Testing human faecal extracts for genotoxic activity with the SOS Chromotest: the importance of controlling for faecal enzyme activity.
Two aqueous extracts of human faeces were prepared from a healthy male donor and assayed in the SOS Chromotest. Both extracts were positive in microtitre fluctuation tests in Salmonella typhimurium TA100 and Escherichia coli WP2uvrA(pKM101). Differences were observed in the induction factors of these samples when p-nitrophenyl-beta-D-galactopyranoside (p-NPG) and o-nitrophenyl-beta-D-galactopyranoside (o-NPG) were used as substrates for the beta-galactosidase assay in the SOS Chromotest. With one sample, a positive induction factor was reproducibly obtained using p-NPG but not o-NPG. When the bacterial cells were washed with fresh LB broth before enzyme assay, the positive induction factor obtained with p-NPG was reduced to an insignificant level. During the 2-h treatment period, both faecal samples enhanced bacterial growth above that of the zero-dose control. When SOS Chromotest assays were performed with no bacteria or with S. typhimurium TA100 or hisG46 (non-lactose fermenting organisms) in place of E. coli PQ37, it was found that the extracts contained significant levels of endogenous beta-galactosidase and alkaline phosphatase, which, due to their carry-over in the bacterial pellet (after centrifugation to remove the coloured extract) gave rise to the positive induction factor obtained with p-NPG. The results obtained in these experiments indicate that where the SOS Chromotest is applied to biological samples, care should be taken in the interpretation of the data and that a washing step should be included to prevent possible errors occurring due to exogenous enzymes in the sample.
Bosworth
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D
Section of Chemical Carcinogenesis, Institute of Cancer Research, Royal Cancer Hospital, Sutton, Surrey, UK.
Venitt
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S
D000469:Alkaline Phosphatase / Q000378:metabolism; D004260:DNA Repair / Q000187:drug effects*; D004926:Escherichia coli / Q000187:drug effects; D005243:Feces / Q000032:analysis*; D006801:Humans; D009152:Mutagenicity Tests / Q000379:methods; D009153:Mutagens / Q000302:isolation & purification* / Q000494:pharmacology; D009154:Mutation; D013014:SOS Response, Genetics / Q000187:drug effects*; D012486:Salmonella typhimurium / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D001616:beta-Galactosidase / Q000378:metabolism
D009153:Mutagens; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase