Detection of whole chromosomes in micronuclei of cytokinesis-blocked human lymphocytes by antikinetochore staining and in situ hybridization

Abstract

The effect of cytochalasin B (Cyt-B; 3 and 6 micrograms/ml; for the last 28 h) on micronuclei (MN) was studied in 72-h purified lymphocyte cultures of three male donors. The frequency of MN was much higher in multinucleate cells (mean 100-204 MN per 1000 cells) than in binucleate cells (mean 8.2-21.0 MN per 1000 cells), tetranucleate cells containing more MN than trinucleate cells. The presence of whole chromosomes in the MN was studied in two separate experiments by immunofluorescence using antikinetochore (CREST) serum and by a centromeric alphoid DNA oligomer probe (in situ hybridization, ISH). In the tri- and tetra-nucleate cells produced by Cyt-B, MN were clearly more often kinetochore-positive (K+) (mean 82-86%) and centromere-positive (C+) (mean 73-83%) than in mononucleate cells of cultures containing no Cyt-B (mean 63% for CREST and 50% for ISH), indicating that most of the excess MN in the multinucleate cells were due to whole chromosomes. The binucleate lymphocytes had about as high prevalence of K+MN (mean 79-84%) as the tri- and tetra-nucleate cells, despite their low MN count. Also in the ISH analysis, the majority of MN in binucleate cells were positively stained (mean 58-62%). If it is assumed that the extra labelled MN are due to Cyt-B, the present findings suggest that Cyt-B could be responsible for approximately 45-57% (CREST data) or approximately 17-23% (ISH data) of MN in binucleate cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytogenetic markers of susceptibility: influence of polymorphic carcinogen-metabolizing enzymes.

“…MN can also be scored in exfoliated cells of buccal, nasal, esophageal, bronchial, or urothelial mucosa (15). The presence of whole chromosomes in MN can be checked by identifying centromeric DNA sequences or kinetochore proteins in the MN (16,17). MN analysis appears to be a good in vitro tool to investigate the effects of clastogens and agents (aneuploidogens) that induce numerical abnormalities of chromosomes.…”

Instabilidade cromossômica induzida por agroquímicos em trabalhadores rurais na região de Passo Fundo, Rio Grande do Sul, Brasil

“…As análises de micronúcleos em linfócitos de sangue periférico de seres humanos e em linhagens de células cultivadas, mostraram ser efetivas para medir danos citogenéticos de agentes com diferentes mecanismos de genotoxicidade (Eastmond & Tucker, 1989;Fenech & Morley, 1985;Norppa et al, 1993b). Várias pesquisas foram realizadas utilizando cultura de linfó-citos de sangue periférico in vitro, para avaliar o efeito de diferentes praguicidas.…”

12th Meeting of the Scientific Group on Methodologies for the Safety Evaluation of Chemicals: Susceptibility to Environmental Hazards

“…Micronuclei can also be scored in exfoliated cells of buccal, nasal, or urothelial mucosa (41). The presence of whole chromosomes in MN can be checked by identifying centromeric DNA sequences (using FISH) or kinetochore proteins in the MN (72,73). In vivo, increased MN frequencies in lymphocytes have been associated with exposure to ionizing radiation, aging, and gender (74).…”