Simultaneous detection of X-chromosome loss and non-disjunction in cytokinesis-blocked human lymphocytes by in situ hybridization with a centromeric DNA probe; implications for the human lymphocyte in vitro micronucleus assay using cytochalasin B.
A methodology for the simultaneous detection of chromosome loss and gain in mammalian cells has been developed which is based upon the analysis of chromosome distribution in daughter nuclei of binucleated human lymphocytes. X-chromosome distribution was followed by in situ hybridization, using a commercial biotinylated DNA probe specific for the centromeric alphoid sequences of human X-chromosome. In order to optimize the experimental protocol for the use of cytokinesis-blocked lymphocytes in aneuploidy assays, the effect of harvest time and cytochalasin B (Cyt B) dosage upon chromosome distribution was investigated. To this end, lymphocyte cultures were treated 44 h after mitogen stimulation with different dosages of Cyt B and collected at 60, 66 and 72 h. High rates of binucleated cells with unbalanced chromosome distribution (two spots in one nucleus and none in the other in male cells; three spots in one nucleus and one in the other in female cells) and abnormal spot number (more than or less than two per male cell or four per female cell) were observed at 66 and 72 h in cultures treated with the lowest Cyt B dose (3 micrograms/ml). In contrast, low frequencies of unbalanced or abnormal binucleated cells were observed at 60 h with both 3 and 6 micrograms/ml Cyt B. These results indicate that binucleated lymphocytes with low background frequencies of malsegregation (required for the analysis of induced aneuploidy), can be obtained by harvesting lymphocyte cultures 60 h after stimulation (16 h after Cyt B block).(ABSTRACT TRUNCATED AT 250 WORDS)
Zijno
A
A
Laboratory of Comparative Toxicology and Ecotoxicology, Istituto Superiore di Sanità, Rome, Italy.
Marcon
F
F
Leopardi
P
P
Crebelli
R
R
D000328:Adult; D000782:Aneuploidy; D002455:Cell Division; D003571:Cytochalasin B; D015342:DNA Probes; D005069:Evaluation Studies as Topic; D005260:Female; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D066298:In Vitro Techniques; D008214:Lymphocytes / Q000187:drug effects* / Q000648:ultrastructure*; D008297:Male; D015162:Micronucleus Tests / Q000379:methods*; D008957:Models, Genetic; D009153:Mutagens / Q000633:toxicity; D013997:Time Factors; D014960:X Chromosome
D015342:DNA Probes; D009153:Mutagens; D003571:Cytochalasin B