Predicting human neurotoxicity of propylene glycol methyl ether (PGME) by implementing in vitro neurotoxicity results into toxicokinetic modelling.

Although organic solvents have been associated with CNS toxicity, neurotoxicity testing is rarely a regulatory requirement. We propose a strategy to assess the potential neurotoxicity of organic solvents and predict solvent air concentrations that will not likely produce neurotoxicity in exposed individuals. The strategy integrated an in vitro neurotoxicity, an in vitro blood-brain barrier (BBB), and an in silico toxicokinetic (TK) model. We illustrated the concept with propylene glycol methyl ether (PGME), widely used in industrial and consumer products. The positive control was ethylene glycol methyl ether (EGME) and negative control propylene glycol butyl ether (PGBE), a supposedly non-neurotoxic glycol ether. PGME, PGBE, and EGME had high passive permeation across the BBB (permeability coefficients (Pe) 11.0 × 10-3, 9.0 × 10-3, and 6.0 × 10-3 cm/min, respectively). PGBE was the most potent in in vitro repeated neurotoxicity assays. EGME's main metabolite, methoxyacetic acid (MAA) may be responsible for the neurotoxic effects reported in humans. No-observed adverse effect concentrations (NOAECs) for the neuronal biomarker were for PGME, PGBE, and EGME 10.2, 0.07, and 79.2 mM, respectively. All tested substances elicited a concentration-dependent increase in pro-inflammatory cytokine expressions. The TK model was used for in vitro-to-in vivo extrapolation from PGME NOAEC to corresponding air concentrations (684 ppm). In conclusion, we were able to predict air concentrations that would not likely result in neurotoxicity using our strategy. We confirmed that the Swiss PGME occupational exposure limit (100 ppm) will not likely produce immediate adverse effects on brain cells. However, we cannot exclude possible long-term neurodegenerative effects because inflammation was observed in vitro. Our simple TK model can be parameterized for other glycol ethers and used in parallel with in vitro data for systematically screening for neurotoxicity. If further developed, this approach could be adapted to predict brain neurotoxicity from exposure to organic solvents.

Cyclosporine A kinetics in brain cell cultures and its potential of crossing the blood-brain barrier.

There is an increasing need to develop improved systems for predicting the safety of xenobiotics. However, to move beyond hazard identification the available concentration of the test compounds needs to be incorporated. In this study cyclosporine A (CsA) was used as a model compound to assess the kinetic profiles in two rodent brain cell cultures after single and repeated exposures. CsA induced-cyclophilin B (Cyp-B) secretion was also determined as CsA-specific pharmacodynamic endpoint. Since CsA is a potent p-glycoprotein substrate, the ability of this compound to cross the blood-brain barrier (BBB) was also investigated using an in vitro bovine model with repeated exposures up to 14 days. Finally, CsA uptake mechanisms were studied using a parallel artificial membrane assay (PAMPA) in combination with a Caco-2 model. Kinetic results indicate a low intracellular CsA uptake, with no marked bioaccumulation or biotransformation. In addition, only low CsA amounts crossed the BBB. PAMPA and Caco-2 experiments revealed that CsA is mostly trapped to lipophilic compartments and exits the cell apically via active transport. Thus, although CsA is unlikely to enter the brain at cytotoxic concentrations, it may cause alterations in electrical activity and is likely to increase the CNS concentration of other compounds by occupying the BBBs extrusion capacity. Such an integrated testing system, incorporating BBB, brain culture models and kinetics could be applied for assessing neurotoxicity potential of compounds.

Accelerated Caco-2 cell permeability model for drug discovery.

INTRODUCTION: By culturing Caco-2 cells according to a new and optimized protocol, it has been possible to accelerate the cell culture process in such a way that the cells can be used for experiments after only 6 days. The accelerated Caco-2 model has been compared to the traditional model (requiring 21-25 days of culture) in terms of tightness of the junctions, ability to rank chemical compounds for apparent permeability, active efflux and to discriminate P-gp substrates. METHODS AND RESULTS: In the new protocol, Caco-2 cells were cultured with the classical Caco-2 medium supplemented with puromycin. The initial cell seeding density was increased two times compared to the traditional procedure and the presence of a low concentration of puromycin in the culture medium reduced the Caco-2 permeability of mannitol. Bi-directional studies were performed with known P-gp substrates (rhodamine 123, digoxin and saquinavir) and with a total of 20 marketed drugs covering a wide range of physicochemical characteristics and therapeutic indications. Strong correlations were obtained between the apparent permeability in absorptive (Papp A→B) or secretory (Papp B→A) of the drugs in the accelerated model and in the traditional models and comparable efflux ratios were observed in the two studied models. DISCUSSION: The new protocol reduces costs for screening and leads to higher throughput compared to traditional Caco-2 cell models. This accelerated model provides short time-feedback to the drug design during the early stage of drug discovery.

In vitro blood-brain barrier model adapted to repeated-dose toxicological screening.

Toxicity to the central nervous system (CNS) is a key feature in the toxicological profile of compounds and there is a growing interest to use in vitro cell assays. The blood-brain barrier (BBB) is a highly restrictive barrier that preserves homeostasis within the brain microenvironment. By modelling the BBB it is possible to investigate whether a compound is likely to compromise its functionality, which would cause unwanted effects on brain cells. These investigations are usually performed using a single exposure to drugs, whereas CNS side effects usually result from repeated exposures. The main objective of this study was to adapt our established BBB model to the evaluation of repeated-dose toxicity at the BBB. Studies were undertaken within the European Predict-IV consortium to study the effect on BBB permeability of 12 selected drugs after 14 days of repeated treatment to a single pre-selected concentration. Compared to single exposure, a 100-fold lower colchicine concentration in 14 days repeated-dose treatment was toxic. This demonstrates the importance to evaluate the BBB toxicity in repeated-dose testing. Finally, the potentiating effects of cyclosporin A on the BBB toxicity of colchicine illustrate the possibility to use in vitro BBB models to make risk assessment of drug-drug interactions.

Environmental aspects in plant protection practices of non-agricultural pesticide users: case study of communes and the ministry of public works and transport (MET) of the Walloon Region (Belgium).

In order to gain a better understanding of non-agricultural pesticide use and to prepare the legislative and technical dossiers required under the Water Framework Directive, between October 2006 and March 2007, two surveys were conducted of 97 Walloon communes and 65 districts of the Walloon Ministry of Public Works and Transport (MET) (General Directorates for Motorways and Roads and for Waterway Infrastructure). The questionnaire (26 questions on six topics) was sent by e-mail or fax, with a response rate of 60 out of 97 communes and 33 out of 65 districts. This article describes the environmental aspects of the surveys (health-related aspects are the subject of separate article). The surveys have brought to light a number of good practices (including zero pesticides) and a growing awareness of environmental issues among non-agricultural users. However, bad habits, legislation infringements and a failure to follow good plant protection practice are still a problem and pose major environmental risks (in the form of water pollution from pesticides). Information, awareness-raising and training therefore remain a priority for non-agricultural users.

Impact of the plant protection practices on the operators' exposure: survey by the communes and ministry of equipment and transport (MET-RW).

In order to gain a better understanding of non-agricultural pesticide use and to prepare the legislative and technical dossiers required under the Water Framework Directive, between October 2006 and March 2007, two surveys were conducted of 97 Walloon communes and 65 districts of the Walloon Ministry of Public Works and Transport (MET) (General Directorates for Motorways and Roads and for Waterway Infrastructure). The questionnaire (26 questions on six topics) was sent by e-mail or fax, with a response rate of 60 out of 97 communes and 33 out of 65 districts. This article describes the health-related aspects of the surveys (environmental aspects are the subject of separate article). The surveys have brought to light a number of bad practices and a growing awareness of the non-agricultural users with respect to health risks. However, bad habits, legislation infringements and a failure to follow good plant protection practice are still a problem and pose major health risks (which endanger the operator and the public). Information, awareness-raising and, especially, training of people therefore remain a priority for non-agricultural users.

Biofilters to treat the pesticides wastes from spraying applications: results after 4 years of study.

Biofilters were developed in order to eliminate or reduce the quantity of pesticides from rinsing and cleaning waters of sprayers. Biofilters consist in 1, 2 or 3 plastic containers of 1 m3 placed in a vertical pile and containing a substrate elaborated from a homogenised mixture of local soil, chopped straw and compost able to absorb and degrade the pesticides. Biofilters are installed near the area for cleaning and rinsing the sprayer and the waste waters are pumped into the system. Since 2002, a pilot study is carried out in Belgium in order to evaluate the efficacy of these systems. Twenty pilot systems were installed since 2002 until 2004 in several farms, agricultural technical centres or schools and in a municipal maintenance service. The efficacy of the biofilters was studied for several chemical classes of herbicides (sulfonylurea, aryloxyalcanoic acids, chloroacetanilides), insecticides (pyrethroids, carbamates) and fungicides (dicarboximides, phenylamides, triazoles and strobilurines). The balance of the inputs and the outputs of the pesticides was determined by monitoring the elutes. The degradation kinetic of pesticides into the substrate was evaluated by analysing the pesticides into the substrate. The microbiological activity of the substrate was also evaluated by measuring respiration and some indirect parameters like dry matter content, Kjeldahl nitrogen content, organic carbon content and biological oxygen demand (BOD). Results obtained until now after four years of experiments have showed an overall good efficacy (retention) of pesticides by the biofilter and a high degradation rate for the majority of pesticides. Biofilters permit to reduce highly the quantity of pesticides from rinsing and cleaning waters of sprayers and contribute significantly to the reduction of the contamination of surface water. Biofilters are now registered by the Ministry of Agriculture and Environment of the Walloon Region in Belgium and are recommended to pesticides users.

Development of biofilters to treat the pesticides wastes from spraying applications.

Several studies carried out in Europe showed the importance of direct losses to the contamination of surface water by pesticides. These pesticides losses can occur at the farm site when the sprayer equipment is filled with the pesticide formulation (spills, overflowing, leaking) and during the clean-up (rinsing) of the sprayer after the treatment. In Belgium studies are carried out on biofilters to treat in an efficient way effluents containing pesticides. The biofilter substrate is elaborated from a homogenised mixture of local soil, chopped straw and peat or composted material, able to absorb or degrade the active substances. Biofilters consist in systems of 2 or 3 units depending on the spray equipment of the farmer and on the configuration of the farmyard. Each unit is made from a 1 m3 plastic container and the different units are stacked in a vertical pile and connected between them using plastic valves and pipes. Eight pilot systems were installed in March 2002 in seven farms and in one agricultural school, all selected in the loamy region of Belgium specialised in arable crops such as cereals, sugar beets and vegetables. The efficacy (yield) of the systems was determined by measuring the balance of the inputs and outputs of the pesticides. Results were expressed in percent of pesticide retained on the biofilters. The results obtained after two years with 5 tracer pesticides (atrazine, carbofuran, diuron, lenacil and simazine) brought on the biofilter installations are very satisfactory since the percentage of retention is generally higher than 95% of the amount applied. In the beginning of 2004, ten new pilot biofilters were installed in several farms or agricultural technical centres (producing cereals, sugar beets, potatoes, vegetables, fruits or ornamental plants), and in a municipal maintenance service. Some biofilters were installed in duplicate in order to compare the efficacy of different substrates. The efficacy of the biofilters was studied for the 5 classical tracer pesticides but also for other chemical classes of herbicides (sulfonylurea, aryloxyalcanoic acids, chloroacetanilides), insecticides (pyrethroids, carbamates) and fungicides (dicarboximides, phenylamides, triazoles and strobilurines). To monitor these pesticides in elutes and substrates, two analytical methods were developed, optimised and validated : the first one by Gas Chromatography with Mass Spectrometry Detection (GC-MS), and the second one by High Performance Liquid Chromatography with UV Diode Array Detection (HPLC-DAD). The micro-organisms activity in the substrate was also measured in some situations.

Database "SIGMA Pro" information system and bibliographical management of active substances of plant protection products and biocides.

In order to satisfy its obligations as stipulated in European legislative, in 1998, the Management of Surface Waters in the Walloon Region modified its monitoring of plant protection products in surface waters in co-operation with the Regional Phyto Committee (LLN). Notably, this monitoring network takes into account the agricultural specifics of the main watersheds in Wallonia. It has led to the publication of a specific decree (A.G.W. of June 29th, 2000), which, among others, specifies the list of dangerous relevant substances in Walloon Region as well as their investigation methods (quality, monitoring network, action programmes). Together with the implementation of Directive 76/464/EEC concerning the pollution caused by some dangerous substances in aquatic environment, the Management of Surface Waters is also finalizing a database connected with plant production products. It contains technical data on active ingredients and relevant metabolites (nomenclature, toxicology, physico-chemical properties, ...), bibliographical information referring to these (synthesis of documents, table with results, ...) and some Internet connections. It has been elaborated so as to help Walloon public authorities make their decision.

Theoretical conformational analysis and synthesis of analogues of the heptapeptide antibiotic K-582 A.

A detailed theoretical conformational analysis of the linear heptapeptide antibiotic [Arg2]K-582 A (Arg-Arg-D-Orn-Thr-D-Orn-Lys-D-Tyr) was carried out. The results of the computer simulation suggest that the linear peptide has a high propensity to fold in solution into a quasi-cyclic conformation in equilibrium with pi(L-D) helices. The synthesis of two inactive analogues with an L-Lys in place of D-Orn3 or D-Orn5 confirms the importance of the proposed folding pattern for the occurrence of the antimicrobial activity of K-582 A.

Molecular cloning of the tsm0185 gene responsible for adenylate cyclase activity in Saccharomyces cerevisiae.

The gene of Saccharomyces cerevisiae responsible for adenylate cyclase activity was cloned by complementation of a thermosensitive tsm0185 mutation in yeast; it was also shown to complement the yeast cyr1 mutation. Preliminary results indicate the presence of a repeated sequence on the same genomic fragment.

L'Echinococcose Pulmonaire Et Hepatique.

Report of two cases of echinococcosis of lung and liver and review of the literature.