Heritable translocations induced by dermal exposure of male mice to acrylamide.

Acrylamide (AA) is an important industrial chemical used mainly in the production of polymers. It can be absorbed through the skin. AA was shown to be a germ cell clastogen that entails a genetic risk for exposed workers. The genetic risk calculation was based on mouse heritable translocation test data obtained after acute intraperitoneal (ip) exposure (Adler et al., 1994). To obtain a correction factor between ip and dermal exposure, dominant lethal and heritable translocation tests were carried out with dermal exposure of male mice to AA. In the dominant lethal test, male (102/El x C3H/El)F1 mice were exposed by dermal application to the shaved backs of 50 mg/kg AA per day on five consecutive days or to five daily ip injections of 50 mg/kg AA. One day after the end of exposure, the males were mated to untreated females of the same hybrid stock for four days and females were changed every four days for a total of five matings. Dominant lethal effects were found during matings 1-3. For ip exposure, these values were 81.7, 85.7 and 45.4%, respectively; for dermal exposure the corresponding values were 22.1, 30.6 and 16.5%, respectively. In the heritable translocation assay, male C3H/El mice were treated with five dermal exposures of 50 mg/kg AA and mated 1.5-8.5 days after the end of exposure to untreated female 102/El mice. Pregnant females were allowed to come to term and all offspring were raised to maturity. Translocation carriers among the F1 progeny were selected by a sequential fertility testing and cytogenetic analysis including G-band karyotyping and M-FISH. A total of 475 offspring were screened and 41 translocation carriers were identified. The observed translocation frequency after dermal exposure was 8.6% as compared to 21.9% after similar ip exposure (Adler, 1990). The calculated ratio of ip vs. dermal exposure of 0.39 can be applied to obtain a more realistic calculation of genetic risk for dermally exposed workers.

Seven-fluorochrome mouse M-FISH for high-resolution analysis of interchromosomal rearrangements.

The mouse has evolved to be the primary mammalian genetic model organism. Important applications include the modeling of human cancer and cloning experiments. In both settings, a detailed analysis of the mouse genome is essential. Multicolor karyotyping technologies have emerged to be invaluable tools for the identification of mouse chromosomes and for the deciphering of complex rearrangements. With the increasing use of these multicolor technologies resolution limits are critical. However, the traditionally used probe sets, which employ 5 different fluorochromes, have significant limitations. Here, we introduce an improved labeling strategy. Using 7 fluorochromes we increased the sensitivity for the detection of small interchromosomal rearrangements (700 kb or less) to virtually 100%. Our approach should be important to unravel small interchromosomal rearrangements in mouse models for DNA repair defects and chromosomal instability.

Induction of chromosomal aberrations by dacarbazine in somatic and germinal cells of mice.

Dacarbazine (DTIC) is a chemotherapeutic agent that has been successfully applied to treat various types of cancer such as Hodgkin's disease, malignant melanomas, soft tissue sarcomas and advanced neuroblastomas. Many of the patients are of reproductive age and express concern over the genetic risk of the treatment they receive. Therefore, DTIC was tested for its clastogenic effects in somatic and germinal cells of mice. In the bone marrow micronucleus assay DTIC induced micronuclei that increased linearly in the dose range 0-125 mg/kg. In a dominant lethal study DTIC gave a positive response at the dose of 500 mg/kg when conceptions occurred 5-16 days after treatment, corresponding to treated spermatids and early spermatozoa. The induction of heritable translocations was tested in that sensitive period. The observed translocation rate among the F(1) progeny of male mice treated with 500 mg/kg DTIC was 2.13% (P < 00.1 against the historical control of 0.05%). Assuming linearity of the dose-response effect, the point estimate was used to calculate a doubling dose for the induction of heritable translocations of 12 mg/kg. Alternatively, an induced translocation rate of 41.6x10(-6) per unit dose was calculated. Both figures indicate that an increased genetic risk may exist for male patients after chemotherapy with DTIC under the assumption that germ cells of mice and humans are equally sensitive to the clastogenic effects of DTIC. However, the genetic risk is restricted to conceptions within a period of 40 days after the end of chemotherapy, since the sensitive stages of spermatogenesis are spermatids and early spermatozoa.

Facilitating haplotype analysis by fully automated analysis of all chromosomes in human-mouse hybrid cell lines.

Recent evidence suggests that haplotype analysis is essential in recognizing genetic factors involved in the tendency toward a particular disease or pharmacogenetic phenotype, as well as to identify genes involved in multigenic disorders. Because of the increasing need for efficient haplotype tests, a new hybrid system, called conversion technology, was developed. Conversion technology aims at converting the diploid chromosome content into a haploid state so that hybrids contain a single copy of any desired chromosome. A number of mutations can now be identified easily, as they are no longer obscured by the normal sequence present on the other copy of the chromosome. However, the efficient use of this hybrid system depends on a complete analysis of both human and mouse chromosome complements in order to assess the stability of the hybrid cells and to accurately determine their human chromosome content. We describe a new multicolor FISH-based method capable of analyzing both genomes simultaneously in a single hybridization. This new technique should become an instrumental part of inexpensive, reliable haplotype tests.

cDNA cloning, chromosome assignment, and genomic structure of a human gene encoding a novel member of the RBM family.

We have cloned and characterised a novel human gene mapping to chromosome 20q11.2. A partial transcript was initially isolated from a human cDNA library transcribed from RNA of the colon carcinoma cell line T-84. In order to determine the full coding sequence of this novel mRNA, we isolated seven cDNA clones from a human cDNA library transcribed from RNA of the acute monocytic leukemia cell line THP1 by colony hybridization. On Northern blot analysis of four human cell lines, the cDNAs isolated hybridize with an abundantly expressed mRNA species of 3.5 kb. A full-length cDNA transcript of this novel mRNA has an open reading frame of 2,796 bp encoding a protein with a calculated molecular weight of 97 kDa. Two repetitive structural consensus motifs are contained within the deduced protein sequence, namely five distinct RNA binding motifs and two proline rich regions. The derived protein sequence also contains putative transmembrane domains. These structural motifs identify this novel protein as a member of an expanding protein family containing RNA binding motifs (RBM). As seen from recently completed sequence of the genomic area encoding this novel mRNA by the Sanger Centre Human Genome Project, the coding region of this gene, RBM12, is intronless.

Karyotyping mouse chromosomes by multiplex-FISH (M-FISH).

Karyotyping of mouse chromosomes is a skillful art, which is laborious work even for experienced cytogeneticists. With the growing number of mouse models for human diseases, there is an increasing demand for automated mouse karyotyping systems. Here, such a karyotyping system for mouse chromosomes based on the multiplex-fluorescence in-situ hybridization (M-FISH) technology is shown. The system was tested on a number of individual mice with numerical and structural aberrations and its reproducibility and robustness verified. Mouse M-FISH should be a valuable tool for the analysis of chromosomal rearrangements in mice.

Analysis of chromosomal alterations in non-small cell lung cancer by multiplex-FISH, comparative genomic hybridization, and multicolor bar coding.

Lung cancer has a considerable impact on morbidity and mortality throughout the world. Despite extensive effort, no lung cancer-specific cytogenetic changes, such as lineage-specific translocations or inversions, have been described to date. In this study we used multiplex fluorescence in situ hybridization (M-FISH), comparative genomic hybridization, and multicolor bar coding to analyze eight cell lines derived from non-small cell lung cancers. M-FISH did not identify any balanced translocations, which are the dominating feature in leukemias and lymphomas. Instead, M-FISH unraveled an enormous number of numerical and structural aberrations, with each tumor having its own "private" pattern of chromosomal changes. In contrast, comparative genomic hybridization demonstrated similarities between tumors, because each cell line shared some chromosomal segments that were commonly gained or lost. One of these involved chromosome 12. Chromosome 12 specific bar code probe sets were constructed and used to demonstrate that breaks on chromosome 12 occur preferentially within specific bands. With the progressive use of higher resolution approaches, more information can be gained about the chromosomal alterations in cancer.