Comparative Assessment of the Inhibitory Potential of the Herbicide Glyphosate and Its Structural Analogs on RGD-Specific Integrins Using Enzyme-Linked Immunosorbent Assays.

Transmembrane glycoprotein integrins play crucial roles in biochemical processes, and by their inhibition or activation, different signal pathways can be disrupted, leading to abnormal physiological functions. We have previously demonstrated the inhibitory effect of glyphosate herbicide's active ingredient on cell adhesion and its αvß3 integrin antagonist effect. Therefore, it appeared particularly exciting to investigate inhibition of glyphosate and its metabolites on a wider range of Arg-Gly-Asp (RGD) binding integrins, namely αvß3, α5ß1 and αllbß3. Thus, the purpose of this study was to assess how extended the inhibitory effect observed for glyphosate on the integrin αvß3 is in terms of other RGD integrins and other structurally or metabolically related derivatives of glyphosate. Five different experimental setups using enzyme-linked immunosorbent assays were applied: (i) αvß3 binding to a synthetic polymer containing RGD; (ii) αvß3 binding to its extracellular matrix (ECM) protein, vitronectin; (iii) α5ß1 binding to the above polymer containing RGD; (iv) αllbß3 binding to its ECM protein, fibrinogen and (v) αvß3 binding to the SARS-CoV-2 spike protein receptor binding domain. Total inhibition of αvß3 binding to RGD was detected for glyphosate and its main metabolite, aminomethylphosphonic acid (AMPA), as well as for acetylglycine on α5ß1 binding to RGD.

Utilization of a Novel Immunofluorescence Instrument Prototype for the Determination of the Herbicide Glyphosate.

An enzyme-linked fluorescent immunoassay (ELFIA) method has been developed for the quantitative analytical determination of the herbicide active ingredient glyphosate in environmental matrices (surface water, soil, and plant tissues). Glyphosate, as a ubiquitous agricultural pollutant, is a xenobiotic substance with exposure in aquatic and terrestrial ecosystems due its extremely high worldwide application rate. The immunoassay developed in Project Aquafluosense is part of a fluorescence-based instrumentation setup for the in situ determination of several characteristic water quality parameters. The 96-well microplate-based competitive immunoassay method applies fluorescence signal detection in the concentration range of 0-100 ng/mL glyphosate. Application of the fluorescent signal provides a limit of detection of 0.09 ng/mL, which is 2.5-fold lower than that obtained with a visual absorbance signal. Beside the improved limit of detection, determination by fluorescence provided a wider and steeper dynamic range for glyphosate detection. No matrix effect appeared for the undiluted surface water samples, while plant tissues and soil samples required dilution rates of 1:10 and 1:100, respectively. No cross-reaction was determined with the main metabolite of glyphosate, N-aminomethylphosphonic acid, and related compounds.

Hormesis, the Individual and Combined Phytotoxicity of the Components of Glyphosate-Based Formulations on Algal Growth and Photosynthetic Activity.

The occurrence of the market-leading glyphosate active ingredient in surface waters is a globally observed phenomenon. Although co-formulants in pesticide formulations were considered inactive components from the aspects of the required main biological effect of the pesticide, several studies have proven the high individual toxicity of formulating agents, as well as the enhanced combined toxicity of the active ingredients and other components. Since the majority of active ingredients are present in the form of chemical mixtures in our environment, the possible combined toxicity between active ingredients and co-formulants is particularly important. To assess the individual and combined phytotoxicity of the components, glyphosate was tested in the form of pure active ingredient (glyphosate isopropylammonium salt) and herbicide formulations (Roundup Classic and Medallon Premium) formulated with a mixture of polyethoxylated tallow amines (POEA) or alkyl polyglucosides (APG), respectively. The order of acute toxicity was as follows for Roundup Classic: glyphosate < herbicide formulation < POEA. However, the following order was demonstrated for Medallon Premium: herbicide formulation < glyphosate < APG. Increased photosynthetic activity was detected after the exposure to the formulation (1.5-5.8 mg glyphosate/L and 0.5-2.2 mg POEA/L) and its components individually (glyphosate: 13-27.2 mg/L, POEA: 0.6-4.8 mg/L), which indicates hormetic effects. However, decreased photosynthetic activity was detected at higher concentrations of POEA (19.2 mg/L) and Roundup Classic (11.6-50.6 mg glyphosate/L). Differences were demonstrated in the sensitivity of the selected algae species and, in addition to the individual and combined toxicity of the components presented in the glyphosate-based herbicides. Both of the observed inhibitory and stimulating effects can adversely affect the aquatic ecosystems and water quality of surface waters.

Ecotoxicological Evaluation of Safener and Antimicrobial Additives in Isoxaflutole-Based Herbicide Formulations.

The environmental load by isoxaflutole and its formulated herbicide products has increasingly become apparent because, after the ban of atrazine, isoxaflutole has become its replacement active ingredient (a.i.). Obtaining information regarding the fate of this a.i. in environmental matrices and its ecotoxicological effects on aquatic organisms is essential for the risk assessment of the herbicide. In this study, the effects of Merlin Flexx- and Merlin WG75 formulated isoxaflutole-based herbicide products and two selected additives (cyprosulfamide safener and 1,2-benzisothiazol-3(2H)-one antimicrobial agent) were investigated on Raphidocelis subcapitata in growth inhibition assays. In ecotoxicological tests, two conventional (optical density and chlorophyll-a content) and two induced fluorescence-based (Fv*/Fp: efficiency of the photosystem PSII and Rfd* changes in the observed ratio of fluorescence decrease) endpoints were determined by UV-spectrophotometer and by our FluoroMeter Module, respectively. Furthermore, dissipation of isoxaflutole alone and in its formulated products was examined by an HPLC-UV method. In ecotoxicological assays, the fluorescence-based Rfd* was observed as the most sensitive endpoint. In this study, the effects of the safener cyprosulfamide and the antimicrobial agent 1,2-benzisothiazol-3(2H)-one on R. subcapitata is firstly reported. The results indicated that the isoxaflutole-equivalent toxicity of the mixture of the isoxaflutole-safener-antimicrobial agent triggered lower toxicity (EC50 = 2.81 ± 0.22 mg/L) compared to the individual effect of the a.i. (EC50 = 0.02 ± 0.00 mg/L). The Merlin Flexx formulation (EC50 = 27.04 ± 1.41 mg/L) was found to be approximately 50-fold less toxic than Merlin WG75, which can be explained by the different chemical characteristics and quantity of additives in them. The additives influenced the dissipation of the a.i. in Z8 medium, as the DT50 value decreased by approximately 1.2- and 3.5-fold under light and dark conditions, respectively.

Potential Risk of Pollen from Genetically Modified MON 810 Maize Containing Cry1Ab Toxin to Protected Lepidopteran Larvae in the Pannonian Biogeographical Region-A Retrospective View.

A credible risk analysis of maize pollen containing Cry1Ab toxin must include the assessment of (i) pollen production and its Cry1 toxin content; (ii) distribution of the pollen grains in the surroundings; (iii) pollen-catching capacity of the weeds on field edges; (iv) the lifestyle of protected lepidopteran larvae living on weeds; (v) Cry1 toxin sensitivity of non-target caterpillars; and (vi) Cry1 toxin resistance of individual non-target populations. The concentration range of 5-4300 ng Cry1Ab toxin/g dry pollen determined in MON 810 pollen batches is too diverse for handling it as a single set in any mathematical modeling. Within the work carried out mainly with the DK-440 BTY cultivar, the seed samples officially received from the variety owner produced significantly different (250-470 vs. 5-15 ng/g) Cry1Ab toxin concentrations in the pollen. Nymphalis io L1-L3 larvae were nearly six times more sensitive for Dipel than Nymphalis c-album. Feeding on the back side and in a leaf nest, Vanessa atalanta may be subject to lower pollen exposures. N. io larvae may actively attempt to avoid patches with high pollen contamination. Cry1Ab toxin resistance also partially emerged in N. io populations reared in the Pannonian Biogeographical Region (Hungary).

Herbivorous Juvenile Grass Carp (Ctenopharyngodon idella) Fed with Genetically Modified MON 810 and DAS-59122 Maize Varieties Containing Cry Toxins: Intestinal Histological, Developmental, and Immunological Investigations.

Feeding experiments with juvenile grass carp (Ctenopharyngodon idella) fed with genetically modified maize MON 810 or DAS-59122 dried leaf biomass were carried out with 1-, 3- and 6-month exposures. Dosages of 3-7 µg/fish/day Cry1Ab or 18-55 µg/fish/day Cry34Ab1 toxin did not cause mortality. No difference occurred in body or abdominal sac weights. No differences appeared in levels of inorganic phosphate, calcium, fructosamine, bile acids, triglycerides, cholesterol, and alanine and aspartame aminotransferases. DAS-59122 did not alter blood parameters tested after 3 months of feeding. MON 810 slightly decreased serum albumin levels compared to the control, only in one group. Tapeworm (Bothriocephalus acheilognathi) infection changed the levels of inorganic phosphate and calcium. Cry34Ab1 toxin appeared in blood (12.6 ± 1.9 ng/mL), but not in the muscle. It was detected in B. acheilognathi. Cry1Ab was hardly detectable in certain samples near the limit of detection. Degradation of Cry toxins was extremely quick in the fish gastrointestinal tract. After 6 months of feeding, only mild indications in certain serum parameters were observed: MON 810 slightly increased the level of apoptotic cells in the blood and reduced the number of thrombocytes in one group; DAS-59122 mildly increased the number of granulocytes compared to the near-isogenic line.

An Optical Planar Waveguide-Based Immunosensors for Determination of Fusarium Mycotoxin Zearalenone.

A planar waveguide (PW) immunosensor working as a polarisation interferometer was developed for the detection of mycotoxin zearalenone (ZON). The main element of the sensor is an optical waveguide consisting of a thin silicon nitride layer between two thicker silicon dioxide layers. A combination of a narrow waveguiding core made by photolithography with an advanced optical set-up providing a coupling of circular polarised light into the PW via its slanted edge allowed the realization of a novel sensing principle by detection of the phase shift between the p- and s-components of polarised light propagating through the PW. As the p-component is sensitive to refractive index changes at the waveguide interface, molecular events between the sensor surface and the contacting sample solution can be detected. To detect ZON concentrations in the sample solution, ZON-specific antibodies were immobilised on the waveguide via an electrostatically deposited polyelectrolyte layer, and protein A was adsorbed on it. Refractive index changes on the surface due to the binding of ZON molecules to the anchored antibodies were detected in a concentration-dependent manner up to 1000 ng/mL of ZON, allowing a limit of detection of 0.01 ng/mL. Structurally unrelated mycotoxins such as aflatoxin B1 or ochratoxin A did not exert observable cross-reactivity.

Food loss in the Hungarian food industry - Examining quantification problems and attitudes of managers and their impact on policy.

The food industry is estimated to be the second biggest generator of food losses after households. The novelty of this paper is that it focuses on this less analysed sector, conducting exploratory research based on a detailed primary survey among Hungarian food processors with a uniquely large sample of 175 respondents. A SPSS program was used to analyse the data, generating descriptive statistics, statistical tests for exploring relationships between variables and cluster analysis for classifying respondents based on their attitudes towards the problem. In-depth interviews (19) and a workshop were also applied, refining the results. Only half of the respondents maintain exact records of food loss. The leaders of the companies usually do not consider the generation of losses as a serious problem, but instead tend to see it as a natural phenomenon related to technological processes. Based on the cluster analysis carried out, the companies could be classified into two distinguishable groups. Larger companies perceived the loss to be a significantly more serious problem, they recorded it more accurately than the other group and they had more important motivations such as the desire to achieve cost reduction and better allocation of resources. Food processors face several barriers during food loss management: regulatory issues, the lack of a developed market for by-products, insufficient stakeholder collaboration along the food chain and currently, insufficient incentive to reduce losses. Reduction and better utilisation of food loss can only be achieved if there is a fundamental change in food loss regulation: incentives should be provided to drive companies to avoid losses and to make the best use of the by-products.

Development of an Immunofluorescence Assay Module for Determination of the Mycotoxin Zearalenone in Water.

Project Aquafluosense is designed to develop prototypes for a fluorescence-based instrumentation setup for in situ measurements of several characteristic parameters of water quality. In the scope of the project an enzyme-linked fluorescent immunoassay (ELFIA) method has been developed for the detection of several environmental xenobiotics, including mycotoxin zearalenone (ZON). ZON, produced by several plant pathogenic Fusarium species, has recently been identified as an emerging pollutant in surface water, presenting a hazard to aquatic ecosystems. Due to its physico-chemical properties, detection of ZON at low concentrations in surface water is a challenging task. The 96-well microplate-based fluorescence instrument is capable of detecting ZON in the concentration range of 0.09-400 ng/mL. The sensitivity and accuracy of the analytical method has been demonstrated by a comparative assessment with detection by high-performance liquid chromatography and by total internal reflection ellipsometry. The limit of detection of the method, 0.09 ng/mL, falls in the low range compared to the other reported immunoassays, but the main advantage of this ELFIA method is its efficacy in combined in situ applications for determination of various important water quality parameters detectable by induced fluorimerty-e.g., total organic carbon content, algal density or the level of other organic micropollutants detectable by immunofluorimetry. In addition, the immunofluorescence module can readily be expanded to other target analytes if proper antibodies are available for detection.

Detection of Cry1Ab toxin in the leaves of MON 810 transgenic maize.

The distribution of Cry1Ab toxin was detected in the leaves of genetically modified maize of genetic event MON 810 by enzyme-linked immunosorbent assay. Cry1Ab toxin contents in the leaves at reproductive (milk, R3) phenological stage were measured to be between 3,878 and 11,148 ng Cry1Ab toxin/g fresh weight. Toxin content was significantly lesser (significant difference (SD) = 1,823 ng Cry1Ab toxin/g fresh leaf weight, p < 0.01) in leaves at the lowest leaf level, than at higher leaf levels, probably due to partial leaf necrotisation. A substantial (up to 22%) plant-to-plant variation in Cry1Ab contents in leaves was observed. When studying toxin distribution within the cross and longitudinal sections of single leaves, lesser variability was detected diagonally, with approximately 20% higher toxin concentrations at or near the leaf vein. More significant variability (SD = 2,220 ng Cry1Ab toxin/g fresh leaf weight, p < 0.01) was seen lengthwise along the leaf, starting at 1,892 ng Cry1Ab toxin/g fresh weight at the sheath and rising to maximum concentration at the middle of the lamella. Cry1Ab toxin content may suffer significant (SD = 2,230 ng Cry1Ab toxin/g fresh leaf weight, p < 0.01) decreases in the leaf due to necrotisation. The results indicate that the longitudinal dimension of the leaf has more significance for sampling purposes than the diagonal position.

Appearance of Thiacloprid in the Guttation Liquid of Coated Maize Seeds.

Thiacloprid (TCL) uptake by maize plants that emerge from coated seeds has been investigated and characterized via measurements of the compound in the guttation liquid. TCL levels were determined in the guttation liquid: (a) under field and semi-field conditions, (b) for different maize varieties, (c) applying different dosages, and (d) as affected by cross-contamination between maize seeds via soil. Cross-contamination was described by uptake interactions between seeds coated with TCL and neighboring seeds not coated or coated with other neonicotinoids, e.g., either thiamethoxam (TMX) or clothianidin (CLO). TCL levels remained under 100 µg/mL in the guttation liquid under field conditions, and were quantifiable even on the 39th day after planting of coated seeds. Higher levels up to 188.6 µg/mL were detected in plants grown under semi-field conditions in pots. Levels in the guttation liquid were also found to be influenced by the applied dosages. The uptake of TCL was found to vary for different maize varieties. Appearance of TCL as a cross-contaminant in the guttation liquid of neighboring plants emerging from non-coated maize seeds indicates translocation of the compound via soil. Peak levels of TCL cross-contamination were found to be lower (43.6 µg/mL) than the corresponding levels in the parent maize plants emerging from coated seeds (107.5 µg/mL), but values converge to each other. Similar trends were observed with neighboring seeds coated with other neonicotinoids (TMX or CLO). The translocation rate of TCL and its uptake by other plants seem to be lower than that of TMX or CLO.

Neonicotinoids: Spreading, Translocation and Aquatic Toxicity.

Various environmental and ecotoxicological aspects related to applications of neonicotinoid insecticides are assessed. Dosages of neonicotinoids applied in seed coating materials were determined and are compared to other applications (spray and granule). Environmental levels in soils and affecting factors in translocation are discussed. Excretion of neonicotinoids via guttation from coated maize seeds up to two months upon emergence, as well as cross-contamination of plants emerged from non-coated seeds or weeds nearby have been demonstrated. Contamination of surface waters is discussed in scope of a worldwide review and the environmental fate of the neonicotinoid active ingredients and the formulating surfactant appeared to be mutually affected by each other. Toxicity of neonicotinoid active ingredients and formulations on Daphnia magna completed with some investigations of activity of the detoxifying glutathione S-transferase enzyme demonstrated the modified toxicity due to the formulating agents. Electrophysiological results on identified central neurons of the terrestrial snail Helixpomatia showed acetylcholine antagonist (inhibitory) effects of neonicotinoid insecticide products, but no agonist (ACh-like) effects were recorded. These data also suggested different molecular targets (nicotinergic acetylcholine receptors and acetylcholine esterase enzyme) of neonicotinoids in the snail central nervous system.

Commercial glyphosate-based herbicides effects on springtails (Collembola) differ from those of their respective active ingredients and vary with soil organic matter content.

Glyphosate-based herbicides (GBH) are currently the most widely used agrochemicals for weed control. Environmental risk assessments (ERA) on nontarget organisms mostly consider the active ingredients (AIs) of these herbicides, while much less is known on effects of commercial GBH formulations that are actually applied in the field. Moreover, it is largely unknown to what extent different soil characteristics alter potential side effects of herbicides. We conducted a greenhouse experiment growing a model weed population of Amaranthus retroflexus in arable field soil with either 3.0 or 4.1% soil organic matter (SOM) content and treated these weeds either with GBHs (Roundup LB Plus, Touchdown Quattro, Roundup PowerFlex) or their respective AIs (isopropylammonium, diammonium or potassium salts of glyphosate) at recommended dosages. Control pots were mechanically weeded. Nontarget effects were assessed on the surface activity of the springtail species Sminthurinus niger (pitfall trapping) and litter decomposition in the soil (teabag approach). Both GBHs and AIs increased the surface activity of springtails compared to control pots; springtail activity was higher under GBHs than under corresponding AIs. Stimulation of springtail activity was much higher in soil with higher SOM content than with low SOM content (significant treatment x SOM interaction). Litter decomposition was unaffected by GBHs, AIs or SOM levels. We suggest that ERAs for pesticides should be performed with actually applied herbicides rather than only on AIs and should also consider influences of different soil properties.

Aquatic toxicity and loss of linear alkylbenzenesulfonates alone and in a neonicotinoid insecticide formulation in surface water.

Substance losses of linear alkylbenzene sulfonates (LASs) in a neat surfactant mixture, in an insecticide formulation Mospilan 20 SG, and in solutions with different neonicotinoid active ingredients (AIs) was studied in distilled water and in surface water samples originated from River Danube. Analytical measurements were performed both by HPLC-UV and commercial ELISA methods. Loss rates of LASs were found different in these aqueous matrices, with decomposition rates higher for the neat surfactant mixture than for Mospilan 20 SG (nearly 2- and 9-fold in distilled water and in surface water from River Danube, respectively). Half-lives determined in surface water from River Danube were shown to be affected by the presence of neonicotinoid AIs thiacloprid > imidacloprid > acetamiprid (ACE), while clothianidin and thiamethoxam did not affect LAS decomposition. Aquatic toxicity of Mospilan 20 SG, along with that of its AI ACE and co-formulant LAS, as well as the mixture of ACE and LAS was also investigated in the 48-h acute immobilisation assay on the water flea (Daphnia magna) aquatic indicator organism. LAS appeared to be significantly (8-fold) more toxic in the D. magna test than ACE, and the toxicity of the formulated insecticide was found to be 1.3 and 19.6 times higher than explained by its AI and LAS content, respectively, indicating synergistic toxicity. The strongest synergy between ACE and LASs was observed, when the neat forms of the two substances were applied in combination at concentrations equivalent to those in Mospilan 20 SG.

Integrin targeting of glyphosate and its cell adhesion modulation effects on osteoblastic MC3T3-E1 cells revealed by label-free optical biosensing.

This study is a discovery of interesting and far reaching properties of the world leading herbicide active ingredient glyphosate. Here we demonstrate the cell adhesion-modifying characteristics of glyphosate affecting cellular interactions via Arg-Gly-Asp (RGD)-dependent integrins. This conclusion was supported by the observations that a glyphosate surface coating induced integrin-specific cell adhesion, while glyphosate in solution inhibited cell adhesion on an RGD-displaying surface. A sensitive, real-time, label-free, whole cell approach was used to monitor the cell adhesion kinetic processes with excellent data quality. The half maximal inhibitory concentration (IC50) for glyphosate was determined to be 0.47 ± 0.07% (20.6 mM) in serum-free conditions. A three-dimensional dissociation constant of 0.352 mM was calculated for the binding between RGD-specific integrins in intact MC3T3-E1 cells and soluble glyphosate by measuring its competition for RGD-motifs binding, while the affinity of those RGD-specific integrins to the RGD-motifs was 5.97 µM. The integrin-targeted affinity of glyphosate was proven using competitive binding assays to recombinant receptor αvß3. The present study shows not only ligand-binding properties of glyphosate, but also illustrates its remarkable biomimetic power in the case of cell adhesion.

Mycotoxin Biosensor Based on Optical Planar Waveguide.

The research aim of this work is to develop a simple and highly sensitive optical biosensor for detection of mycotoxins. This sensor is built on a planar waveguide operating on the polarization interferometry principle, i.e., detecting a phase shift between p- and s-components of polarized light developed during the binding of analyte molecules. The operation of the proposed sensor is similar to that of a Mach⁻Zehnder interferometer, while its design is much simpler and it does not require splitting the waveguide into two arms. The refractive index sensitivity of the polarization interferometer sensor was in the range of 5200 radians per refractive index unit (RIU). Several tests were conducted to detect ochratoxin A (OTA) at different concentrations in direct immunoassay with specific antibodies immobilized in the sensing window. The lowest concentration of OTA of 0.01 ng/mL caused a phase shift of nearly one period. The results obtained prove high sensitivity of the sensors, which are capable of detecting even lower concentrations of mycotoxins at the ppt (part-per-trillion) level.

Label-Free Optical Detection of Mycotoxins Using Specific Aptamers Immobilized on Gold Nanostructures.

This work focuses on the development of the novel label-free optical apta-sensors for detection of mycotoxins. A highly sensitive analytical method of total internal reflection ellipsometry (TIRE) combined with Localized Surface Plasmon Resonance (LSPR) phenomenon in nano-structured gold films was exploited here for the first time for detection of aflatoxin B1 and M1 in direct assay with specific aptamers immobilized on the surface of gold. The achieved detection of low molecular weight molecules, such as aflatoxin B1 and M1, in a wide range of concentrations from 100 ng/mL down to 0.01 ng/mL is remarkable for the LSPR method. The study of binding kinetics of aflatoxin molecules to their respective aptamers using dynamic TIRE measurements yielded the values of affinity constants in the range of 10-8⁻10-7 mol, which is characteristic for highly specific aptamer/target interactions similar to that for monoclonal antibodies. The effect of aptamers' DNA chain length on their binding characteristics was analyzed.

Visible light induces matrix metalloproteinase-9 expression in rat eye.

Up-regulation of matrix metalloproteinase-9 (MMP-9, gelatinase B) in the nervous system has been demonstrated when excitotoxicity-induced tissue remodeling and neuronal death occurs. Induction of MMP-9 by a natural stimulus has not been observed yet. Using RT-PCR and gelatin-zymography we demonstrated MMP-9 induction at transcriptional and protein levels in different structures of the rat eye following over-stimulation with white light. MMP-9 elevation occurred in the retina without reduction in photoreceptor number or major anatomical reorganization. A transient decrease in electroretinogram b-wave indicated the functional recovery. Retrobulbar injection of a broad-spectrum MMP-inhibitor GM6001, slowed the recovery rate of b-wave amplitude. Even room-light applied to dark-adapted awake animals induced MMP-9 increase in the retina, which suggests a role for MMP-9 in physiological functional plasticity of the nervous system, such as light adaptation. This is the first demonstration of MMP-9 induction by a sensory stimulus.

Implication of external price referencing and parallel trade on pharmaceutical expenditure: indirect evidence from lower-income European countries.

External price referencing (EPR) is applied more and more frequently worldwide by payers to control pharmaceutical prices. Together with the parallel trade of pharmaceuticals, EPR may result in lower pharmaceutical prices in higher-income countries and higher prices in lower-income countries, which implies that pharmaceutical expenditure grows more rapidly in the latter than in the former group. Our objective was to assess this hypothesis. We used hierarchical linear models on country-level panel data to show that-after controlling for compounding factors such as GDP, the proportion of the old-age population or life expectancy-the annual growth rate of pharmaceutical expenditure was 2.1% points larger in the lower- than in the higher-income members of the European Union between 2000 and 2008. This difference in trends became non-significant (0.6% points) after the onset of the global economic crisis. There was no significant difference between lower- and higher-income countries in the growth rate of non-pharmaceutical health expenditure in either period. Our results indirectly support the presence of price convergence of pharmaceuticals among European countries, and EPR and parallel trade may provide a reasonable explanation to the observed trend difference of pharmaceutical expenditure in the two groups of countries between 2000 and 2008. This higher growth rate of pharmaceutical expenditure put extra burden on public health care budgets in lower-income countries and resulted in disproportionately more cost-containment measures compared to higher-income countries after 2008. It remains to be seen whether the disappearance of the difference in trend growth rates due to special health policy interventions in countries with economic difficulties is temporary or permanent.

Co-Formulants in Glyphosate-Based Herbicides Disrupt Aromatase Activity in Human Cells below Toxic Levels.

Pesticide formulations contain declared active ingredients and co-formulants presented as inert and confidential compounds. We tested the endocrine disruption of co-formulants in six glyphosate-based herbicides (GBH), the most used pesticides worldwide. All co-formulants and formulations were comparably cytotoxic well below the agricultural dilution of 1% (18-2000 times for co-formulants, 8-141 times for formulations), and not the declared active ingredient glyphosate (G) alone. The endocrine-disrupting effects of all these compounds were measured on aromatase activity, a key enzyme in the balance of sex hormones, below the toxicity threshold. Aromatase activity was decreased both by the co-formulants alone (polyethoxylated tallow amine-POEA and alkyl polyglucoside-APG) and by the formulations, from concentrations 800 times lower than the agricultural dilutions; while G exerted an effect only at 1/3 of the agricultural dilution. It was demonstrated for the first time that endocrine disruption by GBH could not only be due to the declared active ingredient but also to co-formulants. These results could explain numerous in vivo results with GBHs not seen with G alone; moreover, they challenge the relevance of the acceptable daily intake (ADI) value for GBHs exposures, currently calculated from toxicity tests of the declared active ingredient alone.