Further studies on the detection of mutagenic and genotoxic activity in human faeces: aerobic and anaerobic fluctuation tests with S. typhimurium and E. coli, and the SOS Chromotest

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Abstract
The product of six complete bowel movements were collected from one man (consuming a normal western diet) over a period of 5 months. Aqueous faecal extracts were prepared (0.75 g wet wt./ml water) under aerobic and anaerobic conditions. Graded doses were assayed aerobically and anaerobically for mutagenicity in replicate microtitre fluctuation tests employing histidine reversion in Salmonella typhimurium TA100 and tryptophan reversion in Escherichia coli WP2uvrA(pKM101), and in aerobic SOS Chromotests, using E. coli PQ37. The histidine and tryptophan content of each extract was determined by (i) pre-column derivatisation with o-phthaldialdehyde followed by reverse-phase h.p.l.c. and (ii) by aerobic and anaerobic bioassays employing appropriate auxotrophic strains of S. typhimurium and E. coli. All six faecal samples contained enough histidine and tryptophan or their precursors to compromise the interpretation of fluctuation tests. The h.p.l.c. technique, though quantitatively detecting free tryptophan added to faecal extracts, did not account for growth-enhancing effects of faecal extracts detected by bioassay using bacteria with a requirement for tryptophan. However, h.p.l.c. detected histidine more efficiently than bioassays. A combination of reconstruction experiments and bioassays enabled construction of ‘growth windows’ within which, in fluctuation tests, dose-related increases in numbers of positive wells could be ascribed to mutation rather than to feeding of auxotrophic bacteria. Under aerobic conditions of extraction and assay, five out of six samples were mutagenic to S. typhimurium TA100, and all six were mutagenic to E. coli WP2uvrA(pKM101). None of the samples extracted and assayed anaerobically was mutagenic to S. typhimurium, but all six were positive in E. coli WP2uvrA(pKM101). There was an increase both in faecal histidine and tryptophan and in mutagenicity during the period of collection, the association between faecal tryptophan and faecal mutagenicity being statistically significant. All the samples were negative in the SOS Chromotests. We conclude that this donor regularly excretes faeces which contain at least two classes of water-soluble directly-acting mutagens which are not detected in SOS Chromotest at doses five times those giving positive effects in fluctuation tests.