METHODS 

 Acute toxicity 



Before compounds can be evaluated for mutagenic activity, their acute, 

 physiological toxicity must be determined. This is accomplished by measuring 

 the abiUty of single cells to produce macroscopic colonies arising in 

 experimental dishes following exposure to specific concentrations of the test 

 agent for specified periods of time. Relative plating efficiency (RPE), defined 

 as the ratio of macroscopic colonies arising in experimental dishes, to those 

 appearing in controls, may be plotted against dose to yield survival curves of 

 the type show^n in Figure 7-1. Concentrations of compound to be tested for 

 genetic activity are selected from such curves. The exponential portion of each 

 curve is described by equation [1] where (S/S^) is the surviving cell fraction or 

 percent RPE, (n) is the hit or target number (30) and (D/D^) is relative dose 

 (44). The target number is defined operationally by the intersection of the 

 exponential portion of the curve with the ordinate axis when the former is 

 extrapolated back. Relative dose is defined as the ratio of the experimental 

 molar concentration of toxicant (D) to that increase in molar concentration 

 (D ) required to reduce the cell population by the fraction (1/e). The value of 

 D is, for each compound, obtained from a plot of molar concentration versus 

 surviving cell fraction. Because chemicals differ in their molar toxicity by 

 orders of magnitude," relative dose provides a convenient way to depict survival 

 data for many compounds simultaneously. It is noted that the random hit 

 model expressed by equation [1] was derived for radiation effects (30), and 

 requires interpretive modifications when describing cell inactivation by 

 chemicals (32). The use of plating efficiency to assess acute chemical toxicity 

 has been described elsewhere (34). 



^=[l-(l-e->/Oo)"] [l] 



The CHO Cell/BrdU-VL System 



Figure 7-2 represents a simplified and generalized protocol for inducing, 

 isolating and characterizing mutant cells. Initially, cells are inoculated into 

 dishes or flasks and allowed to attach to the plastic substratum. Following 

 attachment, cells are exposed to the test agent at one or more concentrations. 

 The cells are then washed free of the test compound and fresh medium added. 

 During the expression period, cells are grown under nonselective conditions, 

 permitting induced genetic damage to become fixed into DNA, and ultimately 

 to become expressed at the cellular level. The length of the expression period is 

 a function of the system employed, the genetic markers involved, and the 

 conditions of the experiment. Selection represents the application of a set of 

 conditions permitting mutant cells to survive while eliminating wild-type cells. 

 Selective conditions employed are specific for the type of mutant sought. Once 

 potential mutants have been isolated, they may be subjected to genetic analysis 

 for confirmatory purposes and for further characterization. 



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