Analysis of the effects diclofenac has on Japanese medaka (Oryzias latipes) using real-time PCR.

The expression levels of cytochrome P450 1A, p53 and vitellogenin were investigated in three different tissues of male medaka fish after exposure to diclofenac that is one of the main concerns among pharmaceuticals frequently found in sewage treatment plant (STP) effluents. The results showed that cytochrome P450 1A, p53 and vitellogenin were highly expressed in tissue-specific gene expression patterns after exposure to 8 mg/l and 1 microg/l of diclofenac. These elevated expression levels of three biomarkers suggested that diclofenac has potential to cause cellular toxicity, p53-related genotoxicity and estrogenic effects. It is also noteworthy that diclofenac has the potential to cause these effects even at an environmentally relevant concentration of diclofenac, 1 microg/l.

Gene expression analysis and classification of mode of toxicity of polycyclic aromatic hydrocarbons (PAHs) in Escherichia coli.

Escherichia coli is known to respond to certain toxic chemicals through an increased expression of various stress genes. In this study, therefore, the expression of recA, katG, fabA and grpE genes was used as a representative for DNA, oxidative, membrane and protein damage, respectively, after E. coli was exposed to different polycyclic aromatic hydrocarbons (PAHs), i.e., phenanthrene, naphthalene and benzo[a]pyrene. To accomplish this, the expression levels of these four genes were quantified using a real-time RT-PCR analysis when E. coli cultures were under stressful conditions, such as those caused by an exposure to mitomycin C, hydrogen peroxide and phenol. It was found that the primary toxic effect of each chemical is clearly seen when the expression levels of the different genes are compared. Tests with the PAHs showed naphthalene and benzo[a]pyrene to be genotoxic, while phenanthrene had no clear effect on the expression of any of these genes. Based on these results, the effects due to these toxic chemicals and the extent of each stress can be evaluated with ease using the expression levels of different stress responsive genes.

Analysis of the stress effects of endocrine disrupting chemicals (EDcs) on Escherichia coli.

In this study, three of the representative EDCs, 17beta-estradiol, bisphenol A, and styrene, were employed to find their mode of toxic actions in E. coli. To accomplish this, four different stress response genes, recA, katG, fabA, and grpE genes, were used as a representative for DNA, oxidative, membrane, or protein damage, respectively. The expression levels of these four genes were quantified using a real-time RT-PCR after challenge with three different EDCs individually. Bisphenol A and styrene caused high-level expression of recA and katG genes, respectively, whereas 17beta-estradiol made no significant changes in expression of any of those genes. These results lead to the classification of the mode of toxic actions of EDCs on E. coli.

Evaluation of a high throughput toxicity biosensor and comparison with a Daphnia magna bioassay.

A high throughput toxicity biosensor has been designed and constructed using recombinant Escherichia coli cells, containing stress specific promoters (recA, fabA, or katG) or constitutive promoters (lac) fused to luciferase genes originating from Vibrio fisheri. These genetically engineered cells were immobilized in 96 well plates. By optimizing cell immobilization conditions and the strains' response specificity to toxic chemicals, bioluminescent outputs decreased or increased dose-dependently upon adding test chemicals. However, to date the toxicity data obtained using this biosensor have not been compared with the results of other toxicity tests. Phenolics were chosen to evaluate the correlation between the LD50 and the EC50 (GC2) or EC120 (DPD2540) of Daphnia magna and E. coli, respectively. Toxicity data obtained from constitutive strains by bioluminescent level decrements were compared with the results from D. magna as a standard. LD50 values were used as parameters of D. magna toxicity and EC50 of EC120 values were used for the immobilized biosensor. In the DPD2540 test, phenolics, membrane damaging toxic chemicals, for testing immobilized stress specific bacterial strains trigger dose-dependant bioluminescence increase within specific concentration. Although the stress specific responsiveness from the strains could not be compared with D. magna's LD50 values, these responses offer additional information, such as upon the mode of toxic action in the sample, in addition to the cellular toxicity results as indicated by the EC50. This novel high throughput toxicity biosensor can be implemented to investigate the toxicity of any other soluble materials, and can be used as a standardization tool for the evaluation of toxicity.

Immobilization as a technical possibility for long-term storage of bacterial biosensors.

For applications in field experiments, the recombinant strain Salmonella typhimurium TA1535 was immobilized to permit its immediate utilization after long storage periods. Salmonella typhimurium TA1535 cells contain the plasmid that has an inducible SOS promoter fused to a promoterless luxCDABFE operon from Photobacterium leiognathi. The induction of bioluminescence occurs in the presence of the DNA-damaging agent mitomycin C which stimulates the bacterial SOS response. Early stationary phase cells were immobilized at a cell concentration of 10(10) CFU/ml in microtiter plates and stored up to 6 weeks at 4 degrees C in a sealed container. Even after 4 weeks of storage, the bioluminescence kinetics and yield in response to different concentrations of mitomycin C were not significantly different from those of freshly prepared samples.