410
A. Roda . P. Pasini . M. Guardigli
monitoring (King et al. 1990). A bioluminescent whole-cell biosensor for specific online monitoring of naphthalene and its degradation product salicylate was developed
by immobilizing the reporter bacteria in calcium alginate (Heitzer et al. 1994). A microbial sensor for the detection of middle-chain alkanes was constructed by introducing in Escherichia coli a plasmid containing a fusion between the promoter of alkB and
luxAB of V. fischeri and a separate plasmid bearing the regulatory gene alkS from Pseudomonas oleovorans. The biosensor was induced by n-alkanes from pentane to decane and was
applied to the analysis of contaminated ground water samples (Sticher et al. 1997).
As concerns the detection of some organic compounds, their ability to bind specific nuclear receptors is exploited to construct recombinant cell-based biosensors.
Upon binding an active ligand, the ligand-occupied receptor interacts with specific
DNA sequences (responsive elements) carried by an expression plasmid to modulate
gene transcription. This causes the expression of the reporter gene and the synthesis
of the reporter protein, which is detected by luminescent techniques.
A very wide range of synthetic chemicals and natural compounds have been found
to be oestrogenic. These endocrine-disrupting compounds (EDCs) include pesticides,
plasticizers, breakdown products of surfactants, synthetic oestrogens used as drugs
and phyto-oestrogens, most of which are often detectable in many surface waters. The
majority of these molecules exhibit different molecular structures, while sharing the
ability to bind the human oestrogen receptors (hER). A reporter gene bioassay was
developed using a breast cancer cell line (MCF-7), which naturally expressed the endogenous oestrogen receptor and was stably transfected with an oestrogen-regulated
firefly luciferase gene (Pons et al. 1990). Recently, this cell line along with newly developed ones, with detection limits of 1-5 pM oestradiol, was used to detect oestrogenic
activity in wastewater sewage treatment plant influents and effluents. The direct analysis of the water samples, after sterile filtration on 0.2 flm membranes, showed that the
oestrogenic activity was equivalent to 60-100 pM oestradiol in the influent and was
reduced by a factor 4-10 in the effluent, thus suggesting that effluents from sewage
treatment works are an important potential source of oestrogenic compounds contaminating the aquatic environment (Balaguer et al. 1999). An oestrogen receptormediated, chemical-activated luciferase reporter gene-expression bioassay (ERCALUX) was developed by transfection of the firefly luciferase luc gene in the T47D
breast cancer cell line. The system was tested on single oestrogen-like compounds or
combinations and exhibited a detection limit of 0.5 pM oestradiol (Legler et al.1999).
Other reporter gene bioassays for oestrogenic activity assessment are based on
recombinant yeast cells. This cellular model is often used because yeast cells are easily engineered and cultured, and have rapid growth rates, thus providing a very robust model suitable for wide-scale screening. Most yeast-based systems use the f3-galactosidase coding sequence as a reporter gene with the enzyme activity detected by
colourimetric methods, which are often time-consuming and poorly sensitive, providing detection limits inadequate for the direct analysis of environmental aqueous
samples (Rehmann et al. 1999). In our laboratory, a chemiluminescent whole cell biosensor based on recombinant yeast cells was developed and optimized for environmental monitoring of oestrogen-like compounds. A recombinant yeast strain (Saccharomyces cerevisiae) was used in which the DNA sequence of the human oestrogen receptor was stably integrated into the main chromosome. The yeast cells also contained
an expression plasmid carrying oestrogen-responsive sequences and the reporter gene
A. Roda . P. Pasini . M. Guardigli
monitoring (King et al. 1990). A bioluminescent whole-cell biosensor for specific online monitoring of naphthalene and its degradation product salicylate was developed
by immobilizing the reporter bacteria in calcium alginate (Heitzer et al. 1994). A microbial sensor for the detection of middle-chain alkanes was constructed by introducing in Escherichia coli a plasmid containing a fusion between the promoter of alkB and
luxAB of V. fischeri and a separate plasmid bearing the regulatory gene alkS from Pseudomonas oleovorans. The biosensor was induced by n-alkanes from pentane to decane and was
applied to the analysis of contaminated ground water samples (Sticher et al. 1997).
As concerns the detection of some organic compounds, their ability to bind specific nuclear receptors is exploited to construct recombinant cell-based biosensors.
Upon binding an active ligand, the ligand-occupied receptor interacts with specific
DNA sequences (responsive elements) carried by an expression plasmid to modulate
gene transcription. This causes the expression of the reporter gene and the synthesis
of the reporter protein, which is detected by luminescent techniques.
A very wide range of synthetic chemicals and natural compounds have been found
to be oestrogenic. These endocrine-disrupting compounds (EDCs) include pesticides,
plasticizers, breakdown products of surfactants, synthetic oestrogens used as drugs
and phyto-oestrogens, most of which are often detectable in many surface waters. The
majority of these molecules exhibit different molecular structures, while sharing the
ability to bind the human oestrogen receptors (hER). A reporter gene bioassay was
developed using a breast cancer cell line (MCF-7), which naturally expressed the endogenous oestrogen receptor and was stably transfected with an oestrogen-regulated
firefly luciferase gene (Pons et al. 1990). Recently, this cell line along with newly developed ones, with detection limits of 1-5 pM oestradiol, was used to detect oestrogenic
activity in wastewater sewage treatment plant influents and effluents. The direct analysis of the water samples, after sterile filtration on 0.2 flm membranes, showed that the
oestrogenic activity was equivalent to 60-100 pM oestradiol in the influent and was
reduced by a factor 4-10 in the effluent, thus suggesting that effluents from sewage
treatment works are an important potential source of oestrogenic compounds contaminating the aquatic environment (Balaguer et al. 1999). An oestrogen receptormediated, chemical-activated luciferase reporter gene-expression bioassay (ERCALUX) was developed by transfection of the firefly luciferase luc gene in the T47D
breast cancer cell line. The system was tested on single oestrogen-like compounds or
combinations and exhibited a detection limit of 0.5 pM oestradiol (Legler et al.1999).
Other reporter gene bioassays for oestrogenic activity assessment are based on
recombinant yeast cells. This cellular model is often used because yeast cells are easily engineered and cultured, and have rapid growth rates, thus providing a very robust model suitable for wide-scale screening. Most yeast-based systems use the f3-galactosidase coding sequence as a reporter gene with the enzyme activity detected by
colourimetric methods, which are often time-consuming and poorly sensitive, providing detection limits inadequate for the direct analysis of environmental aqueous
samples (Rehmann et al. 1999). In our laboratory, a chemiluminescent whole cell biosensor based on recombinant yeast cells was developed and optimized for environmental monitoring of oestrogen-like compounds. A recombinant yeast strain (Saccharomyces cerevisiae) was used in which the DNA sequence of the human oestrogen receptor was stably integrated into the main chromosome. The yeast cells also contained
an expression plasmid carrying oestrogen-responsive sequences and the reporter gene