Engineering of dense arrays of Vertical Si1-xGexnanostructures.

Vertical nanostructure technologies are becoming more important for the down scaling of nanoelectronic devices such as logic transistors or memories. Such devices require dense vertical nanostructured channel arrays (VNCA) that can be fabricated through a top-down approach based on group IV materials. We present progresses on the top-down fabrication of highly anisotropic and ultra-dense Si1-xGex(x= 0, 0.2, 0.5) VNCAs. Dense nanowire and nanosheet patterns were optimized through high resolution lithography and transferred onto Si1-xGexsubstrates by anisotropic reactive ion etching with a fluorine chemistry. The right gas mixtures for a given Ge content resulted in perfectly vertical and dense arrays. Finally we fabricated oxide shell/SiGe core heterostructures by dry- and wet-thermal oxidation and evaluated their applicability for nanostructure size engineering, as already established for silicon nanowires. The impact of the nanostructured shape (wire or sheet), size and Ge content on the oxide growth were investigated and analysed in detail through transmission electron microscopy.

Nanowire based bioprobes for electrical monitoring of electrogenic cells.

The continuous miniaturization of electronic components and the emergence of nano-biotechnology has opened new perspectives to monitor electrical activities at the single cell level. Here, we describe the creation of very high surface-to-volume ratio passive devices (vertical nanowire probes) using large-scale fabrication process, allowing to follow the electrical activity of mammalian neurons. Based on conventional silicon processing, the silicon nanowires were silicided in platinum in order to improve their electrochemical performances and to guarantee their biocompatibility. Very high signal to noise ratio was achieved (up to 2000) when measuring spontaneous action potentials. Moreover, this bio-platform was used to record the impact of various bio-chemical and electrical stimulations on neuronal activity. To conclude, this study proposes a thorough comparison of the characteristics and performances of these new nanowire-based nanoprobes with the main alternative systems published up to now.

Thin-dielectric-layer engineering for 3D nanostructure integration using an innovative planarization approach.

Three-dimensional (3D) nanostructures are emerging as promising building blocks for a large spectrum of applications. One critical issue in integration regards mastering the thin, flat, and chemically stable insulating layer that must be implemented on the nanostructure network in order to build striking nano-architectures. In this letter, we report an innovative method for nanoscale planarization on 3D nanostructures by using hydrogen silesquioxane as a spin-on-glass (SOG) dielectric material. To decouple the thickness of the final layer from the height of the nanostructure, we propose to embed the nanowire network in the insulator layer by exploiting the planarizing properties of the SOG approach. To achieve the desired dielectric thickness, the structure is chemically etched back with a highly diluted solution to control the etch rate precisely. The roughness of the top surface was less than 2 nm. There were no surface defects and the planarity was excellent, even in the vicinity of the nanowires. This newly developed process was used to realize a multilevel stack architecture with sub-deca-nanometer-range layer thickness.

Vertical nanowire array-based field effect transistors for ultimate scaling.

Nanowire-based field-effect transistors are among the most promising means of overcoming the limits of today's planar silicon electronic devices, in part because of their suitability for gate-all-around architectures, which provide perfect electrostatic control and facilitate further reductions in "ultimate" transistor size while maintaining low leakage currents. However, an architecture combining a scalable and reproducible structure with good electrical performance has yet to be demonstrated. Here, we report a high performance field-effect transistor implemented on massively parallel dense vertical nanowire arrays with silicided source/drain contacts and scaled metallic gate length fabricated using a simple process. The proposed architecture offers several advantages including better immunity to short channel effects, reduction of device-to-device variability, and nanometer gate length patterning without the need for high-resolution lithography. These benefits are important in the large-scale manufacture of low-power transistors and memory devices.

High yield of self-catalyzed GaAs nanowire arrays grown on silicon via gallium droplet positioning.

We report and detail a method to achieve growth of vertical self-catalyzed GaAs nanowires directly on Si(111) with a near-perfect vertical yield, using electron-beam-defined arrays of holes in a dielectric layer and molecular beam epitaxy. In our conditions, GaAs nanowires are grown along a vapor-liquid-solid mechanism, using in situ self-forming Ga droplets. The focus of this paper is to understand the role of the substrate preparation and of the pre-growth conditioning. Without changing temperature or the V/III ratio, the yield of vertical nanowires is increased incrementally up to 95%. The possibility to achieve very dense arrays, with center-to-center inter-wire distances less than 100 nm, is demonstrated.

A glucuronidation pathway of capecitabine occurs in rats but not in mice and humans.

Glucuronidation of 5'-DFCR, a metabolite of capecitabine, was confirmed in experimental models from rats whereas 5'-DFCR glucuronide was detected neither in bile or liver from mice nor in liver microsomes from human. Metabolic interactions at the level of the glucuronidation pathway between CAP and other drugs are unlikely in patients.

Enhancement of moxidectin bioavailability in lamb by a natural flavonoid: quercetin.

Moxidectin is an antiparasitic drug widely used in cattle, sheep and companion animals. Due to the involvement of P-glycoprotein (P-gp) and cytochrome P450 3A in the metabolism of moxidectin, we studied the influence of various P-gp interfering agents (ivermectin, quercetin and ketoconazole) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h. This in vitro study allowed selection of compounds which are able to increase the moxidectin bioavailability in lambs. From this, the modulation of moxidectin pharmacokinetics in plasma of lambs was studied after co-administration of 0.2 mg kg(-1) moxidectin (subcutaneously (SC)) and 0.2 mg kg(-1) ivermectin (SC), or 10 mg kg(-1) quercetin (SC), or 10 mg kg(-1) ketoconazole (orally). Ivermectin and quercetin increased significantly the quantity of 14C moxidectin in the rat hepatocytes. Ketoconazole co-administration led to a higher concentration of moxidectin in the rat hepatocytes. In vivo, only quercetin was able to modify the pharmacokinetics of moxidectin in plasma of lambs by increasing significantly the area under the plasma concentration-time curve. This study allowed the use of a natural agent, quercetin, to improve the bioavailability of moxidectin.

Differential effects of phenobarbital on the constitutive and inducible expression of P450 2B and 3A subfamilies in sheep tissues.

The activity and expression of cytochromes P450 were determined in liver, kidneys, lungs, duodenum, jejunum, ileum, and caecum of adult Lacaune sheep. High expression of total P450, benzphetamine and erythromycin demethylase activities, and P450 2B isoforms, as two distinct proteins that were detected and called P4502 Bm and P4502 Bx, was found in the lungs (in addition to liver). By contrast, the P450 3A subfamily was only expressed in liver and duodenal mucosa of untreated sheep. Phenobarbital (PB) treatment led to significant increases in all measured hepatic parameters and in total P450 of each investigated organ with the exception of ileum and caecum. Benzphetamine demethylase activity increased in liver and kidneys, correlating with the expression of the two P450 2B proteins, which were also induced in duodenum and ileum. By contrast, benzphetamine demethylase activity and expression of the P450 2B isoforms in lungs were unchanged by PB treatment. Erythromycin demethylation activity and P450 3A subfamily expression was increased only in liver of PB-treated sheep.

Influence of verapamil on the efflux and metabolism of 14C moxidectin in cultured rat hepatocytes.

Moxidectin (MOX) is an antiparasitic drug widely used in cattle, sheep and companion animals. As a result of the implication of cytochrome P450 3 A in the metabolism of MOX and the role of competitor substrates of P-glycoprotein (Pgp) in modification of the bioavailability of endectocides, we studied the influence of verapamil (a multidrug-resistance reversing agent) on the metabolism of 14C moxidectin in cultured rat hepatocytes over 72 h. The metabolism of MOX remained low: 10.79 +/- 1.99% of the total 14C moxidectin for the main detected metabolite in verapamil-treated cells and 7.17 +/- 0.74% for the control cells after 24 h. The main detected metabolite in rat hepatocytes was the same as that detected in rat hepatic microsomes (the C29 monohydroxymethyl metabolite). Verapamil increased the quantity of MOX in the cells after 24, 48 and 72 h. Examination of the Area Under the concentration time Curve (AUC) of the main detected metabolite revealed a significant increase in the exposure of cells to MOX after verapamil treatment throughout the experiment. It is hypothesized that verapamil interfered with MOX as a substrate for Pgp during the initial incubation period. After this initial interaction, verapamil metabolites were able to interfere with Pgp. This experiment demonstrated the implication of Pgp in the transport of MOX and allowed prediction of the drug-drug interactions which might modify the bioavailability of endectocides.

Evidence for cytochrome P4501A2-mediated protein covalent binding of thiabendazole and for its passive intestinal transport: use of human and rabbit derived cells.

Thiabendazole (TBZ), an anthelmintic and fungicide benzimidazole, was recently demonstrated to be extensively metabolized by cytochrome P450 (CYP) 1A2 in man and rabbit, yielding 5-hydroxythiabendazole (5OH-TBZ), the major metabolite furtherly conjugated, and two minor unidentified metabolites (M1 and M2). In this study, exposure of rabbit and human cells to 14C-TBZ was also shown to be associated with the appearance of radioactivity irreversibly bound to proteins. The nature of CYP isoforms involved in this covalent binding was investigated by using cultured rabbit hepatocytes treated or not with various CYP inducers (CYP1A1/2 by beta-naphthoflavone, CYP2B4 by phenobarbital, CYP3A6 by rifampicine, CYP4A by clofibrate) and human liver and bronchial CYP-expressing cells. The covalent binding to proteins was particularly increased in beta-naphthoflavone-treated rabbit cells (2- to 4-fold over control) and human cells expressing CYP1A2 (22- to 42-fold over control). Thus, CYP1A2 is a major isoenzyme involved in the formation of TBZ-derived residues bound to protein. Furthermore, according to the good correlation between covalent binding and M1 or 5OH-TBZ production, TBZ would be firstly metabolized to 5OH-TBZ and subsequently converted to a chemically reactive metabolic intermediate binding to proteins. This metabolic activation could take place preferentially in liver and lung, the main biotransformation organs, rather than in intestines where TBZ was shown to be not metabolized. Moreover, TBZ was rapidly transported by passive diffusion through the human intestinal cells by comparison with the protein-bound residues which were not able to cross the intestinal barrier. Consequently, the absence of toxicity measured in intestines could be related to the low degree of TBZ metabolism and the lack of absorption of protein adducts. Nevertheless, caution is necessary in the use of TBZ concurrently with other drugs able to regulate CYP1A2, particularly in respect to liver and lung tissues, recognised as sites of covalent-binding.

Cytochrome P450 decreases are correlated to increased microsomal oxidative damage in rabbit liver and primary cultures of rabbit hepatocytes exposed to AFB1.

Although numerous studies report hepatic drug metabolizing enzyme alterations during aflatoxicosis, the mechanisms involved in P450 decreases remain to be established. The purpose of this work is to investigate whether increased oxidative damage revealed by the detection of malondialdehyde (MDA), lipofuscin substances, and conjugated dienes in microsomes, could explain the decreased P450 content. Studies were conducted with two different doses of aflatoxin B1 (AFB1), both in vivo in rabbits and ex vivo in primary cultures of rabbit hepatocytes, in the presence or absence of beta-naphthoflavone or rifampicin used as respective P450 inducers. Strong negative correlations were observed between MDA and P450 contents, both in vivo and ex vivo, whereas rifampicin appears to protect the hepatocytes from oxidative damage but not AFB1 toxicity. Positive correlation were also obtained between MDA formation and lactate dehydrogenase (LDH), aspartate aminotransferase (ASAT) or alanine amino-transferase (ALAT) releases, used as non-specific markers of AFB1 toxicity. Taken together these results suggest that the dramatic decreases of cytochrome P450 observed in vivo during aflatoxicosis could be linked, at least in part, to microsomal oxidative damage.

Comparative effects of cytokines on constitutive and inducible expression of the gene encoding for the cytochrome P450 3A6 isoenzyme in cultured rabbit hepatocytes: consequences on progesterone 6beta-hydroxylation.

Cultured rabbit hepatocytes were used to compare the relative activities of cytokines to inhibit the constitutive or rifampicin (RIF)-induced expression of the cytochrome P450 3A6 gene (CYP3A6). Human recombinant cytokines tested were interleukin-1beta (IL-1beta) (2 U/mL), interleukin-2 (IL-2) (5,000 U/mL) and interferon-gamma (IFN-gamma) (50 U/mL). Hepatocytes were cultured in the presence or absence of 25 microM RIF for 24 hr, with or without cytokines alone or in combination. All these cytokines inhibited RIF-induced P4503A6 expression without apparent cellular toxicity. By contrast, only IFN-gamma treatment provided a significant decrease (41%) in the constitutive P4503A6 protein level. Moreover, cytokines differed in their ability to repress RIF-dependent transcriptional induction of CYP3A6: IL-1beta and IL-2 were approximately equipotent, causing an almost 40-50% suppression of CYP3A6 mRNA and protein levels, whereas IFN-gamma exerted repressive effects only on P4503A6-related erythromycin N-demethylase activity and inducible protein expression. In fact, although strongly reducing P4503A6 protein content (an approximate 70% decrease), IFN-gamma did not exhibit any influence on CYP3A6 mRNAs with the exception of its association with interleukins. All these results suggest that IL-1beta and IL-2 mainly promote a transcriptional repression mechanism, given the absence of effect of these cytokines on the basal P4503A6 level, whereas IFN-gamma exerts a post-transcriptional suppressive action on both induced and constitutive P4503A6 expression. Consequently, P4503A6-dependent progesterone 6beta-hydroxylase activity also presented a cytokine-specific pattern of inhibition, with a much greater sensitivity than P4503A6 immunoreactive protein to IL-1beta and IL-2 + IFN-gamma treatments. Thus, this study underlines the significant impact of inflammation on steroid metabolism.

Identification of human and rabbit cytochromes P450 1A2 as major isoforms involved in thiabendazole 5-hydroxylation.

This report characterized one of the major cytochrome P450 isozyme involved in thiabendazole metabolism. This study was undertaken by using both cultured rabbit hepatocytes treated or not with drugs known to specifically induced various cytochromes P450 isoenzymes (i.e., P450 1A1/2 by beta-naphthoflavone, P450 2B4 by phenobarbital, P450 3A6 by rifampicine and P450 4A by clofibrate) and human liver (THLE-5) and bronchial (BEAS-2B) epithelial cells expressing or not the major constitutive human cytochromes P450 (i.e., CYP1A2, 2A6, 2B6, 2C9, 2D6, 2E1 or 3A4). Only hepatocytes exposed to beta-naphthoflavone and clofibrate significantly metabolized thiabendazole to 5-hydroxythiabendazole. Extensive biotransformation of this anthelmintic only occurred in human cells expressing CYP1A2. Moreover, experiments performed on rabbit preparations showed good correlations between thiabendazole 5-hydroxylase activity and both ethoxyresorufin and methoxyresorufin O-dealkylase activities. Thus, CYP1A2 is a major isoenzyme involved in thiabendazole 5-hydroxylation.

Comparative Metabolism of Thiabendazole in Cultured Hepatocytes from Rats, Rabbits, Calves, Pigs, and Sheep, Including the Formation of Protein-Bound Residues.

Cultured hepatocytes from rat, rabbit, calf, pig, and sheep were used to study metabolism and formation of protein-bound residues of thiabendazole ([(14)C]TBZ), a benzimidazole anthelmintic and fungicide. In all investigated species, major pathways corresponded to 5-hydroxylation of TBZ and its further conjugation. However, marked interspecies differences in rates of TBZ disappearance and 5-hydroxy metabolite formation were demonstrated. Rabbit hepatocytes presented the fastest TBZ biotransformation and were the most extensive hydroxylators. By contrast, the lowest capacity of oxidation was observed for the rat. Two unidentified minor metabolites, designated M1 and M2, were particularly produced by sheep hepatocytes. Moreover, the protein-bound residues in these cells, which could be related to cytochrome P450-dependent oxidation, were formed in 4 times greater amounts than in the other animal cells. These findings substantiate hepatocytes as an in vitro model for prediction of hepatic metabolism in vivo.

Differential effects of interleukin-1 beta, interleukin-2, and interferon-gamma on the inducible expression of CYP 1A1 and CYP 1A2 in cultured rabbit hepatocytes.

The effects of interleukin-1 beta, interleukin-2 and interferon-gamma and their combinations were investigated on induced cytochrome P 4501A of cultured rabbit hepatocytes considered 72 h after plating. Without apparent cellular toxicity, these cytokines provoke a significant decrease in TBZ- and BNF-induced P4501A1/ 2 expression. However specific patterns of action are revealed: IL-1 beta is the most potent cytokine in regard to CYP1A1/2 mRNA suppression whereas IL-2 exerts repressive effects only on P4501A1 induced expression. Although being a strong inhibitor of induced enzymatic activities and protein contents, IFN-gamma exhibits only a weak influence on CYP1A1/2 mRNAs with the exception of its association with interleukins. All these results suggest that IL-1 beta and IL-2 promote mainly transcriptional repression mechanism whereas IFN-gamma would stimulate a post-transcriptional suppressive pathway.

Dose-related effect of aflatoxin B1 on liver drug metabolizing enzymes in rabbit.

The effects of chronic administration of aflatoxin B1 (AFB1) on liver drug metabolism enzymes were measured in New Zealand rabbits divided into three groups of 5 animals, each receiving over 5 days either arabic gum or AFB1 in arabic gum at a daily oral dose of 0.05 or 0.10 mg/kg. These treatments did not lead to any lethality in any of the treated groups, but the body weight gain was altered. Biochemical exploration of plasma components revealed a dose-dependent hepatotoxicity characterized by cytolysis and cholestasis. At 0.10 mg/kd/day of AFB1, significant decreases were observed in total liver microsomal cytochrome P450, several P450-dependent monooxygenase activities, all individual P450 isoenzymes levels analysed by Western-blotting and glutathione S-transferase activities. By contrast, at 0.05 mg/kg/day of AFB1, even though total cytochrome P450 was decreased by 30%, only P450 1A1 and 3A6 isoenzymes, and aniline hydroxylation, pentoxyresorufin O-depentylation, aminopyrine, erythromycin, ethylmorphine and dimethylnitrosamine N-demethylations were affected. In the same animal group, the only glutathione S-transferase accepting CDNB (1-chloro-2,4-dinitrobenzene) as substrate was decreased by 22%. UDP-glucuronyltransferase accepting p-nitrophenol as substrate was increased in both groups of animals (33-62%). The mechanisms that could contribute to the observed changes in drug metabolizing enzymes are discussed.

Structural requirements for the induction of cytochromes P450 by benzimidazole anthelmintic derivatives in cultured rabbit hepatocytes.

The effect of sulfur-containing benzimidazoles (thiabendazole, 5-hydroxy-thiabendazole, cambendazole) and sulfur-free derivatives (benzimidazole, carbendazim and 5-hydroxycarbendazim) on cytochrome P450 enzymes was investigated in primary cultures of rabbit hepatocytes considered 72 h after plating. Thiabendazole, cambendazole and carbendazim led to a significant dose-dependent increase in both EROD activity and cytochrome P4501A1/2 proteins and mRNA expression. Experiments using actinomycin D strongly suggest that these compounds have a transcriptional control on both CYP1A1 and CYP1A2 genes in primary hepatocytes. Thiabendazole increased both COH activity and P4502A protein levels. We conclude that sulfur is not a prerequisite to the P450 induction potential of benzimidazoles, while 5-hydroxylation leads to inefficient metabolites in terms of inducibility.

Effect of exposure of rabbit hepatocytes to sulfur-containing anthelmintics (oxfendazole and fenbendazole) on cytochrome P4501A1 expression.

The expression of cytochrome P4501A1 and 1A2 was investigated in rabbit hepatocytes maintained in primary cultures for 96 hr in the absence or presence of 100 mum of the benzimidazole anthelmintics oxfendazole or fenbendazole. Total cytochrome P-450, ethoxyresorufin O-deethylase and acetanilide hydroxylase activities were significantly increased in cell cultures receiving benzimidazoles. These increases were more marked after exposure of cultured hepatocytes to oxfendazole (OFZ) than to fenbendazole (FBZ). Western and Northern blot analysis of microsomes and RNA prepared from these cultures revealed increased levels of both protein and specific mRNA for P4501A1. The inhibition of these inductions in the presence of actinomycin D suggests a transcriptional way of activation of this gene. The ability of OFZ to bind to the Ah receptor has been examined. Data obtained from competition experiments with dioxin demonstrated that OFZ and other compounds in the benzimidazole series are not ligand of the Ah receptor. From saturation experiments and Scatchard plot analysis, rabbit hepatocyte Ah receptor (K(d) = 10.6 nm) seems to belong, as does the human Ah receptor, to a low-affinity category. Different induction rates obtained with several benzimidazole drugs suggest that the sulfur atom within the molecule is critical for CYP1A1 induction. The widely used benzimidazole anthelmintics OFZ and FBZ may exert an inducing effect through an original pathway that does not require a specific binding step to the Ah receptor.

Evidence for the ligand-independent activation of the AH receptor.

Benzimidazole derivatives are potent inducers of CYP1A1 in rabbit and human hepatocytes, but apparently do not bind the AH receptor. To resolve this paradoxical behaviour, studies have been concerned with the question of whether an alternative ligand-independent mechanism could explain the activation of the AH receptor. From experiments in cultured rabbit hepatocytes we show that benzimidazoles bind early and transiently to an unknown protein. Moreover, they are able to deplete the AHR in a time- and dose-dependent manner. In contrast, benzimidazoles are unable to induce CYP1A1 mRNA in mouse hepa-1 cells and to deplete the high-affinity AHR form from these cells. Taken together these data suggest that a signal transduction pathway, similar to that involved in the ligand-independent activation of steroid receptors, could only activate the low-affinity forms of AHR as those existing in rabbit and human cells.