DNA damage detected by the alkaline comet assay in the liver of mice after oral administration of tetrachloroethylene.

Induction of DNA damage in the liver and kidney of male CD1 mice was studied by means of the alkaline Comet assay after oral administration of tetrachloroethylene at the doses of 1000 and 2000 mg/kg/day. A statistically significant dose-related increase in tail intensity was established in hepatocytes, indicating that tetrachloroethylene induced DNA damage in the liver. No effect on DNA damage was observed in the kidney. The results are in agreement with carcinogenicity data in mice, in which tetrachloroethylene induced tumours in the liver but not in the kidney, and support that a genotoxic mode of action might be involved in liver carcinogenicity in mice. An alternative interpretation of the results conveyed by the Study director at the test facility, involving that tetrachloroethylene did not induce DNA damage in the liver and kidney of mice, is also presented and discussed.

Mechanisms of human minisatellite mutation in yeast.

Minisatellites are tandem repeat loci, with repeat units ranging in size from 5 bp to 100 bp. The total lengths of repeat arrays vary from about 0.5 kb to 30 kb, and excessive variability in allele length at human minisatellite loci is the result of germline-specific complex recombination events generating new length alleles. Minisatellite alleles also mutate to new lengths in somatic cells, but this occurs at a much lower rate than in the germline. Since recombination is involved in minisatellite mutation, the yeast Saccharomyces cerevisiae is a suitable model organism that has been employed to further dissect the molecular basis of mutation events at human minisatellites. These studies have shown that the mutational behaviour of a minisatellite in meiosis is not determined by the intrinsic properties of the repeat array, but are highly dependent on the position of the minisatellite in the genome. The processes for minisatellite mutation in yeast and humans are identical in the sense that mutation is indeed driven by meiotic recombination, but differ with regard to the types of structural changes that are generated by the recombination events. Tetrad analyses showed that inter-allelic transfers of repeats occur by conversion and not crossing over, and that several chromatids can be involved in successive recombination events in one meiosis, resulting in mutant alleles in several spores. It has been demonstrated that the genes SPO11 and RAD50, involved in the initiation of recombination events, are required for human minisatellite mutation in yeast meiosis. Intrinsic properties of the repeat array appear to determine the stability of human minisatellites in yeast mitosis, since mitotic mutation rates in yeast are highly variable between minisatellites. The repair genes RAD27 and DNA2 stabilise human minisatellites in yeast mitosis, while RAD5 has no effect on mitotic stability. MSH2 depresses human minisatellite frequency in meiotic cells of yeast.

Two modes of germline instability at human minisatellite MS1 (locus D1S7): complex rearrangements and paradoxical hyperdeletion.

Minisatellite MS1 (locus D1S7) is one of the most unstable minisatellites identified in humans. It is unusual in having a short repeat unit of 9 bp and in showing somatic instability in colorectal carcinomas, suggesting that mitotic replication or repair errors may contribute to repeat-DNA mutation. We have therefore used single-molecule polymerase chain reaction to characterize mutation events in sperm and somatic DNA. As with other minisatellites, high levels of instability are seen only in the germline and generate two distinct classes of structural change. The first involves large and frequently complex rearrangements that most likely arise by recombinational processes, as is seen at other minisatellites. The second pathway generates primarily, if not exclusively, single-repeat changes restricted to sequence-homogeneous regions of alleles. Their frequency is dependent on the length of uninterrupted repeats, with evidence of a hyperinstability threshold similar in length to that observed at triplet-repeat loci showing expansions driven by dynamic mutation. In contrast to triplet loci, however, the single-repeat changes at MS1 exclusively involve repeat deletion, and can be so frequent--as many as 0.7-1.3 mutation events per sperm cell for the longest homogeneous arrays--that alleles harboring these long arrays must be extremely ephemeral in human populations. The apparently impossible existence of alleles with deletion-prone uninterrupted repeats therefore presents a paradox with no obvious explanation.

The human minisatellites MS1, MS32, MS205 and CEB1 integrated into the yeast genome exhibit different degrees of mitotic instability but are all stabilised by RAD27.

The yeast Rad27 protein is homologous to mammalian Fen1 and is involved in the processing of replication intermediates. Enhanced instability of various artificial repetitive DNA sequences in RAD27-deficient yeast strains has been observed previously and shown to involve preferentially expansion mutations. In the present investigation, we characterised the mitotic instability of alleles of the naturally occurring human minisatellites MS1, MS32, MS205 and CEB1 and the modified MS1 alleles containing more highly homogeneous repeat regions than the original alleles. These minisatellites demonstrated more pronounced instability in rad27 Delta strains, with increases in the frequencies of both expansion and contraction mutants. In RAD27 strains, MS32 and MS205 were relatively stable, while MS1 and CEB1 were unstable, indicating that the effect of RAD27 on stability is influenced by intrinsic properties of the repeat array. This conclusion received further support from the remarkably high frequency of length-mutants observed for the modified allele of MS1. Thus, our findings emphasise the importance of: (1) comparing results obtained with various naturally occurring minisatellites and (2) manipulating their sequences in attempts to understand the molecular basis for mitotic stability/instability of minisatellite DNA.

The influence of sequence divergence between alleles of the human MS205 minisatellite incorporated into the yeast genome on length-mutation rates and lethal recombination events during meiosis.

Certain minisatellites exhibit hypervariability with respect to the number of repeat units and, thus, allele length. Such polymorphism is generated by germline-specific recombinational events that occur at high frequencies and lead to the gain or loss of repeat units. In order to elucidate the molecular details of mutagenesis in minisatellites, we have integrated human minisatellites into the yeast genome in the vicinity of a hotspot for meiotic double-strand breaks (DSBs). Here, we describe the results of tetrad analyses of mutations in the human MS205 minisatellite in yeast strains heterozygous for alleles composed of 51 and 31 repeat units, as well as in a strain homozygous for the same 51 repeat unit allele. The length-mutation rate was twice as high in the heterozygous strain as in the homozygous strain, suggesting that sequence divergence between alleles enhances the generation of length mutations. In the case of heterozygotes, the frequency of length mutants resulting from inter-allelic exchange was significantly higher in tetrads with three viable spores than in tetrads with four viable spores, indicating that there is a higher probability for spore mortality in tetrads originating from meioses during which inter-allelic exchange of repeat units occurs. In an attempt to explain these findings, we propose a model for minisatellite mutation involving recombination, in which sequence divergence between alleles results in a heteroduplex containing numerous mismatches. We suggest that convergent mismatch-repair tracts in this heteroduplex give rise to a DSB that may be repaired by an additional round of recombination resulting in mutation of a third allele, or be lethal if such recombination fails. It appears probable that the formation of such additional mutants is the major explanation for the difference in meiotic length-mutation rates between the heterozygous and homozygous yeast strains, and that this phenomenon contributes to high germline length-mutation frequencies at minisatellites in humans.