appears to provide protection against oxidative stress and apoptosis. Using the MTT assay, we demonstrated that survival of ZFL cells treated with arsenite for 24h decreased in a dose-dependent manner. The possible mechanisms that promote the cytotoxicity of arsenite were addressed. Cell viability assays revealed that arsenite caused a dose-dependent increase in cell death, and pretreatment of the ZFL cells with antioxidants blunted these effects. Antioxidants such as N-acetyl-cysteine (NAC, 5 mM) and dithiothreitol (DTT, 80 microM) significantly prevented ZFL cells from arsenite-induced death. Nuclear staining was performed using 1 microg/ml Hoechst, and cells were analyzed with a fluorescent microscope. Arsenite (30 microM) induced massive apoptosis that was identified by morphology and condensation and fragmentation of the nuclei of the ZFL cells. Pretreatment with NAC or DTT before arsenite insult effectively protected the cells against oxidative stress-induced apoptosis from the arsenite. Using a transfected human hsp 70 promoter-enhanced green fluorescent protein (EGFP) reporter, pHhsp70-EGFP, the induction of HSP70 against oxidative stress-induced apoptosis by arsenite was observed. The induction of HSP70 by arsenite increased in a dose-dependent manner, and pretreatment of transfected ZFL cells with NAC or DTT before arsenite insult reduced EGFP expression. Taken together, our results provide evidence that stimulation of the heat shock response is a sensitive biomarker of arsenic exposure and that arsenite causes oxidative stress-induced apoptosis in ZFL cells.

Oxidative stress has been suggested to be a major cause of male reproductive failure. Here, we investigated whether arsenic, which impairs male reproductive functions in rodent models, acts by inducing oxidative stress. Male 8-week-old ICR mice were given drinking water containing 20 or 40 mg/l sodium arsenite with or without 0.75 or 1.5 g/l of the antioxidant ascorbic acid for 5 weeks. The arsenic-treated mice showed decreased epididymidal sperm counts and testicular weights compared to untreated mice. These effects were reversed in mice that were co-treated with ascorbic acid. Similarly, arsenic treatment lowered the activities of testicular 3beta-hydroxysteroid dehydrogenase (HSD) and 17beta-HSD, which play important roles in steroidogenesis, and this was reversed by co-treatment with ascorbic acid. The testicles of arsenic-treated mice had decreased glutathione (GSH) levels (which correlate inversely with the degree of cellular oxidative stress) and elevated levels of protein carbonyl (a marker of oxidative damage to tissue proteins). Ascorbic acid co-treatment reversed both of these effects. Thus, ascorbic acid blocks both the adverse effects of arsenic on male reproductive functions and the arsenic-induced testicular oxidative changes. These observations support the notion that arsenic impairs male reproductive function by inducing oxidative stress.

Cytochrome P450 1A1 (CYP1A1) is a heme-containing enzyme involved in metabolism of xenobiotics. CYP1A1 containing a Gly45Asp substitution has not yet been characterized. Escherichia coli expressing the Gly45Asp variant, as well as the purified variant protein, had lower CYP (i.e., holoenzyme) contents than their wild-type (WT) equivalents. The purified variant protein had reduced heme contents compared with their WT equivalents. Enhanced supplementation of a heme precursor during culture did not increase CYP content in E. coli expressing the variant, but did for the WT. Substitution of Gly45 with other residues, especially those having large side chains, decreased CYP contents of E. coli expressing the variants to a considerable extent. A 3D structure of CYP1A1 indicates that Gly45, along with other residues of the PR region, interacts with Arg77 of β- strand 1-1, which indirectly interacts with heme. Substitution analyses suggest the importance of residues of the PR region and Arg77 in holoenzyme expression. E. coli membrane and mammalian microsomes expressing the Gly45Asp variant, as well as the purified variant protein, had reduced ethoxyresorufin O-dealkylation activities, compared with the WT equivalents. These findings suggest the Gly45Asp substitution results in a structural disturbance of CYP1A1, reducing its holoenzyme formation and catalytic activity.

Cytochrome P450 (CYP) 1A1 is a heme-containing enzyme involved in detoxification of hydrophobic pollutants. Its Ala62Pro variant has been identified previously. Ala62 is located in α-helix A of CYP1A1. Residues such as Pro and Gly are α-helix breakers. In this study, the Ala62Pro variant was characterized using heterologous expression. E. coli expressing the Ala62Pro variant, and the purified variant protein, had lower CYP (i.e. holoenzyme) contents than their wild-type (WT) equivalents. The CYP variant from E. coli and mammalian cells exhibited lower 7-ethoxyresorufin O-dealkylation (EROD) and benzo[a]pyrene hydroxylation activities than the WT. Enhanced supplementation of a heme precursor during E. coli culture did not increase CYP content in E. coli expressing the variant, but did for the WT. As for Ala62Pro, E. coli expressing an Ala62Gly variant had a lower CYP content than the WT counterpart, but substitution of Ala62 with α-helix-compatible residues such as Ser and Val partially recovered the level of CYP produced. Microsomes from mammalian cells expressing Ala62Pro and Ala62Gly variants exhibited lower EROD activities than those expressing the WT or Ala62Val variant. A region harboring α-helix A has interactions with another region containing heme-interacting residues. Site-directed mutagenesis analyses suggest the importance of interactions between the two regions on holoenzyme expression. Together, these findings suggest that the Ala62Pro substitution leads to changes in protein characteristics and function of CYP1A1 via structural disturbance of the region where the residue is located.

Heat shock proteins (HSPs) play a central role in cell protection and repair upon stresses, such as that caused by heat and heavy metals. Copper sulfate inducibility of a pHhsp70 construct expressing the enhanced green fluorescent protein (EGFP) gene under the control of the exogenous human hsp70 promoter was tested in transfected CHSE 214 cells and transgenic zebrafish (Danio rerio). We developed a transient expression system, using mosaically transgenic zebrafish, which allows rapid analysis of transgenic expression. Transfected CHSE 214 cells which had been exposed to 250 nM and 2.5 microM copper sulfate for up to 24h showed increased EGFP expression in a dose-dependent manner. The 1.5 microM copper sulfate caused stronger EGFP fluorescence than the 1.0 microM copper sulfate in transgenic zebrafish. Most of the expression was spotty and was detected in the gills, dorsal and ventral retina, myotubes of the trunk, and skin epithelium. Transgenic zebrafish exposed to copper sulfate exhibited gross dysmorphogenesis, edema and trunk abnormalities, such as spinal lordosis, in vertebral development 5 days after fertilization. This transgenic zebrafish system was sensitive enough to detect copper sulfate at doses below the median lethal concentration (the LC50 was calculated to be 1.2 microM (95% confidence interval of 0.6-1.9 microM)). These results indicate that zebrafish could be useful transgenic biosensor systems for the detection of xenobiotic toxicants in the environment.

Iron (Fe) plays essential roles in biological processes, whereas cadmium (Cd) is a toxic and non-essential metal. Two metal transporters, divalent metal transporter 1 (DMT1) and metal transporter protein 1 (MTP1), are responsible for Fe transport in mammals. Here, we studied the effect of dietary Fe on the expression of these metal transporters in peripheral tissues, and the uptake by these tissues of Cd. Mice were fed an Fe-sufficient (FeS: 120 mg Fe/kg) or Fe-deficient (FeD: 2-6 mg Fe/kg) diet for 4 weeks. The total Fe levels in the body were evaluated by measuring tissue Fe concentrations. Tissue Cd concentrations were determined 24 h after the mice received a single oral dose of Cd. Animals fed a FeD diet showed depletion of body Fe levels and accumulated 2.8-fold higher levels of Cd than the FeS group. Quantitative real time RT-PCR revealed that whereas DMT1 and MTP1 were both ubiquitously expressed in all FeS peripheral tissues studied, DMT1 was highly expressed in brain, kidney, and testis, whereas MTP1 was highly expressed in liver and spleen. Depletion of the body Fe stores dramatically upregulated DMT1 and MTP1 mRNA expression in the duodenum as well as moderately upregulating their expression in several other peripheral tissues. The iron response element positive isoform of DMT1 was the most prominently upregulated isoform in the duodenum. Thus, DMT1 and MTP1 may play an important role in not only maintaining Fe levels but also facilitating the accumulation of Cd in the body of mammals.

A plasma enhanced chemical vapor deposition (PECVD) method using plasma polymerized ethylenediamine (PPEDA) was employed to coat glass slides with amine functional groups. Fluorescein isothiocyanate (FITC) labeling indicated that the density of amine functional groups on these coated slides was affected by PECVD conditions, particularly deposition time. The PPEDA-coated slides were used for the development of DNA arrays. 5′-Amino-modified oligodeoxyribonucleotides (ODNs) and cDNA strands were immobilized onto PPEDA-coated slides and used for hybridization to labeled target strands. In addition, patterning of amine groups, which is a very useful technique for preparing high-density DNA arrays, was achieved by selectively depositing PPEDA films on specific areas of a glass surface. These results demonstrate that the formation of amine groups on glass slides by PECVD is an excellent tool for DNA array technology.

A transgenic zebrafish strain was generated expressing the aryl hydrocarbon receptor (AhR)-regulated green fluorescent protein (GFP) reporter gene. Following exposure to β-naphthoflavone (βNF), the transgenic fish exhibited up-regulation of GFP in the face and vertebrae compared to vehicle controls. βNF-exposed fish exhibited gross dysmorphogenesis in vertebral development by 5 days after fertilization.

A microRNA (miRNA) is a small non-coding RNA molecule, which functions in transcriptional and post-transcriptional regulation of gene expression. It has been reported that the expression of miRNAs ...

The role of microRNAs (miRNAs) as a post-transcriptional gene regulator has been elucidated in a broad range of organisms including domestic animals. Characterization of miRNAs in normal tissues is an important step to investigate the functions of miRNAs in various physiological and pathological conditions. Using Illumina Next Generation Sequencing (NGS) technology, we identified a total of 292 known and 329 novel miRNAs in normal horse tissues including skeletal muscle, colon and liver. Distinct sets of miRNAs were differentially expressed in a tissue-specific manner. The miRNA genes were distributed across all the chromosomes except chromosomes 29 and 31 in the horse reference genome. In some chromosomes, multiple miRNAs were clustered and considered to be polycistronic transcript. A base composition analysis showed that equine miRNAs had a higher frequency of A+U than G+C. Furthermore, U tended to be more frequent at the 5′ end of miRNA sequences. This is the first experimental study that identifies and characterizes the global miRNA expression profile in normal horse tissues. The present study enriches the horse miRNA database and provides useful information for further research dissecting biological functions of miRNAs in horse.

Endocrine disrupters are exogenous compounds thought to mimic the action of estrogen or other hormones and influence endocrine activity in the body (Juberg, 2000). These chemicals have adverse effects not only in the reproductive system but also in the central nervous system during development and throughout life. Polychlorinated biphenyls (PCBs) are a class of environmentally persistent and widespread halogenated hydrocarbons. It has been reported that PCBs are potential neurotoxicants. Endosulfan is an organochlorine insecticide that is extensively used to control pests in vegetables, cotton, and fruits. To determine the effect of 2, 2', 4, 4', 5, 5',-hexachlorobiphenyl(2, 4, 5-HCB) and endosulfan on embryo nervous system, we isolated neural stem cells from rat brain at embryonic day 17. Isolated neural stem cells showed pluripotenty. Stem cells could differentiate into neurons and glia. Neurite formation in endosulfan and 2, 4, 5-HCB treated cells. And it appeared to be decreased as compared with that in untreated cells. In order to know the neuro-toxic mechanisms of 2, 4, 5-HCB and endosulfan in neuronal stem cells, we investigated mitogen-activated protein kinase activity (MAPK) and gap junctional intercellular communication (GJIC). Endosulfan decreased the MAPK activity in dose dependent manner. Endosulfan and 2, 4, 5-HCB inhibited GJIC compared to the untreated cell by scrape loading dye transfer (SL/DT). 2, 4, 5-HCB and endosulfan decreased the expression of connexin 43 in dose dependent manner. These results indicated that 2, 4, 5-HCB and endosulfan may inhibit differentiation and proliferation of neural stem cells and gap junctional intercellular communication which play a crucial role in the maintenance of cellular homeostasis.

2-Amino-1-methyl-6-phenylimidazo(4,5-b)pyridine (PhIP), a compound found in cooked meat, is a mammary gland carcinogen in female Sprague-Dawley rats. PhIP-induced rat mammary gland carcinomas were examined for mutations in several genes (exons) known to regulate cell growth and apoptosis, including p53 (4–8), p21Waf1 (coding region), Apc (14, 15), B-catenin (3), E-cadherin (9,13,15), Bcl-x (coding region), Bax (3), IGFIIR (28), and TGFBIIR (3). DNA from 30 carcinomas was examined by single-strand conformation polymorphism analysis, but no mutations were detected in these genes or gene regions. DNA from carcinomas and matching normal tissue were further screened for allelic imbalance by using a polymerase chain reaction–based approach with primers to known microsatellite regions located throughout the rat genome. Of 53 markers examined, 12 revealed allelic imbalance. Microsatellite instability (MSI) was detected at two markers, one on chromosome 4 and one on chromosome 6. Sixty-five percent and 96% of all carcinomas examined (N=23) showed MSI at these loci on chromosomes 4 and 6, respectively, supporting the notion that MSI plays a role in PhIP-induced mammary carcinogenesis. Loss of heterozygosity (LOH), an indication of a possible tumor suppressor gene, was observed at 10 markers distributed on chromosomes 3, 10, 11, 14, and X. The frequency of LOH at these markers was 75–94%, supporting that the regions of allelic imbalance were largely similar for the PhIP-induced carcinomas examined in this study. When PhIP-induced carcinomas from rats placed on high-fat and low-fat diet were compared, no unique regions of allelic imbalance or statistical differences in the frequency of allelic imbalance were observed. Therefore, the high-fat diet, known to be a promoter of PhIP-induced rat mammary carcinogenesis, did not appear to influence allelic imbalance in the carcinomas. Interestingly, 7,12-dimethylbenz[a]-anthracene–induced mammary carcinomas did not show allelic imbalance at 11 of the 12 loci that showed allelic imbalance in PhIP-induced carcinomas. These findings suggest that distinct chemical carcinogens induce different patterns of allelic imbalance during rat mammary carcinogenesis. Since several of the known genes involved in carcinogenesis did not harbor mutations in PhIP-induced carcinomas, further studies are needed to clarify the critical genes involved in PhIP-induced mammary carcinogenesis and to determine whether regions of LOH harbor potentially novel tumor suppressor genes involved in this disease. Mol. Carcinog. 27:76–83, 2000. © 2000 Wiley-Liss, Inc.

Background: Infection by Mycoplasma hyopneumoniae and Actinobacillus pleuropneumoniae, either alone or together, causes serious respiratory diseases in pigs. Results: To develop an efficient multi-disease subunit vaccine against these pathogens, we produced a chimeric protein called Ap97, which comprises a deletion derivative of the N-terminal region of the A. pleuropneumoniae ApxIII toxin (ApxN) and the R1 and R2 repeats of M. hyopneumoniae P97 adhesin (P97C), using an E. coli expression system. The levels of both IgG1 and IgG2a isotypes specific for ApxN and P97C in the sera of Ap97-immunized mice increased, and Ap97 induced the secretion of IL-4 and IFN-gamma. by mouse splenocytes. Antisera from mice and pigs immunized with Ap97 readily reacted with both native ApxIII and P97 proteins. In addition, immunization with the Ap97 vaccine effectively protected pigs against challenge with both pathogens. Conclusions: These findings suggest that Ap97 confers immunogenicity, and is an effective vaccine that protects pigs against infection by M. hyopneumoniae and A. pleuropneumoniae.

Objective—To identify a subantimicrobial dose of doxycycline hyclate (SDD) and for the treatment of periodontitis in dogs. Animals—20 healthy Beagles for measurement of serum doxycycline concentration and 15 Beagles with periodontitis for evaluation of the efficacy of the SDD. Procedures—5 dogs each received doxycycline hyclate PO at a dose of 1, 2, 3, or 5 mg/kg. Blood samples were collected before and after administration, and serum concentrations of doxycycline were measured via high-performance liquid chromatography. Mean serum doxycycline concentrations were calculated, and SDDs were identified. In a separate trial, the identified SDDs (1 or 2 mg/kg) were administered PO once a day for 1 month to dogs with periodontitis (n = 5/group) and a control group (5) was fed vehicle only during the same period. Degree of gingival attachment and bleeding on probing (present or absent) were recorded. Gingival samples were collected before and after the 1-month period from the same anatomic sites. Degree of matrix metalloproteinase inhibition in gingival samples was determined via gelatin zymography and compared among treatment groups. Results—Mean serum doxycycline concentrations in healthy dogs that received 1 or 2 mg of doxycycline/kg were consistently significantly lower than the minimal inhibitory doxycycline concentration for treatment of periodontitis throughout the 24-hour posttreatment period. Zymographic intensities were lower in dogs given 1 and 2 mg/kg than in the control dogs, and the degree of gingival attachment and bleeding significantly improved in dogs given 2 mg/kg, compared with in the control dogs and dogs given 1 mg of doxycycline/kg. Conclusions and Clinical Relevance—A doxycycline dosage of 2 mg/kg daily appeared to be an appropriate subantimicrobial regimen for dogs with periodontitis. Furthermore, this dosage may be suitable for long-term treatment of gelatinolytic inflammatory diseases such as periodontitis in this species.

MicroRNAs (miRNAs) in blood plasma are stable under high levels of ribonuclease activity and could function in tissue-to-tissue communication, suggesting that they may have distinctive structural characteristics compared with non-circulating miRNAs. In this study, the expression of miRNAs in horse plasma and their characteristic nucleotide composition were examined and compared with non-plasma miRNAs. Highly expressed plasma miRNA species were not part of the abundant group of miRNAs in non-plasma tissues, except for the eca-let-7 family. eca-miR-486-5p, -92a, and -21 were among the most abundant plasma miRNAs, and their human orthologs also belong to the most abundant group of miRNAs in human plasma. Uracil and guanine were the most common nucleotides of both plasma and non-plasma miRNAs. Cytosine was the least common in plasma and non-plasma miRNAs, although levels were higher in plasma miRNAs. Plasma miRNAs also showed higher expression levels of miRNAs containing adenine and cytosine repeats, compared with non-plasma miRNAs. These observations indicate that miRNAs in the plasma have a unique nucleotide composition.

Mercury (Hg) is widely distributed in the environment and oral exposure is a main route in the general population. In this study, we estimated the dietary intake of Hg and its relationship with blood Hg levels in Korean adults. The study subjects were recruited from three different districts (rural: 189, coastal: 208 and urban: 184). We used a general questionnaire to collect information about demographic factors, lifestyles and diet. Dietary habits were studied using the 24-h recall method. The estimation of Hg intake was performed using the database of Hg contents in 128 Korean foods based on the previous studies. Blood Hg was analyzed using Direct Mercury Analyzer with the gold-amalgam method. Daily intake of Hg by diet was estimated at 13.57 μg (0.22 μg/kg body weight). The geometric mean Hg concentration in whole blood was 3.92 μg/L. Blood Hg level and Hg intake by diet was higher in coastal areas than in urban or rural areas, respectively. Blood Hg level correlated with the intake of Hg consumed from diet. Seafood was highly responsible and account for 75.6% of total dietary Hg intake. In this study, blood Hg concentrations were found to be significantly affected by sex, age, individual lifestyles and especially the amount of seafood intake, which might play an important role in determining blood Hg levels in Korean adults.

Catalase protects cells from reactive oxygen species-induced damage by catalyzing the breakdown of hydrogen peroxide to oxygen and water. Arsenite decreases catalase activity; it activates phosphatidylinositol 3-kinase (PI3K) and its key downstream effector Akt in a variety of cells. The PI3K pathway is known to inhibit catalase expression. c-Met, an upstream regulator of PI3K and Akt, is also involved in the regulation of catalase expression. To examine the involvement of c-Met and PI3K pathways in the arsenite-induced downregulation of catalase, catalase mRNA and protein expression were analyzed in the human hepatoma cell line HepG2 treated with arsenite and either an inhibitor of c-Met (PHA665752 (PHA)) or of PI3K (LY294002 (LY)). Arsenite treatment markedly activated Akt and decreased the levels of both catalase mRNA and protein. Both PHA and LY attenuated arsenite-induced activation of Akt. PHA and LY treatment also prevented the inhibitory effect of arsenite on catalase protein expression but did not affect the level of catalase mRNA. These findings suggest that arsenite-induced inhibition of catalase expression is regulated at the mRNA and post-transcriptional levels in HepG2 cells, and that the post-transcriptional regulation is mediated via c-Met- and PI3K-dependent mechanisms.

Genomic analysis in the local lymph node assays (LLNAs) is useful for assessing skin sensitization of chemicals and providing insights into mechanisms of sensitization. In this study, we collected 1406 genes from previous microarray findings, validated changes in their expression by RT-PCR analysis in local lymph nodes draining skin exposed to different sensitizers, and interpreted their biological function through pathway-based genomic analysis, in which 468 genes were identified as being in the KEGG pathway database. The top-ranked functions (P 0.01) identified as being affected by the sensitizers were associated with aspects of cell growth, such as DNA replication, cell cycle regulation and pyrimidine metabolism. All the sensitizers tested (DNCB, OXA and TDI) induced significant up-regulation of Psme4, which is associated with DNA replication; Tfdp1, which is related to cell cycle regulation; and Dut, which is involved in pyrimidine metabolism. Specific changes were also shown in functional categories related to the immune response, including cytokines and their receptors. Genes identified in these functional categories, such as Ccl21c, Cxcl9, Cxcl10, Ifng and Il12rb1, were found to have functional relevance. These findings may enhance our understanding and assessment of chemical sensitizers, and enable us to distinguish sensitizers from irritants and to classify chemicals as contact sensitizers.

Silver nanoparticles (AgNPs) may induce deleterious effects in aquatic life on environmental release. The hepatotoxicity of AgNPs was assessed in the liver of adult zebrafish, with the aim of studying the roles of oxidative damage and apoptosis. Zebrafish were exposed to an AgNP solution in which free Ag+ ions were absent at the time of treatment. However, the metal-sensitive metallothionein 2 (MT2) mRNA was induced in the liver tissues of AgNP-treated zebrafish, suggesting that Ag+ ions were released from AgNPs after treatment. It is also possible that MT2 mRNA was induced in the liver tissues by AgNP-generated free radicals. A number of cellular alterations including disruption of hepatic cell cords and apoptotic changes were observed in histological analysis of the liver tissues. The levels of malondialdehyde, a byproduct of cellular lipid peroxidation, and total glutathione were increased in the tissues after treatment with AgNPs. The mRNA levels of the oxyradical-scavenging enzymes catalase and glutathione peroxidase 1a were reduced in the tissues. AgNP treatment induced DNA damage, as demonstrated by analysis with the double-strand break marker γ-H2AX and the expression of p53 protein in liver tissues. In addition, the p53-related pro-apoptotic genes Bax, Noxa, and p21 were upregulated after treatment with AgNPs. These data suggest that oxidative stress and apoptosis are associated with AgNP toxicity in the liver of adult zebrafish.