The Critical Importance of Molecular Biomarkers and Imaging in the Study of Electrohypersensitivity. A Scientific Consensus International Report.

Clinical research aiming at objectively identifying and characterizing diseases via clinical observations and biological and radiological findings is a critical initial research step when establishing objective diagnostic criteria and treatments. Failure to first define such diagnostic criteria may lead research on pathogenesis and etiology to serious confounding biases and erroneous medical interpretations. This is particularly the case for electrohypersensitivity (EHS) and more particularly for the so-called "provocation tests", which do not investigate the causal origin of EHS but rather the EHS-associated particular environmental intolerance state with hypersensitivity to man-made electromagnetic fields (EMF). However, because those tests depend on multiple EMF-associated physical and biological parameters and have been conducted in patients without having first defined EHS objectively and/or endpoints adequately, they cannot presently be considered to be valid pathogenesis research methodologies. Consequently, the negative results obtained by these tests do not preclude a role of EMF exposure as a symptomatic trigger in EHS patients. Moreover, there is no proof that EHS symptoms or EHS itself are caused by psychosomatic or nocebo effects. This international consensus report pleads for the acknowledgement of EHS as a distinct neuropathological disorder and for its inclusion in the WHO International Classification of Diseases.

Interruption of magnesium supply at heading influenced proteome of peripheral layers and reduced grain dry weight of two wheat (Triticum aestivum L.) genotypes.

UNLABELLED: Magnesium (Mg), an indispensable mineral for plant growth, is concentrated in the peripheral layers (PLs) of the mature grain of wheat. The supply of Mg was interrupted from plant heading to maturity and a proteomic approach was used to investigate the PLs at three stages of development. Two genotypes with contrasting concentrations of Mg in the grain were studied: Apache (low Mg) and MgHL (high Mg). The concentration of Mg was significantly reduced in the roots (10-21%), straw (18-50%) and grain (24-10%), respectively. Mg deficiency altered enzymes involved in photosynthesis, glycolysis, respiration, amino acid synthesis, cell division, protein degradation and folding at early stages, especially in MgHL. This latter had smaller grain by reducing grain potential size and dry matter accumulation. By contrast in Apache, few proteins were affected at early stages and proteins related to stress/defense and arginine/proline metabolism were up accumulated resulting in lower number of grains per ear (24.9%). This study showed that Mg in PLs plays an important role in cell division, ATP generation, carbohydrate and amino acid metabolism, and hence may influence grain potential size and assimilates in grain, which determines grain weight. These results should help wheat breeders improve Mg content and hence grain yield. BIOLOGICAL SIGNIFICANCE: Magnesium (Mg) is an abundant cation and is involved in many cell activities. Its role in determining wheat productivity remains unclear. This study is the first to investigate how Mg deficiency influences the physiological characters of wheat and dry matter in the grain in two genotypes with contrasting Mg content. Moreover, Mg is concentrated in peripheral layers of grain, which are known to play a critical role in grain development. In this study, we investigated proteins in the peripheral layers expressed differentially in three development stages to identify the mechanism by which Mg influences grain development. This study revealed that the supply of Mg influences grain yield and that Mg regulates proteins related to cell metabolism and stress defense in grain.

Transformation of the Herbicide Sulcotrione into a Root Growth Enhancer Compound by Sequential Photolysis and Hydrolysis.

Xanthene-1,9-dione-3,4-dihydro-6-methylsulfonyl (1), the main product of sulcotrione phototransformation on plant leaves, was slowly hydrolyzed into 2-hydroxy-4-methylsulfonylbenzoic acid (2) and 1,3-cyclohexanedione (3) in aqueous solution. Interestingly, the rate of hydrolysis was significantly enhanced in the presence of roots of monocotyledonous plants, while the same treatment showed adverse effects on broadleaf weeds. Root growth enhancement varied according to the plant species and concentrations of compound 2, as shown with Zea mays roots. Compound 2 is a derivative of salicylic acid that is known to be a plant signaling messenger. Compound 2 was, therefore, able to mimic some known effects of this phytohormone. This work showed that a pesticide like sulcotrione was transformed into a compound exhibiting a positive impact on plant growth. This study exemplified a rarely reported situation where chemical and biological chain reactions transformed a xenobiotic into a compound exhibiting potential beneficial effects.

Synergistic health effects between chemical pollutants and electromagnetic fields.

Humans and ecosystems are exposed to highly variable and unknown cocktail of chemicals and radiations. Although individual chemicals are typically present at low concentrations, they can interact with each other resulting in additive or potentially synergistic mixture effects. This was also observed with products obtained by radiation actions such as sunlight or electromagnetic fields that can change the effects of chemicals, such as pesticides, and metal trace elements on health. Concomitant presence of various pesticides and their transformation products adds further complexity to chemical risk assessment since chronic inflammation is a key step for cancer promotion. Degradation of a parent molecule can produce several by-products which can trigger various toxic effects with different impacts on health and environment. For instance, the cocktail of sunlight irradiated sulcotrione pesticide has a greater cytotoxicity and genotoxicity than parent molecule, sulcotrione, and questions about the impact of photochemical process on environment. Adjuvants were shown to modify the biological features of pesticides. Addition of other elements, metals or biological products, can differently enhance cell toxicity of pesticides or electromagnetic radiations suggesting a synergy in living organisms. Electromagnetic fields spreading, pesticide by-products and mixtures monitoring become greater for environmental contamination evaluations.

Cytotoxicity on Allium cepa of the two main sulcotrione photoproducts, xanthene-1,9-dione-3,4-dihydro-6-methylsulphonyl and 2-chloro-4-mesylbenzoic acid.

The cytotoxic effects of 2-chloro-4-mesylbenzoic acid (CMBA) and xanthene-1,9-dione-3,4-dihydro-6-methylsulphonyl (XDD), the two main photoproducts of sulcotrione, were investigated on Allium root meristematic cells at different concentrations. Degradation of sulcotrione was correlated to mitotic index decrease, together with increasing anomaly and c-mitosis frequencies. Mitotic index significantly decreased with increasing XDD and CMBA concentrations. Cell frequency with abnormal chromosomes increased with CMBA or XDD application rates. In contrast, CMBA induced a low micronucleus rate even for high concentrations while XDD increased the micronucleus ratio. C-mitoses, chromosomal aberrations due to an inactivation of the spindle, were enhanced by CMBA treatments but not by XDD. The photochemical degradation process of the pesticide can change the risk for the environment.

Toxicity of sulcotrione and grape marc on Vicia faba cells.

The cell toxicity of sulcotrione, a selective triketone herbicide, was evaluated on Vicia faba. Sulcotrione, trademark Mikado, grape marc, and mixtures of sulcotrione or Mikado with grape marc induced cell death. Addition of grape marc to either sulcotrione or Mikado enhanced cell death, especially with Mikado. Addition of grape marc to herbicides, sulcotrione, or Mikado resulted in different expression of genes usually associated with cell stress. Mixtures of grape marc and herbicides enhanced transcript accumulation for ubiquitin, hsp 70, and cytosolic superoxide dismutase, but did not change ascorbate peroxidase transcript accumulation. The results thus provide evidence that sulcotrione, Mikado, and mixtures with grape marc can trigger cell death and specific gene expressions. Cocktails of products with sulcotrione, such as commercial additives and grape marc, can modify biological features of pesticide. Moreover, grape marc differently enhanced cell toxicity of sulcotrione and Mikado, suggesting a synergy between pesticide products and grape marc.

Water extracts from winery by-products as tobacco defense inducers.

Water extracts from winery by-products exhibited significant plant defense inducer properties. Experiments were conducted on three marc extracts containing various amounts of polyphenols and anthocyanins. Infiltration of red, white and seed grape marc extracts into tobacco leaves induced hypersensitive reaction-like lesions with cell death evidenced by Evans Blue staining. The infiltration zones and the surrounding areas revealed accumulation of autofluorescent compounds under UV light. Leaf infiltration of the three winery by-product extracts induced defense gene expression. The antimicrobial PR1, ß-1,3-glucanase PR2, and chitinase PR3 target genes were upregulated locally in tobacco plants following grape marc extract treatments. The osmotin PR5 transcripts accumulated as well in red marc extract treated-tobacco leaves. Overall, the winery by-product extracts elicited an array of plant defense responses making the grape residues a potential use of high value compounds.

Genotoxicity of sulcotrione pesticide and photoproducts on Allium cepa root meristem.

Contamination by toxic agents in the environment has become matters of concern to agricultural countries. Sulcotrione, a triketone herbicide used to control dicotyledonous weeds in maize culture is rapidly photolyzed on plant foliage and generate two main photoproducts the xanthene-1,9-dione-3,4-dihydro-6-methylsulfonyl and 2-chloro-4-mesylbenzoic acid (CMBA). The aim of this study was to analyze the potential toxicity of the herbicide and the irradiated herbicide cocktail. Cytotoxicity and genotoxicity of non irradiated and irradiated sulcotrione were investigated in Allium cepa test. The sulcotrione irradiation was monitored under sunlight simulated conditions to reach 50% of phototransformation. Concentrations of sulcotrione in the range 5 × 10(-)(9)-5 × 10(-)(5)M were tested. Cytological analysis of root tips cells showed that both non irradiated and irradiated sulcotrione caused a dose-dependent decrease of mitotic index with higher cytotoxicity for the irradiated herbicide which can lead to 24.2% reduction of mitotic index compared to water control. Concomitantly, chromosomal aberrations were observed in A.cepa root meristems. Both non irradiated sulcotrione and irradiated sulcotrione induced a dose-dependent increase of chromosomal abnormalities frequencies to a maximal value of 33.7%. A saturating effect in anomaly frequencies was observed in meristems treated with high concentrations of non irradiated sulcotrione only. These data suggest that photolyzed sulcotrione cocktail have a greater cytotoxicity and genotoxicity than parent molecule and question about the impact of photochemical process on environment.

Grape marc extract acts as elicitor of plant defence responses.

Plant protection based on novel alternative strategies is a major concern in agriculture to sustain pest management. The marc extract of red grape cultivars reveals plant defence inducer properties. Treatment with grape marc extract efficiently induced hypersensitive reaction-like lesions with cell death evidenced by Evans Blue staining of tobacco leaves. Examination of the infiltration zone and the surrounding areas under UV light revealed the accumulation of autofluorescent compounds. Both leaf infiltration and a foliar spray of the red grape extract on tobacco leaves induced defence gene expression. The PR1 and PR2 target genes were upregulated locally and systemically in tobacco plants following grape marc extract treatment. The grape extract elicited an array of plant defence responses making this natural compound a potential phytosanitary product with a challenging issue and a rather attractive option for sustainable agriculture and environmentally friendly practices.

Inhibition of herbicide photodegradation by plant products.

Pesticide reactivity toward light is rarely considered at the leaf surface after crop treatment; regardless, these degradation reactions directly impact the pesticide effectiveness. The use of sunscreen adjuvants to overcome photodegradation has presented some limitations so far. Raw hydroalcoholic plant extracts have been recently proposed to be used as photoprotecting adjuvants; on a model system they significantly decreased the photodegradation of pesticide. Here it is demonstrated that their use makes possible a dose reduction. Sulcotrione, a selective herbicide for use in maize, was tested in a growth chamber equipped with simulated solar light against a typical weed in maize. Sprayed weeds were monitored by biometrical and physiological parameters. Sulcotrione minimum dose required for a good herbicidal efficacy (ED(50), corresponding to 50% of chlorophyll content decay) was estimated to be 55 g ha(-1). In the presence of grape extract added in a 3-fold excess compared to the herbicide, the ED(50) decreased to 34 g ha(-1). The use of grape extract allows extension of sulcotrione herbicidal activity and reduction of the dose by 35% in controlled conditions. This is a promising result for the effective dose field adjustment.

Enzymatic adaptations to arsenic-induced oxidative stress in Zea mays and genotoxic effect of arsenic in root tips of Vicia faba and Zea mays.

Agronomic plant species may display physiological and biochemical responses to oxidative stress caused by heavy metals and metalloids. Zea mays plants were grown hydroponically for eight days at different concentrations of As (0, 134 and 668 µM) and at different pH (4, 7 and 9). Metabolic variations in response to As toxicity were measured using physiological parameters and antioxidant enzymatic activities. A significant decrease in SOD activity was observed in the leaves and roots of Z. mays with the majority of As treatments. As decreased G-POX activity less in leaves than in roots. An increase in the concentration of As increased APX activity in leaves and roots, except As(V) at pH 4 and pH 9 in the leaves and As(III) at pH 9 in the roots, when there was a significant decrease in APX activity at low As concentrations. After exposure to As(V), CAT activity was the same as in the control. As(III) led to an increase in CAT activity in leaves and to a decrease in roots. With increasing concentrations of As(III), CAT activity increased in both leaves and roots whatever the pH. To obtain more detailed knowledge on the effects of arsenate and arsenite exposure on Vicia faba and Z. mays, root meristems were also examined. Roots were fed hydroponically with 134, 334, 534 and 668 µM arsenate or arsenite and 4 × 10(-3)M of maleic hydrazide as positive control, at three different pH. Physiological parameters, the mitotic index and micronuclei frequencies were evaluated in root meristems. At all three pH, the highest As(V) and As(III) concentrations induced a substantial modification in root colour, increased root thickness with stiffening, and reduced root length. High concentrations also caused a significant decrease in the mitotic index, and micronucleus chromosomic aberrations were observed in the root meristems of both species.

Photoprotection by plant extracts: a new ecological means to reduce pesticide photodegradation.

A pesticide's reactivity toward light at the leaf surface after crop treatment is rarely considered, although such degradation reactions directly affect the pesticide's effectiveness. To overcome these limitations, the use of plant pigments was proposed as a new class of photoprotecting agent. The photoprotecting properties of seven plant pigments were tested under controlled conditions over herbicide sulcotrione. Grape wine extracts were tested over a panel of pesticides from distinct chemical families. The addition of plant extracts almost systematically reduced the pesticide's photoreactivity. The grape wine extracts improve at least by 38% the half-life of photolysis of almost all of the active ingredients tested, except for the herbicide triclopyr. Fustictree extract increases by 82% the photostability of the herbicide sulcotrione. Plant extracts mainly act as sunscreens; that is, the photostabilization of the active ingredient is due to the competitive energy absorption of UV photon. The use of natural plant extracts is a promising strategy to limit pesticide photodegradation. It is a way to develop sustainable and innovative technology for the plant protection industry, being beneficial from both economic and ecological points of view.

Expression of stress-related genes in tomato plants exposed to arsenic and chromium in nutrient solution.

The molecular responses of hydroponically cultivated tomato plants to As(V) or Cr(VI) were assessed by transcript accumulation analysis of genes coding for products potentially involved in heavy metal tolerance. A quantitative real-time PCR experiment was performed with Hsp90-1, MT2- and GR1-like protein genes using RNA isolated from tomato roots or shoots treated for 24h with As(V) or Cr(VI) at concentrations ranging from 80 to 640 microM. Both transient metallic treatments induced Hsp90-1 transcript accumulation in tomato plants. MT2- and GR1-like transcripts accumulated in tomato roots treated with As(V) but were only slightly affected by Cr(VI) treatment. Tomatoes showed phenotypic symptoms to heavy metal toxicity when plants were exposed to Cr(VI) but not As(V). Plant lethality was observed at 1280 microM Cr(VI), indicating that tomatoes were more tolerant to As than Cr stress under the experimental conditions used here.

Photodegradation product of sulcotrione and the physiological response of maize (Zea mays) and white mustard (Sinapis alba).

One of the strategies for decreasing the consumption of herbicides consists in improving their uptake and efficiency. It was suggested that the photodegradation of herbicides due to sunlight results in a greater demand of herbicides to be introduced into the environment in order to ensure the plant protection activity. Moreover, an ecotoxicological effect of the photoproducts needs to be clarified. The physiological response of Zea mays and Sinapis alba (weed) to sulcotrione and its main photoproduct, called chromone (xanthene-1,9-dione-3,4-dihydro-6-methylsulfonyl), was evaluated under controlled conditions in a growth chamber. The dose-response effects were determined on Z. mays and S. alba. Using the sulcotrione (doses ranging from 1 to 9mg per plant), the physiological parameters indicated a decrease of photosynthesis for the S. alba species while the Z. mays species were only slightly affected. On the contrary, the chromone had no herbicide activity on both species. The sulcotrione is known to block 4-hydroxyphenyl pyruvate dioxygenase (HPPD) enzyme. The differences between the parent herbicide and the photoproduct could be ascribed to drastic structural modifications. We have shown that the chromone probably do not block the HPPD active site.

Effect of chromium species on phytochemical and physiological parameters in Datura innoxia.

In soil, chromium can be found in two main valence states: hexavalent Cr(VI) and trivalent Cr(III). In this study, we investigated the impact of Cr on photosynthetic gas exchange, photosystem II (PSII) activity, Cr translocation and accumulation, proline content and alkaloids production, i.e. scopolamine and hyoscyamine, in Datura innoxia. Cr uptake was influenced by its oxidation state and its concentration in growth medium. The plant roots were determined as being the main organ of Cr accumulation. Cr(VI) was more toxic than Cr(III) as indicated by reduction in plant biomass and net photosynthesis. The stomatal conductance showed a similar trend to that of photosynthetic capacity. Cr(III) and Cr(VI) had a different impact on substomatal CO(2) concentration then Cr toxicity was related to its oxidation states. In plants stressed with a Cr(VI) excess, a down regulation of PSII activity was observed with an impairment of photochemical activity. Indeed, the maximum quantum yield of PSII (F(v)/F(m)), the quantum yield of PSII (PhiPSII) and the efficiency of excitation capture by open centers (F'(v)/F'(m)) decreased. Cr(III) had little effects on PSII primary photochemistry, whatever its form induces an increase of scopolamine content without changes in hyoscyamine content in leaves of D. innoxia. These results provide that chromium contamination can change the secondary metabolites composition of leaves, thereby, impacting the quality, safety and efficacy of natural plant products synthesized by D. innoxia plants.

A possible role for extra-cellular ATP in plant responses to high frequency, low amplitude electromagnetic field.

In parallel to evoking the accumulation of stress-related transcripts, exposure to low level 900 MHz EMF affected the levels of ATP, the main energy molecule of the cell. Its concentration dropped rapidly (27% after 30 min) in response to EMF exposure, along with a 18% decrease in the adenylate energy charge (AEC), a good marker of cell energy status. One could interpret this decrease in ATP and AEC in a classical way, i.e., as the result of an increase in cellular energy usage, but recent work brings exciting new insights in pointing out a signalling function for ATP, especially in the stress physiology context where it could trigger both reactive oxygen species and calcium movement (this latter being involved in plant responses to EMF exposure). In this addendum, we discuss our results within this new perspective for ATP function.

High frequency (900 MHz) low amplitude (5 V m-1) electromagnetic field: a genuine environmental stimulus that affects transcription, translation, calcium and energy charge in tomato.

Using an especially-designed facility, the Mode Stirred Reverberation Chamber, we exposed tomato plants (Lycopersicon esculentum Mill. VFN8) to low level (900 MHz, 5 V m(-1)) electromagnetic fields for a short period (10 min) and measured changes in abundance of three specific mRNA soon after exposure. Within minutes of electromagnetic stimulation, stress-related mRNA (calmodulin, calcium-dependent protein kinase and proteinase inhibitor) accumulated in a rapid, large and 3-phase manner typical of an environmental stress response. Accumulation of these transcripts into the polysomal RNA also took place (indicating that the encoded proteins were translated) but was delayed (indicating that newly-synthesized mRNA was not immediately recruited into polysomes). Transcript accumulation was maximal at normal Ca(2+) levels and was depressed at higher Ca(2+), especially for those encoding calcium-binding proteins. Removal of Ca(2+) (by addition of chelating agents or Ca(2+) channel blocker) led to total suppression of mRNA accumulation. Finally, 30 min after the electromagnetic treatment, ATP concentration and adenylate energy charge were transiently decreased, while transcript accumulation was totally prevented by application of the uncoupling reagent, CCCP. These responses occur very soon after exposure, strongly suggesting that they are the direct consequence of application of radio-frequency fields and their similarities to wound responses strongly suggests that this radiation is perceived by plants as an injurious stimulus.

Intercellular communication in plants: evidence for two rapidly transmitted systemic signals generated in response to electromagnetic field stimulation in tomato.

Exposing all of a wild-type tomato plant to electromagnetic radiation evoked rapid and substantial accumulation of basic leucine-zipper transcription factor (bZIP) mRNA in the terminal leaf (#4) with kinetics very similar to that seen in response to wounding, while in the abscisic acid (ABA) mutant (Sitiens), the response was more rapid, but transient. Submitting just the oldest leaf (#1) of a wild-type plant to irradiation evoked bZIP mRNA accumulation both locally in the exposed leaf and systemically in the unexposed (distant) leaf #4, although systemic accumulation was delayed somewhat. Accumulation of Pin2 mRNA was less than bZIP in both the exposed and distant leaves in wild type, but there was no delay in the systemic response. In Sitiens, bZIP mRNA accumulation was far less than in wild type in both local and distant leaves, while Pin2 mRNA accumulation was stronger in the exposed leaf, but totally prevented in the systemic leaf. In the jasmonic acid (JA) mutant (JL-5) and in wild-type plants treated with the ABA biosynthesis inhibitor, naproxen, responses were similar to those in the ABA mutant, while treatment of the exposed leaf with calcium antagonists totally abolished both local and systemic increases in bZIP transcript accumulation.

Plants Respond to GSM-Like Radiation.

In this article, we propose that an organism's general architecture is of primary importance for its ability to perceive electromagnetic radiation. Animals develop mainly as volumes for internal assimilation and appendages to increase their mobility, while plants develop as surfaces to optimize interaction with the environment. As a consequence, the proportion of cells directly interacting with EMF radiation at the organism/environment interface is much higher in plants than it is in animals, making them especially suited to study EMF effects on life.

Cytotoxic effects of kinetin riboside on mouse, human and plant tumour cells.

The cytotoxicity of two plant hormone compounds, kinetin and kinetin riboside, was studied on tumour cells, by colony forming assay with increased amount of cytotoxic molecules. The concentration of inhibitor required to reduce cell growth to 50% was determined for these molecules. Kinetin riboside was shown to only act on M4 Beu human and B16 murine melanoma cells at low concentration (1.5 and 0.2 microM). On mice with leukaemia P388, this product has no effect on the tumour growth, and it appears to be toxic at the dose of 25 mg/kg. Kinetin riboside was also shown to have a cytotoxic effect on plant tumour cells (crown-gall).