Phosphoramidates of 2'-beta-D-arabinouridine (AraU) as phosphate prodrugs; design, synthesis, in vitro activity and metabolism

2'-Beta-D-arabinouridine (AraU), the uridine analogue of the anticancer agent AraC, was synthesized and evaluated for antiviral activity and cytotoxicity. In addition, a series of AraU monophosphate prodrugs in the form of triester phosphoramidates (ProTides) were also synthesized and tested against a range of viruses, leukaemia and solid tumour cell lines. Unfortunately, neither the parent compound (AraU) nor any of its ProTides showed antiviral activity, nor potent inhibitory activity against any of the cancer cell lines. Therefore, the metabolism of AraU phosphoramidates to release AraU monophosphate was investigated. The results showed carboxypeptidase Y, hog liver esterase and crude CEM tumor cell extracts to hydrolyse the ester motif of phosphoramidates with subsequent loss of the aryl group, while molecular modelling studies suggested that the AraU l-alanine aminoacyl phosphate derivative might not be a good substrate for the phosphoramidase enzyme Hint-1. These findings are in agreement with the observed disappearance of intact prodrug and concomitant appearance of the corresponding phosphoramidate intermediate derivative in CEM cell extracts without measurable formation of araU monophosphate. These findings may explain the poor antiviral/cytostatic potential of the prodrugs.

Effects of the mycotoxin patulin at the level of nuclear receptor transcriptional activity and steroidogenesis in vitro

Patulin (PAT) is a mycotoxin produced by various species of fungi, with Penicillium expansum being the most commonly occurring. Apples and apple products are the main sources of PAT contamination. This mycotoxin has been shown to induce toxic effects in animals, a few of which include reproductive toxicity and interference with the endocrine system. Here the endocrine disrupting potential of PAT has been investigated in vitro to identify disruption at the level of oestrogen, androgen, progestagen and glucocorticoid nuclear receptor transcriptional activity, and to assess interferences in estradiol, testosterone and progesterone steroid hormone production. At the receptor level, 0.5-5000ng/ml (0.0032-32μM) PAT did not appear to induce any specific (ant) agonistic responses in reporter gene assays (RGAs); however, nuclear transcriptional activity was affected. A >6 fold increase in the glucocorticoid receptor transcriptional activity was observed following treatment with 5000ng/ml PAT in the presence of cortisol. At the hormone production level, despite cytotoxicity being observed after treatment with 5000ng/ml PAT, estradiol levels had increased >2 fold. At 500ng/ml PAT treatment, an increase in progesterone and a decrease in testosterone production were observed. The findings of this study could be considered in assessing the health risks following exposure to PAT.

Synthesis, characterization, electrochemical behavior and in vitro protein tyrosine kinase inhibitory activity of the cymene-halogenobenzohydroxamato [Ru(eta(6)-cymene)(bha)Cl] complexes

The ruthenium(II)-cymene complexes [Ru(eta(6)-cymene)(bha)Cl] with substituted halogenobenzohydroxamato (bha) ligands (substituents = 4-F, 4-Cl, 4-Br, 2,4-F-2, 3,4-F-2, 2,5-F-2, 2,6-F-2) have been synthesized and characterized by elemental analysis, IR, H-1 NMR, C-13 NMR, cyclic voltammetry and controlled-potential electrolysis, and density functional theory (DFT) studies. The compositions of their frontier molecular orbitals (MOs) were established by DFT calculations, and the oxidation and reduction potentials are shown to follow the orders of the estimated vertical ionization potential and electron affinity, respectively. The electrochemical E-L Lever parameter is estimated for the first time for the various bha ligands, which can thus be ordered according to their electron-donor character. All complexes exhibit very strong protein tyrosine kinase (PTK) inhibitory activity, even much higher than that of genistein, the clinically used PTK inhibitory drug. The complex containing the 2,4-difluorobenzohydroxamato ligand is the most active one, and the dependences of the PTK activity of the complexes and of their redox potentials on the ring substituents are discussed. (C) 2012 Elsevier B.V. All rights reserved.

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite

A comparative study of in vitro chitin synthase activity in mucoraceous hosts of a mycoparasite: Chitin synthase, the enzyme responsible for the synthesis of chitin in fungal cell wall was extracted from young hyphae of Choanephora cucurbitarum and Phascolomyces articulosus, susceptible and resistant hosts, respectively, to the mycoparasite, Piptocephalis virginiana. Crude enzyme was identified and characterized by measuring the incorporation of the substrate [14C]-UDP-N-acetylglucosamine, into chitin. Most activity occurred in mixed membrane fraction. Inhibition of activity with Polyoxin D and activation with proteases, N-acetyl-glucosamine and magnesium and other ions was observed. Properties of the crude enzyme preparation such as cofactor requirement, Vmax , apparent Km value for UDP-GlcNAc, inhibition by Polyoxin D, response to pH and to temperature, and stability at 4°C were determined. Enzyme activity from both fungi displayed basically the same features as the corresponding enzymes reported from other mucoraceous fungi. However, the two preparations from P. articulosus and C. cucurbitarum differed from each other in their expressed activity (i.e., the preparations from ~ articulosus exhibited higher latency and higher specific chitin synthase activity than the corresponding preparations from ~ cucurbitarum). Trypsin was effective in activation only over a narrow concentration range. Acid protease was the most effec.tive activator. En.dogenous protease estimation indicated higher protease activity in C. cucurbitarum than in P. articulosus. The suggestion is made that regulation of chitin synthase activities may be related to host resistance in the mycoparasitic system.

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growthmedium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett–Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150 ng ml-1, respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53 % metabolic activity and 24.8 % DNA synthesis could be obtained, which suggested that IGF-I and IGFII had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells

Senescence-associated β-galactosidase activity in the in vitro ovarian stromal fibroblasts

Introduction: A growing biological research field is the cellular senescence, a mechanism that has been associated, under certain circumstances, with malignant transformation. Given the high incidence of ovarian cancer and its main origin from the ovarian surface epithelium, as well as the possibility that an epithelial-mesenchymal transition occurs, we evaluated both the in vitro growth of stromal fibroblasts from the ovarian cortex and their β-galactosidase activity at pH 6, enzyme whose expression is considered as a marker of replicative senescence. Methods: 48 samples of ovarian cortical fibroblasts from donors without a history of cancer were serially cultured until the end of their replicative life. β-galactosidase activity at pH 6 was quantified in each passage by the chemiluminiscent method. As control, we used ovarian epithelial cell cultures from the same donors. The enzyme activity was also evaluated in fibroblasts previously induced to senescence by exposure to hydrogen peroxide. Results: The analysis of the enzyme activity and the replicative capacity taken together showed that the fibroblast cultures reached the senescent state at passages 4-5, as what happened with the control epithelial cells. Fibroblasts induced to senescence showed high variability in the values of enzymatic activity. Conclusions: The similarity between both types of cells in reaching the senescent state deserves to be taken into account in relation to the epithelialmesenchymal transition that has been proposed to explain their behavior in the genesis of cancer arising from ovarian surface epithelium. Low β-galactosidase activity values at pH 6 would suggest possible inactivation of the response pathways to oxidative stress.

Further characterization of muscarinic agonist-induced epileptiform bursting activity in immature rat piriform cortex, in vitro

The characteristics of muscarinic acetylcholine receptor agonist-induced epileptiform bursting seen in immature rat piriform cortex slices in vitro were further investigated using intracellular recording, with particular focus on its postnatal age-dependence (P+14-P+30), pharmacology, site(s) of origin and the likely contribution of the muscarinic acetylcholine receptor agonist-induced post-stimulus slow afterdepolarization and gap junction functionality toward its generation. The muscarinic agonist, oxotremorine-M (10 microM), induced rhythmic bursting only in immature piriform cortex slices; however, paroxysmal depolarizing shift amplitude, burst duration and burst incidence were inversely related to postnatal age. No significant age-dependent changes in neuronal membrane properties or postsynaptic muscarinic responsiveness accounted for this decline. Burst incidence was higher when recorded in anterior and posterior regions of the immature piriform cortex. In adult and immature neurones, oxotremorine-M effects were abolished by M1-, but not M2-muscarinic acetylcholine receptor-selective antagonists. Rostrocaudal lesions, between piriform cortex layers I and II, or layer III and endopiriform nucleus in adult or immature slices did not influence oxotremorine-M effects; however, the slow afterdepolarization in adult (but not immature) lesioned slices was abolished. Gap junction blockers (carbenoxolone or octanol) disrupted muscarinic bursting and diminished the slow afterdepolarization in immature slices, suggesting that gap junction connectivity was important for bursting. Our data show that neural networks within layers II-III function as primary oscillatory circuits for burst initiation in immature rat piriform cortex during persistent muscarinic receptor activation. Furthermore, we propose that muscarinic slow afterdepolarization induction and gap junction communication could contribute towards the increased epileptiform susceptibility of this brain area.

Anticholinesterase-induced epileptiform activity in immature rat piriform cortex slices, in vitro

Purpose: Acute in vitro brain slice models are commonly used to study epileptiform seizure generation and to test anti-epileptic drug action. Seizure-like activity can be readily induced by manipulating external ionic concentrations or by adding convulsant agents to the bathing medium. We previously showed that epileptiform bursting was induced in slices of immature (P14–28) rat piriform cortex (PC) by applying oxotremorine-M, a potent muscarinic receptor agonist. Here, we examined whether raising levels of endogenous acetylcholine (ACh) by exposure to anticholinesterases, could also induce epileptiform events in immature (P12–14) or early postnatal (P7–9) rat PC brain slices. Methods: The effects of anticholinesterases were investigated in rat PC neurons using both extracellular MEA (P7–9 slices) and intracellular (P12–14 slices) recording methods. Results: In P7–9 slices, eserine (20 μM) or neostigmine (20 μM) induced low amplitude, low frequency bursting activity in all three PC cell layers (I–III), particularly layer III, where neuronal muscarinic responsiveness is known to predominate. In P12–14 neurons, neostigmine produced a slow depolarization together with an increase in input resistance and evoked cell firing. Depolarizing postsynaptic potentials evoked by intrinsic fibre stimulation were selectively depressed although spontaneous bursting was not observed. Neostigmine effects were blocked by atropine (1 μM), confirming their muscarinic nature. We conclude that elevation of endogenous ACh by anticholinesterases can induce bursting in early postnatal PC brain slices, further highlighting the epileptogenic capacity of this brain region. However, this tendency declines with further development, possibly as local inhibitory circuit mechanisms become more dominant.

Δ9-tetrahydrocannabivarin suppresses in vitro epileptiform and in vivo seizure activity in adult rat

Purpose: We assessed the anticonvulsant potential of the phytocannabinoid Δ9-tetrahydrocannabivarin (Δ9-THCV) by investigating its effects in an in vitro piriform cortex (PC) brain slice model of epileptiform activity, on cannabinoid CB1 receptor radioligand-binding assays and in a generalized seizure model in rats. Methods: Δ9-THCV was applied before (10 μmΔ9-THCV) or during (10–50 μmΔ9-THCV) epileptiform activity induced by Mg2+-free extracellular media in adult rat PC slices and measured using multielectrode array (MEA) extracellular electrophysiologic techniques. The actions of Δ9-THCV on CB1 receptors were examined using [3H]SR141716A competition binding and [35S]GTPS assays in rat cortical membranes. Effects of Δ9-THCV (0.025–2.5 mg/kg) on pentylenetetrazole (PTZ)–induced seizures in adult rats were also assessed. Results: After induction of stable spontaneous epileptiform activity, acute Δ9-THCV application (≥20 μm) significantly reduced burst complex incidence and the amplitude and frequency of paroxysmal depolarizing shifts (PDSs). Furthermore, slices pretreated with 10 μmΔ9-THCV prior to induction of epileptiform activity exhibited significantly reduced burst complex incidence and PDS peak amplitude. In radioligand-binding experiments, Δ9-THCV acted as a CB1 receptor ligand, displacing 0.5 nm [3H]SR141716A with a Ki∼290 nm, but exerted no agonist stimulation of [35S]GTPS binding. In PTZ-induced seizures in vivo, 0.25 mg/kg Δ9-THCV significantly reduced seizure incidence. Discussion: These data demonstrate that Δ9-THCV exerts antiepileptiform and anticonvulsant properties, actions that are consistent with a CB1 receptor–mediated mechanism and suggest possible therapeutic application in the treatment of pathophysiologic hyperexcitability states.

Oestrogenic activity of benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) in MCF7 human breast cancer cells in vitro

Benzyl salicylate, benzyl benzoate and butylphenylmethylpropional (Lilial) are added to bodycare cosmetics used around the human breast. We report here that all three compounds possess oestrogenic activity in assays using the oestrogen-responsive MCF7 human breast cancer cell line. At 3 000 000-fold molar excess, they were able to partially displace [H-3]oestradiol from recombinant human oestrogen receptors ER alpha and ER beta, and from cytosolic ER of MCF7 cells. At concentrations in the range of 5 x 10(-5) to 5 x 10(-4) M, they were able to increase the expression of a stably integrated oestrogen-responsive reporter gene (ERE-CAT) and of the endogenous oestrogen-responsive pS2 gene in MCF7 cells, albeit to a lesser extent than with 10(-8) M 17 beta-oestradiol. They increased the proliferation of oestrogen-dependent MCF7 cells over 7 days, which could be inhibited by the antioestrogen fulvestrant, suggesting an ER-mediated mechanism. Although the extent of stimulation of proliferation over 7 days was lower with these compounds than with 10(-8) M 17 beta-oestradiol, given a longer time period of 35 days the extent of proliferation with 10(-4) M benzyl salicylate, benzyl benzoate or butylphenylmethylpropional increased to the same magnitude as observed with 10(-8) M 17 beta-oestradiol over 14 days. This demonstrates that benzyl salicylate, benzyl benzoate and butylphenylmethylpropional are further chemical components of cosmetic products which give oestrogenic responses in a human breast cancer cell line in culture. Further research is now needed to investigate whether oestrogenic responses are detectable using in vivo models and the extent to which these compounds might be absorbed through human skin and might enter human breast tissues. Copyright (C) 2009 John Wiley & Sons, Ltd.