Sources of variability of the Escherichia coli PQ37 genotoxicity assay (SOS chromotest).
To determine the variability in test results obtained with the Escherichia coli PQ37 genotoxicity assay (SOS chromotest) when varying the test protocol, we examined the influences of sodium dodecylsulfate (SDS) concentrations, of buffer pH and composition on the enzyme assays, the effects of E. coli PQ37 density and culture conditions on the expression and/or determination of alkaline phosphatase (ap) and beta-galactosidase (beta-g) activities, the calculated induction factors (IF) and the SOS-inducing potentials (SOSIP). Initially, we used 0-190 ng (0-1 nmole) 4-nitroquinoline-1-oxide (4-NQO) as a reference compound for the standard procedure in the absence of metabolic activation. Subsequently, to evaluate the results of protocol variations we examined several mutagenic compounds of differing chemical classes using both the standard and a modified assay procedure. We observed the highest enzyme activities using 1 mg SDS per tube and calibrating the ap buffer to pH 8.05 and the beta-g buffer to pH 7.75. The longer the incubation period, the higher the enzyme activities. However, with respect to IF and SOSIP there is no reason to incubate in excess of 90 min. We found no significant differences in the IF and SOSIP values when varying substrate conversion times. There was, however, a definite decrease in beta-g activity when extended substrate incubation times were used. Higher enzyme activities are obtained when the bacterial count is increased. Using lower bacterial counts the enzyme activities decreased, but the sensitivity of E. coli towards genotoxic compounds increased.
Mersch-Sundermann
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Institute of Medical Microbiology and Hygiene, Faculty of Clinical Medicine Mannheim, University of Heidelberg, F.R.G.
Kevekordes
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Mochayedi
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D015112:4-Nitroquinoline-1-oxide / Q000633:toxicity; D000165:Acridine Orange / Q000633:toxicity; D000469:Alkaline Phosphatase / Q000096:biosynthesis; D002021:Buffers; D003114:Colony-Forming Units Assay; D003630:Daunorubicin / Q000633:toxicity; D004305:Dose-Response Relationship, Drug; D004811:Epichlorohydrin / Q000633:toxicity; D004926:Escherichia coli / Q000235:genetics*; D005449:Fluorenes / Q000633:toxicity; D006863:Hydrogen-Ion Concentration; D008741:Methyl Methanesulfonate / Q000633:toxicity; D008748:Methylcholanthrene / Q000633:toxicity; D009152:Mutagenicity Tests; D009153:Mutagens / Q000633:toxicity; D015203:Reproducibility of Results; D013014:SOS Response, Genetics / Q000187:drug effects*; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology; D013997:Time Factors; D001616:beta-Galactosidase / Q000096:biosynthesis
D002021:Buffers; D005449:Fluorenes; D009153:Mutagens; D004811:Epichlorohydrin; D012967:Sodium Dodecyl Sulfate; D008748:Methylcholanthrene; D015112:4-Nitroquinoline-1-oxide; D008741:Methyl Methanesulfonate; C005644:2,4,7-trinitrofluorenone; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D000165:Acridine Orange; D003630:Daunorubicin