Salmonella histidine-reversion assay

Stewart, D.L., E.J. Sass, L.K. Fritz and L.B. Sasser. 1989. Toxicology Studies on Lewisite and Sulfur Mustard Agents Mutagenicity of Sulfur Mustard in the Salmonella Histidine Reversion Assay. Final Report from Pacific Northwest Laboratories (PNL-6873) to U.S. Army Medical Research and Development Command, Fort Detrick, MD, AD A213102. 

StewartDL, Sass EJ, FritzLKetal. (1989). Toxicology studies on Lewisite and sulphur mustard agents mutagenicity of Lewisite in the salmonella histidine reversion assay, Report AD-A213146. Fort Detrick, Frederick, MD, USA US Army Medical Research and Development Command. 

Rao TK, Dorsey GF, Atlen BE, et al. 1982. Mutagenicily of 4,4 -methylenedianiline derivatives in the Salmonella histidine reversion assay. Arch Toxicol 49(3-4) 185-... 

Stewart DL, Sass EJ, Fritz LK, Sasser LB (1989a) Toxicology studies on lewisite and sulfur mustard agents mutagenicity of sulfur mustard in the Salmonella histidine reversion assay. PNL-6873 (DTIC AD A213102). Pacific Northwest Laboratories. Prepared for the U.S. Department of the Army, Medical Research and Development Command, Fort Detrick, MD. 

As summarized in Table 1, aqueous extracts ( leachates ) can be prepared with various materials, largely to mimic the environmental fate of the potential mutagens contained in the solid or semisolid materials. For example, aqueous extracts of solid industrial wastes can be prepared and subjected to mutagenicity testing in an attempt to monitor biohazards. Similarly, fly ash from coal-fired power plantscan be extracted with horse serum for determination of mutagenic activity. Obviously, most of the problems pointed out with organic extracts are also inherent in aqueous extracts. The presence of histidine can also affect the results of the Salmonella histidine-reversion assay. 

TLC has been used to isolate and identify—at least by general chemical class—the potential mutagens. Again, the Salmonella histidine-reversion assay has been used to detect the activities, capitalizing on the ease of coupling metabolic activation to the system. 

TABLE 25.1. Strains Commonly Used in the Salmonella/ Histidine Reversion (Ames) Assay for Detecting Mutations at the Histidine Locus... 

Sulfur mustard has been found to be genotoxic and mutagenic in several microbial assays. The agent caused alkylation of DNA in the yeast Saccharo-myces cerevisiae (Kircher and Brendel 1983) and interstrand DNA cross-links (Venitt 1968) and inhibition of DNA synthesis (Lawley and Brookes 1965) in Escherichia coli. Using the histidine reversion assay, Stewart et al. (1989a) found that sulfur mustard induced point mutations in Salmonella typhimurium strain TA102 and frameshift mutations in TA 97 but neither type of mutation in strains TA98 and TAIOO. 

Extraction methodology, including rapid, simple extraction methods and more complex fractionation procedures, will be considered initially, followed by mutagenicity assay methods with emphasis on the Salmonella histidine-reversion assaytogether with aspects of their applicable procedures. 

The majority of the studies outlined in Section 2 utilized the rapid and inexpensive plate incorporation assay with the Salmonella histidine-reversion system.All strains (missense and frameshift) should be screened with unknown materials. However, in practice, most determinations can be made with strains TAIOO and TA98 (containing the R plasmid pkMlOl). Crude materials or fractions for testing are usually dissolved in DMSO in the range of 5-10 mg of total solids/ml. Stock solutions are stored in the dark at 4°C. Fractions or subfractions are evaporated or lyophilized to dryness or to a concentrated tar before solubilization. In actual practice, many crude fractions are not completely soluble even in DMSO. The treatment can be carried out with the suspended material or the DMSO-soluble materials after suspension and centrifugation. 

Assays that detect reverse mutation are most popular, largely because of the development of the Salmonella/micro-some assay by Bruce Ames and his co-workers.10 13 All five of the strains that sure currently recommended95 contain point mutations that prevent the biosynthesis of histidine. Unless histidine is provided in the growth medium, the bacteria cannot grow. However, if a new mutation occurs at the site of the original mutation and "reverses" its effect, growth without histidine can take place. 

The Ames salmonella-microsome test is a principal sensitive mutagen screening test. Compounds are tested on the mutants of Salmonella typhimurium for reversion from a histidine requirement back to prototrophy. A positive result is seen by the growth of revertant bacteria (which do not require an external histidine source). A microsomal activation system should be included in this assay. The use of five different bacterial test strains are generally required. 

Figure 2-2 Bioluminescent Salmonella assay workflow. The luminescent histidine-dependent cells are exposed to tested compounds in agar overlay containing only traces of histidine in multiwell-plate format. The reverse-mutation events restore endogenous histidine synthesis resulting in luminescent colonies of histidine-independent cells that can be visualized via CCD camera. The fully automated instrument in conjunction with automated image analysis of plates enables the analysis of 100 plates in one run.

Salmonella reverse mutation assay (Ames test) Looks for back mutation to autotrophy (independence from exogenously supplied histidine) of cultured mutant Salmonella (auxotroph, deficient in histidine biosynthesis and thus dependent on exogenous supply of this amino acid) after exposure to the test substance Calf thymus oxidative DNA damage 8-hydroxydeoxyguanosine is measured as a marker of oxidative DNA damage caused by test substance... 

Two in vitro mutagenidty studies were conducted on Glarinol G-80 a reverse mutation assay in five histidine-requiring strains of Salmonella lyphimurium (an Ames test) and an in vitro cytogenetics assay in human lymphocytes (chromosome aberration test) (5). Both studies were conducted in accordance with Good Laboratory Practice (GLP) and in accordanc e with OEGD guidehne 471 and 473 respectively. 

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