Novel biomarkers of genetic damage in humans: use of fluorescence in situ hybridization to detect aneuploidy and micronuclei in exfoliated cells.
Here we describe new techniques that employ fluorescence in situ hybridization (FISH) with centromeric and chromosome-specific DNA probes to detect aneuploidy and micronucleus formation in exfoliated human epithelial cells. Micronuclei arise from chromosome breakage or lagging, which results in chromosome fragments and whole chromosomes being left outside of the main nucleus at telophase. By using a centromeric DNA probe to detect the presence of whole chromosomes in micronuclei and propidium iodide as a general DNA stain in exfoliated nasal, buccal, and bladder cells, we have developed a new fluorescent method that can detect micronuclei and determine the mechanism of formation. The new fluorescent technique gave results that were very similar to those obtained with the standard Feulgen-fast green method. The spontaneous levels of micronuclei in healthy volunteers were buccal, 0.13%, nasal, 0.21%, and urothelial, 0.07%, in approximately 1500 cells per data point. These values are lower than that found in cultured lymphocytes, 0.4-0.8%. Approximately 50% of the exfoliated cell micronuclei contained whole chromosomes (centromeric DNA). FISH was also used to detect aneuploidy in exfoliated buccal and bladder cells. A DNA probe specific for chromosome 9 was used. Average frequencies for 0, 1, 2, 3, and 4 hybridization regions were 4.8, 9.3, 84.8, 0.8, and 0.3% for urothelial cells and 8.2, 9.9, 80.1, 1.4, and 0.4% for buccal cells. The estimated frequency of aneuploidy in exfoliated cells is similar to that found in human lymphocytes analyzed by FISH with the same probe for chromosome 9. These techniques are potentially useful for epidemiological studies of exposed populations and are currently being applied in our laboratory for studies of arsenic- and formaldehyde-exposed populations.
Moore
L E
LE
Department of Biomedical and Environmental Health Sciences, School of Public Health, University of California, Berkeley 94720.
Titenko-Holland
N
N
Quintana
P J
PJ
Smith
M T
MT
D000328:Adult; D000782:Aneuploidy; D002610:Cheek; D002899:Chromosomes, Human, Pair 9; D004848:Epithelium; D005819:Genetic Markers; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008214:Lymphocytes; D015162:Micronucleus Tests
D005819:Genetic Markers