Quantitative image analysis and flow cytometry can improve early detection of cancer.
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DNA↗
Biomedical subjects
M Kirsch-Volders
Publications and source records attributed to M Kirsch-Volders.
Mutagenicity of ethyleneimine.
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Animals↗
An improved method for the 'in vitro' micronucleus test using human lymphocytes.
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Humans↗
Cell population kinetics and ploidy rate of early focal lesions during hepatocarcinogenesis in the rat.
We have studied the changes in cell population kinetics and DNA-content of cycling parenchymal cells during the very early steps of rat hepatocarcinogenesis in Faber's protocol. Adult rats were initiated by a single dose of diethylnitrosamine (DENA, 200 mg kg-1), followed 2 weeks later by a 2-week diet of 0.03% 2-acetylaminofluorene (2-AAF) as selection phase. In the middle of selection time, a single necrogenic dose of carbon tetrachloride (CCl4, 2 ml kg-1) was administered by gavage. Twenty four hours thereafter, radiolabelled thymidine (3H-TdR, 1.5 microCi g-1) was given by repeated injections during 24 h. An emergence of small, pyroninophilic ('tigroid') foci was observed at the second, fifth and eighth days after the proliferative stimulus. The focal putative precancerous cells presented a significant higher labelling index (L1) than the non-affected parenchymal cells for all exposure times. However, the labelling intensity decreased from the second to the eighth day after CCl4, suggesting a dilution of the radiolabelled DNA by repeated divisions within the foci. The nuclei of the same foci were analysed for DNA-content by feulgen microdensitometry on neighbouring sections. A gradual reduction of nuclear DNA-content was observed in 66% of the foci at the fifth day and in 100% of foci at the eight day, as compared to surrounding tissue and untreated animals, where labelling and DNA-content remain in the same ratio.
2-Acetylaminofluorene↗
The micronucleus test in Xenopus: a new and simple 'in vivo' technique for detection of mutagens in fresh water.
The aim of this study was to extend the micronucleus test to Xenopus laevis to screen water for potential mutagens. Up to now, the most commonly used methods for screening fresh water are the bacterial tests (Ames tests). Ames tests are well validated, but are merely in vitro bacterial assays which can only detect point mutations whereas the micronucleus test is an in vivo assay using a vertebrate species, and thus is able to detect both chromosome and genome mutations. This study was designed to optimize the micronucleus test for Xenopus. Three different variables were investigated: (i) the effect of temperature; (ii) the effect of the stage of larval development; and (iii) the effect of varying or continuous exposure concentrations. In addition, a dose-response curve was established for eight concentrations of benzo[alpha]pyrene. The results indicate that the micronucleus test with Xenopus can be used to monitor water pollution.
Animals↗
The mouse bone marrow micronucleus assay can be used to distinguish aneugens from clastogens.
The occurrence of aneuploidy can be measured in several assays. However, none of them have been sufficiently validated. The bone marrow micronucleus test may be considered as a method for aneuploidy detection. In this work, micronuclei were induced by two aneugens, vincristine sulfate (0.1 mg/kg) and nocodazole (80 mg/kg), and two clastogens ethylmethanesulfonate (EMS) (300 mg/kg) and cyclophosphamide (60 mg/kg). Three criteria have been examined in order to distinguish micronuclei induced by aneugens and clastogens: the area of the micronuclei, the percentage of micronuclei with C-band-positive material and the DNA content of the micronuclei. C-band-positive micronuclei were found in 47% of the micronuclei for vincristine sulfate, 58% for nocodazole, 17% for EMS and 20% for cyclophosphamide. Areas of micronuclei showed a significant difference when induced by aneugens or by clastogens. Finally, the DNA content of micronuclei also showed a totally different distribution pattern when comparing the aneugen vincristine sulfate with the clastogen EMS. The three methods analysed could thus all make a difference between micronuclei induced by aneugens and those induced by clastogens.
Aneuploidy↗
The influence of phenobarbital and butylated hydroxytoluene on the ploidy rate in rat hepatocarcinogenesis.
The effect of the 'promoters' phenobarbital (PB) and butylated hydroxytoluene (BHT) on the ploidy changes during hepatocarcinogenesis in rats was compared in a densitometric analysis of Feulgen-stained nuclei on paraffin-embedded tissue slices. The triphasic Gerlans protocol for liver-cancer induction was applied. Initiation with a single dose of diethylnitrosamine (DEN), and selection with 2-acetylamino-fluorene (2-AAF) combined with a proliferative stimulus (CCl4 administration), was followed by a treatment with PB or BHT for periods up to 22 weeks. Control animals received no treatment after the initiation and selection procedure. Despite intra- and inter-individual variations, an increase in the amount of 2N nuclei is found in the putative preneoplastic lesions of animals that received initiation and selection (I-S) and 3 weeks basal diet (BD). When the diet is supplemented with PB (after I-S), the increase of diploid nuclei starts earlier. At the time carcinoma arise (22 weeks PB treatment) a decrease in the frequency of 2N nuclei is found. BHT-treated animals which develop no carcinoma within the considered timespan, show a clear increased amount of 2N nuclei in the precancerous lesions only after 14 weeks treatment. It seems that there is a positive correlation between the outgrowth of putative preneoplastic foci and nodules in rat liver and an increase of diploid nuclei in these lesions. PB, as promoter used after initiation and selection, speeds up the development of carcinoma in rat liver, and therefore also the shift to diploidization in these rats starts earlier in comparison with I-S-treated rats. Although BHT does not promote liver carcinogenesis, an increase of diploid nuclei is also observed here during lesion formation. It may, therefore, be concluded that the phenomenon of diploidization is closely linked to and probably necessary for preneoplastic development, but that it is not an absolute indicator for neoplastic transformation.
Animals↗
C-banding as a simple tool to discriminate between micronuclei induced by clastogens and aneugens.
If mouse bone marrow preparations are treated with a classical C-banding procedure, it may be possible to distinguish between micronuclei with or without centromeres. This allows discrimination between micronuclei originating from chromosome breakage and those originating from chromosome loss. Thus, using C-banding, the micronucleus test can be used not only for the detection of clastogens but also aneugens. In this way, more exhaustive methods such as immunological staining using antikinetochore antibodies may not be necessary.
Animals↗
Relationship between frequencies of univalents and meiotic segregation in different mouse strains and their hybrids.
Although univalents in diakinesis are often used as an estimator for chromosome mis-segregation during meiosis, no clear-cut relationship was demonstrated between both phenomena. In this study, the frequencies of autosomal and gonosomal univalents in diakinesis were related to the frequencies of aneuploid metaphase-II gonocytes during spermatogenesis and oogenesis of different mouse strains and their hybrids (inbred strains: DBA/2J, C57Bl; outbred strain; Swiss, inbred x outbred hybrids: Swiss X C57Bl, C57Bl X Swiss, inbred X inbred hybrids: DBA/2J X C57Bl, C57Bl X DBA/2J). As far as the frequencies of univalents are concerned, they were shown to be strain-dependent and similar in both sexes. Moreover, there is a high non-disjunction rate of DBA males and PMSG-HCG-primed DBA females. Aneuploidy in metaphase II is also strain-dependent but different in both sexes; in the male, a clear decrease of aneuploidy frequencies is observed as compared to the frequency of univalents. This decrease does not occur in females.
Aneuploidy↗
Karyotypic analyses of metastatic and non-metastatic mouse tumor cells by G- and C-banding.
Cytogenetic studies using G- and C-banding were performed in order to compare non-metastasizing BWO mouse thymoma cells with their metastatic BW-IS-Li and BW-O-Li1 variants. BW-IS-Li and BW-O-Li1 cells were obtained from liver metastases generated respectively after intra-splenic or intravenous inoculation of mice with BWO cells. BW-O-Li1 cells contained normal chromosomes 4 and 18 which were never found in BWO or BW-IS-Li cells. As they also contained several markers common to the BWO parental cell line, our data provide definite evidence that the BW-O-Li1 cell line results from spontaneous in vivo cell fusion between a BWO cell and a normal host cell. Chromosome 3 was also shown to be "rearrangement-prone" and was less represented among metastatic cell variants. These variants also lack a C-band-deficient marker chromosome which was found in most of the BWO cells. However, as many complex chromosome rearrangements were found in all 3 cell lines, at least some chromosomal material may be present in a hidden form. We were not able in this particular cell system to link the presence or absence of a single chromosome, chromosome fragment or rearrangement to the acquisition or expression of metastatic properties.
Animals↗
Changes in nucleolar transcriptional activity and nuclear DNA content during the first steps of rat hepatocarcinogenesis.
Two complementary genetic parameters were followed in liver parenchymal cells during the first steps of rat hepatocarcinogenesis: the expression of nucleolar genes estimated from their silver stainability and the nuclear DNA content determined after Feulgen staining. Putative preneoplastic lesions as foci and nodules were induced by the triphasic 'Gerlans protocol'. Initiation with a single dose of diethylnitrosamine (DEN) was followed by a selection of initiated cells with 2-acetylaminofluorene (2-AAF) in combination with a single necrogenic dose of CCl4 as a proliferative stimulus. Finally after 1 week of normal diet, the animals were treated or not with phenobarbital (PB) for periods up to 2 months. Serial sections were analysed after silver staining (AgNO3), methyl-green--pyronin staining (Unna-Brachet) and Feulgen staining with densitometric and morphometric methods. Silver staining, which is known to stain an acidic protein associated with rRNA synthesis, increased gradually with the duration of the PB treatment. Morphometry revealed an increase in both nucleolar and nuclear volume; the fraction of nuclei with one nucleolus also increased. These results seem to point towards an increase of nucleolar activity in the early steps of PB promotion. Moreover, this shift cannot be ascribed to an increase of DNA content. Indeed, a parallel study on neighbouring sections stained with Feulgen revealed a shift towards a population of diploid nuclei, in contrast to normal liver cells, which are mostly tetraploid. The observed diploidisation may therefore provide a functional advantage for the expansion of putative preneoplastic cells.
2-Acetylaminofluorene↗
Induction of spindle inhibition and abnormal mitotic figures by Cr(II), Cr(III) and Cr(VI) ions.
A differential staining technique for chromosomes and spindles was used to compare the effects of chromium compounds of different oxidation states with either no, or very low (Cr(II) and Cr(III) or high (Cr(VI) carcinogenic properties. Neither CrCl2, nor CrCl3 (except at 10(-4) M) had any effect on spindle formation and/or chromosome segregation after exposure up to 10(-7) and 10(-5) M, respectively. None of them significantly influenced the mitotic index. In contrast, the Cr(VI) products (Na2CrO4 and CaCrO4) induced a dose-dependent increase of aberrant division patterns up to 10(-4) and 10(-5) M, respectively. Moreover, with CaCrO4, a high frequency of cells with dislocated metaphase chromosomes or malformed spindles with clustered chromosomes were observed. Since these cell types are potential sources of aneuploid daughter cells, they represent a potential risk for cancer induction. These experiments clearly demonstrate that the differential staining methods allows discrimination between spindle modifiers which induce lethal toxicity and those which may lead to possible tumour induction.
Carcinogens↗
Increased silver stainability of metaphase chromosomes from rat hepatocytes during in vivo hepatocarcinogenesis.
Silver stainability of metaphase chromosomes was studied in hepatocytes obtained from rats exposed or not to a partial or complete carcinogenic treatment with diethylnitrosamine and phenobarbital. An increased hyperstaining is reported in the carcinogen-treated animals.
Animals↗
Nucleolar changes during the first steps of experimental hepatocarcinogenesis in rats.
Silver stainability of hepatocytes as an expression of nucleolar activity was studied in vivo during rat hepatocarcinogenesis. Male Wistar rats were injected with one dose of diethylnitrosamine (200 mg/kg body weight dissolved in 0.9% NaCl), followed by a selection procedure with a short exposure to 2-acetylaminofluorene in combination with a proliferative stimulus, such as the administration of CCl4. Finally, after 1 week of a normal diet, some of the rats were treated with phenobarbital. After enzymatic isolation, the hepatocytes were silver stained; the estimation of nucleolar activity was determined by a cytomorphologic analysis of the silver-stained nuclei. It was demonstrated that during the first steps of hepatocarcinogenesis, both diethylnitrosamine, as initiator, and phenobarbital, as promotor, induce modifications of the nucleolar morphology in silver-stained hepatocytes.
2-Acetylaminofluorene↗
NOR variability in twins.
The number of AgNOR (NOR+) and the amount of AgNOR (NORM+) were analysed by means of two multilevel analyses of variance in a total of 12 twin pairs: 3 female and 4 male MZ and 5 male DZ pairs. In the first analysis, only zygosity was controlled; in the second, chromosome types D and G were controlled as well as the interaction between chromosome type and zygosity. For NOR+ and NORM+, when chromosome types D and G are not distinguished, the within-pair variance is greater, though not significantly, in DZ than in MZ pairs; but it is highly significantly greater when chromosome type (D or G type) is under control. This confirms an important genetic determination of NOR+ and NORM+ when in the ANOVA model the D and G types are controlled. However, nongenetic factors also influence the Ag-NOR patterns, but not enough to conceal the genetically defined rDNA pattern. Indeed, about 50% of the cells transcribe their rDNA in a way not closely dependent on the rDNA background and significant intrapair differences of NOR+ pattern exist in MZ twins.
Analysis of Variance↗
Lack of synergistic effect between arsenic, mercury and ethyl methane sulfonate on the frequency of chromosomal aberrations in mice.
The influence of arsenic and mercury on the frequency of chromosome aberrations induced by ethylmethane sulfonate was studied in mice. No synergistic effects could be demonstrated in somatic and germ cells of mice given a combined treatment of arsenic trioxide solution (As2O3, 12 mg per kg body weight) or of mercuric chloride solution (HgCl2, 6 mg per kg body weight) with ethylmethane sulfonate (EMS, 200 mg per kg body weight).
Animals↗
Comparison of metaphase and interphase nucleolar activity in Hela-CCL2 cells and PHA-stimulated human lymphocytes.
Metaphase and interphase nucleolar activity in cultured Hela-CCL2 cells and PHA-stimulated human lymphocytes have been studied with silver nitrate staining. In metaphase, we examined the relationship between the actual number of active NORs (AgNORs) and total number of NOR-bearing acrocentrics. Interphase silver staining over the nucleus was analyzed cytodensitometrically and morphologically. From all investigations, Hela-CCL2 cells and lymphocytes were shown to have similar levels of nucleolar activity. Our results suggest that there is a form of regulation of nucleolar activity in malignant Hela-CCL2 cells as compared to PHA-stimulated human lymphocytes.
Cell Nucleolus↗
Mercury-induced segregational errors of chromosomes in human lymphocytes and in Indian muntjac cells.
Segregational errors of chromosomes were studied in human lymphocytes and in Indian muntjac fibroblasts exposed to methylmercury chloride (CH3HgCl) or mercury chloride (HgCl2). The cells were exposed to the mercury compounds only during a limited period of the pre-DNA synthetic stage of the cell cycle or from that stage up to mitosis. In the lymphocytes we observed a clear increase of C-mitotic figures for both mercury compounds and for both exposure times. Segregational errors were, however, much more important after the shorter exposure period. Muntjac fibroblasts appear to be more sensitive to the mercury than are the lymphocytes so that their suitability for the study of C-mitosis may be questionable. The muntjac cells may be an important tool for the study of polyploidy induction.
Aneuploidy↗