Association of salmonella mutants with germfree rats: site specific model to detect carcinogens as mutagens.

Abstract

An association of the histidine auxotroph of Salmonella typhimurium (strain TA1538) within the gastrointestinal tract of otherwise germfree Sprague-Dawley rats is maintained during periods of observation as ing as long as 7 months. The bacteria are found at levels exceeding 107 per g in the forestomach and at levels greater than 108 per g in the lower bowel and in the feces. Only approximately 104 bacteria per g are found in the posterior stomach and in the upper small intestine. The association of the salmonella mutants is maintained when the bacterial association is increased by the addition of other bacteria characteristic of the gastrointestinal flora. Carcinogenic amines, which cause strain TA1538 to revert to histidine independence in Ames' in vitro assays, increase the number of revertants in the feces when fed to the salmonella-associated rats. In contrast, the number of revertants in the feces does not increase when the rats are fed structurally related compounds which are not mutagenic to the bacteria in vitro and for which no evidence of carcinogenicity exists. Sacrifice of rats after feeding the carcinogen 2-nitrofluorene indicates that the number of revertants is increased in the cecum and colon as well as in the feces. The apparent proximity of the bacterial mutagenic response to the location of the tumor response in the colon suggests that the rat associated with the histidine auxotroph may provide a useful model for further investigation of the possible association between bacterial mutagenesis and carcinogenesis within the gastrointestinal tract. In addition, with this model it may be possible to evaluate selectively the effects of various constituents of the flora on the activation of compounds provoking the revertant response.Concern about exposure to possible mutagenic and carcinogenic agents has stimulated the development of test systems to warn of the presence of such compounds in the environment. Since alterations in nucleic acids are the basis of mutagenesis, and possibly also of carcinogenesis (1), it seemed logical to test compounds in systems previously developed for bacterial genetics in which alterations in nucleic acid structure might be discerned on the basis of easily recognizable phenotypic alterations. Thus, alterations of DNA (2), the detection of mutations in bacteria (3) and phages (4), and the expression of prophages (5) have each served as the basis for identifying chemicals with mutagenic properties.

Biochemical Pharmacology of the Intestinal Flora

“…The normally low number of revertants passed in the feces was greatly increased when known carcinogens were administered to these rats. Structurally related compounds without carcinogenic activity failed to increase the number of revertants in the feces (70). Although the concentration of revertants in the feces of these animals was correlated with that in the lower bowel, it is not clear that revertants actually form in the colon.…”

Detection of carcinogens as mutagens in the Salmonella/microsome test: assay of 300 chemicals: discussion.

“…We suspect that even a higher percentage of carcinogens will be detected in the test after further improvements in the in vitro activation system or in the tester strains. The Salmonella test has been adapted for the testing of urine (31,32,35) and colonic contents (36) as a supplemental way of detecting mutagenic metabolites and some of the carcinogens negative in the standard test might also be detected in this way (see safrole).…”

“…(4) Complex mixtures or natural products with carcinogenic activity can be investigated, using the test as a bioassay for identifying the mutagenic ingredients; e.g., cigarette smoke condensate is mutagenic (55) and numerous tobacco companies are trying to identify the chemicals responsible. (5) Colonic contents (36) and human feces (W. R. Bruce, personal communication) and urine (31,32,35) can be monitored to see if ingested products or drugs are giving rise to mutagens. (6) The variety of substances that humans are exposed to, both pure chemicals and mixtures, is being assayed for mutagenicity by hundreds of laboratories: e.g., water supplies; soot from city air; hair dyes (51) and cosmetics; drugs; food additives; food; mold toxins; pesticides; industrial chemicals; fumigants.…”

Mutagenesis within the gastrointestinal tract determined by histidine auxotrophs ofSalmonella typhimurium

“…Only about lo4 bacteria per g were found in the contents of the posterior stomach and in that of the upper small intestine. 12 Ordinarily, the concentration of histidine-independent (his+) revertants appearing in the feces were less than 100 per g. There was a sharp rise in the number of his+ revertants, however, in response to the ingestion of a number of carcinogens administered in doses comparable to those that caused tumors in experimental animals ( Table 1). It was encouraging that the ingestion of structurally related compounds, which are not considered to be carcinogenic, did not provoke this revertant response in the feces ( Table 1).…”