Mechanisms involved in the induction of aneuploidy : the significance of chromosome loss

The induction of aneuploidy by physical and chemical agents using different test systems was evaluated. The effect of X-rays, caffeine, acetaldehyde, ethanol, diethylstilbestrol, propionaldehyde, and chloral hydrate was studied by chromosome counting in Chinese hamster embryonic diploid cells. Aneugenic ability of cadmium chloride, cadmium sulfate, potassium dichromate, chromium chloride, nickel chloride, and nickel sulfate was assessed by means of anaphase-telophase analysis in Chinese hamster ovary cells. Chromosome counting in human fibroblasts (MRC-5 cell line) was employed to evaluate the effect of cacodilic acid, cadmium chloride, cadmium sulfate, and potassium dichromate. Finally, the induction of kinetochore-positive and kinetochore negative micronuclei by cadmium chloride, cadmium sulfate, potassium dichromate, chromium chloride, and nickel chloride was studied using CREST antibodies. When the effect of different agents was determined by chromosome counting, an increase of hypoploid but not of hyperploid cells was observed. Anaphase-telophase analysis showed that metal salts increased the frequency of lagging chromosomes. This finding has been confirmed by the increment of kinetochore-positive micronuclei using CREST antibodies. Therefore, chromosome loss could be considered as the main cause of induced aneuploidy.


INTRODUCTION
Although aneuploidy is a serious health problem, the experimental methodology used to investigate the condition has not been completely validated.Development of a comprehensive test battery is necessary for the evaluation and detection of aneugenic chemicals.The reliability of any aneuploidy test is always challenged by the fact that the mechanisms involved in aneuploidy induction are poorly understood due, in part, to the multiple factors related with the occurrence of chromosome disjunction and nondisjunction.
Various test systems have been used to study chemical-induced aneuploidy.Chromosome counting in diploid cell lines (Danford, 1984(Danford, , 1985;;Dulout and Natarajan, 1987) was a validated test although it is time consuming.
In the present study, results obtained by chromosome counting, anaphase-telophase analysis and cytokinesisblocked micronucleus assay were compared.Experiments were carried out to test the suspected aneugenic ability of different compounds or to validate the assay by studying known aneugenic chemicals.Chromosome counting reliability and usefulness was demostrated by Dulout and Natarajan (1987).The aneugenic ability of the synthetic estrogen diethylstilbestrol (DES), acetaldehyde, propionaldehyde, chloral hydrate, cadmium chloride, cadmium sulfate, potassium dichromate and cacodilic acid has been evaluated by employing this assay (Dulout and Natarajan, 1987;Dulout and Furnus, 1988;Furnus et al., 1990, Güerci et al., 2000).Anaphase-telophase analysis was employed to test the aneugenic ability of propionaldehyde, cadmium chloride, cadmium sulfate, potassium dichromate, chromium chloride (III), nickel chloride and nickel sulfate (Seoane and Dulout, 1994;Seoane, 1999).Cytokinesisblocked micronucleus assay using anti-kinetochore antibodies was employed to evaluate the above mentioned heavy metal salts (Seoane, 1999).

MATERIAL AND METHODS
Chinese hamster embryo cells (CHED) and human diploid fibroblasts (MRC-5) were employed to carry out Mechanisms involved in the induction of aneuploidy: the significance of chromosome loss * A.I. Seoane, A.M. Güerci

Abstract
The induction of aneuploidy by physical and chemical agents using different test systems was evaluated.The effect of X-rays, caffeine, acetaldehyde, ethanol, diethylstilbestrol, propionaldehyde, and chloral hydrate was studied by chromosome counting in Chinese hamster embryonic diploid cells.Aneugenic ability of cadmium chloride, cadmium sulfate, potassium dichromate, chromium chloride, nickel chloride, and nickel sulfate was assessed by means of anaphase-telophase analysis in Chinese hamster ovary cells.Chromosome counting in human fibroblasts (MRC-5 cell line) was employed to evaluate the effect of cacodilic acid, cadmium chloride, cadmium sulfate, and potassium dichromate.Finally, the induction of kinetochore-positive and kinetochore negative micronuclei by cadmium chloride, cadmium sulfate, potassium dichromate, chromium chloride, and nickel chloride was studied using CREST antibodies.When the effect of different agents was determined by chromosome counting, an increase of hypoploid but not of hyperploid cells was observed.Anaphase-telophase analysis showed that metal salts increased the frequency of lagging chromosomes.This finding has been confirmed by the increment of kinetochore-positive micronuclei using CREST antibodies.Therefore, chromosome loss could be considered as the main cause of induced aneuploidy.
the chromosome counting assay.Chinese hamster ovary cells were employed in the anaphase-telophase test.MRC-5 cells were used in the cytokinesis-blocked micronucleus assay.Experimental procedures, compound dilutions and doses assayed have been described in previous papers (Dulout and Furnus, 1988;Furnus et al., 1990;Seoane and Dulout, 1994;Seoane and Dulout, 1999;Seoane, 1999;Güerci et al., 2000) RESULTS Irradiation increased the frequency of aneuploid cells with a corresponding decrease of diploid cells, except for the dose of 100 rad (Figure 1).Pulse treatments with different doses of DES also increased the frequency of aneuploid cells in relation with the dose employed but not with the duration of the treatment.
In cells treated with acetaldehyde, ethanol and DES the frequency of aneuploid cells increased significantly compared to controls (Figures 2,3).In addition, the frequency of aneuploid cells observed in the three treatments with acetaldehyde was higher than that detected in cells treated with DES.The increase of aneuploid metaphases was mainly due to the increment of hypoploid cells.Whereas the relation between hypoploid/hyperploid cells was 0.81 in the controls, in treated cells this relation varied from 2.72 to 3.82.
In the treatments with propionaldehyde and chloral hydrate a significant increase of aneuploid cells compared to untreated controls was observed (Figure 4).Although the frequencies of hypoploid cells were higher than the frequencies of hyperploid cells in all treatments, the differences were not significant.
An increase of lagging chromosomes was found in cells treated with cadmium salts, although significant differences from untreated controls were only found with the highest dose of cadmium sulfate.Whereas potassium dichromate increased the frequencies of lagging chromosomes at all the doses employed, only the two highest doses of chromium chloride gave positive results.Increments of lagging chromosome frequencies induced by nickel salts were lower than those induced by the other heavy metal salts.However, significant differences from controls were found only with the two highest doses (Figure 5).
Results of chromosome counting using MRC-5 cells (Figure 6) showed significant increments of aneuploidy in cacodilic acid-and cadmium chloride-treated cells.Cadmium sulfate induced significant increases at the two highest doses.Potassium dichromate induced significant increments of aneuploidy at all doses tested although at a lower degree than the other compounds.
Cadmium salts induced significant increases of kinetochore-positive and kinetochore-negative micronuclei when the cells were treated with the two highest concentrations of each compound.The lowest doses induced borderline increases in the frequency of kinetochore-posi-

Hyperploid cells Hypoploid cells
tive micronuclei but no differences with respect to control values were found in the frequency of kinetochorenegative micronuclei.The highest increments of total micronuclei frequency were induced by chromium chloride.Most of these micronuclei were kinetochore-positive. Potassium dichromate also induced higher increases of kinetochore-positive micronuclei than kinetochorenegative ones.Nickel salts induced statistically significant increases of kinetochore-positive micronuclei with the three doses employed.On the other hand, increases in kinetochore negative micronucleus frequency were only slight (Figure 7).

DISCUSSION
When a normal disjunction occurs in a mitotic division, the chromatids of a chromosome segregate to each cellular pole.Chromosomal missegregation can be produced by: 1) multipolar mitoses arisen from centriole alterations, 2) nondisjunction, when the chromatids of a chromosome do not separate correctly and the entire chromosome migrates to one pole, 3) chromosome loss, when a chromosome (or a chromatid) remains lagged at the equator and does not migrate to the corresponding pole.

h 8 h 16 h 2h
micronuclei containing either the lagged chromosome or the lagged chromatid are formed.When cells were analyzed by chromosome counting, higher frequencies of hypoploid than hyperploid cells were found.These results seem to be in conflict with the belief that aneuploidy is a result of the induction of equal numbers of these two kinds of cells.Nevertheless, other evidence indicated that the increase of hypoploid cells is not a result of chromosome loss caused by technical factors: a) the chromosome counts were only made in metaphases surrounded by cytoplasm, as an indication of the integrity of the plasma membrane; b) the average chromosome number per cell in the different treatments was constant (data not shown); c) as the frequency of aneuploid cells increased, a correlative increment of multinucleated interphase cells was observed in the cultures treated with the chemicals.When the frequencies of hypoploid cells were compared with the frequency of hyperploidy plus the frequency of multinucleated cells a positive correlation and a good fit with the regression line were found.
The kinetochore-stained micronucleus test is based upon the assumption that a micronucleus containing a kinetochore presumably contains the centromere and the entire chromosome.This micronucleus will segregate with only one of the daughter cells but both have a high probability of being aneuploid (Thompson and Perry, 1988;Eastmond and Tucker, 1989).It must be considered that only malsegregation events (when a chromosome or a chromatid fails to migrate correctly and remains at the metaphase plate) could be detected in the cytokinesis-blocked micronucleus assay by using CREST antibodies.When nondisjunction occurs, two aneuploid daughter cells are formed (one hypoploid and one hyperploid), but neither lagging chromosomes nor micronuclei are induced if the entire chromosome migrates correctly to one pole.Positive-kinetochore micronuclei are formed only when a chromo-

Cadmium chloride
Cadmium sulfate
Figure 7 -Frequencies of kinetochore-negative and kinetochore-positive micronuclei in MRC-5 cells detected by using CREST antibodies.

Potassium dichromate Chromium chloride
Nickel chloride Nickel sulfate some (or chromatid) remains lagged as a consequence of segregation errors (Ford and Correl, 1991;Van Hummelen et al., 1992).Neither lagging chromosomes nor positive kinetochore micronuclei are indicative of nondisjunction (Lynch and Parry, 1993).These considerations are in agreement with findings obtained by chromosome counting and anaphase-telophase analysis.Recently, Natarajan and coworkers (1993) found that hypoploidy was induced more frequently than hyperploidy in cadmium chloridetreated CHED cells.Similar results were reported by Warr and coworkers (1993) in LUC2 cells treated with other aneuploidy-inducing agents.Taking into account these facts, it appears that nondisjunction is not the main mechanism in the generation of aneuploidy and it can be proposed that malsegregation is at least partially involved.

Figure 5 -
Figure 5 -Frequencies of lagging chromosomes induced by different metal salts in anaphase-telophase of CHO cells.