Mutagenesis vol. 18 no. 5 pp. 471-475,
September 2003
© 2003 UK Environmental Mutagen Society/Oxford University Press
Detection by dual color fluorescence in situ hybridization of in vivo chromosome damage in rat hepatocyte nuclei
Laboratory of Genetic Toxicology, The Institute of Environmental Toxicology, 4321 Uchimoriya-machi, Mitsukaido-shi, Ibaraki 303–0043, Japan
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Abstract |
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Our previous study using a multicolor fluorescence in situ hybridization (FISH) technique revealed that region-specific DNA probes for rat chromosome 1 enabled the detection of structural chromosome damage in rat interphase nuclei from peripheral blood and bone marrow cells. In the present study, this FISH technique was modified for application to a non-hematopoietic organ (liver) and the usefulness of the system was tested using diethylnitrosamine (DEN) as a model hepatocarcinogen. Male Sprague–Dawley rats were orally treated once with DEN at 200 mg/kg. Their livers were removed at 4, 7 or 14 days after treatment and homogenized with a tissue grinder to isolate hepatocyte nuclei. The nucleus suspension was fixed in methanol:acetic acid and air dried. Dual color FISH with two probes, one labeled with tetramethylrhodamine and one with digoxigenin, demonstrated that the maximum increase in the frequency of nuclei with spatially abnormal signals was observed 7 days after treatment. A dose–response relationship for induction of abnormal nuclei was observed. This improved dual color FISH system is potentially valuable for assessing in vivo clastogenicity in all rat organs.
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Introduction |
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The liver is the most active drug metabolizing and detoxifying organ (Leffert et al., 1982
). An important predictor of hepatocarcinogenicity of a chemical is its ability to induce in vivo genetic damage in the mammalian liver. Damage to liver DNA can be tested for in the unscheduled DNA synthesis assay (Ashby et al., 1985) and the alkaline single cell gel electrophoresis assay (Sasaki et al., 1997). Gene mutational damage in the liver can be assessed in the transgenic animal mutation assay (Gossen et al., 1989; Kohler et al., 1990; Nohmi et al., 1996). In vivo cytogenetic damage can be tested for in a micronucleus assay that utilizes freshly isolated hepatocytes from livers of hepatectomized rats (Tates et al., 1980), but that is a difficult, time-consuming and complicated procedure.
Fluorescence in situ hybridization (FISH) techniques can be used with a variety of DNA probes and labeling strategies in metaphase or interphase cells (Pinkel et al., 1986, 1988; Cremer et al., 1988; Kuo et al., 1991). We have developed a FISH system that uses region-specific DNA probes to detect chromosome damage in rat interphase nuclei of hematopoietic tissue (Matsumoto and Tucker, 1998). In this system, a sequence of regions along chromosome 1 is labeled by multicolor FISH and chromosome damage is determined by signal spatial relationships in the targeted chromosome regions. With some modifications this system, theoretically, should be able to detect chromosome damage in rat liver.
The present paper describes two improvements of the conventional FISH system, physical isolation of nuclei from cells and the use of DNA probes labeled with tetramethylrhodamine, and demonstrates the detection of structural chromosome damage in hepatocyte nuclei obtained from diethylnitrosamine (DEN)-treated rats.
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Materials and methods |
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DNA probes
Two different region-specific DNA probes for rat chromosome 1 were generated by chromosome microdissection (Lüdecke et al., 1989
). Single copies of the upper (1q12
q21) and middle (1q31q35) regions of the long arm were dissected from normal metaphase chromosomes prepared from peripheral blood cultures. The microdissection was performed with a glass microneedle controlled by a micromanipulator (PB-10; Narishige, Tokyo, Japan) attached to an inverted microscope (Diaphot; Nikon, Tokyo, Japan). Dissected DNA was amplified by a modified procedure based on the methods of Bohlander et al. (1992) and Guan et al. (1993). One copy of the dissected fragment was transferred to a 5 µl collection drop [containing 40 mM Tris–HCl, pH 7.5, 50 mM NaCl, 20 mM MgCl2, 200 µM each dNTP, 1 U topoisomerase I (Gibco BRL, Life Technologies Inc., Gaithersburg, MD) and 1 µM DOP primer (5'-GATCAAGCTTNNNNNNATGTGG-3')] in a 200 µl PCR tube. The DOP primer was synthesized by Amersham Pharmacia Biotech Inc. (Tokyo, Japan). The drop was incubated at 37°C for 30 min. Topoisomerase I was inactivated by heating at 96°C for 10 min. The chromosome DNA was preamplified for eight cycles as follows: denaturation at 94°C for 1 min, annealing at 30°C for 2 min and extension at 37°C for 2 min. For each cycle, 
0.3 U T7 DNA polymerase (Sequenase version 2.0; Amersham Life Science, Arlington Heights, IL) were added. Following preamplification, 50 µl of the PCR reaction mixture [10 mM Tris–HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl2, 200 µM each dNTP, 1 µM DOP primer and 2.5 U Taq DNA polymerase (Perkin Elmer, Foster City, CA)] was added to the tube. A conventional PCR reaction was performed with heating to 95°C for 5 min followed by 35 cycles at 94°C, 56°C and 72°C for 30 s each. From this ‘first generation’ material, a 2 µl aliquot of the DNA products was added to a second PCR reaction mixture [10 mM Tris–HCl, pH 8.3, 50 mM KCl, 2 mM MgCl2, 200 µM each dNTP, 2 µM DOP primer and 5 U Stoffel fragments of Taq DNA polymerase (Perkin Elmer)] and amplified for 30 cycles at 94°C, 56°C and 72°C for 30 s each. For labeling with tetramethylrhodamine, a 2 µl of aliquot of this ‘second generation’ material was added to a third PCR reaction mixture [10 mM Tris–HCl, pH 8.3, 50 mM KCl, 4 mM MgCl2, 200 µM dATP, dGTP and dCTP, 180 µM dTTP, 20 µM tetramethylrhodamine-5-dUTP (Roche Diagnostics, Mannheim, Germany), 2 µM DOP primer and 5 U Stoffel fragments] and amplified for 25 cycles at 95°C for 30 s, 56°C for 30 s and 72°C for 40 s. For digoxigenin labeling, the amount of dTTP was reduced to 160 µM, and 20 µM tetramethylrhodamine-5-dUTP was replaced with 40 µM digoxigenin-11-dUTP (Roche Diagnostics) in the third PCR reaction mixture.
Tetramethylrhodamine-labeled probes were used for the upper chromosomal region and digoxigenin-labeled probes were for the middle region. Probe size and quantity were determined in 2% agarose gels in 1x TAE.
DEN treatment
DEN (CAS no. 55-18-5; Tokyo Kasei Kogyo Ltd, Tokyo, Japan) was dissolved in physiological saline. Six-week-old male Sprague–Dawley rats were obtained from Charles River Breeding Laboratories (Tokyo, Japan) and acclimatized in the animal facility with a controlled environment (room temperature 22 ± 3°C, relative humidity 50 ± 20%). The animals were used 1 week later and had a mean body weight of 245 g. To determine the time course of clastogenesis, the rats were given DEN at 200 mg/kg body wt (maximum tolerance dose) by a single i.p. injection. Two rats per sampling time were anesthetized with diethyl ether at 0, 4, 7 or 14 days after treatment. To determine the dose–response relationship, two rats were given DEN at 0, 50, 100 or 200 mg/kg body wt by a single i.p. injection. After 7 days all rats were anesthetized for sampling.
Preparation of liver nuclei
The peritoneal cavity of the rats was opened, the portal vein cannulated, the post-caval vein cut and the liver perfused with Hanks’ balanced salt solution, pH 7.4, at 37°C. Approximately 150 mm3 of liver tissue was removed and minced into pieces of 1 mm3. The mince was suspended in cold buffer (0.25 M sucrose, 25 mM HEPES, 0.1 mM Na2EDTA, pH 7.4) and homogenized gently in a Dounce tissue grinder (Wheaton) with a loose fitting pestle. The homogenate was filtered through a cell strainer (40 µm mesh; Becton Dickinson, Franklin Lakes, NJ) and the nucleus suspension was fixed in 3:1 methanol:acetic acid and dropped onto glass slides. After air drying, the slides were stored at –20°C in a N2 atmosphere in the presence of a desiccant.
Dual color FISH
To label two regions of chromosome 1 in different colors, dual color FISH was performed according to the procedure of Pinkel et al. (1988), with modifications (Breneman et al., 1995; Matsumoto and Tucker, 1998). Slide preparations of liver nuclei were immersed in phosphate-buffered saline at 37°C for 15 min and then dehydrated in a 70%, 85%, 100% ethanol series. They were then denatured in 70% formamide/2x SSC at 70°C for 5 min, followed by another dehydration series. The probe cocktail consisted of 1.5 µl tetramethylrhodamine-labeled probe, 1.5 µl digoxigenin-labeled probe, 3 µl sonicated herring sperm DNA (1 mg/ml) and 3 µl rat whole genomic DNA (1 mg/ml). The cocktail was micro-vacuumed down to a volume of 4.5 µl. Then 10.5 µl hybridization mix was added to the cocktail (50% formamide/2x SSC/10% dextran sulfate). The probe mixture was denatured at 70°C for 5 min, applied to the slides, covered with a 22 x 22 mm glass coverslip, sealed with rubber cement and hybridized in a 100% humidity incubator at 37°C for 2–3 days.
Unbound probe was removed with three 5 min washes in 50% formamide/2x SSC, pH 7.0 (45°C), followed by a single 5 min wash in 2x SSC (45°C), a 5 min wash in 2x SSC with 0.1% Triton X-100 (45°C) and a 5 min wash in PT buffer (0.1 M sodium phosphate, pH 8.0, 0.1% Triton X-100) at room temperature. The slides were treated with PTM (2% powdered non-fat milk in PT buffer) at room temperature for 5 min and antibodies were applied. The probe labeled with tetramethylrhodamine was detected with a rabbit anti-tetramethylrhodamine antibody (1:1000 in PTM; Molecular Probes Inc., Eugene, OR) and visualized with Alexa Fluor 594-conjugated chicken anti-rabbit IgG (H + L) (1:1000 in PTM; Molecular Probes). The digoxigenin-labeled probe was detected by a sequential application of fluorescein-conjugated sheep anti-digoxigenin, Fab fragments (1:100 in PTM; Roche Diagnostics), Alexa Fluor 488-conjugated goat anti-fluorescein (1:50 in PTM; Molecular Probes) and Alexa Fluor 488-conjugated donkey anti-goat IgG (H + L) (1:133 in PTM; Molecular Probes). The slides were mounted in p-phenylenediamine antifade solution.
A preliminary check for the above dual color FISH technique was performed on the metaphase chromosomes of rat hepatoma HTC cells, which are pseudo-triploid. HTC cells were cultured in Eagle’s minimum essential medium supplemented with 0.1 mM non-essential amino acids and 10% fetal bovine serum (Gibco BRL). Colcemid was added to the culture 2 h prior to harvest. The cells were treated with 0.075 M KCl, fixed in 3:1 methanol:acetic acid and air dried on glass slides.
Nucleus scoring
All slides were coded before scoring and observed with a fluorescence microscope (BX-50; Olympus, Tokyo, Japan) at 400x magnification. A dual bandpass filter for FITC and Texas Red was used to simultaneously visualize the green (Alexa Fluor 488) and red (Alexa Fluor 594) fluorescent signals. The fluorescence spectra of the Alexa Fluor 488 and 594 dyes are almost identical to those of FITC and Texas Red, respectively, i.e. the absorption/emission maximum is 495/518 nm for Alexa Fluor 488 and 591/618 nm for Alexa Fluor 594. Photographs were taken with a CCD camera (CoolSNAP-HQ; Nippon Roper, Chiba, Japan) and no enhancement of brightness or contrast of digital images was done.
To be eligible for inclusion in the analysis, each nucleus had to have two to four signals for each of the two hybridization colors, because the absence of signals could arise not only from chromosome deletions, but also from failure of hybridization or overlapping signals. Structural chromosome abnormalities were defined empirically on the basis of physical distance of dual color signals as viewed in two dimensions. Nuclei were judged to be abnormal if the distance between two adjacent color signals was greater than the diameter of the signals. Over 2000 nuclei were scored for each rat.
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Results |
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Dual color FISH of interphase nuclei
The conditions of the dual color FISH were optimized using the metaphase chromosomes of rat hepatoma HTC cells (Figure 1). When two region-specific DNA probes were hybridized to the rat hepatocyte interphase nuclei by the optimized dual color FISH, the corresponding chromatin domains exhibited red and green fluorescent signals (Figure 2). Background fluorescence was negligible and the signals were bright enough to be readily identifiable with a conventional fluorescence microscope. Red and green signals were similar in size and brightness. These two signals appeared closer than the actual distance between 1q21 and 1q31 (
36 Mb or 1.3% of the whole genome) (Baylor College of Medicine, 2003; National Center for Biotechnology Information, 2003), due to the fact that the chromatin loops were slightly dispersed in interphase. Overlapping signals appeared yellow. The morphology of FISH signals in the interphase nucleus, which depends on cell cycle stage, was mostly uniform, probably because mitotic activity in the liver was so low (Carriere, 1969) that most of the hepatocytes could be considered to be in the G1 stage.
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Approximately 40% of nuclei from the control rats had multiple pairs of dual color signals, indicating polyploidy. The Sprague–Dawley rats used in this study have a lower incidence of polyploid hepatocytes than Wistar or Fischer rats, where 70–80% of hepatocytes are polyploid (Schwarze et al., 1991
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Detection of abnormal nuclei induced by DEN
Abnormal diploid, triploid and tetraploid nuclei with separated dual color signals were clearly distinguishable from normal nuclei (Figure 3). The mean frequency of abnormal nuclei immediately after treatment with DEN was 0.23%. At 4 days after treatment the frequency had almost doubled (Figure 4). After 7 days, the frequency peaked at 1.15%, while at 14 days it was decreased but was still significantly higher than the control level. There was no change in the incidence of polyploid nuclei after DEN treatment (data not shown). The mean frequency of abnormal nuclei in the 0 mg/kg group was 0.15% 7 days after treatment and increased dose-dependently; the frequency of abnormal nuclei in the 200 mg/kg group was 1.39% (Figure 5).
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Discussion |
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Bright, clear signals are important for accurate scoring in FISH analyses. Interphase FISH, however, can be heavily influenced by cellular phenomena and hybridization artifacts, which makes performance and interpretation of results difficult, especially with single copy DNA probes (Eastmond et al., 1995
). To ensure that fluorescent signals were clear in this study, we used two improvements, isolation of cell nuclei and tetramethylrhodamine labeling of DNA probes. The isolation of the nuclei removed all traces of cytoplasm, which always affects the quality of FISH. Tetramethylrhodamine labeling of the probe enhanced the fluorescent signal. Unlike biotin, which is an endogenous ligand in cells, use of dye-based haptens usually permits background-free staining of cells (Harmer and Samuel, 1989; Wiegant et al., 1996), and non-specific background fluorescence was remarkably reduced in the present FISH technique.
In this system, structural chromosome aberrations cannot be recognized until chromosomes move and the red and green signals are consequently separated in nuclei with chromosome damage. Each chromosome occupies its own micrometer scale territory during interphase (Cremer and Cremer, 2001). Many researchers have examined whether the chromosome maintains that territory. Large chromosomal movements are observed during interphase of Drosophila (Csink and Henikoff, 1998) and neuronal cells from epileptics (Borden and Manuelidis, 1988). On the other hand, chromosome territories show only small movements during several hours of interphase in cultured human cells (Edelmann et al., 2001). More recent studies using HeLa cells with GFP-tagged chromatin demonstrate that the arrangements of the chromosome territories are stably maintained from G1 to late G2, whereas major changes in chromosomal positions occur during mitosis (Walter et al., 2003). In the case of rat hepatocytes, the main period of chromosomal movements is unknown at present. Further studies on the dynamics of chromosome territories in hepatocytes are necessary to understand the mechanisms of formation of a nucleus with separated signals.
The frequency of abnormal hepatocyte nuclei was decreased 14 days after DEN treatment. The reason for the decrease is not clear, but it may be that the severely damaged hepatocytes underwent programmed cell death, since serious genetic damage leads to activation of apoptosis, involving genes such as p53 (Schulte-Hermann et al., 1999). It is likely, however, that not all the damaged cells died out, because the clastogenic effects of DEN can remain constant for 56 days (Tates et al., 1983) or up to 1 year (Herens et al., 1995). The alkylation of rat liver DNA by DEN results in the formation of ethylated bases and phosphotriesters, with the phosphotriester lesions being much more persistent than the ethylated bases (den Engelse and Philippus, 1977).
There are several disadvantages to this system (Matsumoto and Tucker, 1998). One is the possibility of false negatives and/or false positives because, for example, air drying converts the nuclei into two-dimensional structures. Fixation with paraformaldehyde might maintain the three-dimensional architecture and measuring the signal-to-signal physical distances in each nucleus with a confocal laser scanning microscopy might enable three-dimensional reconstruction (Solovei et al., 2002), though that would require advanced technology and considerable effort.
In conclusion, dual color FISH using region-specific DNA probes detected chromosome damage in the liver of rats that had been treated with DEN. This suggests that the system could serve as an in vivo testing system for substances that, because they do not reach hematopoietic tissues, show negative results in conventional micronucleus tests. This system has a potential for assessing in vivo clastogenicity in all rat organs. Thus the most valuable advantage of this system is that it can detect cytogenetic damage in any organ, even if metaphase cells are difficult to observe. Moreover, this system may be useful in predicting organ-specific carcinogenicity of test substances.
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Acknowledgements |
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The author would like to thank Drs M.Hayashi and T.Ohta for their valuable suggestions throughout this study. This work was performed under the auspices of the Ministry of Agriculture, Forestry and Fisheries and supported by a grant from the Tutikawa Memorial Fund for Study in Mammalian Mutagenicity.
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1Tel: +81 297 27 4539; Fax: +81 297 27 4518; Email: matsumoto{at}iet.or.jp
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References |
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Received on April 15, 2003; accepted on July 10, 2003.
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