Low dose evaluation of the antiandrogen flutamide following a Mode of Action approach.

The dose-response characterization of endocrine mediated toxicity is an on-going debate which is controversial when exploring the nature of the dose-response curve and the effect at the low-end of the curve. To contribute to this debate we have assessed the effects of a wide range of dose levels of the antiandrogen flutamide (FLU) on 7-week male Wistar rats. FLU was administered by oral gavage at doses of 0, 0.001, 0.01, 0.1, 1 and 10mg/kg/day for 28 days. To evaluate the reproducibility, the study was performed 3 times. The molecular initiating event (MIE; AR antagonism), the key events (LH increase, Leydig cell proliferation and hyperplasia increases) and associated events involved in the mode of action (MOA) of FLU induced testicular toxicity were characterized to address the dose response concordance. Results showed no effects at low doses (<0.1mg/kg/day) for the different key events studied. The histopathological changes (Leydig cell hyperplasia) observed at 1 and 10mg/kg/day were associated with an increase in steroidogenesis gene expression in the testis from 1mg/kg/day, as well as an increase in testosterone blood level at 10mg/kg/day. Each key event dose-response was in good concordance with the MOA of FLU on the testis. From the available results, only monotonic dose-response curves were observed for the MIE, the key events, associated events and in effects observed in other sex related tissues. All the results, so far, show that the reference endocrine disruptor FLU induces threshold effects in a standard 28-day toxicity study on adult male rats.

Thyroid tumor formation in the male mouse induced by fluopyram is mediated by activation of hepatic CAR/PXR nuclear receptors.

Fluopyram, a broad spectrum fungicide, caused an increased incidence of thyroid follicular cell (TFC) adenomas in males at the highest dose evaluated (750ppm equating to 105mg/kg/day) in the mouse oncogenicity study. A series of short-term mechanistic studies were conducted in the male mouse to characterize the mode of action (MOA) for the thyroid tumor formation and to determine if No Observed Effect Levels (NOELs) exist for each key event identified. The proposed MOA consists of an initial effect on the liver by activating the constitutive androstane (Car) and pregnane X (Pxr) nuclear receptors causing increased elimination of thyroid hormones followed by an increased secretion of thyroid stimulating hormone (TSH). This change in TSH secretion results in an increase of TFC proliferation which leads to hyperplasia and eventually adenomas after chronic exposure. Car/Pxr nuclear receptors were shown to be activated as indicated by increased activity of specific Phase I enzymes (PROD and BROD, respectively). Furthermore, evidence of increased T4 metabolism was provided by the induction of phase II enzymes known to preferentially use T4 as a substrate. Additional support for the proposed MOA was provided by demonstrating increased Tsh ß transcripts in the pituitary gland. Finally, increased TFC proliferation was observed after 28days of treatment. In these dose-response studies, clear NOELs were established for phase 2 liver enzyme activities, TSH changes and TFC proliferation. Furthermore, compelling evidence for Car/Pxr activation being the molecular initiating event for these thyroid tumors was provided by the absence of the sequential key events responsible for the TCF tumors in Car/Pxr KO mice when exposed to fluopyram. In conclusion, fluopyram thyroid toxicity is mediated by activation of hepatic Car/Pxr receptors and shows a threshold dependent MOA.

Liver tumor formation in female rat induced by fluopyram is mediated by CAR/PXR nuclear receptor activation.

Fluopyram is a broad spectrum fungicide targeting plant pathogenic fungi (eg. white dot, black mold, botrytis). During the general toxicity evaluation of fluopyram in rodents, the liver was identified as a target organ (hepatomegaly and liver hypertrophy were observed in all studies). At the end of the guideline carcinogenicity study, an increased incidence of hepatocellular adenomas and carcinomas was observed in female Wistar rats following exposure to the highest fluopyram dose evaluated (1500ppm). Short-term mechanistic studies (3, 7 or 28days of exposure) were conducted in the female rat to identify the initial key events responsible for the tumor formation and to establish thresholds for each of the early hepatic changes. Increased expression of constitutive androstane receptor (CAR) and pregnane X receptor (PXR) inducible genes was recorded after each exposure period. Further confirmation of CAR/PXR activation was provided by increased activity of specific Phase I enzymes (PROD/BROD respectively). Increased hepatocellular proliferation (measured by Ki67) was observed after each exposure period with the greatest proliferative response occurring after 3days of treatment. In these studies, dose responses and clear thresholds were established for gene expression, enzyme activity and cell proliferation. Furthermore, these early hepatic changes were shown to be reversible following compound withdrawal. Other modes of action for liver tumor formation such as DNA damage, cytotoxicity and peroxisome proliferation were excluded during the investigations. In conclusion, fluopyram is a threshold carcinogen and the resultant hepatocellular carcinomas in the female rat are due to hepatocellular proliferation mediated by CAR/PXR activation.

Inter-laboratory comparisons of assessment of the allergenic potential of proteins in mice.

Assessment of the potential allergenicity of novel proteins, including those expressed in genetically modified plants, is an important issue. In previous studies, we have shown that the IgE measurement induced by systemic exposure of BALB/c mice to a range of proteins correlates broadly with what is known of their allergenic potential in humans. The approach used a homologous passive cutaneous anaphylaxis (PCA) assay that reflects IgE-dependent biological activity and is of sufficient sensitivity to detect IgE production in the absence of adjuvant. In previous studies, the immunization phase was conducted independently in two separate facilities, and the subsequent analytical work (PCA) conducted in a single facility. The purpose here was to further evaluate the transferability of this approach. To this end, BALB/c mice were exposed to a range of doses of peanut agglutinin or ovalbumin, allergenic proteins of peanut and hen's egg, respectively, in two independent laboratories. Serial doubling dilutions of serum pooled for each treatment group were analyzed for specific IgE. At higher doses of allergen very similar, or identical, IgE titers were achieved in both laboratories, although at lower doses, responses were somewhat more variable. These data demonstrate that, although technically demanding, the measurement of protein allergen-induced IgE antibody production in mice using PCA is relatively robust and is transferable and reproducible between laboratories. This approach may provide a useful tool for the safety assessment of novel proteins and suggests that continued evaluation of the approach with a wider range of protein allergens and non-sensitising proteins is justified.

[Complete (R0) resection is the only valid prognostic factor in abdominoperineal resection for recurrent cancer of the anal canal (a consecutive series of 95 patients)]. / Résection R0, seul facteur pronostique dans les amputations abdominopérinéales de rattrapage des cancers du canal anal (série consécutive de 95 patients).

INTRODUCTION: When radiation therapy fails to control cancer of the anal canal, the only therapeutic alternative is salvage abdomino-perineal resection (APR). Its role remains debatable since very few long-term survivals have been reported. No prognostic factors have yet been identified in the limited series of reported cases. PATIENTS: 95 APR's performed over a 20 year period are reviewed and analyzed. RESULTS: Median follow-up was 5.5 years. Only one prognostic factor was identified: an R0 resection (n=76) versus either R1 (n=9) or R2 (n=9) resection. Median survival for R0 APR was more than 10 years versus 1 year for R1 and R2 resections (p=0.001). There was no prognostic difference between salvage APR for disease progression (n=55) or for late recurrence (n=40). The sub-group of women<45 years of age (n=5) had a particularly poor prognosis with no survivors beyond 2 years. CONCLUSION: When anal cancer recurs after radiation therapy, a salvage APR is indicated. If an R0 resection can be achieved, median survival is greater than 10 years. However, the justification for APR when only an R1 or R2 resection can be achieved is much less clear; in such cases there was no survival beyond 3 years.

[Fast track rehabilitation in colonic surgery]. / Réhabilitation rapide en chirurgie colique.

In France, the total hospital stay after colorectal resection by laparoscopy or laparotomy varies between 10 and 20 days. For several years, the concept of fast track rehabilitation in colonic surgery has been developed. In addition to a specific surgical and anaesthetic management, this concept relies on postoperative measures, particularly an early food intake and ambulation, which allow a spectacular reduction of the hospital stay to only 2 days. Two remarks temper initial optimism: all patients do not wish a short hospital stay and the wards do not always have the available resources necessary to set up this concept.

Large-scale analysis of mRNA translation states during sucrose starvation in arabidopsis cells identifies cell proliferation and chromatin structure as targets of translational control.

Sucrose starvation of Arabidopsis (Arabidopsis thaliana) cell culture was used to identify translationally regulated genes by DNA microarray analysis. Cells were starved by subculture without sucrose, and total and polysomal RNA was extracted between 6 and 48 h. Probes were derived from both RNA populations and used to screen oligonucleotide microarrays. Out of 25,607 screened genes, 224 were found to be differentially accumulated in polysomal RNA following starvation and 21 were found to be invariant in polysomal RNA while their total RNA abundance was modified. Most of the mRNA appears to be translationally repressed (183/245 genes), which is consistent with a general decrease in metabolic activities during starvation. The parallel transcriptional analysis identifies 268 regulated genes. Comparison of transcriptional and translational gene lists highlights the importance of translational regulation (mostly repression) affecting genes involved in cell cycle and cell growth, these being overrepresented in translationally regulated genes, providing a molecular framework for the arrest of cell proliferation following starvation. Starvation-induced translational control also affects chromatin regulation genes, such as the HD1 histone deacetylase, and the level of histone H4 acetylation was found to increase during starvation. This suggests that regulation of the global nuclear transcriptional activity might be linked to cytoplasmic translational regulations.

[Malignant-like angiomyolipoma of the liver: report of one case and review of the literature]. / Angiomyolipome épithélioïde hépatique potentiellement malin: à propos d'un cas et revue de la littérature.

Frequently found in kidney, angiomyolipoma is a rare mesenchymal tumor when diagnosed in the liver and usually benign composed of proliferative blood vessels, fatty tissue and smooth muscle. We report the case of a 67-year-old woman who underwent a left hepatectomy for a 4th segment tumor unidentified after imaging and fine needle biopsy. Final anatomopathologic examination revealed an epithelioïd hepatic angiomyolipoma with signs of malignant behaviour as vascular and lymphatics embolus and invaded left portal vein thrombosis. During the subsequent 24-month follow-up, no recurrence was observed. A review of the literature found only two cases of malignant hepatic angiomyolipoma with fatal issue, however, their incidence must be underrated because of their scarcity and the difficulty of their diagnosis, which needs immunohistochemical confirmation with HMB 45 in particularly. Advances in imaging and anatomopathology in particular with the concept of PEComa (Perivascular-Epithelioïd Cell) as the unifying feature should lead to the recognition of the various variant patterns and cell types. The latter which are important for a correct diagnosis, in order to obtain reliable data about frequency, possible malignant behaviour and therefore consensus management for hepatic angiomyolipoma.

[Thoracic sympathectomy: treatment for hyperhidrosis]. / Sympathectomie thoracique: un traitement de l'hyperhidrose.

Hyperhidrosis is a benign functional anomaly which is highly stressful for the patient. Active management is required. Several medical options are available but are often ineffective. The thoracic sympathic system plays a fundamental role in propagating stimulation of sudoral gland secretion. Endoscopic thoracic sympatecomy thus provides a radical treatment for severe palmar and axillary hyperhidrosis. We describe the technique used in our unit and present results and possible complications. This method has been used by many teams for several Years and despite some differences, most confirm major patient benefit. Phenomena of transferred sudation are frequent by are usually not invalidating. Patients should however be informed of this possibility because the effect is often irreversible.

Large scale characterization of plant plasma membrane proteins.

After a brief review of the strategies used to date to identify systematically plasma membrane (PM) proteins, emphasis was given to the proteomic approach of PM proteins from the model plant Arabidopsis thaliana. Comparative analysis of two-dimensional gels from PM and cytosolic fractions was used to assess the cellular origin of proteins found in PM fraction. The classification obtained was confirmed by protein sequencing that showed, in addition, that most analyzed proteins were peripheral proteins. A large proportion of these appeared to correspond to PM-constitutive proteins that were present in the PM from different plant organs, but were not uniquely located at the PM depending on the organ. In addition, the presence of organ-specific sets of PM-specific proteins was also demonstrated. Additional procedures were developed to identify integral PM proteins. The combined use of PM washes with alkaline carbonate buffer or Triton X-100/KBr, and of a new detergent to solubilize protein, resulted in improved recovery of hydrophobic proteins on gels. Results are discussed in terms of construction of comprehensive proteomes for PM and other membranes and organelles.

Towards the recovery of hydrophobic proteins on two-dimensional electrophoresis gels.

An extensive proteomic approach relies on the possibility to visualize and analyze various types of proteins, including hydrophobic proteins which are rarely detectable on two-dimensional electrophoresis (2-DE) gels. In this study, two methods were employed for the purification of hydrophobic proteins from Arabidopsis thaliana leaf plasma membrane (PM) model plants, prior to analysis on 2-DE immobilized pH gradient (IPG) gels. Solubilization efficiency of two detergents, (3-[(3-cholomidopropyl)-1-propanesulfonic acid (CHAPS) and C8phi, were tested for the recovery of hydrophobic proteins. An immunological approach was used to determine the efficiency of the above methods. Fractionation of proteins by Triton X-114 combined with solubilization with CHAPS resulted in the inability to detect hydrophobic proteins on 2-DE gels. The use of C8phi for protein solubilization did not improve this result. On the contrary, after treatment of membranes with alkaline buffer, the solubilization of PM proteins with detergent C8phi permitted the recovery of such proteins on 2-DE gels. The combination of membrane washing and the use of zwitterionic detergent resulted in the resolution of several integral proteins and the disappearance of peripheral proteins. In the resolution of expressed genome proteins, both large pH gradients in the first dimension and various acrylamide concentrations in the second dimension must be used. Notwithstanding, it is important to combine various sample treatments and different detergents in order to resolve soluble and hydrophobic proteins.

Cloning of the V-ATPase subunit G in plant: functional expression and sub-cellular localization.

A 13-kDa tobacco plasma membrane protein was isolated from two-dimensional electrophoresis gels. After microsequencing, RT-PCR techniques and cDNA library screening allowed for the cloning of two cDNAs. These cDNAs encoded for the subunit G of the vacuolar H+-ATPase, the first one identified in plants. Analysis of mRNA distribution showed a maximum level in the leaves and in the stem of the apical part of the tobacco plant. Heterologous functional complementation of the yeast mutant (deltavma10::URA3) was achieved with the two cDNAs. After fractionation of microsomal membranes on linear sucrose gradient, Western blots were performed using antibodies against recombinant protein and three peaks were identified: one which comigrated with the tonoplast marker and the others at slightly higher density corresponding to endoplasmic reticulum and to plasma membrane fractions.

New zwitterionic detergents improve the analysis of membrane proteins by two-dimensional electrophoresis.

Severe quantitative loss of protein is often observed in high-resolution two-dimensional electrophoresis of membrane proteins, while the resolution is usually not affected. To improve the solubility of proteins in this technique, we tested denaturing cocktails containing various detergents and chaotropes. Best results were obtained with a denaturing solution containing urea, thiourea, and zwitterionic detergents, synthesized for this purpose. Among the dozen detergents synthesized and tested, amidosulfobetaines with an alkyl tail containing 14-16 carbons proved most efficient, solubilizing previously undetected membrane proteins.

Use of a proteome strategy for tagging proteins present at the plasma membrane.

A plasma membrane (PM) fraction was purified from Arabidopsis thaliana using a standard procedure and analyzed by two-dimensional (2D) gel electrophoresis. The proteins were classified according to their relative abundance in PM or cell membrane supernatant fractions. Eighty-two of the 700 spots detected on the PM 2D gels were microsequenced. More than half showed sequence similarity to proteins of known function. Of these, all the spots in the PM-specific and PM-enriched fractions, together with half of the spots with similar abundance in PM fraction and supernatant, have previously been found at the PM, supporting the validity of this approach. Extrapolation from this analysis indicates that (i) approximately 550 polypeptides found at the PM could be resolved on 2D gels; (ii) that numerous proteins with multiple locations are found at the PM; and (iii) that approximately 80% of PM-specific spots correspond to proteins with unknown function. Among the later, half are represented by ESTs or cDNAs in databases. In this way, several unknown gene products were potentially localized to the PM. These data are discussed with respect to the efficiency of organelle proteome approaches to link systematically genomic data to genome expression. It is concluded that generalized proteomes can constitute a powerful resource, with future completion of Arabidopsis genome sequencing, for genome-wide exploration of plant function.

Construction of a directory of tobacco plasma membrane proteins by combined two-dimensional gel electrophoresis and protein sequencing.

The polypeptide pattern of the plasma membrane from tobacco was studied by two-dimensional gel electrophoresis. When using classical carrier ampholyte isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis (IEF/SDS-PAGE) approximately 400 polypeptide spots were detected after silver staining and computer analysis using the QUEST software. This resolution was sufficient to assess physiological effects such as changes in a phytohormone concentration. By using pH 4-8 immobilized pH gradient (IPG)-IEF and 10%T SDS-PAGE gels, approximately 600 polypeptides, corresponding to ca. 80% of the total population expected, were resolved. This cross-section of the plasma membrane polypeptide population was mainly constituted by low or intermediate molecular mass (25 to 45 kDa) and acidic (5.2 < pI < 6.1) polypeptides. After sample application by in-gel rehydration, large amounts of plasma membrane protein (between 5 mg and 10 mg protein) were analyzed using IPG-IEF, and N-terminal protein sequencing was performed for polypeptides collected from one gel. Internal protein sequences were also obtained. Nearly all protein sequences corresponded to unidentified proteins but several of them matched translated sequences from unidentified plant expressed sequence tags (ESTs). It is concluded that the combined use of IPG-IEF gels and in-gel rehydration allows, in the case of plant membrane protein, both analytical and micropreparative separations with an efficiency comparable to that demonstrated for soluble proteins. Finally, it is suggested that a systematic investigation of plant plasma membrane polypeptides is feasible and would constitute a source of new and plant-specific genes.