Mechanisms Workshop
Chair: Professor J Metcalfe
The mechanisms session included presentations
from research groups funded by both the MTHR and Home office TETRA
programmes. These teams were using a diverse range of model systems
with varying degrees of organisational complexity (cultured mammalian
cells, nematode worms, brain slices and mice) to explore possible
interaction mechanisms. Endpoints being investigated included changes
in intracellular concentrations of important signalling second messengers
such as calcium ions and redox species, changes in expression of
specific genes, changes in gross electrical activity of nervous
tissue, and altered animal behaviour. In general research projects
were at relatively early stages with most of the effort to date
having gone into the development of relevant exposure and analysis
systems. Experimental data were being accumulated, but sample analysis
was either incomplete or results were still blinded. Despite the
great diversity of work in progress some common trends emerged.
As potential changes are expected to be relatively modest, assay
techniques must offer considerable sensitivity. One technique is
the use of reporter constructs, where the effect on the promoter
(controlling element) of a given gene is assessed by replacing the
gene’s coding sequences with those of an enzyme such as b-galactosidase
or other sensitive markers such as green fluorescent protein. Such
systems offer significant amplification of the initial change. In
the absence of accepted interaction mechanisms, automated approaches
may be employed to carry out large-scale screening. Hence, one study
is employing powerful gene chip technology capable of analysing
changes in the expression of 20,000 genes analysed in a single assay.
The use of automated imaging systems offers the possibility for
screening changes in a number of endpoints such as intracellular
calcium ion concentration, redox signalling intermediates, and cell
morphology. Because the systems are automated, changes can be quantified
in large numbers of individual cells and it is possible to analyse
the time course of any fluctuations in intracellular concentrations.
As the indicator dyes generally chelate or otherwise trap the species
being assayed, caution is needed to avoid ‘buffering out’
the very changes that are sought.
The use of automated systems that permit analysis of changes in
large numbers of individual cells permits the generation of results
with great statistical confidence. Confidence in the validity of
experimental data can also be endangered by the lack of consistency
of the response. In contrast, identification of changes occurring
at different levels of functional organisation (expression of genes
in brain cells, electrophysiological activity of brain tissue, and
animal behaviour) may provide convincing evidence for an effect.
An important theme to come out of the meeting is that when trying
to identify small effects, great care is needed to ensure tight
control of all variables in order to improve consistency. Good experimental
practice dictates that physical variables, such as temperature,
humidity and gaseous environment must be tightly controlled. However,
it is important to recognise that the status and responsiveness
of biological materials may be affected by many other factors, including
nutrient availability, or prior or concurrent exposure to other
stressors. It may sometimes be desirable to introduce changes to
an experimental protocol to reflect best practice in a particular
field of study, but caution is needed as such changes could themselves
introduce unwanted variability. When investigating the effects of
electromagnetic fields it is important to consider the possibility
that the system may respond to frequencies outside the range of
interest. For example, low frequencies associated with power supplies
and motors should be minimised and characterised.
Where experiments are investigating the effects of exposure to a
modulated signal (GSM, 3G, and TETRA) they include a comparison
with an unmodulated signal at the carrier frequency in addition
to a sham exposure. Two studies are achieving simultaneous sham
and exposed conditions through the use of paired transverse electromagnetic
(TEM) cells. A third study is employing a stripline waveguide, which
is effectively half a TEM cell. The mouse study is using loop antennas
tuned to the frequency of interest. All of the studies have used
numerical modelling techniques to assess the magnitude and homogeneity
of specific energy absorption rate (SAR) in exposed materials. Numerical
models have generally been validated by means of physical measurements.
Overall the session illustrated the amount of careful preliminary
work that is needed in order to establish tightly controlled experimental
conditions that can be used as a basis for future work.
Summaries
of the closed sessions of the MTHR Research Seminar - 4th of November
2003 |
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