Primary DNA Damage

29 May

The measurement of DNA damage and repair induced by exogenous chemicals, both in vivo and in vitro, has been a relatively common technology used in short-term tests for potential carcino- genicity. The most generally utilized technology  involves the analysis of nonreplicative  DNA synthesis with appropriately labeled precursor nucleotides (cf. Harbach et al., 1991). More so- phisticated techniques involve the measurement of DNA strand breakage by eluting DNA frag- ments from columns to which the DNA is bound with an alkaline solution (Sina et al., 1983; Miyamae et al., 1997). These techniques have been applied to a variety of tissues in cell culture, both primary and cell lines. Primary liver cell cultures have been among the most popular of the tissues utilized (Williams et al., 1989; Strom et al., 1981; Swierenga et al., 1991). While primary hepatocyte cultures have the advantage of an extensive endogenous metabolic apparatus, other workers have attempted to obviate the problem of metabolism of the agent to the active form by administration  of the test chemical in vivo, with subsequent explantation of specific organ tis- sues to culture and measurement of unscheduled DNA synthesis in such cultures (cf. Furihata and Matsushima, 1987). Just as with all of the short-term tests indicated above, the use of DNA repair analysis has limitations, as evidenced by the fact that in an extensive investigation by Wil- liams and associates (1989) of 167 chemicals testing negative, 44 were carcinogenic. This and the other points raised in this section demonstrate both the usefulness and limitations of short- term tests of mutagenicity and DNA damage for indicating potential carcinogenicity. Regulatory agencies have chosen to approach this problem by requiring a number of different tests to be performed during the study of a particular compound, and these data are taken into account with all of the other information, especially that developed by studies in vivo, as discussed below.

Figure 13.6 Example  of Chinese  hamster  cells grown for two generations  in BrdU and stained  with Giemsa  stain after appropriate  treatments.  The differential  staining  of the two sister chromatids  can be readily noted, and the exchanges are indicated by arrows. (Adapted from Takayama and Sakanishi, 1977, with permission of the authors and publisher.)

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