A semi-automated, microplate version of the SOS Chromotest for the analysis of complex environmental extracts.
Environmental monitoring for genotoxicity requires that a large number of measurements be made across space and time. This requirement demands a rapid and efficient bioassay system. The SOS Chromotest is a rapid, efficient bacterial system for the detection of DNA damaging agents. Over 100 publications have described its use on a variety of samples. Relatively few studies have used the test to examine complex mixtures. Effective testing of complex samples poses a variety of problems. Although solutions have been proposed, few have validated the resulting protocol. In this work we present a semi-automated microplate version of the SOS Chromotest for the examination of complex mixtures. Experiments were conducted to determine the optimal cell concentration, exposure time, substrate conversion time and S9 enzyme concentration. The performance of the method was evaluated using 6 reference genotoxins and 3 complex mixtures. The complex mixtures examined are extracts of diesel particulate matter, urban dust and coal tar. The results obtained indicate that optimal responses often require fewer cells (approximately equal to 5-10 x 10(6) CFU/ml) and a longer exposure (3 h) than that recommended in the original protocol. Interfering effects of colored and turbid samples are removed using centrifugation and initial optical density readings taken 60 min after cell resuspension and lysis. The performance of the established protocol was evaluated using mitomycin C and benzo[a]pyrene results for 207 microplates and solvent control results for 293 microplates. The results indicate that the established method is accurate, sensitive and precise. Coefficient of variation on mean SOSIP values for mitomycin C and benzo[a]pyrene are < 5%. Solvent control data indicate that the standard threshold for determination of a positive response (induction factor > 1.5) is excessively conservative. All liquid transfers were automated using the Biomek automated laboratory workstation. Automation permits a throughput of up to 72 samples per day and maintains excellent precision and accuracy.
White
P A
PA
Department of Biology, McGill University, Montréal, Québec, Canada.
Rasmussen
J B
JB
Blaise
C
C
D001331:Automation; D001564:Benzo(a)pyrene / Q000633:toxicity; D002863:Chromogenic Compounds / Q000378:metabolism; D003033:Coal Tar / Q000633:toxicity; D004249:DNA Damage / Q000235:genetics; D004784:Environmental Monitoring / Q000295:instrumentation / Q000379:methods*; D004785:Environmental Pollutants / Q000633:toxicity*; D004926:Escherichia coli / Q000187:drug effects / Q000378:metabolism; D005742:Gasoline / Q000633:toxicity; D016685:Mitomycin / Q000633:toxicity; D009152:Mutagenicity Tests / Q000295:instrumentation / Q000379:methods*; D009153:Mutagens / Q000633:toxicity*; D013014:SOS Response, Genetics; D012680:Sensitivity and Specificity
D002863:Chromogenic Compounds; D004785:Environmental Pollutants; D005742:Gasoline; D009153:Mutagens; D001564:Benzo(a)pyrene; D016685:Mitomycin; D003033:Coal Tar