A Simple Method for the Detection of Mutagens in Urine: Studies with the Carcinogen 2-Acetylaminofluorene

Abstract

We described previously a simple test on petri plates for detecting many carcinogens as mutagens using an especially sensitive set of bacterial strains to detect mutagens and a rat, or human, liver homogenate for carcinogen activation. We now extend the utility of the method for the detection of mutagenic metabolites in urine. The addition of commercial 6-glucuronidase (EC 3.2.1.31) to the petri plates along with the urine, liver homogenate, and bacteria allows detection of metabolites that are excreted in urine as jB-glucuronide conjugates.By this method mutagenic activity is readily demonstrated with urine of rats that were administered as little as 200 jug (1.6 mg/kg) of the carcinogen, 2-acetylaminofluorene.In this case the major urinary metabolite that we detect appears to be a glucuronide conjugate. We propose that the method be used for the screening of human urines in order to detect mutagenic metabolites of drugs and of dietary components. It may also be useful for testing of urinary metabolites of drugs and food additives in experimental animals.We have previously described a very sensitive and simple bacterial test system for detecting chemical mutagens (1-4). The compounds are tested on petri plates with specially constructed mutants of Salmonella typimurium as tester strains. Four tester strains have been selected for sensitivity and specificity in being reverted from a histidine requirement back to prototrophy by a variety of mutagens. One strain can be used to detect mutagens causing base-pair substitutions and three to detect various kinds of frameshift mutagens. In addition to the histidine mutation, each tester strain has two additional mutations that greatly increase their sensitivity to mutagens: one causes loss of the excision repair system and the other loss of the lipopolysaccharide barrier that coats the surface of the bacteria. We have shown that by adding a microsomal activation system of rat (or human) liver to the petri plates a wide variety of carcinogens can be activated to mutagens and thus detected easily (3). Thus, an important aspect of mammalian metabolism can be duplicated in an in vitro test. The present study examines the testing of urine in the combined bacterial/liver system. A wide variety of metabolites of drugs and other ingested compounds appear in the urine. Since many metabolites are excreted in urine as glucuronides, we added fl-glucuronidase (EC 3.2.1.31) to our test plates. The method is demonstrated by administering the carcinogen, 2-acetylaminofluorene (AcAF), to rats and detecting mutagenic metabolites in the urine.AcAF, like many other compounds, gives rise to a large number of metabolites (5-li). The oxidation products Nhydro.xy-2-acetylaminofluorene (N-OH-AcAF), N-hydroxy-2-aminofluorene (N-OH-AF), and 2-nitrosofluorene (NOF), are thought to be proximate or ultimate carcinogens formed from AcAF and 2-aminofluorene (AF) by the microsomal systems of liver (5). They are more carcinogenic than the parent compounds at the site of application in rodents an...

Detection of carcinogens as mutagens: bacterial tester strains with R factor plasmids.

“…One of the old frameshift tester strains, TA1536, can now be dropped from the set of tester strains, as extremely few mutagens revert it and those that do can be detected well with the other frameshift tester strains. The (28,29) and several groups are starting to screen the human population. It is also being used to detect mutagenic activity in complex mixtures such as cigarette smoke and its fractions (30).…”

Testing the Environment for Dispersed Mutagens: Use of Plant Bioconcentrators Coupled with Microbial Mutagen Assays

“…Call number x 104/core Another way to minimize the effect of feeding is to extract in a solvent in which the interfering metabolite is insoluble. Thus, in the Ames test, which depends on reversion from histidine auxotrophy, an organic solvent is used because histidine is a polar molecule (36,37). Another method is to remove histidine using resin adsorption techniques (38) (41) tested many water weeds, including Nuphar variegatum, Nymphaea tuberosa, and Lemna minor, and found that none of these extracts inhibited E. coli.…”

The urine mutagenicity assay system. Studies related to recovery, storage and concentration procedures.

“…However, both acti ve and inactive forms may be excreted in the urine. Several animal studies have shown that, when animals are treated with mutagenic carcinogens or chemotherapeutic agents, mutagenic activity can be detected in their urine (3,5,II ,17). Increases in mutagenic activity have also been found in urine samples from smokers and from pa tients treated with chemotherapeutic agents (11,13 ,18,19, 20, 22, 24) .…”