METHODS 
Acute toxicity 
Before compounds can be evaluated for mutagenic activity, their acute, 
physiological toxicity must be determined. This is accomplished by measuring 
the ability 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 shown 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 Q ) 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 increased in molar concentration 
(D q ) required to reduce the cell population by the fraction (1/e). The value of 
D q 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 asses acute chemical toxicity 
has been described elsewhere (34). 
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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