András Székács

No scientific consensus on GMO safety

A broad community of independent scientific researchers and scholars challenges recent claims of a consensus over the safety of genetically modified organisms (GMOs). In the following joint statement, the claimed consensus is shown to be an artificial construct that has been falsely perpetuated through diverse fora. Irrespective of contradictory evidence in the refereed literature, as documented below, the claim that there is now a consensus on the safety of GMOs continues to be widely and often uncritically aired. For decades, the safety of GMOs has been a hotly controversial topic that has been much debated around the world. Published results are contradictory, in part due to the range of different research methods employed, an inadequacy of available procedures, and differences in the analysis and interpretation of data. Such a lack of consensus on safety is also evidenced by the agreement of policymakers from over 160 countries - in the UN’s Cartagena Biosafety Protocol and the Guidelines of the Codex Alimentarius - to authorize careful case-by-case assessment of each GMO by national authorities to determine whether the particular construct satisfies the national criteria for ‘safe’. Rigorous assessment of GMO safety has been hampered by the lack of funding independent of proprietary interests. Research for the public good has been further constrained by property rights issues, and by denial of access to research material for researchers unwilling to sign contractual agreements with the developers, which confer unacceptable control over publication to the proprietary interests.The joint statement developed and signed by over 300 independent researchers, and reproduced and published below, does not assert that GMOs are unsafe or safe. Rather, the statement concludes that the scarcity and contradictory nature of the scientific evidence published to date prevents conclusive claims of safety, or of lack of safety, of GMOs. Claims of consensus on the safety of GMOs are not supported by an objective analysis of the refereed literature.

Cry1Ab toxin production of MON 810 transgenic maize

Levels of Cry1Ab toxin were detected in genetically modified maize of genetic event MON 810 against near isogenic maize as negative control by two commercial immunoassays. The immunoassays were characterized for their cross-reactivity (CR) between Cry1Ab protoxin and activated toxin, and were compared with each other for toxin detection in a reference plant sample. Cry1Ab toxin levels, corrected for active toxin content using the CR values obtained, were monitored in maize DK-440 BTY through the entire vegetation period. The toxin concentration was found to show a rapid rise in the leaves to 17.15 +/- 1.66 microg/g by the end of the fifth week of cultivation, followed by a gradual decline to 9.61 +/- 2.07 microg/g by the 16th week and a slight increase again to 13.51 +/- 1.96 microg/g during the last 2 weeks due to partial desiccation. Similar but lesser fluctuation of toxin levels was seen in the roots between 5.32 +/- 0.49 microg/g at the less differentiated V1 stage and 2.25 +/- 0.30 microg/g during plant development. In contrast, Cry1Ab toxin levels appeared to be stably 1.36 +/- 0.45, 4.98 +/- 0.31, 0.47 +/- 0.03, and 0.83 +/- 0.15 microg/g in the stem, anther wall, pollen, and grain, respectively. Toxin concentrations produced at the VT-R4 phenological stages under actual cultivation conditions were compared with each other in three different years within an 8-year period.

Forty Years with Glyphosate

Indeed, the two boasted pesticides show certain similarities in their history of discovery and fate. Both were synthesised first several decades prior to the discovery of their pesticide action. DDT and glyphosate were first described as chemical compounds 65 and 21 years before their discovery as pesticides, respectively. Both fulfilled extensive market need, therefore, both burst into mass application right after the discovery of their insecticide/herbicide activity. They both were, to some extent, connected to wars: a great part of the use of DDT was (and remains to be) hygienic, particularly after Word War II, but also the Vietnam War; while glyphosate plays an eminent role in the “drug war” (Plan Colombia) as a defoliant of marijuana fields in Mexico and South America. And last, not least, ecologically unfavourable characteristics of both was applauded as advantageous: the persistence of DDT had been seen initially as a benefit of long lasting activity, and the zwitterionic structure and consequent outstanding water solubility of glyphosate, unusual among pesticides, also used to be praised, before the environmental or ecotoxicological disadvantages of these characteristics were understood.

Cytotoxicity on human cells of Cry1Ab and Cry1Ac Bt insecticidal toxins alone or with a glyphosate‐based herbicide

The study of combined effects of pesticides represents a challenge for toxicology. In the case of the new growing generation of genetically modified (GM) plants with stacked traits, glyphosate‐based herbicides (like Roundup) residues are present in the Roundup‐tolerant edible plants (especially corns) and mixed with modified Bt insecticidal toxins that are produced by the GM plants themselves. The potential side effects of these combined pesticides on human cells are investigated in this work. Here we have tested for the very first time Cry1Ab and Cry1Ac Bt toxins (10 ppb to 100 ppm) on the human embryonic kidney cell line 293, as well as their combined actions with Roundup, within 24 h, on three biomarkers of cell death: measurements of mitochondrial succinate dehydrogenase, adenylate kinase release by membrane alterations and caspase 3/7 inductions. Cry1Ab caused cell death from 100 ppm. For Cry1Ac, under such conditions, no effects were detected. The Roundup tested alone from 1 to 20 000 ppm is necrotic and apoptotic from 50 ppm, far below agricultural dilutions (50% lethal concentration 57.5 ppm). The only measured significant combined effect was that Cry1Ab and Cry1Ac reduced caspases 3/7 activations induced by Roundup; this could delay the activation of apoptosis. There was the same tendency for the other markers. In these results, we argue that modified Bt toxins are not inert on nontarget human cells, and that they can present combined side‐effects with other residues of pesticides specific to GM plants. Copyright

Development of a non-labeled immunosensor for the herbicide trifluralin via optical waveguide lightmode spectroscopic detection

A highly sensitive immunosensor using optical waveguide lightmode spectroscopy (OWLS) was developed for the detection of the herbicide trifluralin. OWLS as an in situ and label free method of detection, based on the measurement of the diffraction of a linearly polarized laser beam (He-Ne laser, 632.8 nm) on a diffraction grating in a thin waveguide layer (SiO 2 -TiO 2 ), offered means to produce immunosensors utilizing immobilized antibodies raised against trifluralin allowing a non-competitive biosensor, or immobilized trifluralin conjugate allowing a competitive biosensor for this analyte. Immobilization of molecules sensitizing the sensor was undertaken on amino silanized waveguide surfaces in a two-step procedure using glutaraldehyde. Within the immobilized antibody (Ab) based immunosensor the signal measured was proportional to the trifluralin content in the samples, but the method allowed detection of trifluralin only above 100 ng ml -1 due to the small molecular size of the antigen (Ag). In the immobilized antigen based immunosensor, a trifluralin-bovine serum albumin (BSA) conjugate was covalently linked to the waveguide surface. During measurements the standard solutions and samples were mixed in 1:1 ratio with antiserum, containing constant amounts of antibodies. The amount of free antibodies bound to the surface was inversely proportional to the trifluralin content of the solutions measured. The immobilized antigen based method allowed detection of trifluralin in the concentration range of 2 x 10 -7 to 3 x 10 -5 ng ml -1 . Results of trifluralin determinations were compared to those obtained in parallel enzyme-linked immunosorbent assay (ELISA) tests and in gas chromatorgraphic-mass spectrometric (GC-MS) analyses, and indicated an increase of six orders of magnitude in the limit of detection (LOD).

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.

Monitoring Pesticide Residues in Surface and Ground Water in Hungary: Surveys in 1990–2015

Over 2000 surface, ground and raw drinking water samples have been analyzed in the frame of different monitoring projects in Hungary and watercourses in neighboring countries between 1990 and 2015. Effects of pesticide contamination on ecological farming and drinking water supply have been assessed. Main water pollutant ingredients of agricultural origin in Hungary are herbicides related to maize production. After EU pesticide re-registration, diazinon, atrazine, and trifluralin gradually disappeared as contaminants. High levels of water soluble pollutants (e.g., acetochlor) in surface water result in temporarily enhanced levels in raw drinking water as well. Extreme levels observed for herbicide residues were of agrochemical industrial origin.

Immunoassays for plant cytokinins as tools for the assessment of environmental stress and disease resistance.

The level of cytokinin type hormones present in plant tissues, such as N 6 -(2-isopentenyl)-adenosine (IPA), N 6 -(2isopentenyl)-adenosine (2-iP), trans-zeatin riboside (ZR) and trans-zeatin (Z) is a good indicator of the resistance of plants to abiotic environmental stresses and to necrotic pathogens. Hapten-homologous and hapten-heterologous competitive indirect enzyme-linked immunosorbent assays (ELISAs) were developed, allowing the use of minute amounts of plant extracts for cytokinin analysis. These assays were used for the detection of members of the cytokinin plant hormone family including IPA and ZR types. The assays, in optimized formats, readily detected these plant hormones at concentration levels of 2‐5 ng ml 1 , and showed high specificity for selected cytokinins. Certain assay parameters (e.g. the type of tracer enzyme, incubation and preincubation time, etc.) had a strong influence on detection sensitivity. Nonetheless, the assays appear robust showing tolerance to pH and to several water-miscible organic solvents. The described ELISA systems were sensitive enough to detect endogenous hormone levels in crude plant extracts without intense purification. In vitro selected transgenic tobacco and tomato lines showing tolerance to several stress factors proved to have higher levels of cytokinins than the corresponding control plants indicating that the developed immunoassay is suitable for the determination of stress resistance of plants by monitoring their cytokinin content.

Characterization of a spectrophotometric assay for juvenile hormone esterase

Two surrogate substrates, methyl 1-heptylthioacetothioate (HEPTAT) and methyl 1-hexylthioacetothioate (HEXTAT) were utilized to compare a new spectrophotometric assay with the standard radiochemical partition assay used to quantify juvenile hormone esterase (JHE) activity. The surrogate substrates were made with one common factor being a thiol ester moiety substituting for the ester moiety found in juvenile hormones (JHs) and a thioether replacing the 2,3-olefin of the JHs. As a result, nucleophilic attack by the serine residue of JHE at the carbonyl functional group results in a hydrolytic reaction and release of methanethiol. In the presence of Ellmans Reagent (DTNB) methanethiol will cleave the disulfide bond of DTNB resulting in a chromophore detectable at 405 nm. Methyl 1-hexylthioacetothioate and its oxygen ester analogue, methyl-1-hexylthioacetate, were compared for JHE activity. Statistical analysis of the slopes indicated a very small but significant difference between the hydrolytic rates for the thiol ester and oxygen ester. However, the data indicate that thiol esters can replace oxygen esters to quantify hydrolytic activity by the JHEs examined. Results gathered from different preparations of JHE including tissue culture media from a baculovirus expression system, affinity- and DEAE-purified enzyme, as well as insect hemolymph indicate an excellent correlation between the two assays. Isoelectric focusing of pure and crude JHE preparations resulted in coinciding peaks of hydrolytic activity when using the standard partition assay and the spectrophotometric assay, with no other peaks of activity found in the crude preparations with either substrate. Several esterase bands were found at different isoelectric points when gels were stained with alpha-naphthyl acetate.(ABSTRACT TRUNCATED AT 250 WORDS)

Neonicotinoid insecticides inhibit cholinergic neurotransmission in a molluscan (Lymnaea stagnalis) nervous system

Neonicotinoids are highly potent and selective systemic insecticides, but their widespread use also has a growing impact on non-target animals and contaminates the environment, including surface waters. We tested the neonicotinoid insecticides commercially available in Hungary (acetamiprid, Mospilan; imidacloprid, Kohinor; thiamethoxam, Actara; clothianidin, Apacs; thiacloprid, Calypso) on cholinergic synapses that exist between the VD4 and RPeD1 neurons in the central nervous system of the pond snail Lymnaea stagnalis. In the concentration range used (0.01-1 mg/ml), neither chemical acted as an acetylcholine (ACh) agonist; instead, both displayed antagonist activity, inhibiting the cholinergic excitatory components of the VD4-RPeD1 connection. Thiacloprid (0.01 mg/ml) blocked almost 90% of excitatory postsynaptic potentials (EPSPs), while the less effective thiamethoxam (0.1 mg/ml) reduced the synaptic responses by about 15%. The ACh-evoked membrane responses of the RPeD1 neuron were similarly inhibited by the neonicotinoids, confirming that the same ACh receptor (AChR) target was involved. We conclude that neonicotinoids act on nicotinergic acetylcholine receptors (nAChRs) in the snail CNS. This has been established previously in the insect CNS; however, our data indicate differences in the background mechanism or the nAChR binding site in the snail. Here, we provide the first results concerning neonicotinoid-related toxic effects on the neuronal connections in the molluscan nervous system. Aquatic animals, including molluscs, are at direct risk while facing contaminated surface waters, and snails may provide a suitable model for further studies of the behavioral/neuronal consequences of intoxication by neonicotinoids.