As noted in Chapter 6, chromosomal alterations are extremely common if not ubiquitous in all malignant neoplasms, as was originally suggested by Boveri (1914). Therefore, the induction of chromosomal abnormalities by chemicals in relatively short-term in vivo and in vitro methodol- ogies would logically be considered as an excellent test for carcinogenic potential. Although this has been true in general, the application of various tests for clastogenicity, aneuploidy, and chro- matid alterations has not formed the basis for determining the potential carcinogenicity of chem- icals. In part, the technology involved is more complicated and expensive than most of the gene mutation assays, and the molecular basis for at least one of the more common tests, that of sister chromatid exchange, is not fully understood. Theoretically, from considerations discussed in Chapter 9, short-term assays for the induction of clastogenicity and related abnormalities would allow the rapid identification of potential progressor agents.
Chromosomal alterations in vivo were studied in germ cells two decades ago by Generoso et al. (1980) in mice. As carried out by these workers, this procedure involves the administration of an agent to male mice shortly before breeding and subsequent examination of male offspring
for sterility and/or chromosomal abnormalities in both germ and somatic cells. The test is some- what complex, and thus far only a few very potent mutagenic agents have been found positive in it. A more commonly employed short-term test for clastogenesis is the micronucleus test, which measures induced clastogenesis in rodent bone marrow in vivo by morphological evaluation of micronuclei containing chromosome fragments in cell preparations from bone marrow (Heddle et al., 1983). However, this assay also has an occasional false positive, such as vitamin C (Tin- well and Ashby, 1994). With the LEC rat, which exhibits a defect in copper metabolism leading to hepatitis and hepatomas, an increased frequency of chromosome aberrations was seen in the bone marrow after administration of direct-acting alkylating agents that did not need metabolic activation (Ito et al., 1994). However, carcinogenic agents requiring metabolic activation, espe- cially in the liver, induced fewer chromosomal abnormalities in the bone marrow of these rats than in normal rats.
Studies in vitro of chromosomal alterations have been carried out both in yeast and in cul- tured mammalian cells. In the former, various genetic end points are studied, the abnormalities seen being the result of chromosomal alterations (Wintersberger and Klein, 1988). In mamma- lian cell lines, most of the systems used the same lines as for the gene mutation assays, e.g., Galloway et al. (1985). Relatively few analyses of induced chromosomal alterations have been carried out in normal diploid cells in culture. This test is used much more extensively than most of the other short-term tests involving chromosomal alterations (cf. Ishidate et al., 1988). As might be expected, some discrepancies have arisen between the mutagenic and clastogenic ef- fects of chemicals by these two different systems (cf. Ashby, 1988). Furthermore, chromosomal alterations in these cell lines are sensitive to oxidants (Gille et al., 1993; Shamberger et al., 1973; Kirkland et al., 1989), and preferential targets of chemicals in these aneuploid cell lines are chromosomes bearing amplified genes, already indicative of the karyotypic instability of the cell lines being used (Ottagio et al., 1993).
Another short-term test involving changes in chromosomal structure by mechanisms not entirely understood is the technique of “sister chromatid exchange” (SCE). During metaphase, sister chromatids, each of which is a complete copy of the chromosome, are bound together by mechanisms that involve specific proteins (Nasmyth, 1999). SCE reflects an interchange be- tween DNA molecules within different chromatids at homologous loci within a replicating chro- mosome (Latt, 1981). The detection of SCEs requires methods of differentially labeling sister chromatids. The usual technique is to allow a cell to incorporate a label, usually a halogenated pyrimidine such as bromodeoxyuridine (BrdU), for one replication cycle and then letting it un- dergo a second replication cycle in which the presence of the labeled precursor is actually op- tional. Results of such a technique are seen in Figure 13.5. The degree of staining is noted by the shading of the boxes symbolizing the individual sister chromatids. The diagram in Figure 13.5 indicates the normal staining pattern that would be seen in the absence of SCE. In Figure 13.6 may be seen an example of cells in culture subjected to the technology seen in Figure 13.5, wherein several SCEs can readily be noted. Although the exact mechanism of this phenomenon is not understood, the frequency of induced SCEs has been related linearly to the induction of mutation in the same cell (Carrano and Thompson, 1982). On the other hand, clastogenic events do not parallel SCE formation (Galloway and Wolff, 1979). SCEs occur in normal individuals (Sinha et al., 1985), and the increased levels of SCEs induced in cultured cell lines many times disappear after a number of cell divisions in the absence of the inducing agent (Muscarella and Bloom, 1982). Increases in SCEs may also be induced in cell culture by altering amino acid levels (Zhang and Yang, 1992) and under conditions of nucleotide-pool imbalance (Kaufman,1986). The procedure has been used in vivo as well as in vitro (DuFrain et al., 1984). In an extensive examination and comparison of the SCE method with cytogenetic changes, the two
Figure 13.5 Sister chromatid differentiation by BrdU-dye techniques. Cells incorporate BrdU for one cycle shown on the left, followed by a second cycle of replication in which BrdU may be present or absent, the only difference being the intensity of labeling difference. Sister chromatids in metaphase chromosomes from such second-division cells will exhibit unequal staining intensity either by fluorescence or direct ob- servation by visible light, depending on the stain utilized. Solid, hatched, and open areas around each rect- angle symbolizing the sister chromatid represent intense, intermediate, and pale staining respectively. (Adapted from Latt, 1981 with permission of the author and publisher.)
methods were about 70% congruent, again indicating that clastogenesis and SCEs are not identi- cal phenomena (Gebhart, 1981).