p xiv c.9 - p xiv c.9 molecular characterization of micronuclei in peripheral blood reticulocytes of...

aberrations in bone marrow cells of Swiss mice. Four to six mice (equalnumber of male and female) for each dose were sacrificed at I, 7, 21 and100 days after exposure. Metaphase chromosomes preparations were madeaccording to the conventional method. Two-colour FISH was performed todetect stable as well as unstable chromosome aberrations (i.e. translocationsand dicentrics, respectively). The frequencies of chromosome aberrations(dicentrics, fragments and translocations) at I day after exposure were thehighest and they decreased with time. The frequencies of dicentrics andfragments decreased very fast, while translocations decreased at a slowerrate. The majority of the translocations were reciprocal and terminal types.Radiation sensitivity and the persistence of translocations were found to bedifferent among the chromosomes studied,

Keyword(s): FISH; Mouse chromosome-specific probes; X-rays

Ip XIV c.71 Validation of multl-color FISH methods for the de­tection of chromosome abnormalities In human androdent sperm

Andrew Wyrobek", Paul Van Hummelen', Francesco Marchetti", JackBishop2, XIU Lowe I . / Lawrence Livermore National Laboratory, CA, USA;2NIEHS. RTp, NC. USA

Multi-color sperm FISH technology is a promising tool for investigatingthe mechanisms of induction of chromosomally abnormal sperm and foridentifying paternal risk factors for abnormal pregnancies and birth defects.There have been steady advances in the number of chromosomes detectedsimultaneously in sperm. Currently, we mark four chromosomes (X, Y, 18,and 21), each in different colors, for the detection of sperm aneuploidyand diploidy. In addition, a newly developed multi-color FISH procedureallows for the detection of sperm carrying duplication/deficiency products ofchromosome breakage and/or rearrangements that occurred before or duringmale meiosis. These FISH methods have been validated utilizing semen fromhealthy men who were previously analyzed for sperm cytogenetics by thehamster-egg technique. Using strict sconng criteria, we obtain frequencies ofchromosomally defective human sperm similar to those found by the hamstertechnique, but which are generally lower than those reported by others.

Recently, we developed corollary multi-color FISH methods for detectinganeuploidy in the epididymal sperm ofmouse and rat. The mouse assays werevalidated for aneuploidy using cytogenetic analyses of meiotic metaphaseII, first cleavage, and offspring. These validated FISH assays allow forinterspecies investigations of genetic and environmental risk factors for theproduction of chromosomally abnormal sperm.

[Work was performed under the auspices of the U.S. DOE by the LawrenceLivermore National Laboratory under contract W-7405-ENG-48 with supportfrom NIEHS YOI-ES-10203-o0j

Keyword(s): Sperm aneuploidy; FISH; sperm aberrations

Ip XIV c.sl Study of proliferation of T-Iymphocytes In CFg.patients

Sofie Van Impel, Inge Hauspie', Micheline Klrsch-Volders", Kenny DeMeirleirl. /Free University of Brussels (VUB). Laboratory of Human Ge­netics, Pleinlaan 2. 1050 Brussels, Be/gium; 2Free University of Brussels(VUB). Laboratory of Human Physiology. Laarbeeklaan 1OJ, 1090 Jette,Belgium

Chronic Fatigue Syndrome (CFS) is an illness characterized by generalized,disabling fatigue associated with complaints of a physical and neuropsychi­atric nature. Modest dysfunctions of multiple organ systems, including theimmune, endocrine, and muscular systems, have been identified in cases ofCFS. Many CFS patients show an intolerance to alcoholic beverages. Thisdisorder presents dilemmas for diagnosis, aetiology, pathology and treatment.

Many characteristics of CFS suggest a viral triggered onset, followed byan abnormal immune reaction. Viral infections, among which entero-, retro­and herpesviruses, are frequently mentioned in association with the onset ofCFS. Yet no particular virus has been linked to CFS.

S-XIV: New mutagenicity tests and their evaluation S137

Taking into account the recent findings about the role of viral proteinsin taking over the control of cell-proliferation, our aim was to test the linkbetween viral infection, abnormal cell proliferation and abnormal fatigue.

Human lymphocytes from II patients and II concurrent controls werecultured in the presence and absence of (co-) mutagens (cyclophosphamide,alcohol). The proliferation index was assessed by discrimination betweencells having divided once or more in culture (cytokinesis-block method) andthe chromosomal aberrations with the in vitro micronucleus assay.

The data show an accelerated proliferation of T-Iymphocytes in CFSpatients compared to healthy donors. No differential effect of alcohol orcyclophosphamide was observed.

Keyword(s): CFS; cell-cycle control; in vitro micronucleus assay

Ip XIV e.91 Molecular characterization of micronuclei In periph­eral blood retlculocytes of the mouse by means of anIn situ RT-PCRlPRINS approach

Antonella Russo, Cristiano Salata. Department of Biology, University ofPadooa, Via U Bassi 58/B. 35121 Padooa, Ita/y

We developed a new assay for the molecular characterization of MN inmouse peripheral blood reticulocytes. To distinguish young from mature ery­throcytes, in situ RT-PCR was applied to amplify and label a cDNA sequencefrom the beta-globin transcript. The presence/absence of centromere in MNwas evaluated by a subsequent PRINS cycle allowing the labelling of thepericentromeric DNA sequence. Analysis of blood samples obtained aftercolchicine treatment or from untreated mice demonstrated that: I) Labellingis highly specific since it is obtained only in the presence of primers. 2)The proportion of cells labelled after in situ RT-PCR was consistent withthat observed in preparations supravitally stained with acridine orange. 3)As expected, MN frequencies in labelled or unlabelled cells were different;in particular, in the erythrocytes the MN frequency corresponded to controllevel. 4) C+ MN in reticulocytes were 65%, in very good agreement withthe observed proportion of CREST + MN in bone marrow, while in theerythrocytes only 18% of C+ MN were found. These results indicate thatthe methodology proposed here IS a reliable technique for the molecularcharacterization of MN in mouse peripheral blood reticulocytes; we arepresently testing the possibility to detect also centromeric and telomericsequences. In addition, the data suggest that C+ MN are preferentially lostin the erythrocyte population. Finally, we demonstrated the possibility toidentitY specific RNA sequences into the cells by in situ RT-PCR; thisapproach can be of general interest for the study of cell response to induceddamage.

Keyword(s): In situ RT-PCR; PRINS (Primed In situ DNA Synthesis);peripheral blood reticulocyte MN assay