In vivo effect of a TLR5 SNP (C1205T) on Salmonella enterica serovar Typhimurium infection in weaned, specific pathogen-free Landrace piglets.

Toll-like receptor 5 is a pattern-recognition receptor for bacterial flagellin. We previously reported that a single nucleotide polymorphism (SNP) of swine TLR5, C1205T, impairs recognition of Salmonella typhimurium (ST) flagellin and ethanol-killed Salmonella Choleraesuis (SC). In the present study, weaned, specific pathogen-free (SPF) Landrace piglets with CC, CT or TT genotypes were orally infected with ST (L-3569 strain) to determine the effect of this specific SNP on ST infection in vivo.

In vitro screening of lactobacilli isolated from chicken excreta to control Salmonella Enteritidis and Typhimurium.

1. The aim of this work was to select lactic acid bacteria (LAB) strains from chicks and hens of egg-laying strains for potential use to control Salmonellae. 2.

Iron availability alters ascorbate-induced stress metabolism in Glehnia littoralis root cultures.

Our previous study indicated that formation of furanocoumarin phytoalexins could be induced in Glehnia littoralis root cultures by treatment with 10-40 mM ascorbic acid (AsA). This furanocoumarin production is much less evident when G. littoralis roots are treated with AsA under iron-deficient conditions.

New short term prediction method for chemical carcinogenicity by hepatic transcript profiling following 28-day toxicity tests in rats.

We have previously shown the hepatic gene expression profiles of carcinogens in 28-day toxicity tests were clustered into three major groups (Group-1 to 3). Here, we developed a new prediction method for Group-1 carcinogens which consist mainly of genotoxic rat hepatocarcinogens. The prediction formula was generated by a support vector machine using 5 selected genes as the predictive genes and predictive score was introduced to judge carcinogenicity.

Discrimination of carcinogens by hepatic transcript profiling in rats following 28-day administration.

This study aimed at discriminating carcinogens on the basis of hepatic transcript profiling in the rats administrated with a variety of carcinogens and non-carcinogens. We conducted 28-day toxicity tests in male F344 rats with 47 carcinogens and 26 non-carcinogens, and then investigated periodically the hepatic gene expression profiles using custom microarrays. By hierarchical cluster analysis based on significantly altered genes, carcinogens were clustered into three major groups (Group 1 to 3).

High-accuracy prediction of carcinogenicity by global quantitative analysis of post-translational modifications in a 28-day in vivo rat study.

A global quantitative analysis of post-translational modifications (PTMs) of distinct proteins was executed at the proteomic level using two-dimensional fluorescence differential gel electrophoresis. We evaluated the effects of 66 chemical compounds, including 15 genotoxic carcinogens, 28 non-genotoxic carcinogens, and 23 non-carcinogens, in the male F344 rat liver in a 28-day repeated dose study. In the master gel of rat liver protein, we identified 728 spots by hybrid quadrupole time-of-flight mass spectrometry.

Development and application of a stable HeLa cell line capable of site-specific transgenesis using the Cre-lox system: establishment and application of a stable TNFRI knockdown cell line to cytotoxicity assay.

Mammalian cell models for gene knock-out/knock-in experiments are important for functional analysis of genes and have a potential of useful tool for toxicological studies. However, uncontrolled insertion of transgenes has raised significant concerns over unwanted side effects. To address this issue, we established a stable HeLa55 cell line capable of site-specific transgenesis by means of Cre-mediated cassette exchange at a site on the long arm of human chromosome 9 containing no constitutive transcripts.

Direct measure of fluorescence intensity for efficient receptor-binding assay: conjugates of ethinylcarboxyestradiol and 5(and 6)-carboxyfluorescein via alpha, omega-diaminoalkanes as a tracer for estrogen receptor.

Steroidal nuclear receptors (NRs) have been acknowledged as a target binding protein of so-called endocrine disruptors. It is therefore necessary to develop an efficient assay system for screening these endocrine-disrupting chemicals. We here describe the first exemplification of a direct measure of fluorescence intensity for a binding assay of NRs.

Comprehensive identification of cytochrome p450 isoforms from solubility-based fractionated rat liver microsomes.

Cytochrome P450 isoforms from male rat liver microsomes were comprehensively identified using nano liquid chromatography tandem mass spectrometry (nanoLC-MS/MS). The enrichment of P450, an endomembrane-anchored heme protein, was achieved by solubility-based protein fractionation, and greatly improved the total number of identified P450 isoforms. LC-MS/MS analysis of fractions resulted in the identification of total 36 P450 isoforms.

A comparative study of gene expression profiles in rat liver after administration of alpha-hexachlorocyclohexane and lindane.

Alpha-hexachlorocyclohexane (alpha-HCH) is a stereoisomer of gamma-HCH, the active ingredient of lindane (> 99% gamma-HCH). In the present study, cDNA microarray technology was employed to identify changes in gene expression associated with toxicity in livers of male Fischer 344 rats after treatment with alpha-HCH (2, 20 mg/kg/day) and lindane (1, 10 mg/kg/day) by daily oral gavage for up to 28 days. Liver samples were obtained after 1, 3, 7, 14 and 28 days and compared for gene expression profiles.

Effects of orally administered glycine on myofibrillar proteolysis and expression of proteolytic-related genes of skeletal muscle in chicks.

We examined the effects of orally administered glycine on myofibrillar proteolysis in food-deprived chicks. Food-deprived (24 h) chicks were orally administered 57, 113, and 225 mg glycine/100 g body weight and killed after 2 h. The plasma N(tau)-methylhistidine concentration, used as myofibrillar proteolysis, was decreased by glycine.

AMPK activation stimulates myofibrillar protein degradation and expression of atrophy-related ubiquitin ligases by increasing FOXO transcription factors in C2C12 myotubes.

In skeletal muscle, AMP-activated protein kinase (AMPK) is a metabolic master switch regulating glucose and lipid metabolism. Recently, AMPK has been implicated in the control of protein synthesis in skeletal muscle, but the effect of AMPK activation on myofibrillar protein degradation has yet to be elucidated. The present study was designed to examine the effect of 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside (AICAR)-induced AMPK signaling on effector mechanisms of myofibrillar protein degradation and the expression of atrophy-related genes (atrogin-1/MAFbx, MuRF1, proteasome C2 subunit, calpains, cathepsin B, and caspase-3) in C2C12 myotubes.

Quantitative proteomic analysis of rat liver for carcinogenicity prediction in a 28-day repeated dose study.

The potential of quantitative proteomic analysis to predict carcinogenicity of chemical compounds was investigated. Using 2D-DIGE, we analyzed the effects of 63 chemical compounds on protein expression in the rat liver after 28 daily doses. Types of carcinogens were categorized depending on the species and organ specificity.

Effects of fasting and refeeding on expression of atrogin-1 and Akt/FOXO signaling pathway in skeletal muscle of chicks.

This experiment was conducted to study the effects of fasting and refeeding on expression of the atrogin-1 and Akt/FOXO signaling pathway in skeletal muscle of chicks. Chicks were fasted for 24 h and refed for 2 h. Atrogin-1 mRNA expression was increased by fasting, and their increment was reduced by refeeding.

Differences in gene expression profiles in the liver between carcinogenic and non-carcinogenic isomers of compounds given to rats in a 28-day repeat-dose toxicity study.

Some compounds have structural isomers of which one is apparently carcinogenic, and the other not. Because of the similarity of their chemical structures, comparisons of their effects can allow gene expression elicited in response to the basic skeletons of the isomers to be disregarded. We compared the gene expression profiles of male Fischer 344 rats administered by daily oral gavage up to 28 days using an in-house oligo microarray.

Suppression of myofibrillar proteolysis in chick skeletal muscles by alpha-ketoisocaproate.

We previously reported that L-leucine suppresses myofibrillar proteolysis in chick skeletal muscles. In the current study, we compared the effects of L- and D-enantiomers of leucine on myofibrillar proteolysis in skeletal muscle of chicks. We also assessed whether leucine itself or its metabolite, alpha-ketoisocaproate (alpha-KIC), mediates the effects of leucine.

Effects of orally administrated amino acids on myofibrillar proteolysis in chicks.

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N(tau)-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks.

Application of RNAi inducible TNFRI knockdown cells to the analysis of TNFalpha-induced cytotoxicity.

RNA interference (RNAi) has become a popular tool for downregulating in many species including mammalian cells. Therefore, suppression of target genes in mammalian cultured cells using RNAi may represent an ideal alternative to knockout studies for understanding the molecular mechanisms of chemical toxicity. Here, we assessed the potential of RNAi mediated gene knockdown in HeLa and HepG2 cells to cytotoxicity studies.

Two-step models to predict binding affinity of chemicals to the human estrogen receptor alpha by three-dimensional quantitative structure-activity relationships (3D-QSARs) using receptor-ligand docking simulation.

Binding of chemicals to the estrogen receptor (ER) is known to be a key mode of action of endocrine disruption effects. In this study, combined quantitative structure-activity relationship (QSAR) models from discriminant and multilinear regression (MLR) analyses, termed a two-step model, were developed. These were used to predict the binding potency to human ERalpha of four chemical groups, namely alkylphenols, phthalates, diphenylethanes and benzophenones.

Effects of 4-tert-pentylphenol on the gene expression of P450 11beta-hydroxylase in the gonad of medaka (Oryzias latipes).

Alkylphenols including 4-tert-pentylphenol (4-PP) have been shown to alter sexual differentiation in fish due to their estrogenic properties. Medaka (Oryzias latipes) is so sensitive to these substances because morphological sex reversal and testis-ova induction are well developed in the exposed males. However, little work has been done to characterize the molecular effects of estrogenic substances on the morphological and gonadal feminization in male fish.

Comparison of the Hershberger assay and androgen receptor binding assay of twelve chemicals.

We performed the Hershberger assay of 12 chemicals based on the OECD draft protocol. The chemicals tested by the Hershberger assay were phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, phthalic acid di-n-propyl ester, diethylstilbestrol, 17beta-estradiol, tamoxifen, 5alpha-dihydrotestosterone, dichlorodiphenyldichloroethane, cyproterone acetate, 6alpha-methyl-17alpha-hydroxy-progesterone, atrazine, and spironolactone. Phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, and phthalic acid di-n-propyl ester are phthalates; diethylstilbestrol and 17beta-estradiol are estrogenic chemicals; tamoxifen is partial estrogen receptor antagonist with mainly estrogenic properties; 5alpha-dihydrotestosterone is an androgen derivatives; dichlorodiphenyldichloroethane is a reference androgen antagonistic chemical; cyproterone acetate, 6alpha-methyl-17alpha-hydroxy-progesterone, and spironolactone have an androgenic steroid structure and are known as androgen antagonistic chemicals; and atrazine is a reference endocrine disruptor.

Comparative study of the uterotrophic potency of 14 chemicals in a uterotrophic assay and their receptor-binding affinity.

We performed an immature rat uterotrophic assay of 14 chemicals having various receptor-binding affinities in order to assess the relationship between their uterotrophic potency and receptor-binding affinity. The chemicals tested were phthalic acid di-n-hexyl ester, phthalic acid di-n-amyl ester, phthalic acid di-n-propyl ester, 2-ethylhexyl-p-hydroxybenzoate, 4,4'-biphenol, 4,4'-sulfonyldiphenol, 4,4'-dihydroxydiphenylmethane, 2,4-dihydroxybenzophenone, 4,4'-cyclohexylidenebisphenol, 4-t-butylpyrocatechol, clomiphene citrate, 4,4'-(1,3-phenylenediisopropylidene)bisphenol, p-t-butylphenol, and diallylterephthlate. Two of the 14 chemicals, phthalic acid di-n-propyl ester and diallylterephthlate, exhibited no receptor-binding affinity, and the receptor-binding affinity of phthalic acid di-n-hexyl ester and phthalic acid di-n-amyl ester was lower than that of the other chemicals.

Comparison of the reporter gene assay for ER-alpha antagonists with the immature rat uterotrophic assay of 10 chemicals.

We performed a reporter gene assay for estrogen receptor (ER)-alpha agonists and antagonists of 10 chemicals that showed both estrogen agonistic and reduced the estrogenic effect of ethinyl estradiol in a rat uterotrophic assay. The chemicals tested by the immature uterotrophic assay were p-(tert-pentyl)phenol, 4,4'-thiobis-phenol, 4,4'-(hexafluoroisopropylidene)diphenol, 2,2-bis(4-hydroxyphenyl)-4-methyl-n-pentane, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol, 4-(phenylmethyl)phenol, 4,4'-dihydroxybenzophenone, 2,2',4,4'-tetrahydroxybenzophenone, 4-hydroxybenzophenone and 2,4,4'-trihydroxybenzophenone. Although all chemicals examined in this study were positive in the reporter gene assay for ER-alpha agonists, 4,4'-(octahydro-4,7-methano-5H-inden-5-ylidene)bisphenol was only positive in the reporter gene assay for ER-alpha antagonists.

Unique bioconcentration characteristics of new aryl fluoroalkyl ethers in common carp (Cyprinus carpio).

The bioconcentration factors (BCFs) of seven new aryl fluoroalkyl ethers--four bis-4-tetrafluoroethoxyphenyl-type (bis-type) compounds and three mono-4-tetrafluoroethoxyphenyl-type (mono-type) compounds--were obtained by bioconcentration tests using common carp. The BCFs of 4 of the 7 ethers were higher than 5000, indicating their high bioconcentration potential. The bioconcentration characteristics of the bis-type compounds were different from those of the mono-type compounds and non-fluoro diphenylmethanes with a similar skeleton structure to the bis-type compounds, in taking longer to reach a plateau and having a slower elimination rate and in their distribution patterns in the fish body.

Comparison of reporter gene assay and immature rat uterotrophic assay of twenty-three chemicals.

We performed a reporter gene assay for ERalpha-mediated transcriptional activation and an immature rat uterotrophic assay of 23 chemicals, to study the relationship between these two assays and to examine the usefulness of the reporter gene assay. The chemicals analyzed in the study were as follows: benzophenone, bisphenol A, bisphenol B, bisphenol F, p-cumyl phenol, dibutyl phthalate, dicyclohexylphthalate, dihydrotestosterone, equilin, 17alpha-estradiol, estrone, ethynyl estradiol, genistein, hematoxylin, nonylphenol mixture, 4-n-nonylphenol, norethindrone, norgestrel, octachlorostyrene, 4-n-octylphenol, 4-tert-octylphenol, tributyltin-chloride and zearalenone. To perform the reporter gene assay, HeLa cells were transfected with a rat ERalpha expression construct and an estrogen-regulated luciferase reporter construct.