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Health Risks from Exposure to Low Levels of Ionizing Radiation: Beir VII Phase 2
more stable true minisatellites in the mouse genome (Bois and others 1998a, 1998b). The ESTRs are highly unstable (i.e., they manifest high spontaneous mutation rates) in both somatic and germ cells. The mutational changes are manifest as changes in the number of tandem repeat cores and, hence, allele length. The available data suggest that the ESTR instability is a replication- or repair-based process involving polymerase slippage similar to mechanisms suggested for microsatellite instability (Ellegren 2000).
In contrast to mouse ESTRs, the minisatellites in humans consist of longer repeats (10 to 60 bp) that may span from about 0.5 kb to several kilobases and show considerable sequence variation along the array (Jeffreys and others 1991; 1994; May and others 1996; Buard and others 1998; Tamaki and others 1999; Stead and Jeffreys 2000; Vergnaud and Denoeud 2000). The majority of the classical minisatellites are GC rich. The fact that some of the human minisatellite loci studied are highly unstable and have very high spontaneous mutation rates of the order of a few percent is now well documented (Jeffreys and others 1985, 1988, 1995; Smith and others 1990; Vergnaud and Denoeud 2000). Mutation at these loci is almost completely restricted to the germline and is attributed to complex gene conversion-like events involving recombinational exchanges of repeat units between alleles (Jeffreys and others 1994; May and others 1996; Jeffreys and Neumann 1997; Tamaki and others 1999; Buard and others 2000; Stead and Jeffreys 2000; Vergnaud and Denoeud 2000).
Radiation Studies with Mouse ESTR Loci
The Loci Used
Two ESTR loci have been used thus far in mouse mutation studies, namely, the Ms6-hm, and Hm-2, both of which show multiallelism and heterozygosity within inbred strains. The Ms6-hm is <10 kb in size (varying greatly between different mouse strains) and consists of tandem repeats of the motif GGGCA. Linkage analysis localized Ms6-hm near the brown (b) coat color gene on chromosome 4. The germline mutation rate is about 2.5% per gamete (Kelly and others 1989). The Hm-2 locus is located on chromosome 9 and consists of GGCA tetranucleotide repeats with alleles containing up to 5000 repeat units (i.e., up to 5 kb). The germline mutation rate of this locus is estimated to be of the order of at least 3.6% (Gibbs and others 1993). As discussed below, Dubrova and colleagues studied mutation induction at both of the above loci, whereas the Japanese workers focused their attention only on the Ms6-hm locus.
Low-LET Radiation Studies
In the studies of Dubrova and colleagues (1993) involving irradiation of spermatagonial stem cells (0.5 and 1 Gy of γ-rays; CBA/H strain), significant increases in the frequencies of mutations at the Ms6-hm and Hm-2 loci were found. Subsequent work with X-irradiation doses of 0.5 and 1 Gy established that for mutations induced in the above cell stage, the dose-effect relationship was consistent with linearity (y = 0.111 + 0.338D), where D is the dose in grays (Dubrova and others 1998a, 1998b). From these data, the authors estimated that the DD for ESTR mutations induced in spermatogonia was 0.33 Gy for acute X-irradiation, similar to that reported for specific locus mutations in mice.
In the above work, spermatids were found to be insensitive to mutation induction, a finding at variance with those of Sadamoto and colleagues (1994) and Fan and coworkers (1995) with the C3H/HeN mouse strain. These authors showed that for Ms6-hm locus mutations, all male germ cell stages were sensitive (3 Gy of γ-irradiation). Nonetheless, both sets of studies demonstrated that increases in mutation frequencies could be detected at radiation doses and sample sizes substantially smaller than those used in conventional genetic studies with specific locus mutations.
High-LET Radiations Studies
Niwa and collegues (1996) found that acute neutrons from a 252Cf source (65% neutrons + 35% γ-rays) were 5.9, 2.6, and 6.5 times more effective, respectively, in spermatozoa, spermatids, and spermatogonia, than acute γ-irradiation in inducing mutations at the Ms6-hm locus. In similar studies, Dubrova and colleagues (2000a) noted that in spermatogonial cells, chronic neutrons also from a 252Cf source had a relative biological effectiveness of about 3 relative to chronic γ-irradiation (regression equations: y = 0.136 + 1.135D, neutrons; doses of 0.125, 0.25, and 0.5 Gy; y = 0.110 + 0.373D, γ-rays; doses of 0.5 and 1 Gy). Additionally (and not unexpectedly), they found that at the above γ-ray doses of 0.5 and 1 Gy, there was no dose-rate effect. It should be remembered that the lower effectiveness of chronic γ-irradiation recorded in earlier specific locus mutation studies (Russell and others 1958) occured at total doses of 3 and 6 Gy. This observation is in contrast to earlier results with specific locus mutations (Russell and others 1958) at 3 and 6 Gy showing that chronic γ-irradiation was only one-third as effective as acute X-irradiation in inducing specific locus mutations.
Mutation Induction at the ESTR Loci—An Untargeted Process Arising as a Result of Radiation-Induced Genomic Instability
One important conclusion that emerges from these studies is that mutation frequencies in the progeny of irradiated animals are too high to be accounted for by the direct induc-