Influence of S9 mix composition on the SOS response in Escherichia coli PQ37 by polycyclic aromatic hydrocarbons.
To investigate the variability in test results obtained with the SOS chromotest (Escherichia coli PQ37 genotoxicity assay) when varying the composition of the exogenous metabolizing system (S9 mix), we examined the influence of different S9 and NADP concentrations, of buffer pH value, of SDS concentrations, the effects of E. coli PQ37 density and centrifugation steps on the expression of beta-galactosidase (beta g) and alkaline phosphatase (ap) activity, the calculated induction factors (IFs) and SOS-inducing potencies (SOSIPs). Additionally we examined the metabolic potency (stability) of S9 mix when stored at 37 degrees C before use. Initially, we used 0-5000 ng (= 0-20 nmole) benzo[a]pyrene (B[a]P) as a reference compound for the test procedure in the presence of standard S9 mix. Subsequently, to evaluate the results of S9 mix variations we examined several polycyclic aromatic hydrocarbons (PAHs) using both the standard and a modified S9 mix composition and test protocol. We observed the highest beta g and ap activities and/or IFs using only 11-27 microliters 9000 x g liver supernatant (S9) from Aroclor 1254-induced rats per assay (20-50% of standard amount) and calibrating the S9 mix Tris buffer to pH 7.8-8.0. 60-300 micrograms NADP/assay (10-50% of standard) was sufficient for optimum activation of PAHs. In contrast to previous investigations about the variability of the SOS chromotest in the absence of a metabolizing system, higher induction factors were obtained when using higher bacterial densities (12-18 x 10(6) cfu/assay). Centrifugation steps as recommended by other investigators were not necessary when using optimum S9 amounts. The metabolic activity of S9 mix remained nearly constant approximately 20 min after preparation, but decreased to 80% of its activity in about 1 h.
Mersch-Sundermann
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Department of Medical Microbiology and Hygiene, Faculty of Clinical Medicine Mannheim, Ruprecht-Karls-University of Heidelberg, Germany.
Wintermann
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Kern
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Hof
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D000818:Animals; D001564:Benzo(a)pyrene / Q000633:toxicity; D004926:Escherichia coli / Q000187:drug effects* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008099:Liver / Q000378:metabolism*; D009249:NADP / Q000494:pharmacology; D011083:Polycyclic Compounds / Q000378:metabolism / Q000633:toxicity*; D051381:Rats; D017207:Rats, Sprague-Dawley; D013014:SOS Response, Genetics / Q000187:drug effects*; D001616:beta-Galactosidase / Q000032:analysis
D011083:Polycyclic Compounds; D001564:Benzo(a)pyrene; D009249:NADP; D001616:beta-Galactosidase