Flow cytometric measurement of nuclear DNA content variations as a potential in vivo mutagenicity test

Abstract

Mutagen-induced variations of the cellular DNA content have been studied in mouse bone marrow cells in uiuo using high resolution flow cytometric techniques. During the first days after a single injection of the chemical mutagen cyclophosphamide an increased coefficient of variation in the GI peak of the flow histograms was observed. The magnitude and duration of this effect were dose-dependent. The reproducibility of the measurements was high, indicating that individual variability between animals and instrumental dispersion is small. The results demonstrate that on the basis of the flow cytometric measurement of cellular DNA content, a short-term in uiuo test for mutagenicity can be established which is much faster than conventional cytogenetic methods.Key terms: Mouse bone marrow, chemical mutagens, flow cytometry, DNA content variations, DNA staining method, mutagenicity testing.The need to develop rapid, reproducible and reliable methods for the detection of mutagens has been increasingly felt in recent years because of enhancement in environmental polstructural chromosome aberrations and dysfunction of the mitotic apparatus can cause an unequal distribution of the DNA in the daughter cells. It has been shown that by using high resolution flow cytometric techniques an increased dispersion Of the DNA 'Ontent produced in this way can be measured in X-irradiated Chinese hamster cells zn uztro access to food and drinking water. Cyclophosphamide (analytical grade, (Heidelberg, Germany) No. 17681) was administered by a single ip injection (0.56-0.7 ml of 0.075-0.6% solution in physiological saline). Control animals received an equivalent volume of saline. After cells, harvested by flushing out the femur with 5 ml of Ca-and Mgfree phosphate buffered saline containing 0.5 mM EDTA disodium salt, were fixed in 70% ethanol and stored under refrigeration.For accurate and reproducible measurements of DNA content a procedure based on a method described by Pinaev et al, 15) was developed. Fixed cells were pelleted by centrifugation at 200 X g for 10 min, resuspended at a concentration of 2-to 6 x 10" cells in 1 ml lutants. In a proliferating population, mutagen-induced different intervals of time, mice were sacrificed and bone m m o w and mouse bone marrow cells in uiuo (4). Hopefully, the flow cytometric measurement of the dispersion in the cellular DNA content induced by interactions with environmental agents can provide a powerful tool for cytogenetic investigations and mutagenicity testing. In the present work, time dependence, dose-effect relationship and reproducibility of DNA dispersion induced by the chemical mutagen cyclophosphamide were studied in mice. Materials and MethodsMale NMRI mice weighing 28-35 g were used in these experiments. The animals were maintained in standard plastic boxes and had free of 0.2 M citric acid at pH 1.8 with 0.5% Tween 20, and incubated at room temperature for 20 min. Subsequently, the pH was raised to 7.4 and the cells were stained by addition of 9 ml of disodium hydrogen phosphate...

Heavy Metal Pollution, Selection, and Genome Size: The Species of the Žerjav Study Revisited with Flow Cytometry

“…Following the chopping method of Galbraith et al [21], about 25 mg fresh leaves (dry fruits in case of Euphorbia amygdaloides) from each plant sample were cochopped in Otto's buffer I (Otto et al [22]) together with Pisum sativum (1C = 4.42 pg DNA; Greilhuber and Ebert [23]), Zea mays (1C = 2.73 pg DNA; Doležel et al [24]), Secale cereale (1C = 7.79 pg DNA; Doležel et al [24]), or Solanum pseudocapsicum (1C = 1.29 pg DNA; Temsch et al [25]) as the internal standard organisms. The resulting isolated nuclei were filtered through a 30 µm mesh and subsequently incubated with RNase A for 30 minutes at 37…”

“…The nuclei were then stained in Otto's buffer II (Otto et al [22]) containing the fluorochrome propidium iodide (PI, 50 µg/ml) for 1 hour or overnight in the refrigerator. For measurement, a CyFlow ML flow cytometer or a PAII (both Partec, Muenster, Germany) was used.…”

Protocol to cryopreserve and isolate nuclei from adipose tissue without dimethyl sulfoxide

“…Propidium iodide, the fluorochrome used in flow cytometry analysis, intercalates into double-stranded DNA without base-dependent bias, making it suitable for estimating DNA amount (Dolezel et al, 2007). High heterogeneity in DNA amount suggests a lower quality of adipocyte nuclei because it represents higher dispersion in the DNA amount, which may be because of structural and numerical chromosomal aberrations as well as the formation of micronuclei (Otto et al, 1981). It was previously shown that treatment with a mutagen drug increased the CV in a dose-dependent manner, as measured by flow cytometry (Otto et al, 1981).…”

Flow‐cytometric analysis of the effects of triethylenemelamine on somatic and testicular tissues of the rat

“…Recently, flow cytometry was shown to be a highly sensitive procedure to detect the effects of mutagens on DNA content both in vivo (15) and in vitro using intact nuclei (7) and isolated chromosomes (14). The advantages of flow cytometry over other cytological techniques applicable to mammalian tissues include the rapidity with which the test can be performed, the large number of cells that can be counted, and the potential diversity of tissues that can be analysed (1,5).…”