Assay Forward

A typical mutagen assay experiment proceeds as follows  

One week later, the cells have grown to full culture size (300 ml at 5 X 10 cells/ml). Subcultures are prepared from it, one or more for each concentration of the compound to be tested, including untreated controls. All cultures are adjusted to a cell concentration of 3 X lO /ml. 

During the expression period, records pertaining to the dilution of cell cultures with fresh medium are maintained to permit accurate assessment of each culture s growth rate and hence an estimate of viability relative to a control culture. 

Optimal expression time is generally 48 hr (about four generations), but it may vary markedly from one mutagen to another. Following expression, 1.6 x lO cells are centrifuged from each culture and resuspended in 200 ml CM (final concentration of 0.8 x 10 Coulter particle counts per milliliter CM). From this point onward, all cultures are handled identically so that no distinction will be made between control and treated. 

A typical mutagen assay experiment is comprised of four groups of plated Falcon flasks referred to below as series A through D. 

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Reversion Mutation Assays Forward Mutation Assays... 

TK or HPRT forward mutation assays in cultured mammalian ceils Drosophila sex-linked recessive lethal assay... 

McGregor DB, Brown A, Cattanach P, et al. 1988. Responses of the L5178Y tk+/tk- mouse lymphoma cell forward mutation assay III. 72 coded chemicals. Environ Mol Mutagen 12 85-154. 

Another issue is the development and refinement of the testing protocols used in mesocosms. Mesocosms could have a more important role in environmental risk assessment if the data coming from them could be better interpreted. The use of biomarker assays to establish toxic effects and, where necessary, relate them to effects produced by chemicals in the field, might be a way forward. The issues raised in this section will be returned to in Chapter 17, after consideration of the individual examples given in Part 2. 

Salmonella typhimurium (reverse mutation) Escherichia coli (forward mutation, DNA modification) Saccharomyces cerevisia (reverse mutation) Bacillus subtilis (rec assay) Gene mutation or DNA modification Bruce and Heddle 1979 Dunkel et al. 1984 Fukunaga et al. 1982 Kharab and Singh 1985 Nestmann et al. 1979 Nishioka 1975 Rosenkranz and Poirier 1979 Simmon 1979b... 

McGregor, D.B., A.G.Brown, S.Howgate, D.McBride, C.Riach, and W.J.Caspary. 1991. Responses of the L5178Y mouse lymphoma cell forward mutation assay. V. 27 coded chemicals. Environ. Mol. Mutagen. 17 196-219. 

Macgregor, J.T., D.H. Gould, A.D. Mitchell, and G.P. Sterling. 1979. Mutagenicity tests of diflubenzuron in the micronucleus test in mice, the L5178Y mouse forward mutation assay, and the Ames Salmonella reverse mutation test. Mutat. Res. 66 45-53. 

Fig. II.8 Assays for 10 ng/mL PSA on IL Class BioCDs. (a) Forward label free assay at 10 ng/mL showing the histrogram of approximately 10,000 spots pairs, (b) Sandwich assay shows clear separation...

A plethora of chemical reactions that are intimately associated with the quantitative analysis essentially belong to the class of reversible reactions. These reactions under certain prevailing experimental parameters are made to proceed to completion, whereas in certain other conditions they may even attain equilibrium before completion. In the latter instance, erroneous results may creep in with regard to the pharmaceutical substance under estimation. Hence, it has become absolutely necessary first to establish the appropriate conditions whereby the reactions must move forward to attain completion so as to achieve the ultimate objective in all quantitative assays. 

Theory In general, quaternary nitrogen containing compounds like—choline chloride, acetylpyridinium chloride, benzethonium chloride, and bethanechol chloride readily form insoluble salts quantitatively with tetraphenyl boron and this puts forward the basis for the gravimetric assay of the above cited pharmaceutical substances. 

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