Heat shock induces variably the major heat shock proteins of CV1 clones

AbstractCV1 cells have been subcloned several times. Five of these clones were studied for the induction of the major heat shock proteins. These CV1 clones exhibit morphological differences as well as differences in SDS-PAGE protein profiles. These clones responded to heat shock variably as judged from the induction of the major heat shock proteins, 70, 72 and 92 kDa. Variable expression of the heat shock proteins suggests that the selective pressure for isolation of cell clones may affect gene expression differently

Effect of 910-MHz Electromagnetic Field on Rat Bone Marrow

Aiming to investigate the possibility of electromagnetic fields (EMF) developed by nonionizing radiation to be a noxious agent capable of inducing genotoxicity to humans, in the current study we have investigated the effect of 910-MHz EMF in rat bone marrow. Rats were exposed daily for 2 h over a period of 30 consecutive days. Studying bone marrow smears from EMF-exposed and sham-exposed animals, we observed an almost threefold increase of micronuclei (MN) in polychromatic erythrocytes (PCEs) after EMF exposure. An induction of MN was also observed in polymorphonuclear cells. The induction of MN in female rats was less than that in male rats. The results indicate that 910-MHz EMF could be considered as a noxious agent capable of producing genotoxic effects

The use of premature chromosome condensation to study in interphase cells the influence of environmental factors on human genetic material

Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental factors to which humans are exposed in their natural and anthropogenic environment. These factors exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environment that constitute part of the anthropogenic environment. It is well known that genetic damage due to these factors has dramatic implications for human health. Since most of the environmental genotoxic factors induce arrest or delay in cell cycle progression, the conventional analysis of chromosomes at metaphase may underestimate their genotoxic potential. Premature Chromosome Condensation (PCC) induced either by means of cell fusion or specific chemicals, enables the microscopic visualization of interphase chromosomes whose morphology depends on the cell cycle stage, as well as the analysis of structural and numerical aberrations at the G1 and G2 phases of the cell cycle. The PCC has been successfully used in problems involving cell cycle analysis, diagnosis and prognosis of human leukaemia, assessment of interphase chromosome malformations resulting from exposure to radiation or chemicals, as well as elucidation of the mechanisms underlying the conversion of DNA damage into chromosomal damage. In this report, particular emphasis is given to the advantages of the PCC methodology used as an alternative to conventional metaphase analysis in answering questions in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics and experimental therapeutics

Dynamic Analysis of DNA Damage by Flow Cytometry and FISH

The micronucleus assay, developed to assess DNA damage induced by noxious agents, supplies information on whether the damage is due to clastogenic or aneugenic action. Although it is the test that can be used to assess agents' toxicity, it cannot provide information on the molecular events that result in the induction of micronuclei. To study the molecular events, the combination of both microscopic and analytical techniques is required. Flow-sorting induced micronuclei, based on their DNA content, in combination with chromosomal FISH and other molecular techniques, may provide information on these events

Assessment of the genotoxicity of imidacloprid and metalaxyl in cultured human lymphocytes and rat bone marrow.

Abstract Imidacloprid and metalaxyl are two pesticides that are widely used in agriculture, either separately, or in combination. These agents were studied for their possible genotoxic effects with respect to the following cytogenetic end-points: (1) in vitro micronucleus (MN) formation and sister-chromatid exchange (SCE) induction in human lymphocytes and (2) in vivo micronucleus induction in polychromatic erythrocytes (PCEs) of the rat bone-marrow. The results of the MN analysis indicate that MN frequencies after treatment with both pesticides, separately or as a mixture, do not significantly differ from those in the controls except after treatment with metalaxyl alone at 50 g/ml (p < 0.05). The results of the SCE analysis show that SCE frequencies after treatment with imidacloprid do not differ significantly from those in the controls. A statistically significant increase (p < 0.05) in SCE frequency resulted from treatments with metalaxyl at 5, 10 and 100 g/ml and with the combination of imidacloprid and metalaxyl at 100 and 200 g/ml. Finally, the in vivo micronucleus assay with rat bone-marrow polychromatic erythrocytes showed a statistically significant effect upon separate treatments with imidacloprid and metalaxyl at doses of 300 mg/kg body weight (b.w.) (p < 0.01) or upon combined treatment with 200 mg/Kg b.w. (p < 0.001) and 400 mg/kg b.w. (p < 0.05)

Differential Micronuclei Induction in Human Lymphocyte Cultures by Imidacloprid in the Presence of Potassium Nitrate

Humans are exposed to pesticides as a consequence of their application in farming or their persistence in a variety of media, including food, water, air, soil, plants, animals, and smoke. The interaction of pesticides with environmental factors may result in the alteration of their physicochemical properties. Square wave cathodic stripping voltammetry (SW-CSV), a technique that simulates electrodynamically the cellular membrane, is used to investigate whether the presence of potassium nitrate (KNO 3 ) in the culture medium interferes with the genotoxic behavior of imidacloprid. The cytokinesis block micronuclei (CBMN) method is used to evaluate imidacloprid's genotoxicity in the absence or presence of KNO 3 in the culture medium and, as a consequence, its adsorption by lymphocytes. Comparing micronuclei (MN) frequencies in control and imidacloprid-treated blood cell cultures, statistically significant differences were not detected. KNO 3 did not induce MN frequencies compared to control. Statistically significant differences in MN frequencies were observed when blood cell cultures were treated with imidacloprid in the presence of increasing concentrations of KNO 3 . SW-CSV revealed that by increasing KNO 3 molarity, imidacloprid's concentration in the culture medium decreased in parallel. This finding indicates that imidacloprid is adsorbed by cellular membranes. The present study suggests a novel role of a harmless environmental factor, such as KNO 3 , on the genotoxic behavior of a pesticide, such as imidacloprid. KNO 3 rendered imidacloprid permeable to lymphocytes, resulting in elevated MN frequencies. KEYWORDS: genotoxicity, micronuclei (MN), pesticides, imidacloprid, potassium nitrate (KNO 3 ), square wave cathodic stripping voltammetry (SW-CSV) INTRODUCTION The human diet is characterized by the presence of numerous naturally occurring chemicals in trace amounts. All these chemicals, constituents of processed and nonprocessed food, have been mainly considered as harmless to human health. Among the chemicals, potassium nitrate (KNO 3 ) is found as a preservative in processed food Humans are exposed to a large number of industrially made chemicals, designed for various purposes, among which are those to be used for crop protection. Several of these chemicals are responsible for serious negative side effects, such as biochemical malfunctions or genetic instability Pesticides constitute a heterogeneous class of chemicals representing an important group of environmental pollutants. In Europe, more than 3 million tons are released into the environment yearly Imidacloprid, a systemic chloronicotinyl insecticide with soil, seed, and foliar uses, blocks the nicotinergic neuronal pathway that is more abundant in insects than in warm-blooded animals. It is a General Use Pesticide (GUP) classified by the EPA as a class II and class III toxicity agent The genotoxic and mutagenic activities of certain pesticides have been studied both in in vitro and in vivo systems using cytogenetic end points, such as the cytokinesis block micronuclei (CBMN) method A rather important technique to simulate electrodynamically the adsorptive ability of cellular membranes is square wave cathodic stripping voltammetry (SW-CSV), using a hanging mercury drop electrode (HMDE). The HMDE is a capillary electrode with a mercury drop at its edge. The mercury drop acts as an electrode surface. It has been widely used in environmental and biological samplings. Its function is based on the application of a fixed voltage between 200 and -1200 mV, targeted at the redox of the electroactive species, such as imidacloprid, which has been adsorbed on its surface. Electrons flowing between the adsorbed electroactive species and the electrode surface are expressed in current (measured in nanoamperes) that corresponds with these species in solution. Electroactive species move towards the electrode surface by diffusion, while they are adsorbed via electrostatic interactions The present work focuses on the synergistic action between an environmental factor, such as KNO 3 , and a pesticide, such as imidacloprid. The possible side effects of KNO 3 on imidacloprid genotoxicity towards cultured human lymphocytes were investigated, using both the CBMN method and the SW-CSV. MATERIALS AND METHODS Reagents Ultrapure Milli-Q water, Millipore Academic system, was used throughout the experiments. Imidacloprid (RDH 37894; purity: 99.9% by HPLC) obtained from Sigma-Aldrich and KNO 3 (GA 18517; purity: 99.9% by HPLC) obtained from Fluka were used without further purification. Stivaktakis et al.: Potassium Nitrate Alters Imidacloprid's Genotoxicity TheScientificWorldJOURNAL (2010) 10, 80-89 82 Imidacloprid stock solution (100 μM = 25.6 ppm) was prepared by dissolving 1.18 mg of insecticide in 46.07 ml of Milli-Q water and stored in the dark at 4 o C. This imidacloprid concentration is well below its solubility limit [26], while it corresponds to the mean value of previously reported concentrations The buffer solution used for voltammetric measurements was Britton-Robinson, prepared from a stock solution containing 10 mM phosphoric acid, 10 mM boric acid, and 10 mM acetic acid adjusted to the desired pH value, with NaOH. This buffer system is commonly used in analogous voltammetry experiments Apparatus and Software For the voltammetric measurements of imidacloprid, an electrochemical analyzer (Model TraceLab50, Radiometer Analytical) was used to control the voltage of a three-electrode system. As a working electrode, a Radiometer HMDE was used with a drop area of 3 mm 2 controlled by pneumatic connection with nitrogen (99.999%) at p = 1 bar. The reference electrode (Ag/AgCl-KCl = 3 M), type TR020, and auxiliary platinum electrode, type TM020, for POL150 were also from Radiometer. This system was connected with a Pentium II PC. All voltammetric measurements were carried out at 2 o C. As shown previously, low temperature facilitates the adsorption of imidacloprid on the mercury drop surface Instrumental Parameters The SW-CSV technique was used with: Cell parameters -Electrode HMDE, stirrer at 525 rpm, purge time 600 sec, accumulation time (t acc ) 60 sec, waiting time 10 sec, Hg-drop growth time 0.7 sec Signal parameters -Accumulation potential (E acc ) = -1060 mV[25], step duration 0.04 sec, step amplitude 1 mV, pulse amplitude +50 mV Current range -Minimum 10 nA, maximum 10 mA, under our experimental conditions the detection limit for imidacloprid was 0.25 μM. The pH measurements were carried out with a CRISON GLP21 pH-meter. Micronucleus Test in Human Lymphocytes In Vitro Blood samples were obtained from two healthy nonsmokers, without previous known contact with pesticides. Donors aged between 20 and 25 years. Whole blood (0.5 ml) was added to 6.5 ml Ham's F-10 medium (Invitrogen), 1.5 ml fetal calf serum (Invitrogen), and 0.3 ml phytohemagglutinin (Invitrogen) to stimulate cell division. The appropriate Stivaktakis et al.: Potassium Nitrate Alters Imidacloprid's Genotoxicity TheScientificWorldJOURNAL (2010) 10, 80-89 83 chemicals were added 41 h postculture initiation to final imidacloprid concentration of 20 μM, and of KNO 3 to final concentrations of 2, 10, and 20 μM in separate treatments. In mixed treatments, the final concentrations were 20 μM for imidacloprid plus 5, 10, 15, and 20 μM for KNO 3 . Mitomycin-C (Sigma) at final concentration of 1.5 μM served as positive control. Cultures were incubated at 37 o C for 72 h. Three hours after the addition of the chemicals, 6 μg/ml cytochalasin-B (Sigma) was added at 44 h postculture initiation. Cells collected by centrifugation at 72 h postculture initiation, fixed with freshly made methanol/acetic acid (Riedel-de Haen/Merck) mixture (3:1 v/v) after mild hypotonic treatment, were stained with Giemsa (Fluka) The Cytokinesis Block Proliferation Index (CBPI), given by the equation: CBPI = M 1 + 2M 2 + 3(M 3 + M 4 )/N, where M 1 , M 2 , M 3 , and M 4 correspond to the numbers of cells with one, two, three, and four nuclei and N is the total number of cells Analytic Determination of Imidacloprid from Human Lymphocyte Cultures One milliliter of supernatant, derived from 72-h blood cultures in the presence of imidacloprid, was mixed with 9 ml of absolute ethanol Statistical Analysis Statistical analysis of MN data was made by the G-test for independence on 2x2 tables. This test is based on the general assumption of the χ 2 analysis, but offers theoretical and computational advantages RESULTS SW-CSV The effect of adsorbed imidacloprid on cultured cells was further investigated by the CBMN method. Stivaktakis et al.: Potassium Nitrate Alters Imidacloprid's Genotoxicity TheScientificWorldJOURNAL Micronucleus Test in Human Lymphocytes In Vitro Stivaktakis et al.: Potassium Nitrate Alters Imidacloprid's Genotoxicity TheScientificWorldJOURNAL (2010) 10, 80-89 85 Our data of 20 + 5 combined mixtures indicated marginally no significant statistical differences in both BNMN and MN frequencies (p = 0.057) compared to the control value. In the meantime, the same data appear to have a possible inducible effect, as their absolute values are almost twofold increased from their corresponding control values. A similar condition was observed at the concentration of 20 + 10 μΜ with regard to BNMN frequencies. The imidacloprid and KNO 3 cytotoxic effect was evaluated by the determination of CBPI. Regarding the cytotoxic index in all tested cytogenetic end points (see The reported control and positive control frequencies of MN were consistent to the literature, and authenticate our experimental procedure and treatment observations The presence of KNO 3 at concentrations higher than 10 μΜ in combination with 20 μΜ imidacloprid in culture media induces a statistically significant increase of MN frequencies (p < 0.05 and 0.01, respectively) compared to control, while in separate treatments no statistical differences are observed, as seen in DISCUSSION Skepticism has gradually developed over studying the synergistic action of pesticides and their genotoxic side effects to humans and other organisms. Many variables intervene in the study system, rendering it too complex, not easily reproducible, and increasing the workload exponentially. In addition, scientists agree that the synergistic effects of agricultural chemical mixtures on human health are controversial and further studies on this issue are needed The present study poses an additional speculation on studying the synergistic effects between pesticides and environmental factors. For this purpose, the CBMN method was used in parallel to the SW-SCV electroanalytical technique. There is a relationship between polarographic and/or voltammetric behavior and genotoxic properties of organic compounds. By studying the mechanisms of their electrode reactions, useful clues can be provided to elucidate the mechanism of their interaction with living cells and their fate in the environment SW-CSV was used to study the effect of KNO 3 , a -harmless‖ chemical, on imidacloprid's genotoxicity to human lymphocyte cultures in vitro. This analytical voltammetric technique simulates electrodynamically the cellular membrane by applying a certain potential to a HMDE. By this system, the adsorptive ability of the cellular membrane towards imidacloprid, in the presence or absence of KNO 3 , could be evaluated. The CBMN method was used to evaluate imidacloprid genotoxicity in the absence or presence of KNO 3 . Neonicotinoids, including imidacloprid, are the most important new class of insecticides of the past 3 decades. They are increasingly replacing the organophosphates and methylcarbamates. Neonicotinoids have low activity on mammals relative to insect nicotinic acetylcholine receptors, providing a mechanistic basis for their safety 87 Controversial results regarding imidacloprid's genotoxic effect to cultured human lymphocytes were previously reported The negative in vitro genotoxic result in our findings from the separate treatment of 20 μM imidacloprid and the absence of imidacloprid's genotoxic effect in separate treatments on human lymphocytes in vitro at concentrations up to 400 μM, as well as the weak genotoxic effect on rat bone marrow in vivo A gradual reduction of imidacloprid's SW signal in culture medium with increasing KNO 3 concentration was observed by voltammetry. This is an indication that imidacloprid interacting with KNO 3 is adsorbed by lymphocytes. In the absence of KNO 3 , this reduction is not observed, while the SW signal is not altered by the presence of KNO 3 (see The calculated data from the electroanalytical method and genotoxicity experiments using the combination of imidacloprid and KNO 3 in cultured human lymphocytes denote the possibility of imidacloprid becoming genotoxic upon its interaction with a -harmless‖ chemical such as KNO 3 (see Our present data indicate that the combination of -harmless‖ environmental factors with pesticides that have not been classified as genotoxic to humans may interact, resulting in the alteration of the pesticides' physicochemical properties. Such alteration may convert harmless-to-humans pesticides into harmful ones with unidentified side effects. The latter could be an indication of reconsidering our classification of natural or man-made chemicals as harmless or potentially harmful ones. Thus, further studies, including genotoxic, electroanalytical, physicochemical, and molecular assessment, are required before drawing final conclusions on the separate and/or combined with environmental factors action of pesticides. ACKNOWLEDGMENT

The use of premature chromosome condensation to study in interphase cells the influence of environmental factors on human genetic material

Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental factors to which humans are exposed in their natural and anthropogenic environments. These factors exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environments that constitute part of the anthropogenic environment. It is well known that genetic damage due to these factors has dramatic implications for human health. Since most of the environmental genotoxic factors induce arrest or delay in cell cycle progression, the conventional analysis of chromosomes at metaphase may underestimate their genotoxic potential. Premature chromosome condensation (PCC) induced either by means of cell fusion or specific chemicals, enables the microscopic visualization of interphase chromosomes whose morphology depends on the cell cycle stage, as well as the analysis of structural and numerical aberrations at the G 1 and G 2 phases of the cell cycle. The PCC method has been successfully used in problems involving cell cycle analysis, diagnosis, and prognosis of human leukemia, assessment of interphase chromosome malformations resulting from exposure to radiation or chemicals, as well as elucidation of the mechanisms underlying the conversion of DNA damage into chromosomal damage. In this report, particular emphasis is given to the advantages of the PCC methodology used as an alternative to conventional metaphase analysis in order to answer questions in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics, and experimental therapeutics. KEYWORDS: premature chromosome condensation, cell fusion, calyculin-A, lymphocytes, DNA damage, genotoxicity, sister chromatid exchanges, chromosomal damage, chromosome aberrations, cell cycle delay, chemicals, ionizing radiation Hatzi et al.: Interphase genotoxicity assessment using PCC TheScientificWorldJOURNAL (2006TheScientificWorldJOURNAL ( ) 6, 1174TheScientificWorldJOURNAL ( -1190 INTRODUCTION Nowadays, there is a constantly increasing concern regarding the mutagenic and carcinogenic potential of a variety of harmful environmental agents to which humans are exposed in their natural and anthropogenic environments. Harmful environmental agents, exerting their effect on living organisms from the beginning of life, developed the appropriate evolutionary pressure that resulted in their defense mechanisms. Human technological development as well as human lifestyle introduced new series of harmful agents that, either alone or in combination, affect humans and all other organisms. These new agents exert their hazardous potential in humans' personal (diet, smoking, pharmaceuticals, cosmetics) and occupational environments that constitute part of the anthropogenic environment. The most important consequences of human exposure to environmental hazards are DNA damage induced either by direct or indirect binding to DNA. Direct-acting chemicals like DNA alkylating agents (e.g., mitomycin-C), crosslinking agents (e.g., methyl methanesulfonate), and oxygen radicals (e.g., hydrogen peroxide), bind covalently to DNA The exposure of cells to genotoxic factors may result in changes at the chromatid level, such as sister chromatid exchanges (SCEs), as well as in structural chromosomal alterations such as chromosomal aberrations Until now, the conventional cytogenetic analysis of chromosomal damage as a result of exposure to environmental chemicals is mainly based on the microscopic analysis of chromosomes in metaphase In this report, the advantages of the PCC methodologies used as an alternative to conventional metaphase analysis or in combination with other cytogenetic techniques are reviewed. Furthermore, the use of PCC in overcoming problems that could not be solved with the conventional metaphase analysis is Hatzi et al.: Interphase genotoxicity assessment using PCC TheScientificWorldJOURNAL (2006) 6, 1174-1190 1176 presented, and particular emphasis is given to the potential use of PCC to elucidate the mechanisms underlying conversion of DNA damage into chromosomal damage. Additional applications of the PCC methodologies in the fields of radiobiology, biological dosimetry, toxicogenetics, clinical cytogenetics, and experimental therapeutics are described, and future perspectives are discussed. PCC METHODOLOGIES PCC Induction Using Cell Fusion Central for the PCC assay is the fusion of interphase "test" cells with mitotic "inducer" cells that can be mediated either by incubation with Sendai virus As an alternative to the Sendai virus, the well-known fusogen polyethylene glycol (PEG) was applied to PCC-induction procedures Using the PCC assay by means of cell fusion, interphase cells that are either cycling, noncycling, or arrested can be visualized and analyzed. In the hybrids formed by cell fusion, the mitotic factors present in the donor mitotic cell dissolve the nucleus membrane and condense chromatin of the interphase "test" cell. More specifically, cells that have undergone PCC assume a morphology that is characteristic of the position of the interphase cell in the cell cycle: single chromatid per chromosome in G 1 phase, double chromatids per chromosome in G 2 , and pulverized chromosome regions in S phase Chemically Induced PCC Chemically induced PCCs can be obtained by the use of the chemical compounds such as calyculin-A. Calyculin-A was initially isolated from the marine sponge Discodermia calyx 1177 APPLICATIONS OF PCC METHODOLOGIES Biodosimetry and Biomonitoring of Exposure to Ionizing Radiation Exposure of cells to ionizing radiation has been shown to cause a wide variety of phenomena, the most prominent of which are the induction of mutations, the induction of transformation, cell cycle arrest, and cell death. As a result, the study of phenomena elicited by radiation are of particular importance to human health and, therefore, elucidation of the underlying biochemical mechanisms and cytogenetic effects of ionizing radiation are a high priority in radiation biology research. In the field of biological dosimetry, numerous methods capable of detecting radiation-induced changes at the molecular, cytogenetic, and cellular level have been used in order to obtain absorbed dose estimates For these reasons, the PCC methodology was proposed as an alternative biodosimetric tool by Pantelias and Maillie in 1984[50] and since then it has been extensively used to assess and evaluate the induction and repair of chromosome damage after in vivo or in vitro exposure of human cells to ionizing radiation Mechanisms Underlying Conversion of DNA Damage into Chromosomal Damage Visualizing cells with conventional analysis at metaphase can give information concerning only the residual damage after exposure to genotoxic environmental factors. The analysis of such interactions is based, therefore, only on those cells that proceed to mitosis. Therefore, it is difficult to elucidate the mechanism underlying the conversion of DNA damage into chromosomal damage. With the PCC methodologies, it is possible to gain valuable information not only to understand the biochemical mechanisms that affect the conversion of DNA damage into chromosomal damage, but also to determine possibly chromosomal radiosensitivity in G 2 phase, as well as variability in radiosensitivity at various stages of the cell cycle. The Hatzi et al.: Interphase genotoxicity assessment using PCC TheScientificWorldJOURNAL (2006) 6, 1174-1190 1181 onset and the efficiency of chromatin condensation-decondensation are important determinants of these processes. Data obtained so far demonstrate the important role of cdk1/cyclin-B complex and of the G 2 checkpoint control mechanism in affecting chromatin conformation changes and conversion of DNA damage into chromosomal damage. Using the PCC method, it was realized specifically that changes in chromatin conformation soon after irradiation, presumably as a result of histone-H1 phosphorylationdephosphorylation, strongly affected the conversion of DNA lesions into visual PCC fragments. The cdk1/cyclin-B complex was originally defined as the mitosis promoting factor (MPF), identified in mitotic frog eggs as a factor capable of inducing mitosis in G 2 -phase cells. Regulation of cdk1/cyclin-B complexes at multiple levels ensures the tight regulation of the timing of mitotic entry In early reports, G 0 human lymphocytes were irradiated and analyzed at various times after fusion with mitotic CHO cells, i.e., as chromatin condensation proceeded. The yield of fragments observed was directly related to the amount of chromosome condensation allowed to take place after irradiation and inversely related to the extent of chromosome condensation at the time of irradiation. From these experiments, it was concluded that changes in chromosome conformation interfered with repair processes of DNA damage. In contrast, resting chromosomes (as G 0 lymphocytes irradiated before fusion) showed efficient repair of chromosomal damage. These results supported the hypothesis that DNA damage is converted into cytogenetic lesions and becomes observable when chromatin conformation changes occur during the cell cycle G 2 checkpoint facilitates repair of chromosomal damage, and the hypothesis that G 2 -checkpoint defects during the G 2 -to M-phase transition can also affect G 2 -chromosomal sensitivity. This was tested using caffeine to abolish G 2 checkpoint by inhibiting ATM protein (Ataxia Telangiectasia mutated protein) Concerning the variability of radiosensitivity to ionizing radiation at various stages of the cell cycle, it is already known that middle to S phase and G 1 phase are resistant stages, while mitosis, G 1 /S, and G 2 /M transition are very sensitive to radiation Genotoxicity of Exposure to Chemical Agents It is well known that increased rates of cell proliferation can escalate the risk of malignancy following exposures to chemical agents 1183 The Uses of PCC in Clinical Cytogenetics and Experimental Therapeutics The fact that chromosomal analysis by means of the PCC method requires a small amount (0.5 ml or less) of sample (i.e., peripheral blood or bone marrow) makes it especially suitable for in vivo and in vitro studies, and also in clinical applications for diagnostic purposes. In particular, the ability to visualize the interphase chromosomes of bone marrow and blood cells using PCC by means of mitotic cell fusion with interphase cells has proved useful and accurate in the study of human acute leukemia Recently, calyculin-A-induced PCC has been combined with multicolor FISH [pq-COBRA-FISH (COmbined Binary RAtio labeling-fluorescence in situ hybridization)] for the cytogenetic analysis of cancer cell lines In the field of prenatal diagnosis, preliminary attempts have been made to combine calyculin-Ainduced PCC with GTG banding for fetus examination Furthermore, a major area of experimental therapeutic research centers on the function of anticancer drugs. In this field, the PCC methodology has been applied to study the effect of several anticancer drugs, such as BCNU, CCNU, Cis-acid, VM-26, adriamycin, and neocarzinostatin, whose mode of action depends on cell cycle arres