Identification of potential mechanisms of toxicity after di-(2-ethylhexyl)-phthalate (DEHP) adult exposure in the liver using a systems biology approach

Phthalates are industrial additives widely used as plasticizers. In addition to deleterious effects on male genital development, population studies have documented correlations between phthalates exposure and impacts on reproductive tract development and on the metabolic syndrome in male adults. In this work we investigated potential mechanisms underlying the impact of DEHP on adult mouse liver in vivo. A parallel analysis of hepatic transcript and metabolic profiles from adult mice exposed to varying DEHP doses was performed. Hepatic genes modulated by DEHP are predominantly PPARalpha targets. However, the induction of prototypic cytochrome P450 genes strongly supports the activation of additional NR pathways, including Constitutive Androstane Receptor (CAR). Integration of transcriptomic and metabonomic profiles revealed a correlation between the impacts of DEHP on genes and metabolites related to heme synthesis and to the Rev-erbalpha pathway that senses endogenous heme level. We further confirmed the combined impact of DEHP on the hepatic expression of Alas1, a critical enzyme in heme synthesis and on the expression of Rev-erbalpha target genes involved in the cellular clock and in energy metabolism. This work shows that DEHP interferes with hepatic CAR and Rev-erbalpha pathways which are both involved in the control of metabolism. The identification of these new hepatic pathways targeted by DEHP could contribute to metabolic and endocrine disruption associated with phthalate exposure. Gene expression profiles performed on microdissected testis territories displayed a differential responsiveness to DEHP. Altogether, this suggests that impacts of DEHP on adult organs, including testis, could be documented and deserve further investigations.

Naturally occurring radioactive material (NORM) from a former phosphoric acid processing plant

In recent years there has been an increasing awareness of the radiological impact of non-nuclear industries that extract and/or process ores and minerals containing naturally occurring radioactive material (NORM). These industrial activities may result in significant radioactive contamination of (by-) products, wastes and plant installations. In this study, scale samples were collected from a decommissioned phosphoric acid processing plant. To determine the nature and concentration of NORM retained in pipe-work and associated process plant, four main areas of the site were investigated: (1) the 'Green Acid Plant', where crude acid was concentrated; (2) the green acid storage tanks; (3) the Purified White Acid (PWA) plant, where inorganic impurities were removed; and (4) the solid waste, disposed of on-site as landfill. The scale samples predominantly comprise the following: fluorides (e.g. ralstonite); calcium sulphate (e.g. gypsum); and an assemblage of mixed fluorides and phosphates (e.g. iron fluoride hydrate, calcium phosphate), respectively. The radioactive inventory is dominated by U-238 and its decay chain products, and significant fractionation along the series occurs. Compared to the feedstock ore, elevated concentrations (<= 8.8 Bq/g) of U-238 Were found to be retained in installations where the process stream was rich in fluorides and phosphates. In addition, enriched levels (<= 11 Bq/g) of Ra-226 were found in association with precipitates of calcium sulphate. Water extraction tests indicate that many of the scales and waste contain significantly soluble materials and readily release radioactivity into solution. (c) 2005 Elsevier Ltd. All rights reserved.

Effects of phthalic acid esters on the liver and thyroid

The effects, over periods from 3 days to 9 months of administration, of diets containing di-2-ethylhexyl phthalate are very similar to those observed in rats administered diets containing hypolipidemic drugs such as clofibrate. Changes occur in a characteristic order commencing with alterations in the distribution of lipid within the liver, quickly followed by proliferation of hepatic peroxisomes and induction of the specialized P-450 isoenzyme(s) catalyzing omega oxidation of fatty acids. There follows a phase of mild liver damage indicated by induction of glucose-6-phosphatase activity and a loss of glycogen, eventually leading to the formation of enlarged lysosomes through autophagy and the accumulation of lipofuscin. Associated changes are found in the kidney and thyroid. The renal changes are limited to the proximal convoluted tubules and are generally similar to changes found in the liver. The effects on the thyroid are more marked. Although the levels of thyroxine in plasma fail to about half normal values, serum triiodothyronine remains close to normal values while the appearance of the thyroid varies, very marked hyperactivity being noted 7 days after commencement of treatment, this is less marked at 14 days, but even after 9 months treatment there is clear cut evidence for hyperactivity with colloid changes which indicate this has persisted for some time. Straight chain analogs of di-2-ethylhexyl phthalate, di-n-hexyl phthalate and di-n-oxtyl phthalate differ entirely in their short-term effects on the liver and kidney but have similar effects on the thyroid. The short-term in vivo hepatic effects of the three phthalate esters can be reproduced in hepatocytes in tissue culture. All three phthalate esters, as well as clofibrate, have early marked effects on the metabolism of fatty acids in isolated hepatocytes. The nature of these changes is such as to increase storage of lipid in the liver. A hypothesis is presented to explain the progress from these initial metabolic effects to the final formation of liver tumors.

Structural and equilibrium studies on mixed ligand complexes of Co(II), Ni(II), Cu(II) and Zn(II) with N-(2-benzimidazolyl)methyliminodiacetic acid and typical N,N donor ligands

Mixed ligand complexes: [Co(L)(bipy)] (.) 3H(2)O (1), [Ni(L)(phen)] (.) H2O (2), [Cu(L)(phen)] (.) 3H(2)O (3) and [Zn(L)(bipy)] (.) 3H(2)O (4), where L2- = two -COOH deprotonated dianion of N-(2-benzimidazolyl)methyliminodiacetic acid (H(2)bzimida, hereafter, H,L), bipy = 2,2' bipyridine and phen = 1,10-phenanthroline have been isolated and characterized by elemental analysis, spectral and magnetic measurements and thermal studies. Single crystal X-ray diffraction studies show octahedral geometry for 1, 2 and 4 and square pyramidal geometry for 3. Equilibrium studies in aqueous solution (ionic strength I = 10(-1) mol dm(-3) (NaNO3), at 25 +/- 1 degrees C) using different molar proportions of M(II):H2L:B, where M = Co, Ni, Cu and Zn and B = phen, bipy and en (ethylene diamine), however, provides evidence of formation of mononuclear and binuclear binary and mixed ligand complexes: M(L), M(H-1L)(-), M(B)(2+), M(L)(B), M(H-1L)(B)(-), M-2(H-1L)(OH), (B)M(H-1L)M(B)(+), where H-1L3- represents two -COOH and the benzimidazole NI-H deprotonated quadridentate (O-, N, O-, N), or, quinquedentate (O-, N, O-, N, N-) function of the coordinated ligand H,L. Binuclear mixed ligand complex formation equilibria: M(L)(B) + M(B)(2+) = (B)M(H-1L)M(B)(+) + H+ is favoured with higher pi-acidity of the B ligands. For Co(II), Ni(II) and Cu(II), these equilibria are accompanied by blue shift of the electronic absorption maxima of M(II) ions, as a negatively charged bridging benzimidazolate moiety provides stronger ligand field than a neutral one. Solution stability of the mixed ligand complexes are in the expected order: Co(II) < Ni(II) < Cu(II) > Zn(II). The Delta logK(M) values are less negetive than their statistical values, indicating favoured formation of the mixed ligand complexes over the binary ones. (c) 2005 Elsevier B.V. All rights reserved.

A facile one step synthesis of N-2 substituted 3-phenyliminoisoindolinones from N-(2-carboxybenzoyl)-anthranilic acid and the design of reverse-turn mimetics

Stirring of N-(2-carboxybenzoyl) anthranilic acid with anilines and amines such as p-toluidine, benzylamine, methyl esters of Leu, Phe, Ile and Val in presence of DCC produces N- 2 substituted 3-phenyliminoisoindolinones in very good yields. Single crystal X-ray diffraction studies and solution phase NMR and CD studies reveal that the 3-phenyliminoisoindolinone moiety is a turn-inducing scaffold which should be useful for reverse-turn mimetics.

Structure and spectra of dichloro(hydroxy-methoxy-di(2-pyridyl)methane)copper(II)

The non-electrolyte dichloro(hydroxy-methoxy-di(2-pyridylmethane)copper(II), resulting from the reaction of di(2-pyridyl)ketone and copper(II) chloride in methanol solution, was isolated and characterized and its structure was determined by X-ray diffraction. The pyridyl nitrogens and the chloride anions virtually from a basal plane in which lies the copper atom, while the oxygen of the methoxy group is in an apical position at a distance of 2.497 (3)Å. The nitrogenous base adopts the boat conformation with the pyridyl rings forming a dihedral angle of 108.72 (14)°. The nearest interatomic copper distance of 3.940(3)Å precludes copper-copper interactions, while the proximity of copper to the out-of-plane chlorine atoms [3.109(3)Å] suggests weakly bound chloro-bridged dimers. Spectral changes indicate that protic molecules displace the methoxy group and water affords the corresponding 1,1-diol.

Complexes of palladium using di-2-pyridyl ketone as ligand: synthesis, structure and, spectral and catalytic properties

During the reaction of di-2-pyridyl ketone (dpk) with Na(2)[PdCl(4)] in alcoholic media, the C=O fragment of dpk undergoes facile solvolysis and the transformed di-2-pyridyl ketone (dpk(ROH), R = Me or H) binds to palladium as NN-donor. When the reaction is carried out in refluxing methanol, a mono-complex of type [Pd(dpk(MeOH))Cl(2)] is obtained. A similar reaction in ethanol affords a bis-complex of type [Pd(dpk(ROH))(2)]Cl(2). Structure of both the complexes have been determined by X-ray crystallography. In acetonitrile solution the [pd(dpk(MeOH))Cl(2)] and [pd(dpk(ROH))(2)]Cl(2) complexes show intense absorptions in the visible and ultraviolet region, origin of which has been probed through uvr calculations. These two palladium complexes are found to be efficient catalysts for Suzuki cross-coupling reactions.

Binuclear and polymeric manganese(II) salicylate complexes: synthesis, crystal structure and catalytic activity of [Mn2(Hsal)4(H2O)4] and [{Mn2(sal)2(Hsal)(H2O)-(H3O)(py)4•2py}n](H2sal = salicylic acid, py = pyridine)

The two air-stable manganese(II) salicylate complexes [Mn2(Hsal)4(H2O)4]1 and polymeric [{Mn2(sal)2(Hsal)(H2O)(H3O)(py)4·2py}n]2(H2sal = salicylic acid and py = pyridine) have been synthesised easily, and their crystal structures determined. Both contain unsymmetrically bridging salicylate ligands. In the presence of added pyridine 1 and 2 vigorously catalyse the disproportionation of H2O2.

Synthesis, structure and properties of cobalt(II/III) seleno-bisphenolate complexes containing NN bidentate co-ligands: kinetic studies on the reduction of a cobalt(III) complex by ascorbic acid in 80% MeCN-20% water

Two cobalt complexes, [Co(L-Se)(phen)]center dot CH2Cl2 (1) and [Co(L-Se)(N,N-Me(2)en)(CH3COO-)] (2) have been synthesized and characterized by elemental analyses, magnetic measurements, i.r. studies etc. Single crystal X- ray studies reveal that in complex (1) cobalt atom is in +2 oxidation state with trigonal bipyramidal geometry, while in complex (2) it is in +3 oxidation state and surrounded octahedrally. The asymmetric unit of complex (2) contains two crystallographically independent discrete molecules. Complex (1) was found to be paramagnetic with mu(eff) = 2.19 BM indicating a low spin cobalt(II) d(7) system, whereas complex (2) is found to be diamagnetic with cobalt(III) in low spin d(6) state. The kinetic studies on the reduction of (2) by ascorbic acid in 80% MeCN-20% H2O (v/v) at 25 degrees C reveal that the reaction proceeds through the rapid formation of inner-sphere adduct, probably by replacing the loosely coordinated AcO- group, followed by electron transfer in a slow step and is supported by a large Q (formation constant) value.

Gap junction-mediated spontaneous Ca(2+) waves in differentiated cholinergic SN56 cells

Neuronal gap junctions are receiving increasing attention as a physiological means of intercellular communication, yet our understanding of them is poorly developed when compared to synaptic communication. Using microfluorimetry, we demonstrate that differentiation of SN56 cells (hybridoma cells derived from murine septal neurones) leads to the spontaneous generation of Ca(2+) waves. These waves were unaffected by tetrodotoxin (1microM), but blocked by removal of extracellular Ca(2+), or addition of non-specific Ca(2+) channel inhibitors (Cd(2+) (0.1mM) or Ni(2+) (1mM)). Combined application of antagonists of NMDA receptors (AP5; 100microM), AMPA/kainate receptors (NBQX; 20microM), nicotinic AChR receptors (hexamethonium; 100microM) or inotropic purinoceptors (brilliant blue; 100nM) was also without effect. However, Ca(2+) waves were fully prevented by carbenoxolone (200microM), halothane (3mM) or niflumic acid (100microM), three structurally diverse inhibitors of gap junctions, and mRNA for connexin 36 was detected by PCR. Whole-cell patch-clamp recordings revealed spontaneous inward currents in voltage-clamped cells which we inhibited by Cd(2+), Ni(2+) or niflumic acid. Our data suggest that differentiated SN56 cells generated spontaneous Ca(2+) waves which are propagated by intercellular gap junctions. We propose that this system can be exploited conveniently for the development of neuronal gap junction modulators.

Protease-activated receptor 2 (PAR2) protein and transient receptor potential vanilloid 4 (TRPV4) protein coupling is required for sustained inflammatory signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR(2)), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR(2) regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR(2) agonist stimulated a transient increase in [Ca(2+)](i). TRPV4 expression led to a markedly sustained increase in [Ca(2+)](i). Removal of extracellular Ca(2+) and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A(2) and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR(2) generates arachidonic acid-derived lipid mediators, such as 5',6'-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR(2)-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR(2) was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR(2) signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR(2)-stimulated neurogenic inflammation. Thus, PAR(2) activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR(2).

Biodegradation of the herbicide mecoprop-p with soil depth and its relationship with class III tfdA genes

Mecoprop-p [(R)-2-(4-chloro-2-methylphenoxy) propanoic acid) is widely used in agriculture and poses an environmental concern because of its susceptibility to leach from soil to water. We investigated the effect of soil depth on mecoprop-p biodegradation and its relationship with the number and diversity of tfdA related genes, which are the most widely known genes involved in degradation of the phenoxyalkanoic acid group of herbicides by bacteria. Mecoprop-p half-life (DT50) was approximately 12 days in soil sampled from <30 cm depth, and increased progressively with soil depth, reaching over 84 days at 70–80 cm. In sub-soil there was a lag period of between 23 and 34 days prior to a phase of rapid degradation. No lag phase occurred in top-soil samples prior to the onset of degradation. The maximum degradation rate was the same in top-soil and sub-soil samples. Although diverse tfdAα and tfdA genes were present prior to mecoprop-p degradation, real time PCR revealed that degradation was associated with proliferation of tfdA genes. The number of tfdA genes and the most probable number of mecoprop-p degrading organisms in soil prior to mecoprop-p addition were below the limit of quantification and detection respectively. Melting curves from the real time PCR analysis showed that prior to mecoprop-p degradation both class I and class III tfdA genes were present in top- and sub-soil samples. However at all soil depths only tfdA class III genes proliferated during degradation. Denaturing gradient gel electrophoresis confirmed that class III tfdA genes were associated with mecoprop-p degradation. Degradation was not associated with the induction of novel tfdA genes in top- or sub-soil samples, and there were no apparent differences in tfdA gene diversity with soil depth prior to or following degradation.

Quantification of ruminal Clostridium proteoclasticum by real-time PCR using a molecular beacon approach

Aims: All members of the ruminal Butyrivibrio group convert linoleic acid (cis-9,cis-12-18 : 2) via conjugated 18 : 2 metabolites (mainly cis-9,trans-11-18 : 2, conjugated linoleic acid) to vaccenic acid (trans-11-18 : 1), but only members of a small branch, which includes Clostridium proteoclasticum, of this heterogeneous group further reduce vaccenic acid to stearic acid (18 : 0, SA). The aims of this study were to develop a real-time polymerase chain reaction (PCR) assay that would detect and quantify these key SA producers and to use this method to detect diet-associated changes in their populations in ruminal digesta of lactating cows. Materials and Results: The use of primers targeting the 16S rRNA gene of Cl. proteoclasticum was not sufficiently specific when only binding dyes were used for detection in real-time PCR. Their sequences were too similar to some nonproducing strains. A molecular beacon probe was designed specifically to detect and quantify the 16S rRNA genes of the Cl. proteoclasticum subgroup. The probe was characterized by its melting curve and validated using five SA-producing and ten nonproducing Butyrivibrio-like strains and 13 other common ruminal bacteria. Analysis of ruminal digesta collected from dairy cows fed different proportions of starch and fibre indicated a Cl. proteoclasticum population of 2-9% of the eubacterial community. The influence of diet on numbers of these bacteria was less than variations between individual cows. Conclusion: A molecular beacon approach in qPCR enables the detection of Cl. proteoclasticum in ruminal digesta. Their numbers are highly variable between individual animals. Signifance and Impact of the Study: SA producers are fundamental to the flow of polyunsaturated fatty acid and vaccenic acid from the rumen. The method described here enabled preliminary information to be obtained about the size of this population. Further application of the method to digesta samples from cows fed diets of more variable composition should enable us to understand how to control these bacteria in order to enhance the nutritional characteristics of ruminant-derived foods, including milk and beef.

A breakthrough in lupin biotechnology: prolific protocolonisation in recalcitrant white lupin (Lupinus albus) triggered by bovine serum albumin

Six nutrient formulations were studied for their efficacy in inducing mitosis in white lupin seedling cotyledon protoplasts of which the formulations of Schafer-Menuhr & Sturmer (AS) and Kao (K8p) were found to be superior over the other four when supplemented with 6-benzylaminopurine and alpha-naphthaleneacetic acid (alpha-NAA). An unltrafiltration treatment of K8p increased mitotic frequency by 130% when compared with the untreated control. Medium enrichment with 0.2% bovine serum albumin (BSA) brought about a dramatic 1341% rise in protoplast division in comparison with BSA-free medium but only when the enrichment was carried out in Kao and Michayluk (KM8p) background containing 2, 4-dichlorophenoxyacetic acid, alpha-NAA and zeatin. A higher number of protocolonies (each proliferating from single protoplast following multiple divisions) were seen in 0.4% BSA. With this breakthrough in white lupin protoplast research, it is now possible to reproducibly obtain protocolonies that was hitherto not possible.