Risks of aneuploidy induction from chemical exposure: Twenty years of collaborative research in Europe from basic science to regulatory implications.

Although Theodor Boveri linked abnormal chromosome numbers and disease more than a century ago, an in-depth understanding of the impact of mitotic and meiotic chromosome segregation errors on cell proliferation and diseases is still lacking. This review reflects on the efforts and results of a large European research network that, from the 1980's until 2004, focused on protection against aneuploidy-inducing factors and tackled the following problems: 1) the origin and consequences of chromosome imbalance in somatic and germ cells; 2) aneuploidy as a result of environmental factors; 3) dose-effect relationships; 4) the need for validated assays to identify aneugenic factors and classify them according to their modes of action; 5) the need for reliable, quantitative data suitable for regulating exposure and preventing aneuploidy induction; 6) the need for mechanistic insight into the consequences of aneuploidy for human health. This activity brought together a consortium of experts from basic science and applied genetic toxicology to prepare the basis for defining guidelines and to encourage regulatory activities for the prevention of induced aneuploidy. Major strengths of the EU research programmes on aneuploidy were having a valuable scientific approach based on well-selected compounds and accurate methods that allow the determination of precise dose-effect relationships, reproducibility and inter-laboratory comparisons. The work was conducted by experienced scientists stimulated by a fascination with the complex scientific issues surrounding aneuploidy; a key strength was asking the right questions at the right time. The strength of the data permitted evaluation at the regulatory level. Finally, the entire enterprise benefited from a solid partnership under the lead of an inspired and stimulating coordinator. The research programme elucidated the major modes of action of aneugens, developed scientifically sound assays to assess aneugens in different tissues, and achieved the international validation of relevant assays with the goal of protecting human populations from aneugenic chemicals. The role of aneuploidy in tumorigenesis will require additional research, and the study of effects of exposure to multiple agents should become a priority. It is hoped that these reflections will stimulate the implementation of aneuploidy testing in national and OECD guidelines.

The measurement of induced genetic change in mammalian germ cells.

In vivo methods are described to detect clastogenic and aneugenic effects of chemical agents in male and female germ cells in vivo. The knowledge of stages of germ cell development and their duration for a given test animal is essential for these experiments. Commonly, mice or rats are employed. Structural chromosome aberrations can be analyzed microscopically in mitotic cell divisions of differentiating spermatogonia, zygotes, or early embryos as well as in first meiotic cell divisions of spermatocytes and oocytes. Numerical chromosome aberrations are scorable during second meiotic divisions of spermatocytes and oocytes. The micronucleus test is applicable to early round spermatids and to first cleavage embryos, and as in somatic cells, it assesses structural as well as numerical chromosome aberrations. In contrast to the somatic micronucleus assay, the timing of cell sampling determines whether the micronuclei scored in round spermatids were formed from structural or numerical aberrations, i.e. with short treatment-sampling intervals the micronuclei are formed by exposed meiotic divisions and represent induced non-disjunction. On the -contrary, after longer intervals of 12-14 days micronuclei are formed from induced unstable structural aberrations in differentiating spermatogonia or during the last round of DNA-synthesis in early spermatocytes. Furthermore, labelling with fluorescent DNA-probes can be used to confirm these theoretical expectations. The mouse sperm-FISH assay is totally based on scoring colour spots from individual chromosomes (e.g. X, Y, and 8) hybridized with specific DNA-probes. The most animal demanding assay described here is the dominant lethal test. It is commonly performed with treated male laboratory rodents and allows the determination of the most sensitive developmental stage of spermatogenesis to a particular chemical under test. Theoretically, unstable structural chromosome aberrations in sperm will lead to foetal deaths after fertilization at around the time of implantation in the uterus wall. These can be scored as deciduomata or early dead foetuses in the uterus wall of the females at mid-pregnancy. None of the tests described in this chapter provide data for a quantitative estimate of the genetic risk to progeny from exposed germ cells. The only tests on which such calculations can be based, the heritable translocation assay and the specific locus test, are so animal and time-consuming that they can no more be performed anywhere in the world and thus are not described here.

The chemotherapeutic agents nocodazole and amsacrine cause meiotic delay and non-disjunction in spermatocytes of mice.

Aneuploidy of germ cells contributes to reduced fertility, foetal wastage and genetic defects. The possible risk of aneuploidy induction by the cancer chemotherapeutic drugs amsacrine (AMSA) and nocodazole (NOC) was investigated in male mice. Two molecular cytogenetic approaches were used: (1) the BrdU-incorporation assay to test the altered duration of meiotic divisions and (2) the sperm-FISH assay to determine aneuploidy induction during meiosis by observing hyperhaploid and diploid sperm. Sperm were sampled from the Caudae epididymes of treated and solvent control males. Single intraperitoneal injections with NOC (35 mg/kg) and AMSA (15 mg/kg) caused a meiotic delay of 24h. The timing of sperm sampling for the sperm-FISH assay was adjusted accordingly, i.e. 23 days after treatment. Mice were treated with 18, 35 and 50 mg/kg of NOC, or 5, 10, 15 and 20 mg/kg of AMSA. Significant dose-dependent increases above the concurrent controls in the frequencies of hyperhaploid sperm were found with both agents. Significant increases in the frequencies of diploid sperm were found only with AMSA. These results provide a basis for genetic counselling of patients under AMSA or NOC chemotherapy. During a period of 3-4 months after the end of chemotherapy, they may stand a higher risk of siring chromosomally abnormal offspring.

Gender differences in the induction of chromosomal aberrations and gene mutations in rodent germ cells.

Germ cell mutagenicity testing provides experimental data to quantify genetic risk for exposed human populations. The majority of tests are performed with exposure of males, and female data are relatively rare. The reason for this paucity lies in the differences between male and female germ cell biology. Male germ cells are produced throughout reproductive life and all developmental stages can be ascertained by appropriate breeding schemes. In contrast, the female germ cell pool is limited, meiosis begins during embryogenesis and oocytes are arrested over long periods of time until maturation processes start for small numbers of oocytes during the oestrus cycle in mature females. The literature data are reviewed to point out possible gender differences of germ cells to exogenous agents such as chemicals or ionizing radiation. From the limited information, it can be concluded that male germ cells are more sensitive than female germ cells to the induction of chromosomal aberrations and gene mutations. However, exceptions are described which shed doubt on the extrapolation of experimental data from male rodents to the genetic risk of the human population. Furthermore, the female genome may be more sensitive to mutation induction during peri-conceptional stages compared to the male genome of the zygote. With few exceptions, germ cell experiments have been carried out under high acute exposure to optimize the effects and to compensate for the limited sample size in animal experiments. Human exposure to environmental agents, on the other hand, is usually chronic and involves low doses. Under these conditions, gender differences may become apparent that have not been studied so far. Additionally, data are reviewed that suggest a false impression of safety when responses are negative under high acute exposure of male rodents while a mutational response is induced by low chronic exposure. The classical (morphological) germ cell mutation tests are not performed anymore because they are animal and time consuming. Nevertheless, information is needed to place genetic risk extrapolations on more solid grounds and thereby to prevent an increased genetic burden to future generations. It is pointed out that modern molecular methodologies are available now to experimentally address the open questions.

Gender differences in germ-cell mutagenesis and genetic risk.

Current international classification systems for chemical mutagens are hazard-based rather than aimed at assessing risks quantitatively. In the past, germ-cell tests have been mainly performed with a limited number of somatic cell mutagens, and rarely under conditions aimed at comparing gender-specific differences in susceptibility to mutagen exposures. There are profound differences in the genetic constitution, and in hormonal, structural, and functional aspects of differentiation and control of gametogenesis between the sexes. A critical review of the literature suggests that these differences may have a profound impact on the relative susceptibility, stage of highest sensitivity and the relative risk for the genesis of gene mutation, as well as structural and numerical chromosomal aberrations in male and female germ cells. Transmission of germ-cell mutations to the offspring may also encounter gender-specific influences. Gender differences in susceptibility to chemically derived alterations in imprinting patterns may pose a threat for the health of the offspring and may also be transmitted to future generations. Recent reports on different genetic effects from high acute and from chronic low-dose exposures challenge the validity of conclusions drawn from standard methods of mutagenicity testing. In conclusion, research is urgently needed to identify genetic hazards for a larger range of chemical compounds, including those suspected to disturb proper chromosome segregation. Alterations in epigenetic programming and their health consequences will have to be investigated. More attention should be paid to gender-specific genetic effects. Finally, the database for germ-cell mutagens should be enlarged using molecular methodologies, and genetic epidemiology studies should be performed with these techniques to verify human genetic risk.

Trichlorfon predisposes to aneuploidy and interferes with spindle formation in in vitro maturing mouse oocytes.

The pesticide trichlorfon (TCF) has been implicated in human trisomy 21, and in errors in chromosome segregation at male meiosis II in the mouse. We previously provided evidence that TCF interferes with spindle integrity and cell-cycle control during murine oogenesis. To assess the aneugenic activity of TCF in oogenesis, we presently analysed maturation, spindle assembly, and chromosome constitution in mouse oocytes maturing in vitro in the presence of 50 or 100 microg/ml TCF for 16 h or in pulse-chase experiments. TCF stimulated maturation to meiosis II at 50 microg/ml, but arrested meiosis in some oocytes at 100 microg/ml. TCF at 100 microg/ml was aneugenic causing non-disjunction of homologous chromosomes at meiosis I, a significant increase of the hyperploidy rate at metaphase II, and a significant rise in the numbers of oocytes that contained a 'diploid' set of metaphase II chromosomes (dyads). TCF elevated the rate of precocious chromatid segregation (predivision) at 50 and 100 microg/ml. Pulse-chase experiments with 100 microg/ml TCF present during the first 7 h or the last 9 h of maturation in vitro did not affect meiotic progression and induced intermediate levels of hyperploidy at metaphase II. Exposure to > or =50 microg/ml TCF throughout maturation in vitro induced severe spindle aberrations at metaphase II, and over one-third of the oocytes failed to align all chromosomes at the spindle equator (congression failure). These observations suggest that exposure to high concentrations of TCF induces non-disjunction at meiosis I of oogenesis, while lower doses may preferentially cause errors in chromosome segregation at meiosis II due to disturbances in spindle function, and chromosome congression as well as precocious separation of chromatids prior to anaphase II. The data support evidence from other studies that TCF has to be regarded as a germ cell aneugen.

Functional characterization of the mouse organic-anion-transporting polypeptide 2.

We have isolated and functionally characterized an additional murine member of the organic-anion-transporting polypeptide (Oatp) family of membrane transport proteins from mouse liver. The 3.6 kb cDNA insert contains an open reading frame of 2010 bp coding for a 670 amino acid protein. Based on its amino acid identity of 88% to the rat Oatp2, it is considered the mouse Oatp2 orthologue. Functional expression in Xenopus laevis oocytes demonstrated that mouse Oatp2 transports several general Oatp substrates such as estrone-3-sulfate, dehydroepiandrosterone sulfate (DHEAS), ouabain and BQ-123 but hardly any taurocholate nor rocuronium or deltorphin II. The high-affinity rat Oatp2 substrate digoxin is transported with a rather low affinity with an apparent K(m) value of 5.7 microM. Bromosulfophthalein (BSP), a substrate not transported by the rat Oatp2, is transported very well by mouse Oatp2. Northern blot analysis demonstrated a predominant expression in the liver with additional signals in kidney and brain. Using fluorescence in situ hybridization, the Oatp2 gene (gene symbol Slc21a5) was mapped to chromosome 6G1-G3.