Abstract
We show that in vitro toxicity assay based on inhibition of the bioluminescence of recombinant Escherichia coli encoding thermostable luciferase from Photorhabdus luminescens is a versatile alternative to Vibrio fischeri Microtox™ test. Performance of two luxCDABE-transformed E. coli MC1061 constructs (pDNlux) and (pSLlux) otherwise identical, but having 100-fold different background luminescence was compared with the performance of V. fischeri. The microplate luminometer and a kinetic Flash-Assay test format was used that differently from Microtox test is also applicable for high throughput analysis. Toxic effects (30-s till 30-min EC50) of four heavy metals (Zn, Cd, Hg, Cu) and three organic chemicals (aniline, 3,5-dichloroaniline and 3,5-dichlorophenol) were studied. Both E. coli strains had comparable sensitivity and the respective 30-min EC50 values highly correlated (log-log R2 = 0.99; p < 0.01) showing that the sensitivity of the recombinant bacteria towards chemicals analyzed did not depend on the bioluminescence level of the recombinant cells. The most toxic chemical for all used bacterial strains (E. coli, V. fischeri) was mercury whereas the lowest EC50 values for Hg (0.04–0.05 mg/L) and highest EC50 values for aniline (1,300–1,700 mg/L) were observed for E. coli strains. Despite of that, toxicity results obtained with both E. coli strains (pSLlux and pDNlux) significantly correlated with V. fischeri results (log-log R2 = 0.70/0.75; p < 0.05/0.01). The use of amino acids (0.25%) and glucose (0.05%)-supplemented M9 medium instead of leucine-supplemented saline significantly (p < 0.05) reduced the apparent toxicity of heavy metals to both E. coli strains up to three orders of magnitude, but had little or no complexing effect on organic compounds. Thus, P. luminescens luxCDABE-transformed E. coli strains can be successfully used for the acute toxicity screening of various types of organic chemicals and heavy metals and can replace V. fischeri in certain cases where the thermostability of luciferase >30 °C is crucial. The kinetic Flash Assay test format of the bioluminescence inhibition assay facilitates high throughput analysis. The assay medium, especially in case of testing heavy metals should be a compromise: optimal for the viability/luminescence of the recombinant test strain and of minimum complexing potential.
Highlights
Over the last twenty five years, alternative, non-animal test systems have been introduced to supplement and, in some cases, to replace toxicity tests using animals [1], contributing to the 3R’s concept (Replacement, Reduction, Refinement of test animals) [2].For initial toxicity screening of chemicals, bacteria are an additional attractive alternative to eukaryotic organisms
Four heavy metals and three organic compounds with different mechanisms of action were chosen to compare the performance of constitutively luminescent E. coli strains and against that of V. fischeri
As E. coli and V. fischeri are both Gram-negative bacteria with analogous cell envelope structure, their response to most of the chemicals should be theoretically comparable
Summary
For initial toxicity screening of chemicals, bacteria are an additional attractive alternative to eukaryotic organisms. The most well-known bacterial in vitro test is Ames assay with Salmonella typhimurium [3], which may predict genotoxic effects of chemicals to higher organisms (e.g., humans). One of the most widely used bacterial in vitro assays is the MicrotoxTM test, which uses the inhibition of bioluminescence of Vibrio fischeri NRRL-B-11177 as a toxicity endpoint [4]. V. fischeri are naturally luminescent Gram-negative marine bacteria known as Photobacterium phosphoreum. NRRL-B-11177 [5] and/or Aliivibrio fischeri [6] in which luxCDABE genes are responsible for their bioluminescent reaction. A single bacterium may emit 104 or 105 photons s−1 [7]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
