Ames assay method

Benzo(b)-, benzo(k)-, and benzo(j)fluoranthene are common environmental contaminants (38). The tumor-initiating activity of benzo-(b)fluoranthene on mouse skin is about equal to that of DBA (38). All three isomers are mutagenic in the Ames assay (110). Syntheses of the 1,2-, 9,10-, and 11,12-dihydrodiols of benzo(b)fluoranthene by Method I have been reported (111,112). 

Protocols for preparing six environmental sample types prior to the Ames Salmonella assay were proposed at a recent panel discussion sponsored by the U.S. Environmental Protection Agency (USEPA) and the U.S. Army. Air particles, soil-sediment, and solid waste are extracted with dichloromethane, concentrated, and solvent exchanged into dimethyl sulfoxide (DMSO). Organics in water and waste water are absorbed onto XAD columns, then eluted with hexane-acetone, solvent reduced, and exchanged into DM SO. Nonaqueous liquids are assayed directly and as concentrates before they are solvent exchanged to DMSO. If bacterial toxicity or lack of dose response is observed in the Ames assay of extracts, the extracts are fractionated prior to solvent exchange. These are interim methods and have not been subjected to policy review of the USEPA or the U.S. Army. 

It was the consensus of the panel participants that the methods summarized here represent the best available technology to prepare environmental samples for the Ames assay. These methods were chosen, after group discussion, on the basis of the collective laboratory experience of the participants and a thorough review of the literature. However, these are interim protocols subject to laboratory validation. This validation effort has been initiated, and workers in the field of environmental mutagenesis are urged to test these interim protocols in their own laboratories. 

Acute oral toxicity Acute dermal toxicity Primary skin irritation Primary eye irritation Skin sensitization Ames assay (gene mutation) Rat and/or in vitro method Rat and/or in vitro method Rabbit and/or in vitro method Rabbit and/or in vitro method Guinea pig or LLNA Bacteria... 

A new in vitro genotoxicity assay has been developed by the Microbics Corp. This assay, known by the trade name of Mutatox assay, is a convenient method to detect DNA-damaging substances (genotoxins) by measuring light emissions from an isolated dark mutant strain of Photobacterium phosphoreum. The Mutatox assay compares favorably in sensitivity with the Ames test, and it is easier and more rapid to perform [15]. As a toxicity estimation method, one convenient assay method was developed and uses cultured fish cells this is introduced in section 7.4. 

The application of several short-term tests to detect carcinogenic hydrazine derivatives has been of limited success 204a, 260). While a host-mediated assay of 1,2-dimethylhydrazine has been reported to give a positive mutagenic response in S. typhimurium G46 345a), the Ames assay has generally not been sensitive toward this class of compounds. In view of the limited reports on the successful applications of an in vitro metabolic activation system for these compounds, comparisons of such methods are not valid at this time. 

In order to assess the potential toxicological effects of onchidal from a predictive standpoint, the author subjected the two-dimensional molecular structure of onchidal (1) to an in silica QSAR computational analysis. Details regarding the approach of the software, including procedures and model building methods, have been described in recent publications (e.g., Choi et al., 2013 Valencia et al., 2013). The in silica analysis of the molecular structure of onchidal produced predictive information on nonclinical toxicities. The computational QSAR models included bacterial mutagenicity (Salmanella h/pln/murium mutagenicity (Ames) assay), and phospholipidosis (Table 30.3). 

Assays. Protein concentrations were measured by the method of Bradford (18) and the various contractile protein ATPase activities by tRe method of Martin and Doty (19). Gel electrophoresis was carried out by the method of Ames (20) on 1.5 ran polyacrylamide slabs using the discontinuous SDS buffer system of Laemnli (21). Dried gels were scanned at 550 nm for densiometry measurements. 

In vitro assays are increasingly being used. Some of the reasons are cost, availability of more rapid results, and avoidance of negative publicity. Assays such as cytochrome P-450 enzymes, the Ames test, and the mouse lymphoma tk test are in vitro methods. For absorption studies, Caco-2 (Exhibit 5.9) and Madin-Darby canine kidney cell assays are now routinely used. Hepatocyte cell lines with metabolism capacity are being developed to test drug metabolism and toxicity. All these examples show that, where possible, pharmaceutical firms are gradually dispensing with animal studies. 

In Chapter 1 Bruce N. Ames, the plenary speaker at the symposium, describes the development of the Salmonella/microsome assay, which is better known as the Ames Test. This test was the first proven rehable and rapid in-vitro method for the mutagenic and carcinogenic screening of chemicals. Included is an excellent overview of the potential problem of human exposure to toxic substances as well as a description of how this important method is being used to test chemical substances. 

The use of yeast cells as a eukaryotic complement to the Ames test led to the development of several protocols for the detection of mutation, gene conversion and recombination. The formal introduction of methods [23] followed by much development work from Zimmermarm s laboratory led to large systematic studies [24, 25] and OECD guidelines for the test battery (OECD 480, 481). However the assays are now rarely used, at least in part because of concerns over low sensitivity, thought to reflect limited permeability of the cell wall. 

Salmonella Mutagenicity Test (Ames Test). The methods of bacterial culture, the verification of genetic markers, and the plate incorporation assay were essentially the same as described previously (14, 15). Petri dishes (90 mm) containing about 20 mL of 1.2 Noble agar in minimal Vogel Bonner Medium E supplied with excess biotine and... 

Nouri AME, Mansouri M, Hussain RF, et al. 1995. Super-sensitive epithelial cell line and colorimetric assay to replace the conventional K562 target and chromium release assay for assessment of non-MHC-restricted cytotoxicity. J Immunol Methods 180 63-68. 

Ames BN (1965) Assay of inorganic phosphate, total phosphate and phosphates. In Neufeld EF, Ginsburg V (eds) Methods in Enzymology 8, Academic Press, New York, p 115... 

Busch, R.P. and Virji, M.A. (1985). Serum theophylline assay by Ames Seralyzer compared with Abbott TD, in pediatric care. Clin. Chem. 31, 1247-1248. Castellet, J., Pedromingo, A. and Delgado, A. (1985). Evaluation of the Seralyzer method for the determination of theophylline. Reunion Nacional Soc. Esp. Quimica Clin. 4, 5. 

Acetone (reagent grade) was evaluated by the standard plate incorporation method in the Ames Salmonella reverse mutation assay with strains TA98, TAIOO, TAI535, TAI537, and TAI538. 

In regulatory decisions, the primary standard in vitro methods are the genotoxicity assays, including the Ames test and the mouse lymphoma cell mutagenesis assay. Animal and human respiratory tract cells or tissues in culture are frequently used for... 

We used DEAE-cellulose gel column chromatography at subzero temperature in an ethylene glycol—water mixture to isolate the microsomal cytochrome P-450 in its stable form. The already known stabilizing effect of ethylene glycol and low temperature contribute to the stabilization of the enzyme. The preparation of rat liver microsomes and the assays were carried out as described earlier. The amount of phospholipid was measured by the method of Ames and Dubin (1960). 

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