Attitude of a group of Belgian stakeholders towards proposed agricultural countermeasures after a radioactive contamination: synthesis of the discussions within the Belgian EC-FARMING group.

In the case of radioactive contamination of the environment with an impact on the food chain, the remediation strategy will not only be based on scientific knowledge and technical experience, but will also be dictated by peculiarities of the country. These characteristics include the agro-industrial structure, the local and international economical contexts and the political configuration including the distribution of responsibilities and competencies. This paper identifies and illustrates the most relevant characteristics of the Belgian agricultural system and political environment; it also describes the past experience with food chain contamination, which is expected to influence the attitude of Belgian stakeholders, who would be involved in the setting up of countermeasure strategies for maintaining agricultural production and food safety. The picture drawn explains why several countermeasures aiming to reduce the contamination in food products, although scientifically sound and technically feasible, are hardly acceptable or even not acceptable at all, to the stakeholders.

Evidence of difference between cytotoxicity, cell cycle and morphonuclear effects of polyamines on sensitive and multidrug resistant P388 cell lines.

This paper reports studies on the influence of multidrug resistance (MDR) on the mechanism of polyamine toxicity. The effects of putrescine (PUT), spermidine (SPD) and spermine (SPM) on morphonuclear parameters and cell cycle were studied by means of digital cell image analysis. This reveals that only SPD and SPM condense chromatin inducing a strong decrease in the nuclear area and a cell-cycle arrest in phase G2 in P388/s and the two MDR sublines. A significant difference was observed between the sensitivity of the two phenotypes, which confirms results obtained by means of a microculture tetrazolium test which showed that SPD and SPM were highly, but very differently, cytotoxic on sensitive and MDR sublines, unlike PUT, which was not toxic. This encourages us to study more thoroughly possible differences in polyamine metabolic enzymes and uptake in these cells, to enable us to acquire a better understanding of the impact of MDR phenotype on the polyamine pathway.

Determination of the mechanism of action of anticancer drugs by means of the computer-assisted microscope image analysis of Feulgen-stained nuclei.

The antitumoral effects of 30 drugs was monitored in vitro on three neoplastic cell lines. These 30 drugs belonged to various pharmacological classes which included alkylating agents, antimetabolites, Vinca alkaloids, topoisomerase II inhibitors, and intercalating agents. The aim of the present work is to use the same methodology to characterize the drug-induced effects at several biological levels. The drug-induced modifications were, therefore, monitored by means of the digital cell image analysis of Feulgen-stained nuclei. This methodology enabled the cell cycle kinetics to be studied. Furthermore, the numerical data quantitatively describing chromatin patterns were submitted to multivariate (principal-components) analyses, with the canonical transformation of the data. Statistical analysis shows that each pharmacological class of anticancer drugs induces specific modifications to the chromatin patterns. The present study, therefore, shows that it is possible to identify distinct classes of antineoplastic drugs on the basis of their mechanisms of action by means of the quantitative chromatin pattern description of Feulgen-stained nuclei.

In vivo characterization by means of digital cell image analysis of early-induced fractionated radiotherapy effects on the MXT mouse mammary tumor.

PURPOSE: To study the cell kinetics and chromatin modifications occurring in function of the fractionated irradiation administered to the MXT mouse mammary adenocarcinoma. METHODS AND MATERIALS: The MXT tumor cells were submitted to three fractions of a 4.8 Gy dose delivered at 24-h intervals. MXT tumor cells were collected by means of fine needle aspirations (between 5 and 10 samples were obtained after each irradiation) during treatment and submitted to the computer-assisted microscope analysis of Feulgen-stained specimens. Three groups of parameters has been described: i.e., the geometry of the nucleus, the nuclear DNA content, and the chromatin texture. Furthermore, cell cycle parameters were studied in the aim to know the distribution of the cells within the cell cycle. RESULTS: The mean values relating to geometric parameters (i.e., the nuclear area and its standard deviation) decreased during treatment. Variations in the nuclear DNA content appeared as being cyclical and could be explained in terms of the modifications in the distribution of the cells within the cell cycle. The quantitative analysis of the cell cycle parameters revealed that the percentage of S cells increased regularly after each irradiation. In contrast, the percentage of G2 cells decreased between each irradiation. The parameters describing nuclear texture showed regular variations between each irradiation. These variations consisted in two cycles constituted by a decrease in chromatin condensation, followed by an increase. CONCLUSIONS: The development of the geometric parameters indicates that fractionated radiotherapy leads to the emergence of a more homogeneous population. The effects of the radiotherapy on the distribution of the cells within the cell cycle could be explained through the phenomenon of repopulation and by the high degree of radiosensitivity of the G2 cells (decrease in the percentage of G2 cells). Last, the variations observed at chromatin pattern level could be explained through DNA repair processes.

Cross resistance and collateral sensitivity between cytotoxic drugs and radiation in two human bladder cell lines.

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) test was employed as a means of studying the cross resistance and collateral sensitivities of two bladder cell lines able to grow in the presence of several antineoplastic drugs and/or despite the effects of radiotherapy. This cross resistance and collateral sensitivities were investigated in the context of two antineoplastic drugs (i.e. doxorubicin (an anthracycline) and vinorelbine (a Vinca alkaloid) and radiotherapy. The results show that cell lines able to grow in the presence of anticancer drugs develop a significant degree of resistance to antineoplastic compounds and may also develop resistance to radiotherapy. On the other hand, most cell lines treated first with radiotherapy develop a significant degree of resistance towards ionizing radiation and may also display increased sensitivity towards anticancer drugs. If these results obtained in vitro are clinically relevant, they may have important applications in the treatment of patients. Nevertheless, it will be necessary to validate the results on extensive series of chemo- and/or radioresistant cell lines exhibiting different mechanisms of resistance.

Cytotoxicity, cell cycle kinetics and morphonuclear-induced effects of Vinca alkaloid anticancer agents.

The effects of four Vinca alkaloids (vinblastine, vincristine, vindesine and vinorelbine) on three neoplastic cell lines (the MXT mouse mammary cell line and the T24 and J82 bladder cell lines) were studied at three biological levels, i.e. cell proliferation, cell cycle kinetics and morphonuclear characteristics. These effects were studied by means of digital cell image analysis on Feulgen-stained nuclei. The aim of the present work was to characterize the effects specifically induced by Vinca alkaloids as compared with those obtained previously with other pharmacological classes of anticancer drugs. The results show that Vinca alkaloids inhibit the cell proliferation of neoplastic cell lines at a concentration of 10(-8) M except in the case of the J82 cell line, for which only a slowing down of cell proliferation was observed. Concerning the cell cycle kinetics, the results show that the Vinca alkaloids induce an accumulation of cells in the mitosis phase. This accumulation of mitotic cells was maximal after 15 h incubation in the presence of the drugs. A study of the morphonuclear-induced effects of Vinca alkaloids showed that the variance of the optical density (VOD) is strongly influenced by these Vinca alkaloids. The development of the VOD was parallel with the development of the percentage of mitosis; thus, the VOD enabled the Vinca alkaloid-induced effects to be specifically characterized from a morphonuclear point of view. On the other hand, the results show that the mean value of the variance of the optical density was very highly correlated (P < 0.001) with the efficiency of the Vinca alkaloids in terms of cytotoxicity. In clinical studies, the analysis of the development of this parameter would make it possible to assess the response to chemotherapy in the case of patients treated with Vinca alkaloids.

Chemotherapy-induced nuclear alterations of morphologic and genomic characteristics in a human colon cancer grafted onto nude mice.

PURPOSE: A human Dukes B colonic adenocarcinoma was grafted onto 40 nude mice. The mice were divided into four groups, one control and three representing experimental conditions. Animals in the three experimental groups received either adriamycin (ADR), 5-fluorouracil (5-FU), or camptothecin (CPT) over a 25-day period beginning 34 days after grafting. Control animals received saline on an identical schedule. Animals were killed 105 days after grafting. METHODS: The effect of therapy was assessed by three techniques: 1) tumor size was periodically measured during the life of the animals, 2) modifications of APC, Ki-ras, and p53 genes were studied by polymerase chain reaction, dot-blot analysis, restriction analysis, and DNA sequencing, and 3) image cytometry of Feulgen-stained material was used to characterize 15 parameters describing morphometric, densitometric, and textural features of tumor nuclei. RESULTS: When compared with controls, tumor growth (size) was maximally suppressed by treatment with CPT (P < or = 0.001). Growth was inhibited significantly by treatment with 5-FU (P < or = 0.01); no statistical difference in tumor size was observed between controls and animals treated with ADR. Modifications of APC, Ki-ras, and p53 genes were not observed; however, treatment did inhibit amplification of APC and p53 genes. CONCLUSIONS: The 15 morphonuclear parameters were assessed to define populations of cell nuclei altered by chemotherapy. Although CPT maximally suppressed growth, it did not alter nuclear morphology when compared with controls. Treatment with either 5-FU or ADR resulted in nuclear morphologic alterations defined as distinct populations using multivariate analysis. Nonsupervised linear discriminant analysis was used to quantify the relative proportions of these populations. Four morphonuclear parameters were identified, which discriminated nuclei exposed to either ADR or 5-FU from controls.

Characterization of alkylating versus intercalating anticancer drug-induced effects on cell survival, cell cycle kinetic and morphonuclear pattern of three neoplastic cells lines growing in vitro.

PURPOSE: The influence of three alkylating and three intercalating anticancer drugs on cell survival, cell cycle kinetics and chromatin patterns was monitored in vitro on three neoplastic cell lines. METHODS: This monitoring was carried out by means of the digital cell image analysis of Feulgen-stained nuclei. RESULTS: Results show that in term of cytotoxicity, the intercalating drugs were more potent than the alkylating ones. As for the cell kinetics assessment, most of the experimental conditions led to a blockage of the cells in the G2 phase of the cell cycle. A study of chromatin patterns by means of digital cell image analysis enabled us to describe 15 morphonuclear parameters. The results show that the drugs tested induced specific morphonuclear modifications, e.g. an increase in nuclear size. The 15 morphonuclear parameters were submitted to multivariate analyses, i.e. principal-components analyses followed by the canonical transformation of the data. The results of these multivariate analyses enabled us to discriminate between the alkylating and the intercalating drugs. CONCLUSIONS: We conclude that it would be possible to "diagnose" the mechanism of action of DNA interacting agents (alkylating or intercalating drugs) by means of the combination of digital cell image and multivariate analysis.

Comparative study of multidrug resistance evaluated by means of the quantitative immunohistochemical detection of P-glycoprotein and the functional release of rhodamine 123.

The immunological detection of P-Glycoprotein (P-GP) and the functional release of Rhodamine 123 (R123) have been compared in a number of human and murine cancer cell lines, in chemo- and/or radiotherapy-resistant subclones, and in clinical specimens from patients. The chemoresistance level was established from the viability index (IC50) in the presence of doxorubicin. Cytocentrifuge preparations were immunostained with JSB-1 monoclonal antibody followed by the alkaline phosphatase anti-alkaline phosphatase technique. The strength of the reaction was quantified by a digital image analyser. The kinetic incorporation and release of Rhodamine 123 were evaluated by flow cytometry. The parent cell lines and radiotherapy resistant subclones showed a low IC50, were JSB-1 negative and retained R123 during the whole experiment, while the chemoresistant and radio-chemoresistant cell line mutants had a high IC50, were JSB-1 positive, and actively pumped the R123 out of the cells. Good correlations were obtained between the IC50, the digital image analysis, and flow cytometry. The kinetic profile of the R123 release allowed the distinction between typical and atypical multidrug resistance phenotypes. These findings were confirmed in clinical specimens from patients. We conclude that antigenic and functional studies are complementary and are useful in experimental and clinical approaches to multidrug resistance.

Computer-assisted morphonuclear characterization of radiotherapy-induced effects in MXT mouse mammary adenocarcinomas surviving earlier radiotherapy.

PURPOSE: To present the effects of different radiotherapeutic treatments on the morphonuclear characteristics and growth of the MXT mouse mammary adenocarcinoma. METHODS AND MATERIALS: We collected MXT tumor cells by means of fine-needle aspirations during various radiotherapeutic treatments and analyzed the morphological aspects of the cell nuclei by means of the digital cell image analysis of Feulgen-stained nuclei. In addition, we studied the morphonuclear aspects of cells from MXT tumors that had been radioresistant cell enriched. These radioresistant cell-enriched tumors involved MXT tumors that had survived one or two previous radiotherapies. The radiotherapy-induced effects on the morphonuclear characteristics were monitored by means of both monovariate (one-way variance) and multivariate (principal components and step-wise linear discriminant) analyses. RESULTS: The monovariate analyses showed that radiotherapy significantly influenced the values of the parameters relating to nuclear size (nuclear area--NA), the frequency of small dense chromatin clumps (short run length emphasis--SRL) in the nuclei, and the overall chromatin condensation level (local mean--LM). The global effect corresponded to a decrease in the overall chromatin condensation level in the radioresistant cell-enriched MXT tumors. This decrease occurred concomitantly with an increase in the frequency of the small dense chromatin clumps in the nuclei and a decrease in the nuclear area. The multivariate analyses showed that it was possible to quantitate the proportion of "radiosensitive-like" and "radioresistant-like" cell nuclei in the various MXT tumor types under study. CONCLUSIONS: The development of certain morphonuclear parameters, that is, the NA, the SRL, and the LM, could be proposed to predict the response of human tumors to radiotherapy as, indeed, could the quantitation of the proportion of radioresistant cells.

In vitro digital cell image analysis of morphonuclear modifications induced by natural DNA-interacting anticancer drugs in three neoplastic cell lines.

The effects of seven natural anticancer drugs acting through interaction with DNA were characterized at three levels: cell proliferation, cell cycle kinetics and chromatin texture. These drug-induced effects were monitored in vitro by means of the digital cell image analysis which provides 15 morphonuclear parameters. In terms of cell proliferation, results show that the drug-induced effects ranged from 10(-11)M to 10(-6)M. With respect to the cell cycle kinetics, the results show that the drugs induced an accumulation of the cells in the G2 phase. Concerning the chromatin levels, the results show that it is possible to classify the drugs according to their mechanism of action on the basis of the multivariate analysis of the 15 parameters.

Combination of computerized morphonuclear and multivariate analyses to characterize in vitro the antineoplastic effect of alkylating agents.

The influence of 13 anticancer alkylating agents on cell proliferation, cell cycle parameters, and morphonuclear characteristics was monitored in vitro on three neoplastic cell lines. This monitoring was carried out by means of the digital cell image analysis of Feulgen-stained nuclei. This computer-assisted microscope analysis of chromatin texture made it possible to assess 15 morphonuclear parameters. These 15 parameters were submitted to multivariate analyses, that is, principal-components analyses followed by the canonical transformation of the data. The 13 alkylating agents included four nitrogen mustards (chlormethine, chlorambucil, melphalan, and cyclophosphamide), two nitrosoureas (carmustine and lomustine), two platinum analogues (cisplatine and carboplatine), two ethyleneimine derivatives (thiotepa and investigational PE1001), one antibiotic (mitomycin C), one alkylsulfonate (busulfan), and one triazene (dacarbazine). The mouse MXT mammary and the human J82 and T24 bladder tumor cell lines were used in this study. The results show that these alkylating agents induced specific modifications to the chromatin pattern according to the subclass to which they belong. In other words, the multivariate statistical analyses of the 15 parameters made it possible to identify, at least partly, distinct subclasses of alkylating agents according to their mechanisms of action. As a validation of the methodology, the results also show that most of the alkylating agents induced an increase in the percentage of cells in the G2 phase, while some sometimes induced an increase in the percentage of cells in the S phase of the cell cycle.

Morphonuclear characterization of drug resistance by means of digital cell-image analysis: an in vitro assessment.

The prediction of tumor resistance to antineoplastic drugs remains an important challenge in cancer chemotherapy. Several methods have been proposed in this connection, but they present a number of problems such as clinical relevance and applicability. In the present work we put forward an original methodology to assess the drug sensitivity of cancer cells. For this purpose we submitted chemosensitive and chemoresistant cell lines to different anticancer drugs and monitored the cell growth and the drug-induced morphonuclear effects by means of digital cell-image analysis of Feulgen-stained nuclei. The results showed that drug-induced effects at the morphonuclear level correlated statistically with the effects produced at the cell proliferation level. For example, the mean nuclear size value increased as a function of the drugs' efficiency recorded at the cell proliferation level. In the same way, the frequency of large dense chromatin clumps also increased in accordance with the drugs' efficiency. The present work thus demonstrates that digital cell-image analysis can be applied to monitor the efficiency of chemotherapeutic treatment carried out on cell lines in vitro. The present methodology could possibly be used on solid tumors, from which biological material can be obtained serially by means of fine-needle aspiration. As evidence of this, the present methodology can also be applied to hematological cancers.

Characterization of chemotherapy-induced morphonuclear modifications in the P388 leukaemia and the MXT mammary tumour models of the mouse.

Chemotherapy-induced morphonuclear modifications were monitored in vivo by means of the digital cell image analysis of Feulgen-stained nuclei. Two experimental models were used, i.e. the P388 mouse leukaemia and the MXT mouse mammary carcinoma. The drugs used were doxorubicin, etoposide and cyclophosphamide. The results indicate that the chemotherapy induced a significant decrease in the MXT tumour growth and a significant increase in the survival of the P388 leukaemic mice. These effects were accompanied at the morphonuclear level by an increase in the nuclear area, by modifications in the DNA content in accordance with the effects of the drugs on the cell cycle and by several modifications in the chromatin texture in accordance with the model or the drugs studied. While there were neither homogeneous morphonuclear changes in all treatment groups nor clearcut correlations between the morphonuclear changes and tumour growth or the survival of the animals, the present study nevertheless shows that it is possible, at least partly, to monitor in vivo certain chemotherapy-induced effects occurring at the morphonuclear level, and subsequently to obtain information on the mode of action of the drugs.

Computerized morphonuclear analyses of Feulgen-stained nuclei from 11 chemosensitive and from 11 chemoresistant neoplastic cell lines.

It has previously been demonstrated that the cell nuclei from 1 mouse mammary cancer and 2 neoplastic human bladder cell lines displayed common morphonuclear characteristics when they were made resistant to different anti-neoplastic agents. In the present work this experiment was repeated with a greater number of cell lines from different kinds of tumours. The nuclei from the sensitive and resistant cells were submitted to Feulgen staining and studied by means of computerized nuclear image analysis. This methodology made it possible to characterize the cell nuclei by means of 15 parameters including 1 geometric, 2 densitometric and 12 others quantitatively describing the chromatin pattern. The results showed that the morphonuclear feature of the cell lines significantly varied according to the extent of the resistance. The majority of the observations showed that the direction of the variations seemed to be a function of the origin of the tissue. On the basis of the experimental results reported here, we think that it would be possible to establish a model for the purpose of monitoring the effectiveness of anticancer treatment from a clinical point of view.

Computer-assisted microscope analysis of morphonuclear modifications induced by anticancer antimetabolites in cell lines cultured in vitro.

An original method is proposed for the purpose of discriminating between antimetabolites exhibiting different operating mechanisms. The present work was carried out by means of the digital cell image analysis of Feulgen-stained nuclei cultured in the presence of different antimetabolites. We chose this method because it enables anticancer drug-induced effects to be studied at both morphonuclear and cell cycle levels in the same biological sample. The results show that all the antimetabolites had similar effects on the nuclear texture of the cells and on cell cycle parameters. Furthermore, the use of multivariate analyses made it possible to distinguish between different mechanisms of action among drugs belonging to the same class of antineoplastic, i.e. antimetabolite agents.

In vitro characterization of radiotherapy-induced morphonuclear modifications on chemosensitive as opposed to chemoresistant neoplastic cells.

PURPOSE: We describe by means of digital cell image analysis the influence of X-ray radiation on three in vitro cultured cell lines for which we set up chemosensitive and chemoresistant variants. METHODS AND MATERIALS: The three cell lines correspond to the MXT mouse mammary and the T24 and J82 neoplastic human bladder cells. The digital cell image analysis was carried out by computing morphometric (nuclear size), densitometric (proportion of cells in the G2 cell cycle phase), and textural features (chromatin pattern characteristics) on Feulgen-stained nuclei. RESULTS: The results show that such digital cell image analyses make it possible to monitor radiotherapy-induced effects on these morphonuclear characteristics accurately. X-ray radiotherapy induces a dose-dependent increase in the proportion of cells in the G2 phase of the cell cycle along with a decrease in the overall chromatin condensation level. These two concomitant phenomena lead to a marked radiotherapy-induced increase in nuclear size. We also observed that radiotherapy-induced effects at the morphonuclear level are not only highly specific to the cell type analyzed, that is MXT mouse mammary or J82 or T24 human bladder carcinoma cells, but also to the fact that the cells are either chemosensitive or chemoresistant. CONCLUSION: The digital cell image analyses of Feulgen-stained nuclei is helpful in monitoring the irradiation-induced morphonuclear modifications.

Chemoresistant cell lines: morphonuclear characteristics and chemosensitivity development during long-term culture.

The aims of the present work were first to study the stability of the sensitivity and resistance of various cancer cell lines over the period of one year, and second to attempt to correlate the morphological aspects of the lines with the results obtained. To this end we used one mammary and two human vesical lines, each consisting of three different phenotypes: a primitive phenotype (the sensitive line); a phenotype able to proliferate in the presence of three antineoplastic agents (the resistant line); and a phenotype derived from the resistant line but cultivated in the absence of drugs for more than 50 passages. We studied the stability of these lines not only in terms of their chemosensitivity to drugs, but also from the point of view of the morphological characteristics of their nuclei as described by digital image analysis. The results obtained show that the resistant lines retained their resistance even after treatment by antineoplastic agents had been discontinued. However, in this latter case variations became noticeable in their sensitivity to drugs, variations which shifted the line towards either the sensitive or the resistant phenotype. Results obtained from image analysis show that, for each of the lines studied, the morphological aspects of the continuously treated resistant and sensitive lines were phenotype-specific. However, the resistant lines no longer cultivated in the presence of anti-cancer agents exhibited morphonuclear characteristics varying across those of the other two phenotypes. We attribute these variations to modifications in the quality of the serum used.

The application of computerized analysis of nuclear images and multivariate analysis to the understanding of the effects of antineoplastic agents and their mechanism of action.

The present work involves the creation of a model that makes the rapid study of anticancer drugs possible. In order to make possible the multilevel study of antineoplastic derivatives (their effects on cell proliferation, their cell kinetics, their chromatin texture and the study of their potential operating mechanisms), the computerized analysis of nuclear images linked to multivariate analysis was chosen, and, in so doing, took into account 15 computer-calculated morphonuclear parameters for Feulgen-stained cell nuclei. In order to validate the method it was subjected to a series of well-known cancer drugs. The present results are in agreement with those obtained by conventional methods similar to those used for the study of the cytotoxicity of anticancer molecules or their effect on cell kinetics. Multivariate analysis applied to the parameters described by image analysis should make possible the rapid study of new anticancer drugs together with the rapid orientation of studies undertaken to ascertain their operating mechanisms.

Monitoring of chemotherapy-induced morphonuclear modifications by means of digital cell-image analysis.

We illustrate the potential application of digital cell-image analysis to characterize the morphonuclear modifications induced by various drugs including VP16, a podophyllotoxin derivative, PE1001, an investigational alkylating agent, and doxorubicin, an intercalating agent. Fifteen parameters representative of morphometric (nuclear area), densitometric (nuclear DNA content), and textural (chromatin organization, condensation, and distribution) features were computed on Feulgen-stained nuclei obtained from fine-needle aspirations serially performed during treatment on the MXT mouse mammary cancer model. We observed marked differences between the control and drug-treated MXT cell nuclei. However, mathematical data processing was necessary to improve the ratio of the chemotherapy-induced morphonuclear signal to the control biological morphonuclear signal. This data processing relies upon the use of principal-component analysis followed by the canonical transformation of data. The present method can be applied to all human cancers on which fine-needle aspiration can be performed.