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Aneugen-induced micronuclei (MN) in human lymphocytes may be discerned using image analysis techniques when
P Van Hummelen1, M Nüsse, P Castelain
1Laboratorium voor Antropogenetica, Free University of Brussels, Belgium.
Abstract:
We show that for the in vitro cytochalasin-B human lymphocyte micronucleus (MN) test, the quantification of the DNA content of MN and the difference in DNA content between the two macronuclei in the binucleate cells without MN, as measured by image analysis, gives a first estimation of the aneugenic potential of a test compound. Cultures of isolated human lymphocytes were exposed either to gamma-rays as a clastogen or to carbendazim (MBC) as an aneugen. The lymphocytes were stained with Feulgen stain and the MN were analyzed for DNA content with a Magiscan 2A image analyzer. The mean DNA content of MN induced by MBC were statistically higher than gamma-irradiation-induced MN. It was demonstrated that in culture the lymphocytes, as well as the MN, are in different stages of the cell cycle, but this will not affect the discriminating power of the MN DNA content when only G1 cells are considered, or when DNA content of the MN is expressed relative to the total genome. The identification of G1 and G2 cell populations from image analysis data was performed by extrapolation of DNA content data from G1- and G2-sorted lymphocytes with a FacStar plus flow sorter. It was demonstrated that in MBC-treated cells the DNA rearrangement between the macronuclei in binucleates without MN was on the average higher than in gamma-irradiated and untreated cells, which points to aneugenic effects of MBC without the formation of MN. In contrast to DNA content measurements, the area of the MN is not a reliable measure for discriminating clastogens from aneugens.
Insights
Measuring DNA content in micronuclei (MN) and binucleate cells helps estimate a compound
Area of Science:
- Genotoxicity testing
- In vitro toxicology
- Cell biology
Background:
- The human lymphocyte micronucleus (MN) test is a standard method for assessing genotoxicity.
- Distinguishing between clastogenic (DNA breakage) and aneugenic (chromosome loss/gain) effects is crucial.
- Existing methods for aneugenicity assessment can be complex and time-consuming.
Purpose of the Study:
- To evaluate the potential of measuring DNA content in micronuclei (MN) and binucleate cells for assessing aneugenic potential.
- To compare the DNA content of MN induced by an aneugen (carbendazim) versus a clastogen (gamma-rays).
- To investigate DNA content differences in binucleate cells without MN as an indicator of aneugenicity.
Main Methods:
- Human lymphocytes were cultured and treated with gamma-rays (clastogen) or carbendazim (MBC, aneugen).
- Cells were stained with Feulgen stain, and DNA content of MN and binucleate cells was analyzed using image analysis (Magiscan 2A).
- Cell cycle populations (G1 and G2) were identified using flow cytometry (FacStar plus) for data extrapolation.
Main Results:
- The mean DNA content of MBC-induced MN was significantly higher than that of gamma-irradiation-induced MN.
- Carbendazim-treated cells showed greater DNA rearrangement between macronuclei in binucleates without MN compared to controls and gamma-irradiated cells.
- MN DNA content is a reliable discriminator between clastogens and aneugens, whereas MN area is not.
Conclusions:
- Quantification of DNA content in MN and binucleate cells provides a reliable initial estimation of a compound's aneugenic potential.
- This method can distinguish aneugenic effects, even in the absence of visible micronuclei.
- DNA content measurement is superior to MN area for differentiating clastogenic from aneugenic agents in the MN test.