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It must be pointed out here that classic luciferase reporter
gene analysis and comparable approaches are not the
only way of recording biological effects related to nuclear
receptor and transcription factor activation. Modern
imaging techniques could play a future role within the
scope of the automated recording of the translocation of a
fluorescence-marked toxin-activated receptor. For practical
reasons, however, an infinite number of endpoints cannot
be determined in parallel using methods of this kind either.
Simplification through the use of procaryotic systems
Many laboratories responsible for routine analysis of
food and feed do not currently have the equipment or
the expertise to conduct cell-based assays of effect-based
analytics, in particular those that involve the germ-free
cultivation of mammalian cells. This problem could no
doubt be solved by acquiring the necessary equipment,
and training staff accordingly, but - even though
automated systems are becoming more and more suitable
to take over sub-stages of the experimental workflow -
cultivation of mammalian cell lines is still a more complex
technique than the use of procaryotic systems. In the
field of ecotoxicology, luciferase-expressing strains of
Escherichia coli, Bacillus subtilis and Staphylococcus aureus,
for example, are used to detect various heavy metals,
with each bacteria strain being equipped with a luciferase
reporter construct that is responsive to a particular heavy
metal (9, 10). It must be added at this point, however,
that procaryotic cells differ greatly from eucaryotic cells
in their biochemistry and signal transduction. Because
of a lack of specific interaction partners, such as the
coactivator proteins needed for the proper function
of a certain transcription factor, the functionality of a
mammalian protein introduced into procaryotes cannot
simply be assumed. For this reason, procaryotic systems
are not automatically suited for predicting effects on
the more complex intercellular signaling pathways of a
mammalian organism. Conversely, they could constitute
a time-saving and low-cost alternative to mammalian cellbased test systems for endpoints that are easier to address,
or in cases where corresponding regulation systems
of sufficient specificity exist in procaryotes or can be
implemented in biological systems of this kind.
Limitations of reporter gene-based methods
Only a few nuclear receptors exhibit high substrate
specificity. Instead, a large number of substances whose
chemical structures are only slightly related, if at all, are
accepted by many receptors as ligands. Even the aryl
hydrocarbon receptor examined in the CALUX assay,
which is commonly regarded as a comparatively specific
receptor for dibenzodioxins, also binds some naturally
occurring substances as well as some amino acid derivatives
formed under physiological conditions, in addition to

other contaminants, such as dibenzofurans and several
polychlorinated biphenyls (2). Hormone receptors
like the estrogen receptor show a comparatively broad
substrate specificity, which is additionally accompanied by
pronounced species differences in the affinity to various
ligands (13). The problem of a broad ligand specificity
of a receptor and/or the resultant low specificity for
a single, specific ligand is made even more difficult by
the pronounced crosstalk of various cellular signaling
pathways, i.e., by their mutual influencing. In particular,
signaling cascades with fundamental significance for
cell physiology and development appear to have a high
degree of networking with other signaling pathways. For
example, a reporter system that detects the activity of the
so-called canonical Wnt/β-catenin signaling pathway,
which is important for embryonic development, proved
itself to be responsive in murine embryonic stem cells to a
large number of structurally different chemical substances
whose molecular target structures - at least as far as is
currently known - are not components of the Wnt/βcatenin signaling pathway (17, 18). All in all, this adds up to
immense difficulty for unequivocal substance identification
by means of classic methods of analytical chemistry, which
is currently required by law following a "positive" result
from an effect-based screening test.
In addition, the possible interference of a sample with
the enzymatic reaction of the reporter system has to be
considered. The routinely used firefly luciferase has proven
to be comparatively susceptible to inhibition by many
different chemicals (4), while nonspecific interferences
of the test substance and the transcription factors used in
the reporter gene system can also occur (11). The latter
problems are controllable, however, through the inclusion
of appropriate checks.
A further potential problem of a large battery of
independent reporter gene-based test systems could lie
in the occurrence of isolated "positive" test results in
individual sub-test systems of the battery, which could
ultimately lead to a large number of samples being
categorized as suspicious. The validation of the test battery
as a whole with a clearly prepared strategy for dealing with
"positive" results in individual tests would be required here.
How is the increased likelihood of a coincidentally wrong
positive test result in a single sub-test system, as it is caused
by a larger number of endpoints recorded in parallel, to
be dealt with? Should different types of biological effects
on different signaling cascades with different tasks in the
organism, for example, be given different weightings in the
interpretation of the test results? Or should every finding
be given the same weighting as a matter of principle? Can
an addition of certain values for different test systems in
line with the concept of a hazard index (21) be applied,
and if so, under which preconditions? In addition to these
difficulties, economic aspects would have to be taken into

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