Evaluation of chemically induced cytogenetic lesions in rabbit oocytes: II. A comparison of streptonigrin effects on somatic and germ cells

The dose-response relationships for streptonigrin (NSC-45383)-induced chromosome aberrations in rabbit somatic cells are compared with dose-response data derived from the analysis of inherited structural chromosome abnormalities in preimplantation embryos from female rabbits treated with streptonigrin prior to mating. The incidence of inherited aberrations assessed in over 1000 karyotype preparations from 361 6-day blastocysts obtained from 55 female rabbits is used to derive a measure of the transmissible cytogenetic damage induced in the oocytes. The cytogenetic damage assessed in 2300 lymphoblast metaphases from 23 rabbits and 2750 marrow-derived metaphases from 27 rabbits which were collected and prepared for examination 6 h after the initiation of streptonigrin dosing are used to obtain estimates of the somatic cell insult. A uniform maximum likelihood analysis technique is applied individually to the 3 sets of data to derive the coefficients of the dose-response relationships. The resulting equations are Y = 0.6 ± 28.0 (×10^?5) + 8.2 ± 5.1 (×10^?4χ for inherited aberrations in 6-day blastocysts, Y = 9.7 ± 3.3 (×10^?3 + 1.9 ± (×10^?3)χ for bone-marrow cells, and Y = 2.8 ± 0.7 (×10^?2 + 4.8 ± 0.2 (×10^?3)χ for the lymphoblasts. In the somatic tissues Y is the percentage of cells with chromosome breakage, while in the blastocyst data Y is the percentage of 6-day blastocysts with consistent structural chromosome aberrations, and in all equations χ is the total streptonigrin dose in μg/kg.The study shows that streptonigrin injections in the range of 30–90 μg/kg when given to sexually mature female rabbits cause dose-dependent increases in chromosome aberrations in 2 types of somatic cells and in the incidence of inherited aberrations recovered in 6-day blastocysts. The coefficients of damage recovered in blastocysts versus damage recovered in somatic cells have the ratio of 1:2.3:5.8 (blastocysts: bone marrow: lymphoblasts). The results are discussed in terms of risk assessment and kinetics of aberration loss during meiosis and early embryonic development. The conclusion drawn from the study is that somatic cell cytogenetic damage is in some way predictive of damage incurred by oocytes which can be passed on to preimplantation embryos, at least for agents like streptonigrin.     

Studies on chemically induced dominant lethality. I. The cytogenetic basis of MMS-induced dominant lethality in post-meiotic male germ cells.

Young adult male mice were injected intravenously with doses of methyl methanesulfonate(MMS) ranging from 25 to 100 mg/kg body weight. These males were serially mated to superovulated females from day 1 post injection to day 23 post injection. The morning after mating (about 4-6 h post-copulation) the females were sacrificed and ova flushed from the ampulla. The ova were cultured, in the presence of colchicine, for 26 h and metaphase preparations made of the first cleavage division. Chromosome analysis was done and the types, and extent, of chromosome aberrations correlated to previously published dominant lethal data at the same MMS doses and time intervals. The types of aberrations seen were predominantly double fragments (presumably isochromatid deletions), chromatid interchanges, and some chromatid deletions, as well as shattering effect on the male complement at the highest dose and the time of peak sensitivity to dominant lethal induction. When the frequency of cells containing a cytologically visible aberration is compared to the total dominant lethal data an excellent correlation is obtained. Furthermore, the frequency of highly damaged cells, agrees very well with estimated frequencies of preimplantation loss. These data strongly suggest that chromosome aberrations seen at the first cleavage stage are the basis of MMS-induced dominant lethality.     

The quantitative histochemistry of a chemically induced ependymoblastoma. I. Enzymes

Enzymes from several metabolic pathways were studied quantitatively in homogenates and in homogeneous areas of frozen-dried cryostat sections of an experimental, mouse ependymoblastoma, mouse mammary carcinoma, and mouse melanoma, growing as transplants in mouse brain. Micro analyses were performed in fiveto sixfold replicates on portions of tumour with a dry weight of 0·03-0·2 μg. A close resemblance of the enzyme spectrum of the ependymoblastoma to that of immature brain was noted. Hexokinase and malate dehydrogenase were lower and lactate, glucose-6-phosphate, and NADP⁺-linked isocitrate dehydrogenases, and β-glucuronidase higher in the ependymoblastoma than in whole, adult mouse brain. Mouse mammary carcinoma had a higher level of hexokinase and lower levels of lactate and glucose-6-phosphate dehydrogenases and β-glucuronidase than ependymoblastoma. The concentration of malate dehydrogenase was lower and that of lactate, glucosed-6-phosphate, and NADP⁺-linked isocitric dehydrogenases was higher in the melanoma than in the ependymoblastoma. β-Glucuronidase levels were similar in these two neoplasms. It is suggested that the relatively high levels of several NADP⁺-linked enzymes in the ependymoblastoma may be related to increased capacity for lipid synthesis.     

Influence of acetylcysteine on cytogenetic effects of etoposide in mouse oocytes

The influence of N-acetylcysteine (ACC) on the cytogenetic effects of etoposide in F₁ CBA × C57BL/6 mice was studied. Etoposide introduced intraperitoneally in doses of 10, 20, 40, and 60 mg/kg has a dose-dependent clastogenic activity and has an aneugenic effect with the induction of mainly hypohaploid oocytes. ACC significantly decreases the aneugenic and clastogenic activity of etoposide (20 mg/kg) in oocytes of 6-, 9-, and 12-week-old mice during triple introduction at a dose 200 mg/kg per os. The most pronounced anticlastogenic ACC activity (an 80% decrease) was registered in 9-week-old females; a 100% decrease in aneugenesis was detected in 6-week-old female mice.     

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.     

Molecular cytogenetic analysis in mouse sperm of chemically induced aneuploidy: studies with topoisomerase II inhibitors

The ability of two topoisomerase II (topo II) inhibitors, etoposide (VP-16) and merbarone (MER), to induce meiotic delay and aneuploidy in mouse spermatocytes was investigated. The progression from meiotic divisions to epididymal sperm was determined by injecting male mice with 5-bromo-2′-deoxyuridine (BrdU) and treating the animals 13 days later with the test chemicals. At 20–24 days after treatment, BrdU-containing sperm were identified with a FITC-labelled anti-BrdU antibody and green fluorescent sperm were scored with a laser scanning cytometer (LSC). It was found that VP-16 (50 mg/kg) treatment induced a meiotic delay of about 24 h. A significant reduction of BrdU-labelled sperm was observed at 22 days compared to the controls (VP-16 group: 14.20%; controls: 41.10%, P<0.001). At 23 and 24 days, there were no significant differences between the VP-16 and the control groups. MER (80 mg/kg) treatment did not cause meiotic delay. To determine the frequencies of hyperhaploid and diploid sperm, male mice were treated with 12.5, 25 and 50 mg/kg VP-16 or 15, 30 and 60 mg/kg MER. Sperm were sampled from the Caudae epididymes 24 days after VP-16 treatment or 22 days after MER treatment. Significant increases above the concurrent controls in the frequencies of total hyperhaploid sperm were found after treatment with 25, 50 mg/kg VP-16 (0.074 and 0.122% versus 0.052%) and after treatment with 60 mg/kg MER (0.098% versus 0.044%). Furthermore, significant increases in the frequencies of diploid sperm were found after treatment of mice with all three doses of VP-16 (0.024, 0.032 and 0.056% versus 0.004 and 0.00%, respectively) and with 30 and 60 mg/kg MER (0.022 and 0.05% versus 0.004 and 0.002%, respectively). All dose responses could be expressed by linear equations. The results indicate that cancer patients may stand transient risk for siring chromosomally abnormal offspring after chemotherapy with these topo II inhibitors.     

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

Physical and dosimetric characteristics of the gamma-radiation field which is formed in the room containing the 60Co radiation source were investigated on condition of an unclosed breech mechanism and the presence of the lead layer on the beam pathway. The inverse square law was approximately found for the dependence of the dose rate vs distance on the radiation source both for the unclosed breech mechanism and lead layer (4 and 9 cm wide) in the beam pathway. This finding indicated a non-significant contribution of the radiation scattered from the walls at the point of cytogenetic experiments. The Monte Carlo calculations showed that some changes in the efficient spectrum of gamma-radiation resulted in a decreased average energy of 60Co gamma-rays to 1.03, 1.17 and 1.07 MeV for the unclosed breech mechanism, behind 4 and 9 cm lead layers, respectively.     

Acute and chronic effects of chemically induced unilateral renal disease in rats.

Chemically induced unilateral renal disease was associated with a high incidence of proteinuria, diuresis, a morphological spectrum ranging from perinephritis to acute tubular or cortical necrosis, and unilateral or bilateral glomerular fibrinogen deposition during the first 2 wk after induction. Later, a decrease in proteinuria and return to normal urine output was not infrequently followed by recurrent proteinuria, hypergammaglobulinemia, morphological alterations, and deposition of IgG and beta1C on the glomerular basement membranes and mesangium of the contralateral kidney and the treated kidney. Intercapillary deposition of fibrinogen in association with IgG and beta1C was occasionally observed in one or both kidneys. The morphologic, immunohistologic, serologic, and chemical findings suggest that this model may be useful for further defining the course and prognosis of unilateral renal disease produced by vascular insufficiency.     

Receptor-mediated endocytosis in Xenopus oocytes. I. Characterization of the vitellogenin receptor system

We have investigated the vitellogenin (VTG) receptor system in Xenopus oocytes since these cells are specialized for endocytosis. Oocytes have between 0.2 and 3 X 10(11) receptors per 1-mm cell. There is only a single class of receptors of low affinity (1.3 X 10(-6) M at 22 degrees C and 2-4 X 10(-6) M at 0 degree C), but high specificity (less than 5% nonspecific binding at 2 X 10(-6) M). The specific internalization rate of the VTG receptor (around 2 X 10(-3) s-1) is first order, highly variable, and at the upper end of the range of values reported for mammalian cells. The receptor association rate constant (9.6 X 10(2) M-1 s-1) is extremely low although the dissociation rate constant was immeasurable. Calcium is required for VTG binding, and low pH does not dissociate the VTG-receptor complex. Monensin treatment at 100 microM caused the loss of surface receptors with a t1/2 of 3 h and the accumulation of internalized ligand in a "pre-lysosomal" endocytic compartment. Conversely, the recovery of surface VTG receptors that were removed with trypsin occurred with a t1/2 of about 2 h. These observations indicate that oocytes have very large intracellular pools of receptors and that although surface receptors are internalized on the time scale of minutes, the intracellular pool is recycled on the time scale of hours.     

Aneuploidy in Drosophila, I. Genetic test systems in the female Drosophila melanogaster for the rapid detection of chemically induced chromosome gain and chromosome loss

An account is provided of two genetic schemes in the Drosophila melanogaster female designed as rapid detectors of chemically induced aneuploidy, including both chromosome gain and chromosome loss. One scheme is referred to as FIX, in which the female carried free (heterozygously) inverted X (chromosomes) and the other, ZESTE, where females do not carry inversions and the X-linked sexually dimorphic zeste mutation plays the key role in the detection of aneuploid offspring. The principle attribute of the FIX system is that all euploid offspring are wild-type for body and eye color whereas aneuploid females have a yellow body and aneuploid males white eyes; int he ZESTE system all euploid individuals are wild-type for eye color, aneuploid females possess zeste-colored eyes and aneuploid males white eyes. In addition induced polyploidies (2X:2A gametes) appear as yellow and zeste male intersexes in the FIX and ZESTE systems, respectively. In this way all aneuploids are recognized immediately. Consequently, detection of compounds with weak effects requiring large sample sizes may be made in a fraction of the time associated with more traditional schemes for aneuploidy detection in Drosophila.     

Evaluation of the cytogenetic damage induced by the organophosphorous insecticide acephate

The organophosphorous insecticide acephate was tested for its ability to induce in vitro cytogenetic effect in human peripheral lymphocytes by using the chromosomal aberrations (CAs), sister chromatid exchange (SCE) and micronuclei (MN) assay. The level of nuclear DNA damage of acephate was evaluated by using the comet assay. Concentrations of 12.5, 25, 50, 100 and 200 μg mL⁻¹ of acephate were used. All concentrations of acephate induced significant increase in the frequency of CAs and in the formation of MN dose dependently (r = 0.92 at 24 h, r = 0.95 at 48 h for CAs, r = 0.87 for MN). A significant increase was observed in induction of SCE at 50, 100 and 200 μg mL⁻¹ concentrations during 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) during 48 h treatment period in a dose-dependent manner (r = 0.84 at 24 h, r = 0.88 at 48 h). Acephate did not affect the replicative index and cytokinesis-block proliferation index (CBPI). However, it significantly decreased the mitotic index at all three highest concentrations (50, 100, 200 μg mL⁻¹) for 24 h treatment and at all concentrations (except 12.5 μg mL⁻¹) for 48 h treatment, dose-dependently (r = 0.94 at 24 h, r = 0.92 at 48 h). A significant increase in mean comet tail length was observed at 100 and 200 μg mL⁻¹ concentrations compared with negative control in a concentration-dependent manner (r = 0.94). The mean comet tail intensity was significantly increased at only 200 μg mL⁻¹ concentration. The present results indicate that acephate is a clastogenic, cytotoxic agent and it causes DNA damage at high concentrations in human lymphocytes in culture.     

Studies on chemically induced dominant lethality. II. Cytogenetic studies of MMS-induced dominant lethality in maturing dictyate mouse oocytes.

Young adult female mice were injected intravenously with either 50- or 100- mg/kg doses of methyl methanesulfonate. The females were superovulated and mated to untreated males at intervals ranging from 0.5 to 14.5 days after treatment. The fertilized ova were collected and cultured to the first cleavage mitosis, at which time the female chromosome complement was analyzed for structural chromosomal damage. Chromatid-type aberrations were observed, but at a much lower frequency than previously reported for treatment of post-meiotic male germ cells. The time after treatment at which chromosomal damage was observed and the frequency of affected cells agree, qualitatively, with existing dominant-lethal data derived from treatment of maturing oocytes. Parallel experiments in which metaphase I oocytes were analyzed indicate a lack of MMS-induced chromosomal damage in the meiotic stages. This observation is consistent with the suggestion that an intervening round of DNA synthesis is necessary for MMS-induced lesions to be translated into chromosomal damage. The low yield of chromosomal damage is consistent with the idea that maturing oocytes, unlike later spermatids and spermatozoa, are capable of performing macromolecular repair of premutational lesions.     

The chromosomal normality of in vitro-fertilized rabbit oocytes

The chromosomal normality of rabbit oocytes fertilized in vitro was examined. Ovum donors were superovulated with pregnant mare's serum gonadotropin and human chorionic gonadotropin (hCG). Follicular oocytes were collected laparoscopically 9-10 h after hCG treatment and incubated in vitro with spermatozoa capacitated in vivo. Of 267 aspirated oocytes, 191 (71.4%) were fertilized in vitro and developed to the 2- to 8-cell stage 24-48 h after insemination. In the chromosomal studies, 121 (63.4%) were examined. Of these, 94 (77.7%) had a normal diploid complement of chromosomes (2n = 44) and 6 (5%) showed aneuploidy. Of the remaining 21, 20 were triploid and one was tetraploid. The incidence of triploid oocytes after in vitro fertilization was higher than the rate in vivo (16.5% vs. 9.0%, respectively). These triploid oocytes were suspected to be the result of polyspermic fertilization in vitro. In addition, at the Metaphase II stage, 62 (89.9%) of 69 induced, preovulatory oocytes had a normal number of chromosomes.     

Anti-tumor immunity in B lymphocyte-deprived mice. I. Immunity to a chemically induced tumor.

Immunity to a syngeneic methylcholanthrene-induced tumor was studied in (C57BL/6 X C3H)F1 males, treated continuously from birth with a rabbit anti-mouse IgM serum. Such mice have been shown to be selectively depleted of Ig-bearing lymphocytes and incapable of synthesizing antibodies. In the experiments reported, a heightened resistance of these mice to the syngeneic fibrosarcoma was demonstrated. This resistance was manifest in significantly slower tumor growth at the site of injection, as well as a lower incidence of spontaneous pulmonary metastasis.