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.

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.

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.

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.

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.

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.

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.