Cost-effective experimental design to support modeling of concentration-response functions

Modeling concentration-response function became extremely popular in ecotoxicology during the last decade. Indeed, modeling allows determining the total response pattern of a given substance. However, reliable modeling is consuming in term of data, which is in contradiction with the current trend in ecotoxicology, which aims to reduce, for cost and ethical reasons, the number of data produced during an experiment. It is therefore crucial to determine experimental design in a cost-effective manner. In this paper, we propose to use the theory of locally D-optimal designs to determine the set of concentrations to be tested so that the parameters of the concentration-response function can be estimated with high precision. We illustrated this approach by determining the locally D-optimal designs to estimate the toxicity of the herbicide dinoseb on daphnids and algae. The results show that the number of concentrations to be tested is often equal to the number of parameters and often related to the their meaning, i.e. they are located close to the parameters. Furthermore, the results show that the locally D-optimal design often has the minimal number of support points and is not much sensitive to small changes in nominal values of the parameters. In order to reduce the experimental cost and the use of test organisms, especially in case of long-term studies, reliable nominal values may therefore be fixed based on prior knowledge and literature research instead of on preliminary experiments

Reversible and irreversible pollutant-induced bacterial cellular stress effects measured by ethidium bromide uptake and efflux.

Chemical pollution is known to affect microbial community composition but it is poorly understood how toxic compounds influence physiology of single cells that may lay at the basis of loss of reproductive fitness. Here we analyze physiological disturbances of a variety of chemical pollutants at single cell level using the bacterium Pseudomonas fluorescens in an oligotrophic growth assay. As a proxy for physiological disturbance we measured changes in geometric mean ethidium bromide (EB) fluorescence intensities in subpopulations of live and dividing cells exposed or not exposed to different dosages of tetradecane, 4-chlorophenol, 2-chlorobiphenyl, naphthalene, benzene, mercury chloride, or water-dissolved oil fractions. Because ethidium bromide efflux is an energy-dependent process any disturbance in cellular energy generation is visible as an increased cytoplasmic fluorescence. Interestingly, all pollutants even at the lowest dosage of 1 nmol/mL culture produced significantly increased ethidium bromide fluorescence compared to nonexposed controls. Ethidium bromide fluorescence intensities increased upon pollutant exposure dosage up to a saturation level, and were weakly (r(2) = 0.3905) inversely correlated to the proportion of live cells at that time point in culture. Temporal increase in EB fluorescence of growing cells is indicative for toxic but reversible effects. Cells displaying high continued EB fluorescence levels experience constant and permanent damage, and no longer contribute to population growth. The procedure developed here using bacterial ethidium bromide efflux pump activity may be a useful complement to screen sublethal toxicity effects of chemicals.

Effect of captopril on renal vascular tone in patients with essential hypertension.

1. The effect of acute inhibition of angiotensin-converting enzyme by captopril (50 mg) on renal haemodynamics and function was assessed in nine patients with essential hypertension on unrestricted sodium intake (n = 8) or low sodium diet (n = 1). 2. Captopril induced a rapid and significant decrease in arterial pressure, which was maximal within 60 min. 3. Effective renal plasma flow (ERPF) increased, glomerular filtration rate (GFR) did not change and filtration fraction (FF) decreased after captopril. No change in sodium excretion and a decrease in urinary potassium occurred. 4. In the patient on low sodium diet, captopril induced striking increases in GFR and ERPF (64 and 106% respectively). 5. The logarithm of baseline plasma renin activity was positvely correlated with the change in ERPF and negatively correlated with changes in FF and renal resistance. 6. The results indicate that in patients with essential hypertension angiotensin participates actively in the maintenance of renal vascular tone at the efferent arteriolar level. A possible influence of kinins remains to be defined.

Duloxetine inhibits effects of MDMA ("ecstasy") in vitro and in humans in a randomized placebo-controlled laboratory study.

This study assessed the effects of the serotonin (5-HT) and norepinephrine (NE) transporter inhibitor duloxetine on the effects of 3,4-methylenedioxy-methamphetamine (MDMA, ecstasy) in vitro and in 16 healthy subjects. The clinical study used a double-blind, randomized, placebo-controlled, four-session, crossover design. In vitro, duloxetine blocked the release of both 5-HT and NE by MDMA or by its metabolite 3,4-methylenedioxyamphetamine from transmitter-loaded human cells expressing the 5-HT or NE transporter. In humans, duloxetine inhibited the effects of MDMA including elevations in circulating NE, increases in blood pressure and heart rate, and the subjective drug effects. Duloxetine inhibited the pharmacodynamic response to MDMA despite an increase in duloxetine-associated elevations in plasma MDMA levels. The findings confirm the important role of MDMA-induced 5-HT and NE release in the psychotropic effects of MDMA. Duloxetine may be useful in the treatment of psychostimulant dependence. TRIAL REGISTRATION: Clinicaltrials.gov NCT00990067.

Intracellular ubiquitylation of the epithelial Na+ channel controls extracellular proteolytic channel activation via conformational change.

The epithelial Na(+) channel ENaC is a key player in the maintenance of whole body Na(+) balance, and consequently of blood pressure. It is tightly regulated by numerous signaling pathways including ubiquitylation via the ubiquitin-protein ligase Nedd4-2. This mechanism is itself under the control of several kinases, which phosphorylate Nedd4-2, thereby interfering with ENaC/Nedd4-2 interaction, or by Usp2-45, which binds to and deubiquitylates ENaC. Another, different regulatory mechanism concerns the proteolytic activation of ENaC, during which the channel is cleaved on its luminal side by intracellular convertases such as furin, and further activated by extracellular proteases such as CAP-1. This process is regulated as well but the underlying mechanisms are not understood. Previously, evidence was provided that the ubiquitylation status of ENaC may affect the cleavage of the channel. When ubiquitylation of ENaC was reduced, either by co-expressing Usp2-45, or mutating either the ENaC PY-motifs (i.e. the binding sites for Nedd4-2) or intracellular lysines (i.e. ubiquitylation sites), the level of channel cleavage was increased. Here we demonstrate that lysine-mutated ENaC channels are not ubiquitylated at the cell surface, are preferentially cleaved, and Usp2-45 does not affect their cleavage efficiency. We further show by limited proteolysis that the intracellular ubiquitylation status of ENaC affects the extracellular conformation of αENaC, by demonstrating that non-ubiquitylated channels are more efficiently cleaved when treated with extracellularly added trypsin or chymotrypsin. These results present a new paradigm in which an intracellular, post-translational modification (e.g. ubiquitylation) of a transmembrane protein can affect its extracellular conformation.

Constitutively active mutants of the beta1-adrenergic receptor.

We provide the first evidence that point mutations can constitutively activate the beta(1)-adrenergic receptor (AR). Leucine 322 of the beta(1)-AR in the C-terminal portion of its third intracellular loop was replaced with seven amino acids (I, T, E, F, C, A and K) differing in their physico-chemical properties. The beta(1)-AR mutants expressed in HEK-293 cells displayed various levels of constitutive activity which could be partially inhibited by some beta-blockers. The results of this study might have interesting implications for future studies aiming at elucidating the activation process of the beta(1)-AR as well as the mechanism of action of beta-blockers.

Neuronal nitric oxide synthase does not contribute to the modulation of pulmonary vascular tone in fetal lambs with congenital diaphragmatic hernia (nNOS in CDH lambs).

AIM: The aim of this study was to determine the presence of the neuronal nitric oxide synthase (nNOS) in near full-term lambs with congenital diaphragmatic hernia (CDH) and its role in the modulation of pulmonary vascular basal tone. METHODS: We surgically created diaphragmatic hernia on the 85th day of gestation. On the 135th, catheters were used to measure pulmonary pressure and blood flow. We tested the effects of 7-nitroindazole (7-NINA), a specific nNOS antagonist and of N-nitro-L-arginine (L-NNA), a nonspecific nitric oxide synthase antagonist. In vitro, we tested the effects of the same drugs on isolated pulmonary vessels. The presence of nNOS protein in the lungs was detected by Western blot analysis. RESULTS: Neither 7-NINA nor L-NNA modified pulmonary vascular basal tone in vivo. After L-NNA injection, acetylcholine (ACh) did not decrease significantly pulmonary vascular resistance (PVR). In vitro, L-NNA increased the cholinergic contractile-response elicited by electric field stimulation (EFS) of vascular rings from lambs with diaphragmatic hernia. CONCLUSION: We conclude that nNOS protein is present in the lungs and pulmonary artery of near full-term lamb fetuses with diaphragmatic hernia, but that it does not contribute to the reduction of pulmonary vascular tone at birth

Lack of hypotriglyceridemic effect of gemfibrozil as a consequence of age-related changes in rat liver PPARalpha.

We have investigated if changes in hepatic lipid metabolism produced by old age are related to changes in liver peroxisome proliferator-activated receptor alpha (PPARalpha). Our results indicate that 18-month-old rats showed a marked decrease in the expression and activity of liver PPARalpha, as shown by significant reductions in PPARalpha mRNA, protein and binding activity, resulting in a reduction in the relative mRNA levels of PPARalpha target genes, such as liver-carnitine-palmitoyl transferase-I (CPT-I) and mitochondrial medium-chain acyl-CoA dehydrogenase (MCAD). Further, in accordance with a liver PPARalpha deficiency in old rats, treatment of old animals with a therapeutic dose of gemfibrozil (GFB) (3mg/kg per day, 21 days) was ineffective in reducing plasma triglyceride concentrations (TG), despite attaining a 50% reduction in TG when GFB was administered to young animals at the same dose and length of treatment. We hypothesize that the decrease in hepatic PPARalpha can be related to a state of leptin resistance present in old animals.

Effects of dexamethasone on the metabolic responses to mental stress in humans.

The haemodynamic effects of the sympathetic nervous system (SNS) activations elicited by hypoglycaemia, acute alcohol administration, or insulin can be prevented by a pretreatment with dexamethasone in humans. This suggests a possible role of central corticotropin releasing hormone (GRIT) release. Mental stress activates the SNS, and decreases systemic vascular resistances though a beta-adrenergic-mediated vasodilation thought to involve vascular nitric oxide release. It also increases insulin-mediated glucose disposal, an effect presumably related to vasodilation. In order to evaluate whether activation of SNS by mental stress is glucocorticoid-sensitive, we monitored the haemodynamic and metabolic effects of mental stress during hyperinsulinaemia in healthy humans with and without a 2-day treatment with 8 mg day(-1) dexamethasone. Mental stress decreased systemic vascular resistances by 21.9% and increased insulin-mediated glucose disposal by 2 8.4% without dexamethasone pretreatment. After 2 days of dexamethasone treatment, whole body insulin-mediated glucose disposal was decreased by 40.8%. The haemodynainic effects of mental stress were however, not affected. Mental stress acutely increased insulin-mediated glucose disposal by 28.0%. This indicates that mental stress elicits a stimulation of SNS through dexamethasone-insensitive pathway, distinct of those activated by insulin, alcohol, or hyperglycaemia.

Determinants of vitellogenin B1 promoter architecture. HNF3 and estrogen responsive transcription within chromatin.

The liver-specific vitellogenin B1 promoter is efficiently activated by estrogen within a nucleosomal environment after microinjection into Xenopus laevis oocytes, consistent with the hypothesis that significant nucleosome remodeling over this promoter is not a prerequisite for the activation by the estrogen receptor (ERalpha). This observation lead us to investigate determinants other than ERalpha of chromatin structure and transcriptional activation of the vitellogenin B1 promoter in this system and in vitro. We find that the liver-enriched transcription factor HNF3 has an important organizational role for chromatin structure as demonstrated by DNase I-hypersensitive site mapping. Both HNF3 and the estrogen receptor activate transcription synergistically and are able to interact with chromatin reconstituted in vitro with three positioned nucleosomes. We propose that HNF3 is the cellular determinant which establishes a promoter environment favorable to a rapid transcriptional activation by the estrogen receptor.

The caspase 8 inhibitor c-FLIP(L) modulates T-cell receptor-induced proliferation but not activation-induced cell death of lymphocytes.

The caspase 8 inhibitor c-FLIP(L) can act in vitro as a molecular switch between cell death and growth signals transmitted by the death receptor Fas (CD95). To elucidate its function in vivo, transgenic mice were generated that overexpress c-FLIP(L) in the T-cell compartment (c-FLIP(L) Tg mice). As anticipated, FasL-induced apoptosis was inhibited in T cells from the c-FLIP(L) Tg mice. In contrast, activation-induced cell death of T cells in c-FLIP(L) Tg mice was unaffected, suggesting that this deletion process can proceed in the absence of active caspase 8. Accordingly, c-FLIP(L) Tg mice differed from Fas-deficient mice by showing no accumulation of B220(+) CD4(-) CD8(-) T cells. However, stimulation of T lymphocytes with suboptimal doses of anti-CD3 or antigen revealed increased proliferative responses in T cells from c-FLIP(L) Tg mice. Thus, a major role of c-FLIP(L) in vivo is the modulation of T-cell proliferation by decreasing the T-cell receptor signaling threshold.

A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving.

The epithelial Na+ channel (ENaC) belongs to a new class of channel proteins called the ENaC/DEG superfamily involved in epithelial Na+ transport, mechanotransduction, and neurotransmission. The role of ENaC in Na+ homeostasis and in the control of blood pressure has been demonstrated recently by the identification of mutations in ENaC beta and gamma subunits causing hypertension. The function of ENaC in Na+ reabsorption depends critically on its ability to discriminate between Na+ and other ions like K+ or Ca2+. ENaC is virtually impermeant to K+ ions, and the molecular basis for its high ionic selectivity is largely unknown. We have identified a conserved Ser residue in the second transmembrane domain of the ENaC alpha subunit (alphaS589), which when mutated allows larger ions such as K+, Rb+, Cs+, and divalent cations to pass through the channel. The relative ion permeability of each of the alphaS589 mutants is related inversely to the ionic radius of the permeant ion, indicating that alphaS589 mutations increase the molecular cutoff of the channel by modifying the pore geometry at the selectivity filter. Proper geometry of the pore is required to tightly accommodate Na+ and Li+ ions and to exclude larger cations. We provide evidence that ENaC discriminates between cations mainly on the basis of their size and the energy of dehydration.

Autologous stem cell transplantation: leukapheresis product has anti-angiogenic effects in vivo correlating with neutrophil-derived VEGFR1.

BACKGROUND: High-dose chemotherapy (HDC) followed by autologous stem cell transplantation (ASCT) is used for the treatment of hemato-oncologic malignancies. In this study, we measured the effect of HDC/ASCT on plasma concentrations of antiangiogenic soluble vascular endothelial growth factor receptor 1 (sVEGFR1) and of leukapheresis products (LP) and patient serum on chick chorioallantoic (CAM) angiogenesis. MATERIALS AND METHODS: VEGFR1- and CD34-expressing cells of leukapheresis products were analyzed by flow cytometry. Alternatively spliced isoforms of VEGFR1 mRNA were quantified using reverse transcription PCR. RESULTS: Plasma concentrations of sVEGFR1 decreased after HDC, but significantly increased after ASCT. In the CAM assay, sera of patients elicited a proangiogenic effect before and after HDC, but a strong antiangiogenic response after ASCT, comparable to that of bevacizumab at therapeutic concentrations. LP contains high concentrations of sVEGFR1, and high density of VEGFR1(+) neutrophilic granulocytes, in which mRNA expression is shifted toward the soluble VEGFR1 isoform. CONCLUSION: Neutrophil-derived antiangiogenic sVEGFR1 within the LP may contribute to the therapeutic efficacy of ASCT.