Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1974 Mar;71(3):737–741. doi: 10.1073/pnas.71.3.737

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

William E Durston 1, Bruce N Ames 1
PMCID: PMC388088  PMID: 4522788

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 β-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 β-glucuronide conjugates. By this method mutagenic activity is readily demonstrated with urine of rats that were administered as little as 200 μg (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.

Keywords: carcinogen activation and detection, 2-nitrosofluorene, Salmonella typhimurium, β-glucuronides

Full text

PDF
737

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ames B. N., Durston W. E., Yamasaki E., Lee F. D. Carcinogens are mutagens: a simple test system combining liver homogenates for activation and bacteria for detection. Proc Natl Acad Sci U S A. 1973 Aug;70(8):2281–2285. doi: 10.1073/pnas.70.8.2281. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Ames B. N., Gurney E. G., Miller J. A., Bartsch H. Carcinogens as frameshift mutagens: metabolites and derivatives of 2-acetylaminofluorene and other aromatic amine carcinogens. Proc Natl Acad Sci U S A. 1972 Nov;69(11):3128–3132. doi: 10.1073/pnas.69.11.3128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ames B. N., Lee F. D., Durston W. E. An improved bacterial test system for the detection and classification of mutagens and carcinogens. Proc Natl Acad Sci U S A. 1973 Mar;70(3):782–786. doi: 10.1073/pnas.70.3.782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Belman S., Troll W., Teebor G., Mukai F. The carcinogenic and mutagenic properties of N-hydroxy-aminonaphthalenes. Cancer Res. 1968 Mar;28(3):535–542. [PubMed] [Google Scholar]
  5. CRAMER J. W., MILLER J. A., MILLER E. C. N-Hydroxylation: A new metabolic reaction observed in the rat with the carcinogen 2-acetylaminofluorene. J Biol Chem. 1960 Mar;235:885–888. [PubMed] [Google Scholar]
  6. DeBaun J. R., Smith J. Y., Miller E. C., Miller J. A. Reactivity in vivo of the carcinogen N-hydroxy-2-acetylaminofluorene: increase by sulfate ion. Science. 1970 Jan 9;167(3915):184–186. doi: 10.1126/science.167.3915.184. [DOI] [PubMed] [Google Scholar]
  7. Gabridge M. G., Denunzio A., Legator M. S. Microbial mutagenicity of streptozotocin in animal-mediated assays. Nature. 1969 Jan 4;221(5175):68–70. doi: 10.1038/221068a0. [DOI] [PubMed] [Google Scholar]
  8. IRVING C. C. ENZYMATIC N-HYDROXYLATION OF THE CARCINOGEN 2-ACETYLAMINOFLUORENE AND THE METABOLISM OF N-HYDROXY-2-ACETYLAMINOFLUORENE-9-14C IN VITRO. J Biol Chem. 1964 May;239:1589–1596. [PubMed] [Google Scholar]
  9. IRVING C. C. ON THE STRUCTURE OF THE GLUCURONIDE OF N-HYDROXY-2-ACETYLAMINOFLUORENE. J Biol Chem. 1965 Mar;240:1011–1013. [PubMed] [Google Scholar]
  10. Irving C. C., Veazey R. A., Hill J. T. Reaction of the glucuronide of the carcinogen N-hydroxy-2-acetylaminofluorene with nucleic acids. Biochim Biophys Acta. 1969 Mar 18;179(1):189–198. doi: 10.1016/0005-2787(69)90135-x. [DOI] [PubMed] [Google Scholar]
  11. King C. M., Phillips B. N-hydroxy-2-fluorenylacetamide. Reaction of the carcinogen with guanosine, ribonucleic acid, deoxyribonucleic acid, and protein following enzymatic deacetylation or esterification. J Biol Chem. 1969 Nov 25;244(22):6209–6216. [PubMed] [Google Scholar]
  12. Kriek E. On the interaction of N-2-fluorenylhydroxylamine with nucleic acids in vitro. Biochem Biophys Res Commun. 1965 Sep 22;20(6):793–799. doi: 10.1016/0006-291x(65)90088-4. [DOI] [PubMed] [Google Scholar]
  13. MILLER E. C., MILLER J. A., HARTMANN H. A. N-Hydroxy-2-acetylaminofluorene: a metabolite of 2-acetylaminofluorene with increased carcinogenic activity in the rat. Cancer Res. 1961 Jul;21:815–824. [PubMed] [Google Scholar]
  14. MILLER J. A., CRAMER J. W., MILLER E. C. The N- and ringhydroxylation of 2-acetylaminofluorene during carcinogenesis in the rat. Cancer Res. 1960 Jul;20:950–962. [PubMed] [Google Scholar]
  15. Radomski J. L., Rey A. A., Brill E. Evidence for a glucuronic acid conjugate of N-hydroxy-4-aminobiphenyl in the urine of dogs given 4-aminobiphenyl. Cancer Res. 1973 Jun;33(6):1284–1289. [PubMed] [Google Scholar]
  16. Siebert D. A new method for testing genetically active metabolites. Urinary assay with cyclophosphamide (endoxan, cytoxan) and Saccharomyces cerevisiae. Mutat Res. 1973 Mar;17(3):307–314. doi: 10.1016/0027-5107(73)90002-x. [DOI] [PubMed] [Google Scholar]
  17. Siebert D., Simon U. Cyclophosphamide: pilot study of genetically active metabolites in the urine of a treated human patient. Induction of mitotic gene conversions in yeast. Mutat Res. 1973 Jul;19(1):65–72. doi: 10.1016/0027-5107(73)90114-0. [DOI] [PubMed] [Google Scholar]
  18. Smith D. W. Mutagenicity of cycasin aglycone (methylazoxymethanol), a naturally occurring carcinogen. Science. 1966 May 27;152(3726):1273–1274. doi: 10.1126/science.152.3726.1273. [DOI] [PubMed] [Google Scholar]
  19. WEISBURGER J. H., WEISBURGER E. K., MORRIS H. P. Differences in the metabolism of N-2-fluorenylacetamide in the guinea pig and the rat. Cancer Res. 1958 Oct;18(9):1039–1047. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES