Comparison of the cytogenetic effects of cyclophosphamide on monkey lymphocytes in vivo and in vitro

The comparative in vivo and in vitro study of chromosomal aberrations and SCE induced by cyclophosphamide (CP) in macaca rhesus lymphocytes was performed. The dose of mutagenic exposure for quantitative estimation of effects was determined as a product of concentration of alkylating CP metabolites on the exposure time. The mutagenic effect caused by the same doses of CP (CP metabolites) appeared similar in vivo and in vitro. This suggests that the results obtained in adequate in vitro mutagen-testing experiments may be quantitatively extrapolated for the in vivo conditions.     

Chromosome aberrations produced in human lymphocytes by in vivo and in vitro irradiation

The production of chromosome aberrations in vivo has been studied in lymphocytes from a patient undergoing a wholebody treatment with gamma-radiation up to a cumulative dose of 1.4 Gy. These results were compared with the observations performed on whole blood samples irradiated in vitro with doses from 0.05 up to 2 Gy of gamma-rays. The frequency of chromosome aberrations, particularly the dicentrics, was found to be similar in vivo and in vitro. The yield of dicentrics could be best related to the dose by using a linear-quadratic model in both cases, the ratio of the coefficients a/b being of 0.56 and 0.69 Gy, respectively in vivo and in vitro. These observations confirm that in vitro dose response curves may be used to evaluate accurately an in vivo absorbed dose.     

Cytogenetic effects of cyclophosphamide on human lymphocytes in vivo and in vitro

A comparative study of cytogenetic effects in human lymphocytes caused in vivo by cyclophosphamide (CP) after intravenous injection and in vitro by exposure of plasma of the same patients was carried out. It was found that the frequency of induced chromosome aberrations (CA) and sister-chromatid exchanges (SCE) increased linearly for SCE and exponentially for CA within the 'dose' of alkylating activity of CP metabolites. Parameters of 'cytogenetic effect-dose' in vivo and in vitro coincided. The intensity of cytogenetic effects varied between individuals.     

Cytogenetic effect of barbiturate anesthetizing substances in a human peripheral blood leukocyte culture

It is found that hexenal and sodium thiopental in vitro produced a two-fold increase of frequency of chromosome aberrations as compared with the control and this effect was not dose-dependent. The anesthetics under study affected in vitro 3H-thymidine incorporation into DNA of lymphocytes, and a ten-fold hexenal dose intensified 3H-thymidine incorporation. The frequency of chromosome aberrations in vivo was at the level of the spontaneous mutation after use of sodium thiopental and slightly increased hexenal.     

Chromosome analysis of human spermatozoa following in vitro exposure to cyclophosphamide, benzo(a)pyrene and N-nitrosodimethylamine in the presence of rat liver S9

For the purpose of assessing mutagenic effects (clastogenicity) of metabolites derived from chemical mutagens/carcinogens on human sperm chromosomes, spermatozoa were exposed in vitro to cyclophosphamide (CP), benzo(a)pyrene (BP) or N-nitrosodimethylamine (NDMA) for 2h in the presence or absence of rat liver S9, a metabolic activator of these chemicals. After in vitro fertilization between human spermatozoa and zona-free hamster oocytes, chromosome complements of sperm origin were analyzed cytogenetically.In the absence of S9, none of three chemicals (20 microg/ml CP, 200 microg/ml BP and 20mg/ml NDMA) caused a significant increase in spermatozoa with structural chromosome aberrations (8.6, 10.0 and 7.5%), as compared with their matched controls (10.9, 11.0 and 8.5%). In the presence of S9, however, a significant increase in chromosomally abnormal spermatozoa was observed in CP (37.1%, P < 0.001) and BP (31.0%, P < 0.001), indicating that enzymatic activation of CP and BP induced chromosomal abnormalities in human sperm. In contrast, NDMA did not induce chromosome aberrations in human spermatozoa by S9 treatment, although positive results have been observed in somatic cells. The present results on in vitro clastogenicity of CP, BP and NDMA are consistent with the results in previous in vivo studies with murine spermatozoa. Our S9/human sperm chromosome assay seems to be useful for estimation of hereditary risk of chemicals in human. Because most chemicals need metabolic activation to bind to DNA.     

Mutagenic activity of anticancer agent cis-dichlorodiammine platinum-II.

cis-Dichlorodiamminoplatinum-II (cis-DDP) has been widely used as an anticancer chemotherapeutic agent. The mutagenicity of cis-DDP was investigated in vitro and in vivo using sister-chromatid exchange analysis and the analysis of chromosomal aberrations. Parallel human lymphocyte cultures were incubated with and without the addition of BrdU at 4 concentrations of cis-DDP. Significant increases in SCE rate were observed at 0.25 micrograms/ml and higher, showing a clear dose-response relation between SCE rate and cis-DDP concentration. A significant increase in chromosome breakage and tetraradial figures was observed in BrdU free cultures treated with cis-DDP again showing a dose dependency. Analysis of the distribution of cells in the first, second and third division in cis-DDP treated cultures demonstrated the depressing effect of the drug on mitotic activity. In vivo analysis of SCE and chromosome aberrations in mouse showed that 13.85 mg/kg i.p. of cis-DDP produces significant increases in the rate of SCE and chromosome aberrations in bone-marrow cells.     

Comparison of the frequency of chromosome aberrations induced by thiophosphamide in rabbit lymphocytes in vitro and in vivo

Rabbit lymphocytes were treated with thiophosphamide in vivo and in vitro. In-vitro doses were calculated by multiplying thiophosphamide concentrations by the time of treatment (the doses ranged within 0-1500 mg/min/ml). In-vivo doses were calculated as integral of thiophosphamide concentration function from the time of administration till the time of blood sample collection (the doses ranged within 0-1900 mg/min/ml). It was shown that with the dose increase the rate of chromosome aberrations and the number of disruptions per cell rise exponentially in vivo and in vitro. At the same time the parameters of regression equations coincide. This evidences that thiophosphamide produces the same effect in vivo and in vitro.     

Ratio of the frequency of chromosome aberrations and sister chromatid exchanges to the dose of the mutagenic exposure to cyclophosphamide in vivo and in vitro

Cyclophosphamide (CP) and its metabolites were used to compare the rate of chromosomal aberrations (CA) and sister chromatid exchange (SCE) in the rabbit lymphocytes in vivo and in vitro. The dose-dependent increase of cytogenetic effects rate appeared to be of linear and exponential dependence for SCE and CA, respectively, both in vivo and in vitro. The regression equation coefficients coincided in in vivo and in vitro experiments.     

Comparison of cytogenic response of human lymphocytes to in vivo and in vitro exposure to low doses of gamma-rays. Unstable chromosomal exchanges detected by FPG techniques

On peripheral lymphocytes of eight cancer patients undergone whole-body therapeutic irradiation (at daily dose of 10 cGy up to total dose of 50 cGy of 60Co gamma-rays) the dose-response of unstable chromosome exchanges (dicentrics and centric rings) was studied. This dose response fitted well linear function. The lower slope of dose-response curve was found for in vivo irradiated lymphocytes as compared to the dose response curve obtained for in vitro irradiated lymphocytes of the same patients. This finding seems to provide evidence that in case of protracted irradiation of individuals an absorbed dose could be underestimated if for biological dosimetry an in vitro dose response curve for unstable chromosome aberrations is used as referent one.     

Comparison of the levels of chromosome aberration and sister chromatid exchange induced by chemical mutagens in vitro

Dose dependencies of the induction of sister chromatid exchanges (SCEs) and chromosome aberrations were studied under in vivo exposure of mouse bone marrow cells to 5 alkylating agents. The efficacy of the induction of SCEs for all the substances was 20 to 60 times higher than that of the induction of chromosome aberrations. It was demonstrated that SCEs induced by chemical mutagens in vivo and in vitro are more sensitive tests than chromosome aberrations.     

Genotoxic evaluation of Ara-C by multiple parameters

The cytogenetic effects of the antimetabolite, cytosine arabinoside (Ara-C) are evaluated using in vivo and in vitro test systems and applying multiple parameters. The in vivo assay was carried out on 8-10-week-old inbred Swiss albino male mice using bone marrow as the somatic test system and the cells of testis as the meiotic test system. In vitro human leukocyte cultures were also employed. In vivo experimental doses were computed on surface area basis within the therapeutic dose range and injected intraperitoneally and for in vitro they were calculated on blood volume basis. Evaluation of somatic chromosome mutations included conventional screening for chromosome aberrations, variations in mitotic index and sister-chromatid exchanges (SCEs) by in vivo and in vitro methods besides studies on meiotic test systems using conventional screening for chromosome and sperm-head abnormalities. The quantitative data were subjected to statistical analysis by applying appropriate tests to evaluate their significance. The results of in vivo and in vitro experiments reveal the chromosome mutational activity of the compound. This is further supported by data on SCEs from both systems. However, a comparison of both demonstrated a differential mutagenic response of the drug, more in vivo than in vitro. This is also true for SCEs. Even though the mechanisms involved in causing chromosome aberrations and SCEs are different, the data on both corroborate each other on induction of chromosome mutations.     

Chromosome aberration assays for the study of cyclophosphamide and Bacillus thuringiensis in Oxya chinensis (Orthoptera: Acrididae)

Chromosome aberrations induced by an anti-neoplastic drug, cyclophosphamide (CP) and a bioinsecticide, Bacillus thuringiensis (B.t.) were examined using grasshoppers as an animal model, with injection as the route of exposure. Oxya chinensis (Thunberg), having a small number (2n male symbol =23) of large-sized chromosomes in males, was used for this purpose. The fifth instar nymphs were treated with various concentrations of CP (2, 5 and 10 mg/ml) and B.t. (0.55, 1.83 and 5.50 IU/ml) by injection into the abdomen, using physiological saline and distilled water as negative controls, respectively. The chromosomal preparations were made from the spermatogonia of the specimen testis at different intervals after dosing (24 and 48 h). The effect of the high dose of CP (10 mg/ml) in O. chinensis was also analyzed at the 42-h time point. The chromosome aberrations observed were mainly chromatid and chromosome breaks. CP induced a dose- and time-dependents increase in the number of chromosome aberrations (CAs) per cell and in the percentage of aberrant cells. The strongest effect was seen when grasshoppers were injected with the highest dose and cells were analyzed at the 48-h time point. The results show that CP induced a significant increase in the frequency of CAs in testicular cells of O. chinensis with the three doses employed, compared to the negative control. Our results suggest that there exists in the grasshopper an enzyme system analogous to liver-S9 fraction, and that CP may be used as a positive control in genotoxicity test in this species. In addition, the evaluation of the chromosome aberrations induced by B.t. in the grasshoppers' testicular cells showed that B.t. may induce chromosome aberrations, mainly chromatid and chromosome breaks, in spermatogonia. By statistical analysis, B.t. showed significant dose-effect relationships and it may be mutagenic in this species. Recent research has focused on the development of biological insecticides to protect cereal crops against damage by insect species, such as beetles and grasshoppers. The present studies may contribute to our knowledge of entomological genotoxicity in grasshoppers and provide reference for the research on the mechanism of B.t. toxicity.     

Effect of thyroxine in the G0 stage of the cell cycle on the yield of chromosome aberrations in rat hepatocytes and human lymphocytes after X-irradiation

A study was made of the mechanism of a modifying action of thyroxin (T4) on the chromosome integrity after the application thereof in vivo and in vitro following a single whole-body exposure to X-radiation with a dose of 2.19 Gy. It is concluded that T4 influences the quantity of cells with chromosome aberrations stimulating the recovery of the chromosome integrity: the stimulation can be brought about at the G0 stage of the cell cycle as a result of the direct effect of the hormone on a cell.     

Studies on chromosome aberrations in the eggs of mice fertilized in vitro after irradiation. I. Chromosome aberrations induced in sperm after X-irradiation

The induction of chromosome aberrations in eggs of mice fertilized with X-irradiated sperm was performed by using an in vitro fertilization technique. Capacitated mature sperm was irradiated with various doses of X-rays and cytological analysis of the first cleavage metaphase of in vitro fertilized eggs was made. The frequencies of chromosome aberrations increased exponentially with dose and the dose-response relationship for overall breaks fitted well to a quadratic equation. The chromosome aberrations were mainly chromosome-type (82.1%), and the majority of aberrations were fragments.     

Chromosome aberration assays for the study of cyclophosphamide and Bacillus thuringiensis in Oxya chinensis (Orthoptera: Acrididae)

Chromosome aberrations induced by an anti-neoplastic drug, cyclophosphamide (CP) and a bioinsecticide, Bacillus thuringiensis (B.t.) were examined using grasshoppers as an animal model, with injection as the route of exposure. Oxya chinensis (Thunberg), having a small number (2n♂=23) of large-sized chromosomes in males, was used for this purpose. The fifth instar nymphs were treated with various concentrations of CP (2, 5 and 10 mg/ml) and B.t. (0.55, 1.83 and 5.50 IU/ml) by injection into the abdomen, using physiological saline and distilled water as negative controls, respectively. The chromosomal preparations were made from the spermatogonia of the specimen testis at different intervals after dosing (24 and 48 h). The effect of the high dose of CP (10 mg/ml) in O. chinensis was also analyzed at the 42-h time point. The chromosome aberrations observed were mainly chromatid and chromosome breaks. CP induced a dose- and time-dependents increase in the number of chromosome aberrations (CAs) per cell and in the percentage of aberrant cells. The strongest effect was seen when grasshoppers were injected with the highest dose and cells were analyzed at the 48-h time point. The results show that CP induced a significant increase in the frequency of CAs in testicular cells of O. chinensis with the three doses employed, compared to the negative control. Our results suggest that there exists in the grasshopper an enzyme system analogous to liver-S9 fraction, and that CP may be used as a positive control in genotoxicity test in this species. In addition, the evaluation of the chromosome aberrations induced by B.t. in the grasshoppers’ testicular cells showed that B.t. may induce chromosome aberrations, mainly chromatid and chromosome breaks, in spermatogonia. By statistical analysis, B.t. showed significant dose–effect relationships and it may be mutagenic in this species. Recent research has focused on the development of biological insecticides to protect cereal crops against damage by insect species, such as beetles and grasshoppers. The present studies may contribute to our knowledge of entomological genotoxicity in grasshoppers and provide reference for the research on the mechanism of B.t. toxicity.     

The low dose and low dose rate cytogenetic effects induced by gamma-radiation in human blood lymphocytes in vitro. II. the results of cytogenetic study

The yield of chromosome aberrations induced by gamma-radiation of 60Co in human blood lymphocytes in vitro at low doses (30 divided by 600 mGy) and low dose rates (0.70, 5.05, 59.2 mGy/min) was investigated. It was found that the observed level of chromosomal aberrations induced by gamma-irradiation was unaffected by the value of the dose rate when using constant dose rate and obtaining different doses by altering the exposure time. However, a relatively enhanced level of chromatid aberrations was found at 5.05 and 59.2 mGy/min dose rates in the dose range less than 250 mGy. We have found that the observed level of the sum of chromosomal aberrations induced by gamma-irradiation at doses less than 250 mGy and a dose rate of 59.2 mGy/min was essentially larger compared with the level extrapolated from high doses (above 300 mGy) using a linear-quadratic dose curve. This complied with our previous finding in 1976, 1977 when the enhanced level of dicentrics was only found at a high dose rate approximately 500 mGy/min. Such a non-linear cytogenetic effect does not manifest itself statistically significantly at dose rates of 0.70 and 5.05 mGy/min for the sum of chromosomal aberrations and does not manifest itself at all for dicentrics at all the examined dose rates.     

A comparative study of the micronucleus test and chromosome aberrations in PHA-stimulated human lymphocytes

A dose dependence of the number of cells with chromosome aberrations was studied in PHA-stimulated donor's peripheral blood lymphocytes irradiated in vitro with doses of 10-400 cGy. In studying the number of chromosome aberrations and percentage of cells with micronuclei in parallel cultures no correlation was found between these indices within the groups exposed to a similar radiation dose.     

The effect of ethylnitrosourea on chromosome aberrations in vitro and in vivo.

The effect of ENU on (A) human chromosomes from blood lymphocyte cultures in vitro, and on (B) rat and mouse bone marrow chromosomes in vivo, was investigated. Doses of 25, 50, 100 and 200 mug/ml were tested in vitro and cells with chromosome breakage were found to be dose dependent. Chromosome damage was also dependent on time; maximum damage was seen when cells were treated 2--6 hrs before harvest. Two doses of 100 and 200 mg/kg were studied in rat and mouse in vivo and a dose effect could be shown in both species. The highest number of abnormal cells was found 6 hrs after treatment; there was a sharp decrease at 18 hrs and thereafer. Types of aberrations were also analyzed, in both in vitro and in vivo studies.     

Report on the Eleventh International Workshop on the Identification of Transcribed Sequences 2001. November 9-11, 2001. Washington, DC, USA

Glioblastoma multiforme (GBM) is characterized by intratumoral heterogeneity as to both histomorphology and genetic changes, displaying a wide variety of numerical chromosome aberrations the most common of which are monosomy 10 and trisomy 7. Moreover, GBM in vitro are known to have variable karyotypes within a given tumor cell culture leading to rapid karyotype evolution through a high incidence of secondary numerical chromosome aberrations. The aim of our study was to investigate to what extent this mitotic instability of glioblastoma cells is also present in vivo. We assessed the spatial distribution patterns of numerical chromosome aberrations in vivo in a series of 24 GBM using two-color in situ hybridization for chromosomes 7/10, 8/17, and 12/18 on consecutive 6-microm paraffin-embedded tissue slides. The chromosome aberration patterns were compared with the histomorphology of the investigated tumor assessed from a consecutive HE-stained section, and with the in vitro karyotype of cell cultures established from the tumors. All investigated chromosomes showed mitotic instability, i.e., numerical aberrations within significant amounts of tumor cells in a scattered distribution through the tumor tissue. As to chromosomes 10 and 17, only monosomy occurred, as to chromosome 7 only trisomy/polysomy, apparently as a result of selection in favor of the respective aberration. Conversely, chromosomes 8, 12, and 18 displayed scattered patterns of monosomy as well as trisomy within a given tumor reflecting a high mitotic error rate without selective effects. The karyotypes of the tumor cell cultures showed less variability of numerical aberrations apparently due to clonal adaptation to in vitro conditions. We conclude that glioblastoma cells in vivo are characterized by an extensive tendency to mitotic errors. The resulting clonal diversity of chromosomally aberrant cells may be an important biological constituent of the well-known ability of glioblastomas to preserve viable tumor cell clones under adaptive stress in vivo, in clinical terms to rapidly recur after antitumoral therapy including radio- or chemotherapy.     

Equal induction and persistence of chromosome aberrations involving chromosomes 1, 4 and 10 in thyroid cancer patients treated with radioactive iodine

A number of in vitro studies have questioned the assumption of random distribution of breaks in radiation-induced chromosome aberrations. The therapeutic application of radioactive 131I in thyroid cancer patients offers a good opportunity to study the induction and persistence of cytogenetic damage involving different chromosomes in vivo. Using whole-chromosome painting probes and triple colour painting by fluorescence in situ hybridization (FISH), we have analysed the frequency of chromosomal aberrations (CAs) involving chromosomes 1, 4 and 10 in peripheral blood lymphocytes of 10 thyroid cancer patients sampled before and 1 week, 1 year and 3.5 years after therapeutic application of radioactive iodine in a self-controlled, longitudinal study. A highly significant 3.4-fold increase in the frequency of chromosome breaks was observed 1 week after treatment with a similar representation of all chromosomes analysed. Although a significant decrease in dicentrics was observed during the first year after treatment, the frequency of chromosome aberrations remained over control levels until the last sampling time, 41-47 months post-treatment. The same behaviour, in terms of induction and persistence, was observed for all three chromosomes, confirming our previous results in vitro and rejecting the reported suggestion that chromosome 10 is radiosensitive in vivo. Our finding that the dynamics of radiation-induced CA in vivo is independent on the chromosome of choice suggests that this variable is not important in retrospective studies.