Microwave fields have little effect on α-synuclein aggregation in a Caenorhabditis elegans model of Parkinson's disease.

Potential health effects of radiofrequency (RF) radiation from mobile phones arouse widespread public concern. RF fields from handheld devices near the brain might trigger or aggravate brain tumors or neurodegenerative diseases such as Parkinson's disease (PD). Aggregation of neural α-synuclein (S) is central to PD pathophysiology, and invertebrate models expressing human S have helped elucidate factors affecting the aggregation process. We have recently developed a transgenic strain of Caenorhabditis elegans carrying two S constructs: SC tagged with cyan (C) blue fluorescent protein (CFP), and SV with the Venus (V) variant of yellow fluorescent protein (YFP). During S aggregation in these SC+SV worms, CFP, and YFP tags are brought close enough to allow Foerster Resonance Energy Transfer (FRET). As a positive control, S aggregation was promoted at low Hg(2+) concentrations, whereas higher concentrations activated stress-response genes. Using two different exposure systems described previously, we tested whether RF fields (1.0 GHz CW, 0.002-0.02 W kg(-1); 1.8 GHz CW or GSM, 1.8 W kg(-1)) could influence S aggregation in SC+SV worms. YFP fluorescence in similar SV-only worms provided internal controls, which should show opposite changes due to FRET quenching during S aggregation. No statistically significant changes were observed over several independent runs at 2.5, 24, or 96 h. Although our worm model is sensitive to chemical promoters of aggregation, no similar effects were attributable to RF exposures.

Transcriptomic analysis provides insights on hexavalent chromium induced DNA double strand breaks and their possible repair in midgut cells of Drosophila melanogaster larvae.

Hexavalent chromium [Cr(VI)] is a well known mutagen and carcinogen. Since genomic instability due to generation of double strand breaks (DSBs) is causally linked to carcinogenesis, we tested a hypothesis that Cr(VI) causes in vivo generation of DSBs and elicits DNA damage response. We fed repair proficient Drosophila melanogaster (Oregon R(+)) larvae Cr(VI) (20.0µg/ml) mixed food for 24 and 48h and observed a significant (p<0.05) induction of DSBs in their midgut cells after 48h using neutral Comet assay. Global gene expression profiling in Cr(VI)-exposed Oregon R(+) larvae unveiled mis-regulation of DSBs responsive repair genes both after 24 and 48h. In vivo generation of DSBs in exposed Drosophila was confirmed by an increased pH2Av immunostaining along with the activation of cell cycle regulation genes. Analysis of mis-regulated genes grouped under DSB response by GOEAST indicated the participation of non-homologous end joining (NHEJ) DSB repair pathway. We selected two strains, one mutant (ligIV) and another ku80-RNAi (knockdown of ku80), whose functions are essentially linked to NHEJ-DSB repair pathway. As a proof of principle, we compared the DSBs generation in larvae of these two strains with that of repair proficient Oregon R(+). Along with this, DSBs generation in spn-A and okr [essential genes in homologous recombination repair (HR) pathway] mutants was also tested for the possible involvement of HR-DSB repair. A significantly increased DSBs generation in the exposed ku80-RNAi and ligIV (mutant) larvae because of impaired repair, concomitant with an insignificant DSBs generation in okr and spn-A mutant larvae indicates an active participation of NHEJ repair pathway. The study, first of its kind to our knowledge, while providing evidences for in vivo generation of DSBs in Cr(VI) exposed Drosophila larvae, assumes significance for its relevance to higher organisms due to causal link between DSB generation and Cr(VI)-induced carcinogenesis.

Use of transgenic GFP reporter strains of the nematode Caenorhabditis elegans to investigate the patterns of stress responses induced by pesticides and by organic extracts from agricultural soils.

As a free-living nematode, C. elegans is exposed to various pesticides used in agriculture, as well as to persistent organic residues which may contaminate the soil for long periods. Following on from our previous study of metal effects on 24 GFP-reporter strains representing four different stress-response pathways in C. elegans (Anbalagan et al. Ecotoxicology 21:439-455, 2012), we now present parallel data on the responses of these same strains to several commonly used pesticides. Some of these, like dichlorvos, induced multiple stress genes in a concentration-dependent manner. Unusually, endosulfan induced only one gene (cyp-34A9) to very high levels (8-10-fold) even at the lowest test concentration, with a clear plateau at higher doses. Other pesticides, like diuron, did not alter reporter gene expression detectably even at the highest test concentration attainable, while others (such as glyphosate) did so only at very high concentrations. We have also used five responsive GFP reporters to investigate the toxicity of soil pore water from two agricultural sites in south-east Spain, designated P74 (used for cauliflower production, but significantly metal contaminated) and P73 (used for growing lettuce, but with only background levels of metals). Both soil pore water samples induced all five test genes to varying extents, yet artificial mixtures containing all major metals present had essentially no effect on these same transgenes. Soluble organic contaminants present in the pore water were extracted with acetone and dichloromethane, then after evaporation of the solvents, the organic residues were redissolved in ultrapure water to reconstitute the soluble organic components of the original soil pore water. These organic extracts induced transgene expression at similar or higher levels than the original pore water. Addition of the corresponding metal mixtures had either no effect, or reduced transgene expression towards the levels seen with soil pore water only. We conclude that the main toxicants present in these soil pore water samples are organic rather than metallic in nature. Organic extracts from a control standard soil (Lufa 2.2) had negligible effects on expression of these genes, and similarly several pesticides had little effect on the expression of a constitutive myo-3::GFP transgene. Both the P73 and P74 sites have been treated regularly with (undisclosed) pesticides, as permitted under EU regulations, though other (e.g. industrial) organic residues may also be present.

Five years later: the current status of the use of proteomics and transcriptomics in EMF research.

The World Health Organization's and Radiation and Nuclear Safety Authority's "Workshop on Application of Proteomics and Transcriptomics in Electromagnetic Fields Research" was held in Helsinki in the October/November 2005. As a consequence of this meeting, Proteomics journal published in 2006 a special issue "Application of Proteomics and Transcriptomics in EMF Research" (Vol. 6 No. 17; Guest Editor: D. Leszczynski). This Proteomics issue presented the status of research, of the effects of electromagnetic fields (EMF) using proteomics and transcriptomics methods, present in 2005. The current overview/opinion article presents the status of research in this area by reviewing all studies that were published by the end of 2010. The review work was a part of the European Cooperation in the Field of Scientific and Technical Research (COST) Action BM0704 that created a structure in which researchers in the field of EMF and health shared knowledge and information. The review was prepared by the members of the COST Action BM0704 task group on the high-throughput screening techniques and electromagnetic fields (TG-HTST-EMF).

Transgenic nematodes as biosensors for metal stress in soil pore water samples.

Caenorhabditis elegans strains carrying stress-reporter green fluorescent protein transgenes were used to explore patterns of response to metals. Multiple stress pathways were induced at high doses by most metals tested, including members of the heat shock, oxidative stress, metallothionein (mtl) and xenobiotic response gene families. A mathematical model (to be published separately) of the gene regulatory circuit controlling mtl production predicted that chemically similar divalent metals (classic inducers) should show additive effects on mtl gene induction, whereas chemically dissimilar metals should show interference. These predictions were verified experimentally; thus cadmium and mercury showed additive effects, whereas ferric iron (a weak inducer) significantly reduced the effect of mercury. We applied a similar battery of tests to diluted samples of soil pore water extracted centrifugally after mixing 20% w/w ultrapure water with air-dried soil from an abandoned lead/zinc mine in the Murcia region of Spain. In addition, metal contents of both soil and soil pore water were determined by ICP-MS, and simplified mixtures of soluble metal salts were tested at equivalent final concentrations. The effects of extracted soil pore water (after tenfold dilution) were closely mimicked by mixtures of its principal component ions, and even by the single most prevalent contaminant (zinc) alone, though other metals modulated its effects both positively and negatively. In general, mixtures containing similar (divalent) metal ions exhibited mainly additive effects, whereas admixture of dissimilar (e.g. trivalent) ions often resulted in interference, reducing overall levels of stress-gene induction. These findings were also consistent with model predictions.

Low-intensity microwave irradiation does not substantially alter gene expression in late larval and adult Caenorhabditis elegans.

Reports that low-intensity microwave radiation induces heat-shock reporter gene expression in the nematode, Caenorhabditis elegans, have recently been reinterpreted as a subtle thermal effect caused by slight heating. This study used a microwave exposure system (1.0 GHz, 0.5 W power input; SAR 0.9-3 mW kg(-1) for 6-well plates) that minimises temperature differentials between sham and exposed conditions (< or =0.1 degrees C). Parallel measurement and simulation studies of SAR distribution within this exposure system are presented. We compared five Affymetrix gene arrays of pooled triplicate RNA populations from sham-exposed L4/adult worms against five gene arrays of pooled RNA from microwave-exposed worms (taken from the same source population in each run). No genes showed consistent expression changes across all five comparisons, and all expression changes appeared modest after normalisation (< or =40% up- or down-regulated). The number of statistically significant differences in gene expression (846) was less than the false-positive rate expected by chance (1131). We conclude that the pattern of gene expression in L4/adult C. elegans is substantially unaffected by low-intensity microwave radiation; the minor changes observed in this study could well be false positives. As a positive control, we compared RNA samples from N2 worms subjected to a mild heat-shock treatment (30 degrees C) against controls at 26 degrees C (two gene arrays per condition). As expected, heat-shock genes are strongly up-regulated at 30 degrees C, particularly an hsp-70 family member (C12C8.1) and hsp-16.2. Under these heat-shock conditions, we confirmed that an hsp-16.2::GFP transgene was strongly up-regulated, whereas two non-heat-inducible transgenes (daf-16::GFP; cyp-34A9::GFP) showed little change in expression.

Continuous wave and simulated GSM exposure at 1.8 W/kg and 1.8 GHz do not induce hsp16-1 heat-shock gene expression in Caenorhabditis elegans.

Recent data suggest that there might be a subtle thermal explanation for the apparent induction by radiofrequency (RF) radiation of transgene expression from a small heat-shock protein (hsp16-1) promoter in the nematode, Caenorhabditis elegans. The RF fields used in the C. elegans study were much weaker (SAR 5-40 mW kg(-1)) than those routinely tested in many other published studies (SAR approximately 2 W kg(-1)). To resolve this disparity, we have exposed the same transgenic hsp16-1::lacZ strain of C. elegans (PC72) to higher intensity RF fields (1.8 GHz; SAR approximately 1.8 W kg(-1)). For both continuous wave (CW) and Talk-pulsed RF exposures (2.5 h at 25 degrees C), there was no indication that RF exposure could induce reporter expression above sham control levels. Thus, at much higher induced RF field strength (close to the maximum permitted exposure from a mobile telephone handset), this particular nematode heat-shock gene is not up-regulated. However, under conditions where background reporter expression was moderately elevated in the sham controls (perhaps as a result of some unknown co-stressor), we found some evidence that reporter expression may be reduced by approximately 15% following exposure to either Talk-pulsed or CW RF fields.

Latrophilin is required for toxicity of black widow spider venom in Caenorhabditis elegans.

Black widow spider venom (BWSV) kills Caenorhabditis elegans after injection owing to the presence of heat- and detergent-sensitive components, which are high-molecular-mass latrotoxins. A C. elegans homologue of latrophilin/CIRL (calcium-independent receptor for latrotoxin), B0457.1, was identified and shown to have five conserved domains. RNAi (RNA interference) of this gene rendered C. elegans resistant to BWSV, whereas RNAi for CYP37A1 or a neurexin I homologue, and a deletion mutant of the related B0286.2 gene, had no effect on BWSV toxicity. The latrophilin RNAi mutants exhibit changes in defaecation cycle and alterations in drug sensitivity. These results demonstrate that latrophilin mediates the toxicity of BWSV and provide evidence for a physiological function of this receptor.

A Fluorescence Resonance Energy Transfer Assay For Monitoring α- Synclein Aggregation in a Caenorhabditis Elegans Model For Parkinson's Disease.

The aggregation of α-synuclein (Syn or S) to form insoluble fibrils is important in the pathogenesis of Parkinson's disease, but key risk factors remain ill-defined. We have developed Fluorescence Resonance Energy Transfer (FRET)-based assays for α-synuclein aggregation, using Green Fluorescent Protein variants Cerulean (C) or Venus (V), fused to each other (CV, VC) or to human synuclein (SC, SV etc). Bacterially expressed proteins were purified to homogeneity, and C-terminal fusions SC and SV largely retained their ability to aggregate in vitro. FRET signals from mixtures of SC and SV were used to monitor aggregation. These fusion genes were linked to the C. elegans unc-54 myosin promoter to generate integrated transgenic strains. Increased FRET signals, indicative of S aggregation, were observed following treatment of unc-54::SC + unc-54::SV double transgenic worms with low concentrations of mercury or chlorpyrifos, or with RNAi against hsp-70 and hip-1. Opposite changes in Yellow Fluorescent Protein (YFP) fluorescence in an unc-54::SV strain (NL5901) are likely to reflect FRET from Yellow Fluorescent Protein to aggregates of Syn fusion protein. This could provide the basis for a high throughput screening assay, which could be used for studying the effects of toxic chemicals and environmental pollutants on the aggregation of proteins such as Syn in vivo.

Microwave radiation can alter protein conformation without bulk heating.

Exposure to microwave radiation enhances the aggregation of bovine serum albumin in vitro in a time- and temperature-dependent manner. Microwave radiation also promotes amyloid fibril formation by bovine insulin at 60 degrees C. These alterations in protein conformation are not accompanied by measurable temperature changes, consistent with estimates from field modelling of the specific absorbed radiation (15-20 mW kg(-1)). Limited denaturation of cellular proteins could explain our previous observation that modest heat-shock responses are induced by microwave exposure in Caenorhabditis elegans. We also show that heat-shock responses both to heat and microwaves are suppressed after RNA interference ablating heat-shock factor function.

Biogenic magnetite in the nematode caenorhabditis elegans.

The nematode Caenorhabditis elegans is widely used as a model system in biological research. Recently, examination of the production of heat-shock proteins in this organism in response to mobile phone-type electromagnetic field exposure produced the most robust demonstration to date of a non-thermal, deleterious biological effect. Though these results appear to be a sound demonstration of non-thermal bioeffects, to our knowledge, no mechanism has been proposed to explain them. We show, apparently for the first time, that biogenic magnetite, a ferrimagnetic iron oxide, is present in C. elegans. Its presence may have confounding effects on experiments involving electromagnetic fields as well as implications for the use of this nematode as a model system for iron biomineralization in multi-cellular organisms.

A liquid-based method for the assessment of bacterial pathogenicity using the nematode Caenorhabditis elegans.

Caenorhabditis elegans has previously been used as an alternative to mammalian models of infection with bacterial pathogens. We have developed a liquid-based assay to measure the effect of bacteria on the feeding ability of C. elegans. Using this assay we have shown that Pseudomonas aeruginosa strain PA14, Burkholderia pseudomallei and Yersinia pestis were able to inhibit feeding of C. elegans strain N2. An increase in sensitivity of the assay was achieved by using C. elegans mutant phm-2, in place of the wild-type strain. Using this assay,P. aeruginosa PA01 inhibited the feeding of C. elegans mutant phm-2. Such liquid-based feeding assays are ideally suited to the high-throughput screening of mutants of bacterial pathogens.

Construction and evaluation of a transgenic hsp16-GFP-lacZ Caenorhabditis elegans strain for environmental monitoring.

A novel integrated transgenic Caenorhabditis elegans strain (PC161) incorporates a double reporter construct with green fluorescent protein (GFP) and lacZ genes fused in-frame into the second exon of the hsp16-1 gene. This construct also includes the Simian Virus 40 (SV40) nuclear localization signal such that the fusion protein accumulates in the nuclei of expressing cells. The PC161 strain was used to monitor the effects of several known stressors, including heat, cadmium, and microwave radiation. The time course of induction was similar for both reporters but was strongly influenced by pretreatment conditions. The PC161 worms kept at 15 degrees C beforehand showed a steady increase in reporter expression (up to at least 16 h) when heated to 30 degrees C. However, if washed on ice prior to heat stress at 30 degrees C, PC161 worms showed a much steeper rise in reporter expression, reaching a maximum after 2.5 h and then plateauing. Heat shock induced strong expression of both reporter genes in all tissues apart from the germ line and early embryos. A highly significant linear dose-response relationship was observed for both transgenes with increasing cadmium concentrations (5-100 microg/ml). Prolonged exposure to microwave radiation (750 MHz and 0.5 W for 16 h) also induced expression of both transgenes at 25 and (to some extent) 27 degrees C, but only beta-galactosidase activity was detectable at 23 degrees C, and neither reporter was detectably expressed at 21 degrees C. Throughout all exposures, the lacZ reporter product was more readily detectable than coexpressed GFP. However, the GFP reporter affords opportunities to monitor the stress response in living worms.

Effects of α-synuclein overexpression in transgenic Caenorhabditis elegans strains.

The neural protein α-synuclein aggregates both in vivo and in vitro to form insoluble fibrils that are involved in Parkinson's disease pathogenesis. We have generated α-synuclein/fluorescent-protein fusion constructs overexpressed in muscle cells of the nematode, Caenorhabdtis elegans. Green Fluorescent Protein (GFP) variants, Cerulean (C) or Venus (V), were fused to the C-terminus of human α-synuclein (S); the resultant fusion genes were designated SV and SC, plus a CV fusion as well as S, C and V singly. The aggregation behavior of the purified fusion proteins (expressed in E. coli) will be described elsewhere. These constructs were fused to a C. elegans unc-54 myosin promoter, and integrated transgenic lines generated by microinjection, λ-irradiation, and outcrossing of fluorescent progeny. All transgenic lines expressing α- synuclein showed significant reductions (p <0.05) in lifespan, motility and pharyngeal pumping, as compared to wildtype worms or lines expressing CFP and/or YFP only. We showed that CFP and YFP labels colocalised in granular inclusions throughout the body wall in transgenic lines expressing both SC and SV fusions (SC+SV), whereas SV+C worms displayed YFP-labelled inclusions on a diffuse CFP background. These findings implied that the α-synuclein moieties of these fusion proteins still aggregated together in vivo, whereas CFP or YFP moieties alone did not. This in turn suggested that Foerster Resonanace Energy Transfer (FRET) between CFP and YFP labels in α-synuclein aggregates could allow the extent of aggregation to be quantified. Accordingly, we also showed that net FRET signals increased 2- fold between L4 and adult SC+SV worms.

A small temperature rise may contribute towards the apparent induction by microwaves of heat-shock gene expression in the nematode Caenorhabditis Elegans.

We have previously reported that low intensity microwave exposure (0.75-1.0 GHz CW at 0.5 W; SAR 4-40 mW/kg) can induce an apparently non-thermal heat-shock response in Caenorhabditis elegans worms carrying hsp16-1::reporter genes. Using matched copper TEM cells for both sham and exposed groups, we can detect only modest reporter induction in the latter exposed group (15-20% after 2.5 h at 26 degrees C, rising to approximately 50% after 20 h). Traceable calibration of our copper TEM cell by the National Physical Laboratory (NPL) reveals significant power loss within the cell (8.5% at 1.0 GHz), accompanied by slight heating of exposed samples (approximately 0.3 degrees C at 1.0 W). Thus, exposed samples are in fact slightly warmer (by < or =0.2 degrees C at 0.5 W) than sham controls. Following NPL recommendations, our TEM cell design was modified with the aim of reducing both power loss and consequent heating. In the modified silver-plated cell, power loss is only 1.5% at 1.0 GHz, and sample warming is reduced to approximately 0.15 degrees C at 1.0 W (i.e., < or =0.1 degrees C at 0.5 W). Under sham:sham conditions, there is no difference in reporter expression between the modified silver-plated TEM cell and an unmodified copper cell. However, worms exposed to microwaves (1.0 GHz and 0.5 W) in the silver-plated cell also show no detectable induction of reporter expression relative to sham controls in the copper cell. Thus, the 20% "microwave induction" observed using two copper cells may be caused by a small temperature difference between sham and exposed conditions. In worms incubated for 2.5 h at 26.0, 26.2, and 27.0 degrees C with no microwave field, there is a consistent and significant increase in reporter expression between 26.0 and 26.2 degrees C (by approximately 20% in each of the six independent runs), but paradoxically expression levels at 27.0 degrees C are similar to those seen at 26.0 degrees C. This surprising result is in line with other evidence pointing towards complex regulation of hsp16-1 gene expression across the sub-heat-shock range of 25-27.5 degrees C in C. elegans. We conclude that our original interpretation of a non-thermal effect of microwaves cannot be sustained; at least part of the explanation appears to be thermal.

Toxicity of short-chain alcohols to the nematode Caenorhabditis elegans: a comparison of endpoints.

The toxicities of 4 short-chain alcohols--namely methanol, ethanol, iso-propanol and iso-butanol--were compared in the nematode Caenorhabditis elegans using several different ecotoxicological endpoints. Range-finding tests were conducted using transgenic PC161 worms carrying a double reporter construct (GFP plus lacZ) linked to the stress-inducible hsp16-1 promoter. These tests showed little response from the GFP reporter, but gave good dose-response curves for the lacZ reporter--showing clear induction at 0.5% v/v ethanol in an overnight assay, but only at 4% in a shorter 6-h assay. Comparison of the short-term dose-response curves shows a confusing pattern of differences between the four alcohols tested, although dose-dependence is evident across at least part of the concentration range. Feeding inhibition assays are somewhat inconclusive with regard to alcohol type, although iso-butanol and iso-propanol appear more toxic than ethanol, while methanol is least toxic. To resolve some of the remaining ambiguities, we also used a fecundity assay to show that iso-propanol is more toxic than ethanol, and a lethality assay to show that iso-butanol is more toxic than iso-propanol. Most of the endpoints studied are consistent with the following order of toxicity: iso-butanol > iso-propanol > ethanol > or = methanol.