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. Nineteen LAB strains obtained from culture collections, and 24 strains isolated from excreta of laying hens and chicks, were evaluated for inhibitory capacities against two Salmonella serotypes using a "Spot-the-lawn" technique and other in vitro properties that could be predictive of antimicrobial activity. 3. The size of the inhibition zone differed slightly between Salmonella serotypes, however, the mean size of the Salmonella inhibition zone differed greatly among the LAB strains. Lactobacillus salivarius, L. plantarum, L. rhamnosus and L. reuteri exhibited powerful inhibitory effects to each Salmonella strain. 4. The result of the acid tolerance test showed that all L. salivarius, L. kitasatonis strains and each of L. ingluviei cannot survive in a low pH environment. In the bile acid tolerance assay, growth was inhibited in all strains, except L. kitasatonis HE4, and a large inhibition was observed in most of the L. salivarius and L. crispatus strains. 5. The results demonstrate that some LAB of poultry origin were able to inhibit the growth of Salmonella and survive simulated passage through the gastrointestinal tract. The selected LAB could act in the lower gastrointestinal tract to prevent salmonellosis in poultry.

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. We also examined the expression of proteolytic-related genes by real-time PCR of cDNA from chick skeletal muscles. The mRNA expression of atrogin-1/MAFbx, proteasome C2 subunit, m-calpain large subunit, and cathepsin B was decreased by glycine in a dose-dependent manner. The plasma corticosterone concentration was also decreased by glycine, but the plasma insulin concentration was unaffected. These results indicate that orally administered glycine suppresses myofibrillar proteolysis and expression of proteolytic-related genes of skeletal muscle by decreasing the plasma corticosterone concentration in chicks.

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. Food-deprived (24 h) chicks were orally administered 225 mg/100 g body weight L-leucine, D-leucine or alpha-KIC and were sacrificed after 2 h. L-Leucine administration had an obvious inhibitory effect on myofibrillar proteolysis (plasma N(tau)-methylhistidine concentration) in chicks while D-leucine and alpha-KIC were much more effective. We also examined the expression of the proteolytic-related genes (ubiquitin, proteasome, m-calpain and cathepsin B) by real-time PCR of cDNA in chick skeletal muscles. Ubiquitin mRNA expression was decreased by D-leucine and alpha-KIC but not L-leucine. Proteasome and m-calpain mRNA expressions as well as cathepsin B mRNA expression were likewise decreased by L-leucine, D-leucine and alpha-KIC. These results indicate that D-leucine and alpha-KIC suppress proteolytic-related genes, resulting in an decrease in myofibrillar proteolysis while L-leucine is much less effective in skeletal muscle of chicks, may be explain by conversion of D-leucine to alpha-KIC.

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. These groups are considered to be important chemical classes of ER-binders. The descriptors investigated were calculated following the simulation of docking between the receptor and ligand. Discriminant analysis in the first step of a two-step model was applied to distinguish binders from non-binders. It had a concordance, following leave-one-out (LOO), of greater than 87% for all chemical classes. Binders were defined as chemicals whose IC50 was reliably measured in a competitive binding assay. The MLR analysis in the second step was performed for the quantitative prediction of the binding affinity of chemicals that were previously discriminated as binders. The q2 values for alkylphenols and diphenylethanes were 0.75 and 0.74, respectively. However good MLR relationships were not obtained for phthalates and benzophenones as the observed binding affinities of chemicals in these categories were weak and in a too narrow range.

Experiences on persistent organic pollutants under the Law concerning the Examination and Regulation of Manufacture, etc. of Chemical Substances, Japan, with references to biodegradation and bioaccumulation.

OBJECTIVES: The purpose of this report is to outline current regulations to control chemical environmental pollution in Japan, with special references to internationally defined 12 persistent organic pollutants (POPs). MATERIALS: Law concerning the Examination and Regulation of Manufacture, etc. of Chemical Substances [(LERCS); enacted in 1973] and related administrative activities of monitoring of the environment in Japan. RESULTS: Among the existing chemicals identified by the 1972 Chemicals Inventory, LERCS designates aldrin, chlordanes, DDT, dieldrin, endrin, HCB, PCBs, poly(Cln; n = 3 or more)-chlorinated naphthalene (PCNs) and bis(tributyltin) oxide (TBTO) as Class 1 specified chemical substances which are under strict regulation, such as prohibition of production, import, or use in principle. In addition LERCS designates 23 Class 2 specified chemical substances (including 13 tributyltin and seven triphenyltin compounds) for which notification of scheduled and past production, compliance with technical guidelines and compliance with labeling standards is requested. When compared with the 12 POPs, the designation covers most of them except for mirex and toxaphene, which have never been used in Japan. The regulation has been effective in reducing substantially the levels of the designated chemical substances (and therefore the 12 POPs except for dioxins and furans) in the general environment in Japan. Efforts are currently focused under a newly enacted law to reduce the emission of the two non-intentionally produced pollutants of dioxins and furans, especially from city waste incinerators, so that emission in 2002 should be 10% of that in 1997. CONCLUSION: Regulations to control chemical emissions have been effective in reducing POPs levels in the environment in Japan, and further efforts have been made under a new law to reduce the emission of dioxins, furans and co-planar PCBs.

Biodegradation of poly(tetramethylene succinate-co-tetramethylene adipate) and poly(tetramethylene succinate) through water-soluble products.

Poly(tetramethylene succinate-co-tetramethylene adipate) (PBSA) and poly(tetramethylenesuccinate) (PBS) were hydrolyzed experimentally into water-soluble oligomers and monomers by Chromobacterium extracellular lipase. The oligomers were identified by high-performance liquid chromatography-mass spectrometry and 1H-nuclear magnetic resonance, which indicated that a total of 28 oligomer species were liberated from PBSA, and that 13 of them were identical to the hydrolysates from PBS. Moreover, 20 of the species were polyester-based compounds of monomer units, and the other 8 species were small amounts of diurethane compounds. Bis(hydroxybutyl) succinate (BSB) and bis(hydroxybutyl) hexamethylene dicarbamate (BHB) were the typical oligomers and were chemically synthesized. Biodegradability of BSB and BHB was examined for 28 d in the activated sludge, and analysis of the results of this study indicated that the final conversion rate of constituent carbon to carbon dioxide was estimated at 80 mol% for BSB and 10 mol% for BHB. The remaining amount of carbon in the undegraded BHB was 20 mol%. In the presence of BSB, the biodegradability of BHB was increased by about 1.5 times. The suggestion was made that BSB induced a growth of microorganisms and helped BHB degradation. This is consistent with the observation that the biodegradation of BHB in native soil for 60 d reached > 60%.

Development of non-radio isotopic endpoint of murine local lymph node assay based on 5-bromo-2'-deoxyuridine (BrdU) incorporation.

Allergic contact dermatitis is a serious health problem. Over the last decade, the murine local lymph node assay (LLNA) has been developed to detect chemical allergens, and international validation studies have been conducted. We have tried to establish an alternative non-radioisotopic endpoint for the LLNA by using 5-bromo-2'-deoxyuridine (BrdU) incorporation in place of radioisotopes, such as [3H]thymidine, employed in the standard method. BrdU was given as a single administration at 5 mg/animal 2 days following three consecutive daily applications of a test chemical. BrdU incorporation into draining lymph node cells was measured using an enzyme immunosorbent assay technique. In this study, p-benzoquinone(PBQ), trimellitic anhydride (TMA), citral(CT) and dextran (DEX) were used as pilot chemicals. PBQ, TMA and CT, which are classified as moderate to strong sensitizers in the guinea pig maximization test and were positive in the original LLNA, were also found to elicit positive responses in the alternative LLNA using BrdU incorporation. In contrast, DEX tested negative in the modified assay consistent with previous guinea pig and LLNA data. Consequently, the modified LLNA endpoint using BrdU incorporation may represent a useful alternative to the standard assay in situations, where there is a need to avoid the use of radioisotopes.

Prediction for biodegradability of chemicals by an empirical flowchart.

A method for predicting aerobic biodegradability of chemicals was developed based on empirical knowledge. A flowchart was derived from rule of thumb relationships between the biodegradability and the number of the functional groups and substructures in a certain skeletal structure of chemicals. The flowchart classified chemicals into readily biodegradable, not readily biodegradable and not predictable. It was validated by using MITI data of 177 mono benzene derivatives and 168 acyclic compounds, resulting in correct prediction at 94% and 88% levels, respectively.

Structural essentials of xenoestrogen dialkyl phthalates to bind to the estrogen receptors.

Xenoestrogen dialkyl phthalates, C(6)H(4)(COOC(n)H(m))(2), lack the phenolic hydroxyl group that is an essential structural component of the steroid A ring of 17 beta-estradiol. In order to examine whether dialkyl phthalates imitate the steroid structure, we have synthesized a series of 4-hydroxyl derivatives of dialkyl phthalates. The compounds were examined for their ability to displace [(3)H]17 beta-estradiol from the recombinant human estrogen receptor, which was expressed on Sf9 cells using the vaculovirus expression system. Dialkyl 4-hydroxyl phthalates were found to exhibit several-fold higher binding affinities compared to phthalates without the 4-hydroxyl group. From the analyses of receptor binding modes of dialkyl phthalates with and without the 4-hydroxyl group, it was deduced that the phthalic benzene ring mimics the steroid A ring. A biphasic binding curve observed for dicyclohexyl phthalate was also depicted by its 4-hydroxyl derivative, but it increased binding affinity only at the high affinity binding site. These data suggest that the phthalate benzene moiety recognizes the core of the estrogen receptor binding site and the hydrophobic interaction of the dialkyl moiety substantiates the binding characteristics of the phthalates. The present data indicate that even chemicals with slight structural analogy and weak receptor affinity can perturb the endocrine system when administered in high concentrations.

Evaluation of laboratory-made sludge for an anaerobic biodegradability test and its use for assessment of 13 chemicals.

Laboratory-made sludge for a biogas based anaerobic biodegradability test was prepared as an alternative for digested sludge from wastewater treatment plants (WWTPs). Biodegradation activities and background gas productions of digested sludge from various WWTPs were found to vary significantly depending on the source, which adversely affected test reliability. Subsequently, test conditions such as sludge concentration and sludge washing were examined with the laboratory-made sludge and a sludge concentration of 1.0 g-SS/L without washing was determined to be most suitable. Under these conditions, biodegradability tests were conducted for 13 select chemicals and their relative toxicities to methanogenic bacteria were evaluated. The results of biodegradability tests showed that chemicals with -OH and -CH2OH radicals were readily biodegraded and those with -Cl, -NO2, -NH2, -SO3H and -CH3 had inhibited degradation responses m-nitriphenol and 2,4,6-trichlorophenol were highly toxic to methanogenic bacteria, with m-nitrophenol completely inhibiting methane fermentation as low as 20 mg/L.

Structural requirements of para-alkylphenols to bind to estrogen receptor.

Octyl- and nonylphenols in the environment have been proposed to function as estrogens. To gain insight into their structural essentials in binding to the estrogen receptor, a series of phenols with saturated alkyl groups at the para position, HO-C6H4-CnH2n+1 (n = 0-12), were examined for their ability to displace [3H]17beta-estradiol in the recombinant human estrogen receptor, which was expressed in Sf9 cells using the vaculovirus expression system. All tested para-alkylphenols were found to bind fully to the estrogen receptors in a dose-dependent manner. The interaction of alkylphenols with the receptor became stronger with increase in the number of the alkyl carbons and the activity was maximized with n = 9 of nonylphenol. Phenol (n = 0) exhibited weak but full binding to the receptor, whereas anisole with a protected phenolic hydroxyl group was completely inactive. Also, alkanes such as n-octane, 2,2, 4-trimethylpentane corresponding to tert-octane, and n-nonane exhibited no binding. The results indicate that the binding of para-alkylphenols to the estrogen receptor is due to the effect of covalent bonding of two constituents of the phenol and alkyl groups, which correspond to the A-ring and hydrophobic moiety of the steroid structure, respectively. When alkylphenols were examined for their receptor binding conformation by 1H-NMR measurements and ab initio molecular orbital calculations, it was suggested that nonbranched alkyl groups are in an extended conformation, while branched alkyl groups are in a folded conformation. These results suggest that branched and nonbranched alkyl moieties of alkylphenols interact differently with the lipophilic ligand binding cavity of the estrogen receptor when compared to the binding of 17beta-estradiol.

Binding characteristics of dialkyl phthalates for the estrogen receptor.

Dialkyl phthalates have been suggested to function as xenoestrogen. To explore the structural essentials, a series of ring and alkyl-chain isomers of dialkyl phthalates C6H4(COOCnHm)2 were examined for their ability to displace [3H]17beta-estradiol in the recombinant human estrogen receptor expressed on Sf9 vaculovirus. Compounds with an alkyl chain of more than C3 (n = 3) exhibited a distinct full receptor binding in a dose-dependent manner. When the ring isomers of C3-diallyl (-CH2-CH=CH2) derivatives, namely diallyl phthalate, diallyl isophthalate, and diallyl terephthalate, were examined, the ortho isomer of diallyl phthalate was most potent to bind to the estrogen receptor. The interaction with the estrogen receptor was optimized with dibutyl phthalates of C4. The conformational studies by 1H-NMR measurements and ab initio molecular orbital calculations have suggested that the structure mimics the interface of steroid A and B/C rings of 17beta-estradiol. Dicyclohexyl phthalate bound to the estrogen receptor with a biphasic binding curve, suggesting the compound discriminates two different receptor conformations.