J.E.H. Tattersall

Cell biology: Non-thermal heat-shock response to microwaves

Exposure limits set for microwave radiation assume that any biological effects result from tissue heating: non-thermal effects have been reported but remain controversial. We show here that prolonged exposure to low-intensity microwave fields can induce heat-shock responses in the soil nematode Caenorhabditis elegans. This effect appears to be non-thermal, suggesting that current exposure limits set for microwave equipment may need to be reconsidered.

Ion channel blockade by oximes and recovery of diaphragm muscle from soman poisoning in vitro

1 The actions of oximes and related compounds on the nicotinic acetylcholine receptor ion channel at the adult mouse muscle endplate were investigated by use of single‐channel recording techniques. The aim of the study was to determine whether the channel‐blocking properties of the compounds could contribute to their therapeutic effectiveness against soman poisoning in vitro. 2 Therapeutic effectiveness was assessed in guinea‐pig phrenic nerve‐hemidiaphragm preparations by measuring the degree of recovery of neuromuscular function produced by the compounds following poisoning by soman. A number of the compounds, including some which lacked the oxime group, produced a significant recovery of neuromuscular function which was unrelated to acetylcholinesterase (AChE) reactivation; this was reversed by washing off the compound, and was therefore attributed to a direct pharmacological action on the muscle. 3 Single channel recordings showed that some of the compounds blocked open nicotinic receptor ion channels in preparations of mouse muscle fibres. The compounds which showed the greatest direct pharmacological actions in diaphragms produce a very fast, flickering blockade of the channels. Several quantitative measures of channel‐blocking activity correlated very well with the direct pharmacological action. Furthermore, for two compounds studied in greater detail, the direct action and channel‐blocking showed similar concentration‐response relationships. 4 The results of this study indicate that the direct pharmacological action of oximes and their analogues against neuromuscular blockade by soman in vitro is due to their channel‐blocking activity. The direct action does not correlate well with protection against soman poisoning in vivo, however, which suggests that additional non‐reactivating properties of these compounds, at sites other than the neuromuscular junction, may also be important for their therapeutic effectiveness.

Transgenic nematodes as biomonitors of microwave-induced stress

Transgenic nematodes (Caenorhabditis elegans strain PC72), carrying a stress-inducible reporter gene (Escherichia coli beta-galactosidase) under the control of a C. elegans hsp16 heat-shock promoter, have been used to monitor toxicant responses both in water and soil. Because these transgenic nematodes respond both to heat and toxic chemicals by synthesising an easily detectable reporter product, they afford a useful preliminary screen for stress responses (whether thermal or non-thermal) induced by microwave radiation or other electromagnetic fields. We have used a transverse electromagnetic (TEM) cell fed from one end by a source and terminated at the other end by a matched load. Most studies were conducted using a frequency of 750 MHz, at a nominal power setting of 27 dBm. The TEM cell was held in an incubator at 25 degrees C inside a shielded room; corresponding controls were shielded and placed in the same 25 degrees C incubator; additional baseline controls were held at 15 degrees C (worm growth temperature). Stress responses were measured in terms of beta-galactosidase (reporter) induction above control levels. The time-course of response to continuous microwave radiation showed significant differences from 25 degrees C controls both at 2 and 16 h, but not at 4 or 8 h. Using a 5 x 5 multiwell plate array exposed for 2 h, the 25 microwaved samples showed highly significant responses compared with a similar control array. The wells most strongly affected were those in the rows closest to the source, whereas the most distant row did not rise above control levels, suggesting a shadow effect. These differential responses are difficult to reconcile with general heating effects, although localised power absorption affords a possible explanation. Experiments in which the frequency and/or power settings were varied suggested a greater response at 21 than at 27 dBm, both at 750 and 300 MHz, although extremely variable responses were observed at 24 dBm and 750 MHz. Thus, lower power levels tended, if anything, to induce larger responses (with the above-mentioned exception), which is opposite to the trend anticipated for any simple heating effect. These results are reproducible and data acquisition is both rapid and simple. The evidence accrued to date suggests that microwave radiation causes measurable stress to transgenic nematodes, presumably reflecting increased levels of protein damage within cells (the common signal thought to trigger hsp gene induction). The response levels observed are comparable to those observed with moderate concentrations (ppm) of metal ions such as Zn2+ and Cu2+. We conclude that this approach deserves further and more detailed investigation, but that it has already demonstrated clear biological effects of microwave radiation in terms of the activation of cellular stress responses (hsp gene induction).

Effects of low intensity radiofrequency electromagnetic fields on electrical activity in rat hippocampal slices

Slices of rat hippocampus were exposed to 700 MHz continuous wave radiofrequency (RF) fields (25.2-71.0 V m(-1), 5-15 min exposure) in a stripline waveguide. At low field intensities, the predominant effect on the electrically evoked field potential in CA1 was a potentiation of the amplitude of the population spike by up to 20%, but higher intensity fields could produce either increases or decreases of up to 120 and 80%, respectively, in the amplitude of the population spike. To eliminate the possibility of RF-induced artefacts due to the metal stimulating electrode, the effect of RF exposure on spontaneous epileptiform activity induced in CA3 by 4-aminopyridine (50-100 microM) was investigated. Exposure to RF fields (50.0 V m(-1)) reduced or abolished epileptiform bursting in 36% of slices tested. The maximum field intensity used in these experiments, 71.0 V m(-1), was calculated to produce a specific absorption rate (SAR) of between 0.0016 and 0.0044 W kg(-1) in the slices. Measurements with a Luxtron fibreoptic probe confirmed that there was no detectable temperature change (+/- 0.1 degrees C) during a 15 min exposure to this field intensity. Furthermore, imposed temperature changes of up to 1 degrees C failed to mimic the effects of RF exposure. These results suggest that low-intensity RF fields can modulate the excitability of hippocampal tissue in vitro in the absence of gross thermal effects. The changes in excitability may be consistent with reported behavioural effects of RF fields.

Human plasma-derived BuChE as a stoichiometric bioscavenger for treatment of nerve agent poisoning

Potent organophosphorous (OP) agents, such as VX, are hazardous by absorption through the skin and are resistant to conventional pharmacological antidotal treatments. The residence time of a stoichiometric bioscavenger, human butyrylcholinesterase (huBuChE), in the plasma more closely matches that of VX than do the residence times of conventional therapy drugs (oxime, anti-muscarinic, anticonvulsant). Intramuscular (i.m.) huBuChE afforded almost complete protection when administered prior to the onset of observable cholinergic signs of VX poisoning, but once signs of poisoning became evident the efficacy of i.m. huBuChE decreased. A combination of nerve agent therapy drugs (oxime, anti-muscarinic, anticonvulsant) with huBuChE (i.m.) protected 100% (8/8) of guinea-pigs from a lethal dose of VX (0.74 mg/kg) to 48 h, even when administered on signs of poisoning. Survival was presumed to be due to immediate alleviation of the cholinergic crisis by the conventional pharmacological treatment drugs, in conjunction with bioscavenger that prevented further absorbed agent reaching the AChE targets. Evidence to support this proposed mechanism of action was obtained from PKPD experiments in which multiple blood samples and microdialysate samples were collected from individual conscious ambulatory animals. Plasma concentrations of intramuscularly-administered atropine, diazepam and HI-6 reached a peak within 15 min and were eliminated rapidly within 4h. Plasma concentrations of huBuChE administered by the i.m. route took approximately 24h to reach a peak, but were well-maintained over the subsequent 7days. Thus, the pharmacological therapy rapidly treated the initial signs of poisoning, whilst the bioscavenger provided prolonged protection by neutralising further nerve agent entering the bloodstream and preventing it from reaching the target organs.

Effects of organophosphorus anticholinesterases on nicotinic receptor ion channels at adult mouse muscle endplates

1 The effects of a range of organophosphorus anticholinesterases on the nicotinic acetylcholine receptor ion channel at the adult mouse muscle endplate were investigated by use of single‐channel recording techniques. Diisopropylfluorophosphate (DFP), sarin and soman had no effect on open times at concentrations of up to 100 μm, but ecothiopate (Eco) and O‐ethyl S‐[2‐(diisopropylamino)ethyl]methyl phosphonothiolate (VX) were found to have voltage‐ and concentration‐dependent open channel‐blocking actions at concentrations of 1–50 μm. In addition to its channel‐blocking action, Eco (50 μm) had a weak agonist effect: it is suggested that this may be attributable to thiocholine produced by hydrolysis of Eco. 2 Rate constants for blockade by Eco and VX were determined according to a sequential model. The greater voltage‐dependence of the block by Eco was due to a greater voltage sensitivity of the blocking rate constant compared to VX: the voltage‐dependence of the unblocking rate constant was similar for both compounds. 3 In control recordings, the frequency of channel opening declined exponentially with time after formation of the gigaseal. Sarin and soman both increased the rate of this decline, indicating that they accelerated the rate of desensitization of the receptors. Eco and VX reduced the initial frequency of opening, which may have been due to enhancement of a fast phase of desensitization during gigaseal formation, or to blockade of closed channels. 4 It is concluded that the direct actions of organophosphates on nicotinic receptor ion channels are of little importance for their toxicity under normal conditions, since they occur only at much higher concentrations than those which cause inhibition of acetylcholinesterase. Such actions may become apparent, however, when therapies against the anticholinesterase effects of organophosphates increase their lethal dose sufficiently. These direct actions should also be taken into account when the effects of organophosphates on cholinergic transmission are studied.

Restoration of soman-blocked neuromuscular transmission in human and rat muscle by the bispyridinium non-oxime MB327 in vitro

The standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes is not sufficiently effective against all types of nerve agents. Alternative therapeutic strategies are required and bispyridinium non-oximes, acting as nicotinic antagonists, were identified as promising compounds. A previous study showed that the di(methanesulfonate) salt of the bispyridinium compound MB327 could restore soman-impaired neuromuscular function in vitro and improve survival of sarin, soman and tabun poisoned guinea pigs in vivo. Here, by using the indirect field stimulation technique, the ability of MB327 to counteract soman-impaired neuromuscular transmission was investigated in human intercostal muscle and rat diaphragm preparations. MB327 restored muscle force in a concentration-dependent manner in both species without reactivating soman-inhibited acetylcholinesterase. The therapeutic effect of MB327 could be washed out, indicating a direct effect at the nicotinic receptor level. Also the ability of MB327 to restore respiratory muscle function could be demonstrated for the first time in rat and human tissue. In combination with previous in vitro and in vivo findings MB327 may be considered a promising compound for the treatment of nerve agent poisoning and further studies are needed to identify optimized drug combinations, concentrations and dosing intervals to provide an effective therapy for OP poisoning.

Interaction of bispyridinium compounds with the orthosteric binding site of human α7 and Torpedo californica nicotinic acetylcholine receptors (nAChRs)

Standard treatment of poisoning by organophosphorus (OP) nerve agents with atropine and oximes lacks efficacy with different nerve agents. A direct pharmacologic intervention at the nicotinic acetylcholine receptor (nAChR) was proposed as an alternative therapeutic approach and promising in vitro and in vivo results were obtained with the bispyridinium compound SAD-128. In addition, a number of SAD-128 analogues improved neuromuscular transmission of soman-poisoned diaphragms in vitro. We investigated the interaction of six of these SAD-128 analogues with the orthosteric binding site of the human α7 nAChR and Torpedo californica nAChR with a high-throughput assay using radioactive ligands. The determined affinity constants indicate a weak interaction of three test compounds (K(i) in the micromolar range) with both receptors, but no interaction could be recorded with the other three test compounds. The six SAD-128 analogues showed a low intrinsic inhibitory potency with human acetylcholinesterase (IC₅₀ > 400 μM). In conclusion, the results of the present study do not indicate a correlation between the affinity to the orthosteric binding site and the functional improvement of neuromuscular transmission and it is assumed that other mechanisms contribute to the therapeutic effect of the tested compounds.

Microwave radiation induces a heat-shock response and enhances growth in the nematode Caenorhabditis elegans

This paper shows that prolonged (overnight) exposure to continuous microwave fields (750 MHz, 0.5 W) can induce both a heat-shock response and enhanced growth in the nematode worm Caenorhabditis elegans. Exposures were conducted in a TEM cell with matched load, producing an E-field of approximately 45 V m/sup -1/ at the center (where test worms are placed). Biomonitoring of heat-shock responses has been simplified by using two transgenic strains (PC72 and PC161), which both carry stress-inducible reporter constructs, respectively, placing lacZ (/spl beta/-galactosidase) and lacZ plus green fluorescent protein expression under the control of C. elegans hsp16-1 promoters. In situ localization of reporter expression reveals a minority of test worms, which respond strongly to microwave exposure. Enzyme activity measurements average these reporter responses across many thousands of individual worms, giving a reliable indication of the overall stress imposed on a population. The temperature profile of reporter responses induced by microwave exposure parallels that induced in controls by heat alone, but is displaced down the temperature scale by some 3/spl deg/C. Length measurements were conducted at intervals in synchronized C. elegans cultures seeded with L1 larvae. Using pooled data from nine separate runs, growth was stimulated by 8.5% after overnight microwave exposure (relative to controls), and this disparity increased to 11% after 24 h of further growth without irradiation. Both heat-shock responses and increased growth would be consistent with a modest increase in temperature, raising the possibility that microwave exposure might cause limited heating in this system. However, there is no detectable rise in the temperature of either medium or worms during overnight exposure under these conditions, discounting both generalized and localized (worm-specific) heating effects. It is concluded that both growth and heat-shock responses are induced by microwave exposure through one or more nonthermal routes.

Compared toxicity of the potassium channel blockers, apamin and dendrotoxin

The central toxicities of two potassium ion channel blockers, apamin and alpha-dendrotoxin (DTx), have been compared. Both apamin and dendrotoxin injected intracerebroventricularly produced signs of poisoning, including tremor and ataxia; however, only DTx produced changes in brain electrical activity, with high voltage spikes and epileptiform activity and subsequent brain damage. DTx, but not apamin, increased the amplitude of evoked field potentials and caused repetitive firing of neurones in hippocampal slices. Signs of poisoning following peripheral (intraperitoneal) administration of apamin were similar to those following central administration, including dramatic haemorrhagic effects on the lungs of decedent animals. These results are consistent with dendrotoxin being a centrally-active neurotoxin producing epileptiform activity and brain damage, whilst apamin produces its most significant pathology in the lung, possibly involving a neurogenic mechanism.