Preliminary experiments suggest that these cells show contact inhibition of 

 replication in colonies containing at least 100 cells, as well as in dense 

 monolayers (35). All such clones tested to date appear stable in their ability to 

 express the apparent contact -inhibited state. 



Mutation Frequency and Expression Time 



Mutation is a complex biological process involving much more than 

 interaction of mutagen with DNA, or more generally, with the DNA-replicating 

 system. The mutagen-DNA interaction usually results not in mutation, but in 

 the creation of premutational lesions which become expressed as mutations 

 only after a series of additional events has occured (6). For example, 

 auxotrophic mutants usually become expressed as soon as the cells become 

 depleted of normal gene product. The length of time required for this and 

 other preliminary events to occur may vary with the nature of the gene 

 product, the specific mutagen utilized, and the doses employed (5, 7). To 

 determine the influence of expression time on observed mutation frequency 

 for the mutant phenotypes reported above, experiments employing variable 

 expression time were carried out with EMS and BrdU. Doses used were those 

 gi'/ing maximal observed mutation frequency when the expression time was 

 five days (3 x 10'^ 'molar for EMS, 1 x 10"^ for BrdU). Expression time was 

 varied between two and eleven days. The data are given in Table 7-4, again in 

 terms of the parameters of equation [2] . Observed mutation frequency is 

 plotted in Figure 7-8 as a function of expression time. 



Optimal expression time is defined as the interval between mutagen 

 quenching, and the application of selective conditions yielding maximal 

 mutation frequency when dose is held constant. Tliis is observed to be five 

 days for BrdU and eight days for EMS. Moreover, for a two-day expression 

 period, statistically significant numbers of mutants could always be identified 

 in populations exposed to EMS, whereas none are found in populations treated 

 with BrdU. Once optimal expression time is exceeded, mutant cell frequency 

 decreased rapidly, suggesting that these types of mutants are at a replicative 

 disadvantage relative to wild-type cells under nonselective conditions. The fact 

 that optimal expression time was different for the two mutagens when tested 

 at doses showing similar toxicity, suggests that expression time may be mutagen 

 dependent. This lends support to the contention that assays utilizing variable 

 expression time shall be required when screening compounds for mutagenic 

 activity (4). This may be particularly important for weak mutagens. 



DISCUSSION 



The genetic toxicology of inorganic compounds has largely been ignored, 

 despite the fact that many are ubiquitous, liighly toxic, and impHcated as 



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