Importance of detecting numerical versus structural chromosome aberrations

The aim is to review briefly the key questions related to aneuploidy/polyploidy and to compare the advantages and disadvantages of the in vitro micronucleus test to assess aneuploidy/polyploidy in vitro. The key questions that will be addressed, concern the importance of polyploidy for health, and cancer in particular, the mechanisms leading to aneuploidy and polyploidy, and the survival of aneuploid/polyploid cells.The recently recognised contribution of numerical chromosome changes to carcinogenesis triggered the development and the implementation of tests specifically aiming at the detection of aneugens in the test battery for mutagenicity and carcinogenicity. The validation of the in vitro micronucleus test in combination with the identification of in vitro divided cells with the cytokinesis-block methodology and of centromeres with pancentromeric or chromosome specific centromeric probes fluorescence in situ hybridisation (FISH) provides a sensitive, easy to score and powerful test which allows assessment of cell proliferation, the discrimination between chromosome breaks, chromosome loss and chromosome non-disjunction and polyploidy. Moreover, classic histology permits the estimation of necrosis and apoptosis on the same slide. The cytokinesis-blocked micronucleus assay could be considered as a multi-endpoint test for genotoxic responses to clastogens/aneugens. This methodology has also shown to be capable of identifying threshold values for the induction of chromosome loss and/or non-disjunction by microtubule inhibitors, data which are particularly important for risk calculations. Similar approaches were conducted in vivo on bone marrow in mice and rats (except for identification of chromosome non-disjunction), and are in development for gut in mice.     

The TX;Y test for the detection of nondisjunction and chromosome breakage in Drosophila melanogaster. II. Results of female exposures

The TX; Y test is a short-term assay for the detection of sex-chromosome nondisjunction and chromosome breakage in Drosophila melanogaster. It has been used in previous work following the exposure of males. In this work, females are exposed. When females are the exposed parent, only chromosome gain can be detected. Positive results for the induction of aneuploidy were obtained following exposures of females to X-rays, 10 degrees C cold shock, and colchicine. No increase in aneuploidy was obtained following exposures of females to DMSO and trifluralin. Comparison with similar work in males reveals no consistent pattern concerning the more appropriate sex to use for aneuploidy testing in Drosophila, as colchicine was found to be positive in females only and DMSO and trifluralin were effective in males only. Further work is necessary to validate the TX; Y test and to understand the relative efficacy of female and male exposures to aneuploidy inducing agents in Drosophila.     

Cytogenetic investigation of chemically-induced aneuploidy in mouse spermatocytes

This paper discusses a test system in which mouse spermatocytes are analyzed for aneuploidy induction after mice are treated with various agents. Included in this report are methods and procedures of the assay, criteria for determination of aneuploidy induction, considerations for dose-response and stage-specific actions of agents that cause aneuploidy, and finally, advantages and disadvantages of this test system.     

Aneuploidy in Drosophila, II. Further validation of the FIX and ZESTE genetic test systems employing female Drosophila melanogaster

Two sensitive genetic systems for the detection of germline aneuploidy employing Drosophila melanogaster females were described in the first paper of this series (Zimmering et al., submitted to Mutation Research). Designated FIX and ZESTE, these systems permit the rapid and efficient detection of exceptional offspring derived from aneuploid female germ cells. The current report presents test results from a survey of 8 additional chemicals that have been analyzed in both systems. The tested chemicals include: acetonitrile, cadmium chloride, carbendazim, dimethylsulfoxide (DMSO), methylmercury(II) chloride, methoxyethyl acetate, propionitrile and water. Excluding the negative control, water, only the fungicide carbendazim failed to induce aneuploidy in either test system. Of the remaining 6 chemicals one, methylmercury(II) chloride, was positive in the FIX system but not in ZESTE, while MEA was positive in ZESTE and borderline in FIX. The results provide little evidence of germ-cell stage specificity of response to the tested chemicals. Comparison of the induced rates of aneuploidy i indicates that these can exhibit departures from simple additivity to the spontaneous rates: induced rates in the ZESTE system are generally higher and more variable than those from FIX. Possible reasons for the difference in responsiveness between FIX and ZESTE flies are discussed as is the question of the classification of those chemicals which induce chromosome loss events but not chromosome gains.     

Detection of chemically induced aneuploidy with plant test systems

The utility of plant test systems for detecting chemically induced aneuploidy was evaluated by using papers published in peer-reviewed journals. A total of 147 papers were provided to the group by the Environmental Mutagen Information Center. Based on the criteria established by the Gene-Tox Committee (Waters and Auletta, 1981), 22 papers were selected for in-depth review. Only those papers listing additional, missing, or lagging chromosomes in the meiotic or mitotic cells were included in this review. Although most plant test systems may be developed to utilize either mitotic or meiotic cells for cytogenetic analysis, only a few have been employed for this purpose. In this review, Allium cepa was found to be the most commonly used test system. Other species used less frequently were Vicia faba, Hordeum vulgare, Sorgham vulgare, and Pennisetum americanum. None of the plant test systems have been sufficiently utilized to warrant judgment for its sensitivity and specificity for detecting induced aneuploidy. A suggested protocol for detecting chromosomal malsegregation in meiotic or mitotic cells is presented. Further development and utilization of plant tissue culture techniques and morphological markers identifiable in the seedling stages is recommended for detecting chemically induced aneuploidy.     

Lack of correlation between mutagen-induced chromosomal univalency and aneuploidy in mouse spermatocytes

Aneuploidy represents the predominant type of chromosomal abnormality found in human newborns with birth defects. The concern that environmental agents may cause aneuploidy in germ cells has prompted development of assay systems for detection of potentially aneuploidy-producing agents. One of the most frequently used methods involves cytogenetic analysis of murine spermatogenic cells at the stages of meiotic metaphases. However, criteria for aneuploidy induction have not been standardized in this test system. Many investigators consider the ability of an agent to induce univalents an appropriate measure of its potential to induce aneuploidy. In the present study, the relationship between univalency and aneuploidy was examined in mouse spermatocytes after various mutagen treatments. 45 Swiss mice were treated with 4 different agents; viz., adriamycin vinblastine sulfate, cytosine arabinoside, and radiation (cobalt 60) and 10 untreated animals served as controls. From each animal, 50–200 MIs were examined for both sex-chromosomal and autosomal univalency (X-Y U and AU), and equal numbers of MIIs were examined for aneuploidy (hyperhaploidy). No significant correlations between univalency (either X-Y U or AU) and aneuploidy were found in the mutagen-treated mice; nor were they found in the untreated animals. These results indicate that induction of univalents by a mutagen is not necessarily predictive of the aneuploidy-inducing ability of his agent.     

Test statistics for detecting aneuploidy and hyperdiploidy

Possible approaches to the analytical evaluation of ploidy patterns are discussed and two specific problems are considered: detection of early onset of aneuploidy and detection of moderate hyperdiploidy. A statistical model for a euploid DNA pattern is formulated in terms of a mixture distribution. A test statistic for detecting deviations from this pattern is defined, and its performance is evaluated for simulated data representing differing degrees of severity of aneuploidy. An analysis based on a discriminant function using order statistics of the sample cumulative distribution functions is proposed for detecting hyperdiploidy. This procedure has the advantage of being relatively distribution-free; its performance is evaluated for simulated data and is compared with that of its classical counterparts. Although the results reported are only preliminary, they indicate that tailor-made statistical analyses can provide early detection of aneuploidy and hyperdiploidy with known and acceptable error rates using clinically reasonable sample sizes.     

Characterization of an in vitro micronucleus assay with Syrian hamster embryo fibroblasts

The use of Syrian hamster embryo cells for assessing genotoxicity provides the unique opportunity to determine 5 different end-points (gene mutations, DNA-strand breaks, aneuploidy, DNA repair (unscheduled DNA synthesis, UDS) and neoplastic transformation) in the one cell system. This approach allows direct comparisons of results produced under identical conditions of dose at target, metabolism and bioavailability. We report here on the characterization of an additional end-point in the same cell system: the formation of micronuclei indicating chromosomal changes induced by chemicals. For a preliminary validation of this new test system we have investigated 14 carcinogens and 3 non-carcinogenic structural analogues in order to evaluate the significance of micronucleus induction for carcinogenic properties. All tested carcinogens induced micronuclei in a dose-dependent manner; all non-carcinogens yielded negative results. Correlations between the formation of micronuclei and the Ames test, induction of UDS, cell transformation and the in vivo bone marrow micronucleus test are demonstrated.     

Aneuploidy in carcinomas may be initiated by the acquisition of a single trisomy

Karyotypic studies of eight endometrioid carcinomas of the endometrium in this laboratory, four colorectal polyps (from this laboratory or reported in the literature), and four early carcinomas of the ovary (from the literature), provide evidence that clonal evolution leading to malignant neoplasms at these sites originates when a cell acquires a single additional chromosome. In different tumors, different chromosomes may be involved in this change from euploidy to aneuploidy. Since the resultant clones of trisomic cells occur at an early stage of tumor development, their presence is only likely to be determined when they are at a location that is accessible for study. As aneuploidy is a virtually constant feature of malignancy, the possibility that the concept of a single trisomy as the initial event in the development of all malignant solid neoplasias should be addressed.     

Assessing Discriminative Performance at External Validation of Clinical Prediction Models

Introduction

External validation studies are essential to study the generalizability of prediction models. Recently a permutation test, focusing on discrimination as quantified by the c-statistic, was proposed to judge whether a prediction model is transportable to a new setting. We aimed to evaluate this test and compare it to previously proposed procedures to judge any changes in c-statistic from development to external validation setting.

Methods

We compared the use of the permutation test to the use of benchmark values of the c-statistic following from a previously proposed framework to judge transportability of a prediction model. In a simulation study we developed a prediction model with logistic regression on a development set and validated them in the validation set. We concentrated on two scenarios: 1) the case-mix was more heterogeneous and predictor effects were weaker in the validation set compared to the development set, and 2) the case-mix was less heterogeneous in the validation set and predictor effects were identical in the validation and development set. Furthermore we illustrated the methods in a case study using 15 datasets of patients suffering from traumatic brain injury.

Results

The permutation test indicated that the validation and development set were homogenous in scenario 1 (in almost all simulated samples) and heterogeneous in scenario 2 (in 17%-39% of simulated samples). Previously proposed benchmark values of the c-statistic and the standard deviation of the linear predictors correctly pointed at the more heterogeneous case-mix in scenario 1 and the less heterogeneous case-mix in scenario 2.

Conclusion

The recently proposed permutation test may provide misleading results when externally validating prediction models in the presence of case-mix differences between the development and validation population. To correctly interpret the c-statistic found at external validation it is crucial to disentangle case-mix differences from incorrect regression coefficients.     

Telomere DNA deficiency is associated with development of human embryonic aneuploidy

Aneuploidy represents the most prevalent form of genetic instability found in human embryos and is the leading genetic cause of miscarriage and developmental delay in newborns. Telomere DNA deficiency is associated with genomic instability in somatic cells and may play a role in development of aneuploidy commonly found in female germ cells and human embryos. To test this hypothesis, we developed a method capable of quantifying telomere DNA in parallel with 24-chromosome aneuploidy screening from the same oocyte or embryo biopsy. Aneuploid human polar bodies possessed significantly less telomere DNA than euploid polar bodies from sibling oocytes (−3.07 fold, P = 0.016). This indicates that oocytes with telomere DNA deficiency are prone to aneuploidy development during meiosis. Aneuploid embryonic cells also possessed significantly less telomere DNA than euploid embryonic cells at the cleavage stage (−2.60 fold, P = 0.002) but not at the blastocyst stage (−1.18 fold, P = 0.340). The lack of a significant difference at the blastocyst stage was found to be due to telomere DNA normalization between the cleavage and blastocyst stage of embryogenesis and not due to developmental arrest of embryos with short telomeres. Heterogeneity in telomere length within oocytes may provide an opportunity to improve the treatment of infertility through telomere-based selection of oocytes and embryos with reproductive competence.     

A genetic assay to detect chromosome gain and/or loss in somatic cells of Drosophila melanogaster

A genetic short-term test is described that allows (i) detection and (ii) quantitative evaluation of aneuploidy induced in somatic cells of Drosophila melanogaster. In this somatic aneuploidy test (SAT) larvae of the genotype z w⁻/ w^+JY are exposed to the test compound. Gain and/or loss of the w^+JY chromosome leads to the formation of aneupliod daughter cells: z w⁻/w^+JY and z w⁻/O, respectively. These cells are fully viable, proliferate and, when they are part of an eye primordium, form a yellow/ /white twin spot on the otherwise red background after metamorphosis. The number of eyes screened, the size and number of spots allow for a quantitative estimate of the frequency of induced aneuploidy. Induced aneuploidy was detected after exposure of larvae to X-rays and to vincristine. The somatic aneuploidy test seems to be a simple, sensitive and fast method to screen environmental chemicals for their ability to induce aneuploidy.     

A survey of EPA/OPP and open literature on selected pesticide chemicals. III. Mutagenicity and carcinogenicity of benomyl and carbendazim

The known aneuploidogens, benomyl and its metabolite, carbendazim (methyl 2-benzimidazole carbamate (MBC)), were selected for the third in a series of ongoing projects with selected pesticides. Mutagenicity and carcinogenicity data submitted to the US Environmental Protection Agency's (US EPA's) Office of Pesticide Programs (OPP) as part of the registration process are examined along with data from the open literature. Mutagenicity and carcinogenicity profiles are developed to provide a complete overview and to determine whether an association can be made between benomyl- and MBC-induced mouse liver tumors and aneuploidy. Since aneuploidogens are considered to indirectly affect DNA, the framework adopted by the Agency for evaluating any mode of action (MOA) for carcinogenesis is applied to the benomyl/MBC data.Both agents displayed consistent, positive results for aneuploidy induction but mostly negative results for gene mutations. Non-linear dose responses were seen both in vitro and in vivo for aneuploidy endpoints. No evidence was found suggesting that an alternative MOA other than aneuploidy may be operative. The data show that by 14 days of benomyl treatment, events associated with liver toxicity appear to set in motion the sequence of actions that leads to neoplasms. Genetic changes (as indicated by spindle impairment leading to missegregation of chromosomes, micronucleus induction and subsequent aneuploidy in bone marrow cells) can commence within 1-24h after dosing, well within the time frame for early key events. Critical steps associated with frank tumor formation in the mouse liver include hepatotoxicity, increased liver weights, cell proliferation, hypertrophy, and other steps involving hepatocellular alteration and eventual progression to neoplasms. The analysis, however, reveals weaknesses in the data base for both agents (i.e. no studies on mouse tubulin binding, no in vivo assays of aneuploidy on the target tissue (liver), and no clear data on cell proliferation relative to dose response and time dependency). The deficiencies in defining the MOA for benomyl/MBC introduce uncertainties into the analysis; consequently, benomyl/MBC induction of aneuploidy cannot be definitively linked to mouse liver carcinogenicity at this time.     

Review of literature on chemical-induced aneuploidy in mammalian male germ cells

80 papers published between 1970 and 1984 were evaluated for results pertaining to chemical-induced aneuploidy in mammalian male germ cells. Diverse assays and end points were represented. The assays considered to involve direct measures of aneuploidy were based upon chromosome counts in premeiotic, meiotic, and embryonic cells, and the male pronucleus, or upon phenotypic expression of X-linked genetic markers. Assays in which indirect measures were interpreted as evidence for aneuploidy included those primarily assessing chiasma frequencies, univalent frequencies, and spermatid/sperm sex chromosome body counts. An initial screening to reject studies with insufficient data and those which did not involve a single chemical test agent led to the elimination of 39 papers from further review. The remaining 41 papers reported effects from 46 different chemicals. These papers were rigorously assessed for adequacy of experimental protocols, relevance of end points as direct measures of aneuploidy, and completeness of data presentation and statistical analysis. Criteria specific to each assay were also considered. 4 chemical tests were considered to provide reliable positive or negative aneuploidy data. Cyclophosphamide and chloral hydrate each caused metaphase II hyperploidy when injected into mice. Very limited analyses of trenimon and isoniazid provided negative results. Test findings for 44 chemicals were viewed as inconclusive. It was concluded that standardization of tests to evaluate chemical-induced aneuploidy in male germ cells and the application of these tests towards increasing the data base are badly needed.     

Absence of selection against aneuploid mouse sperm at fertilization.

Is there selection against aneuploid sperm during spermatogenesis and fertilization? To address this question, we used male mice doubly heterozygous for the Robertsonian (Rb) translocations Rb(6. 16)24Lub and Rb(16.17)7Bnr, which produce high levels of sperm aneuploid for chromosome 16, the mouse counterpart of human chromosome 21. The frequencies of aneuploid male gametes before and after fertilization were compared by analyzing approximately 500 meiosis II spermatocytes and approximately 500 first-cleavage zygotes using fluorescence in situ hybridization with a DNA painting probe mixture containing three biotin-labeled probes specific for chromosomes 8, 16, and 17 plus a digoxigenin-labeled probe specific for chromosome Y. Hyperhaploidy for chromosome 16 occurred in 20.0% of spermatocytes and in 21.8% of zygotes. Hypohaploidy for chromosome 16 occurred in 17.0% and 16.7% of spermatocytes and zygotes, respectively. In addition, there was no preferential association between chromosome 16 aneuploidy and either of the sex chromosomes, nor was there an elevation in aneuploidy for chromosomes not involved in the Rb translocations. These findings provide direct evidence that there is no selection against aneuploid sperm during spermiogenesis, fertilization, and the first cell cycle of zygotic development.     

Preimplantation genetic diagnosis--an overview.

Since the early 1990s, preimplantation genetic diagnosis (PGD) has been expanding in scope and applications. Selection of female embryos to avoid X-linked disease was carried out first by polymerase chain reaction, then by fluorescence in situ hybridization (FISH), and an ever-increasing number of tests for monogenic diseases have been developed. Couples with chromosome rearrangements such as Robertsonian and reciprocal translocations form a large referral group for most PGD centers and present a special challenge, due to the large number of genetically unbalanced embryos generated by meiotic segregation. Early protocols used blastomeres biopsied from cleavage-stage embryos; testing of first and second polar bodies is now a routine alternative, and blastocyst biopsy can also be used. More recently, the technology has been harnessed to provide PGD-AS, or aneuploidy screening. FISH probes specific for chromosomes commonly found to be aneuploid in early pregnancy loss are used to test blastomeres for aneuploidy, with the aim of replacing euploid embryos and increasing pregnancy rates in groups of women who have poor IVF success rates. More recent application of PGD to areas such as HLA typing and social sex selection have stoked public controversy and concern, while provoking interesting ethical debates and keeping PGD firmly in the public eye.     

Selfish centromeres and the wastefulness of human reproduction

Many human embryos die in utero owing to an excess or deficit of chromosomes, a phenomenon known as aneuploidy; this is largely a consequence of nondisjunction during maternal meiosis I. Asymmetries of this division render it vulnerable to selfish centromeres that promote their own transmission, these being thought to somehow underpin aneuploidy. In this essay, I suggest that these vulnerabilities provide only half the solution to the enigma. In mammals, as in utero and postnatal provisioning is continuous, the costs of early death are mitigated. With such reproductive compensation, selection can favour a centromere because it induces lethal aneuploidy: if, when taken towards the polar body, it instead kills the embryo via aneuploidy, it gains. The model is consistent with the observation that reduced dosage of a murine drive suppressor induces aneuploidy and with the fact that high aneuploidy rates in vertebrates are seen exclusively in mammals. I propose further tests of this idea. The wastefulness of human reproduction may be a price we pay for nurturing our offspring.

Why do so many human embryos have the wrong number of chromosomes? So-called ’selfish centromeres’ and the fact that new embryos can be produced may provide an answer.     

Aprotic polar solvents inducing chromosomal malsegregation in yeast interfere with the assembly of porcine brain tubulin in vitro

A number of aprotic solvents which had previously been found to induce mitotic aneuploidy in yeast were tested for their effects on re-assembly of twice recycled tubulin from pig brain. Some of the solvents which were strong aneuploidy-inducing mutagens in yeast slowed down tubulin assembly in vitro at concentrations lower than those required for aneuploidy induction. Ethyl acetate, methyl acetate, diethyl ketone and acetonitrile fell into this category. Other strong aneuploidy-inducing agents like acetone and 2-methoxyethyl acetate accelerated tubulin assembly. Non-genetically active methyl isopropyl ketone and isopropyl acetate both accelerated assembly, whereas methyl n-propyl ketone and n-propyl acetate were weak inducers of aneuploidy and slowed down the rate and extent of assembly. Those chemicals which slowed down the assembly rate also reduced the extent of assembly. Most chemicals which accelerated assembly also led to an increased extent of assembly, with the exception of isopropyl acetate. At the higher concentrations, however, a maximum assembly rate was reached which was followed by a slow decline. Although a perfect correlation between effects on the induction of chromosomal malsegregation and the interference with tubulin assembly in vitro was not seen, the experiments with tubulin were carried out using this class of chemicals because some of them strongly induced mitotic aneuploidy under conditions which suggested tubulin to be the prime target. The lack of a perfect coincidence might be due to species differences between the porcine brain and the yeast spindle tubulin, or the test for aneuploidy induction may have been negative because the concentrations required for an effect on yeast tubulin may be greater than the general lethal toxicity limit. Bearing this reservation in mind, the results suggest that the yeast aneuploidy test has a considerable predictive value for mammalian mutagenicity.     

Dichloroacetonitrile, a by-product of water chlorination, induces aneuploidy in Drosophila

Nitriles have been shown to be potent inducers of aneuploidy in yeast and Drosophila test systems. Haloacetonitriles are by-products of water chlorination that have been shown to be mutagenic and carcinogenic following topical application. In this report we show that dichloroacetonitrile, but not dibromoacetonitrile, is an effective inducer of aneuploidy in oocytes of Drosophila melanogaster. Following inhalation exposure of ZESTE adult females, dichloroacetonitrile (8.6 ppm) induced highly significant increments in the frequencies of sex chromosome loss and gain. Sodium cyanide was also found to be a highly effective inducer of germline aneuploidy, suggesting that cyanide toxicity may contribute to potency of nitriles as inducers of aneuploidy.