Translocation Yield Research Articles

Human chromosome-specific changes in a human-hamster hybrid cell line (A L) assessed by fluorescent in situ hybridization (FISH)

Purpose : To quantitatively assess all gamma-ray induced chromosomal changes confined to one human chromosome using fluorescence microscopy and in situ hybridization with a fluorescently labeled human chromosome specific nucleic acid probe. Methods and Materials : Synchronized human-hamser hybrid cells containing human chromosome 11 wer obtained by a modified mitotic shake-off procedure. G1 phase cells (> 95%)_ were irradiated with 137Cs gamma rays (0, 0.5, 1.0, 1.5, 2.0, 4.0, 6.0, 8.0 and 10.0 Gy) at a dose rate of 1.1 Gy/min and mitotic cells colleted 16–20 h later; chromosomal spreads were prepared, denatured,and hybridized with a fluorescein-tagged nucleic acid probe against total human DNA. Chromosomes were examined by fluorescence microscopy and all categories of change involving the human chromosome 11 as target, recorded. Results : Overall, of the 3104 human-hamster hybrid cells examined, 82.1% were euploid, of which 88.6% contained one copy of human chromosome 11, 6.2% contained two copies, and 5.2% contained 0 copies. This is compatiblewith mitotic nondisjunction in a small fraction of cells. Of the remaining 17.9% of cells, 85.2% were tetraploid cells with two copies of human chromosome 11. For all aberrations involving human chromosome 11 there was a linear relationship between yield and absorbed dose of 0.1 aberrations per chromosoem per Gy. The yield of dicentrics, translocations, and terminal deletions that involve one lesion on the human chromosoem was linear, while the yield of interstitial deletions that arise from two interacting lesions on the human chromosoem was curvilinear. The frequencies of dicentrics and translocations were about equal, while there was a high (40–60%) incidence of incomplete exchanges between human and hamster chromosomes. Conclusion : Fluorescent in situ hybridization (FISH) procedures allow for the efficient detection of a broad range of induced changes in target chromosomes. Symmetrical exchanges induced in G1 (translocations) were readily scored and found to equate with the complementary asymmetrical exchanges (dicentrics). That is, nonlethal stable changes, which might be concern ini carcinogenic processes, complement lethal, unstable changes. Interstitial deletions that may contribute to the loss of antioncogenes as well as to lethality are also readily detected with enhanced levels detected at higher doses. The high level of induced terminal deletions and of incomplete dicentrics and translocations indicate a partial failure of interaction between lesions induced in human and hamster DNA, and suggest that such interspecies interactions lack the fidelity of intraspecies DNA lesion interactions. This suggest caution in the use of such model systems as indicators of human cell responsiveness.

Fluorescence in situ hybridization detection of chromosomal aberrations in human lymphocytes: applicability to biological dosimetry.

Human lymphocytes in G0 have been irradiated with X-ray doses from 0 to 4.0 Gy. Metaphase chromosomes 2, 3 and 5 and all centromeres were painted using fluorescence in situ hybridization (FISH) probe libraries. Dicentrics, centric rings and acentrics in the whole genome as well as translocations involving the painted chromosomes were recorded. The translocations were subdivided as complete or incomplete. Interstitial insertions and inversions were also noted. The observations were also recorded according to the Protocol for Aberration Identification and Nomenclature Terminology (PAINT) system of scoring. Given that the painted chromosomes comprise 20.4% of the genome it was found that the yield of bicoloured dicentrics was consistent with the yield of dicentrics in the whole genome. The yield of radiation-induced translocations was not significantly higher than that of bicoloured dicentrics. Of the translocations, 60% were complete and it was concluded that the majority of dicentrics and translocations are complete exchanges. Chromosome 5 took part in exchanges marginally more commonly than its length suggests, but it is not known if this is a property of the chromosome or whether it is a donor-dependent observation. The PAINT system of recording rearrangements was examined and the suggested numerical interpretation of this nomenclature was considered to be unsuitable for use in the estimation of dose for cases of accidental overexposure.

Reduced Recovery of Translocations from X-Irradiated Spermatogonial Stem Cells of Dominant Spotting (W v /+) and Steel (Sl con /Sl con ) Mice

Dose-response relationships for X-ray-induced reciprocal translocations in spermatogonial stem cells of mutant and wild-type mice were established by spermatocyte analysis many cell generations after irradiation. The mutants studied were Wv/+, the viable allele of dominant spotting in the heterozygous state, and Slcon/Slcon, the homozygous contrasted allele of steel. The results show that the recovered yield of translocations was lowered in both mutants with steel being most extreme. Remarkably, however, no indications for enhanced cell killing were obtained in the mutants, and consequently the peak yields of translocations occurred at about the same dose level (6 Gy) as in normal mice. Histological analysis suggested that the postirradiation recovery of the germinal epithelium was retarded in the mutants with the effect in the steel mice again being most extreme. These differences in differentiation-multiplication patterns of regenerating spermatogonia after irradiation are probably responsible for the reduced recovery of translocations from the mutant mice.

The radiosensitivity of spermatogonial stem cells in C3H/101 F 1 hybrid mice

The radiosensitivity of spermatogonial stem cells of C3H/HeH × 101/H F 1 hybrid mice was determined by counting undifferentiated spermatogonia at 10 days after X-irradiation. During the spermatogenic cycle, differences in radiosensitivity were found, which were correlated with the proliferative activity of the spermatogonial stem cells. In stage VIII irr, during quiescence, the spermatogonial stem cells were most radiosensitive with a D 0 of 1.4 Gy. In stages XI irr−V irr, when the cells were proliferatively active, the D 0 was about 2.6 Gy. Based on the D 0 values for sensitive and resistant spermatogonia and on the D 0 for the total population, a ratio of 45:55% of sensitive to resistant spermatogonial stem cells was estimated for cell killing. When the present data were compared with data on translocation induction obtained in mice of the same genotype, a close fit was obtained when the translocation yield ( Y; in % abnormal cells) after a radiation dose D was described by Y = e τD , with τ = 1 for the sensitive and τ = 0.1 for the resistant spermatogonial stem cells, with a maximal e τD of 100.

Analysis of restriction enzyme-induced chromosomal aberrations by fluorescence in situ hybridization.

Fluorescence in situ hybridization and Giemsa staining of metaphase chromosomes were used to determine the relative frequencies of symmetric exchange aberrations (translocations) and asymmetric exchange aberrations (rings, dicentrics, and polycentrics) after exposure of human lymphoblastoid cells to restriction enzymes or X-rays. The yield of symmetric exchanges was determined with the use of chromosome-specific probes for human chromosomes 2 or 4, which were hybridized to metaphase chromosomes from cells exposed to the enzymes PvuII, SacI, or XbaI or 3 or 5 Gy of X-rays. The yield of asymmetric exchanges was determined in Giemsa-stained metaphase chromosomes from the same enzyme-treated or irradiated cell population. About 1.5- to 3-fold more symmetric than asymmetric exchanges were induced after restriction enzyme treatment. However, after X-ray treatment the yield of dicentrics relative to the yield of reciprocal translocations was close to the expected 1:1 ratio.

X-ray induced translocations in premeiotic germ cells of monkeys

The induction of reciprocal translocations by various X-ray exposures was studied in spermatogonial stem cells of rhesus monkeys ( Macaca mulatta) and stump-tailed macaques ( Macaca arctoides) by means of spermatocyte analysis many cell generations after irradiation. The yields of translocations recovered from irradiated stump-tailed macaques were lower than those observed in rhesus monkeys and represent in fact the lowest induction rates per Gy ever recorded for experimental mammals. In the rhesus monkey a humped dose-effect relationship was found with (a) a homogeneous response with (pseudo-)linear kinetics below 1 Gy, (b) much more variability at higher doses, and (c) no induction at all at doses of 4 Gy and above. It is suggested that the post-irradiation proliferation differention pattern of surviving rhesus monkey spermatogonial stem cells is mainly responsible for these characteristics of the dose-response curve.

The effect of sampling time on radiation-induced translocation yield in spermatogonial stem cells of male mice, differing in chromosomal constitution and sexual activity

We have investigated the frequency of reciprocal translocations in the first differentiating spermatogonia entering the first meiotic division after 2 x 2.5 Gy X-rays, given 24 h apart, as well as the development of this parameter in later stem-cell generations by studying multivalent configurations at the first meiotic division. Diakinesis-metaphase I cells were found for the first time between 30 and 40 days after irradiation. Subsequently, meiotic stages were sampled at 120, 180 and 280 days post irradiation. From day 40 post irradiation on, half of the males were allowed to impregnate females which enabled us to estimate the length of the post-irradiation sterile period, the development of litter size and the possible effect of sexual activity on the development of reciprocal translocation-containing stem cells. Half of the males were karyologically normal, the other half were homozygous for a reciprocal translocation (T/T) that affects testis weight and about halves sperm production. Irrespective of male karyotype, the first meiocytes had an induced translocation frequency of 9.00 ± 2.56% ,(n=8 males), followed by frequencies of 20.70 ± 4.87% (n= 15)at 180 days and 20.20 ± 4.30% (n= 20) at 280 days (males with and without mating behavior showing no difference). At 120 days post irradiation, +/+ males had a frequency of 14.59 ± 2.97% irrespective of sexual activity. T/T males (120 days post irradiation) that had mated showed a frequency of 18.63 ± 0.85% (n =4) compared with 13.64 ± 2.36% (n= 7) for those that had not. The observed fise of multivalent-carrying spermatocytes in time was highly significant. Notwithstanding the differences in testis weight and epididymal sperm count between the karyotypes, fertile matings occurred on average 72 days after irradiation, though with relatively wide margins. For the T/T karyotype, the first litter was statistically smaller than the subsequent litters. At 78 days post irradiation, testis weights were back in the subnormal range for both karyotypes and hardly improved in time . Restoration of fertility thus coincidedd with the period just prior to the return to subnormal The first diakinesis-methaphase I cells precede those that are nuumerous enough to accomplish ‘return to fertility’ by about 2 weeks. Thus differen a few dats after irradiation. A pattern of spermatologonial cell division compatible with ‘ return to fertility’ is only established some 2 weeks l

The induction of reciprocal translocations in mouse germ cells by chemicals and ionizing radiations II. Combined effects of mitomycin C or thio-tepa with two doses of γ-rays

The combined effects of mitomycin C (MMC) and thio-tepa (TT) with γ-rays doses of 5 and 9 Gy on mouse stem cells were studied using the spermatocyte test. Both chemicals induced very low yields of translocations after single treatments. In combined treatments with a dose of 5 Gy, a subadditive effect of MMC and an additive effect of TT were found. Combined with a dose of 9 Gy the compounds potentiated the effect of radiations. Up to now, most of the chemicals tested have shown additive effects when combined with doses of the ascending part of the dose-response curve and potentiating effects when combined with doses of its descending part. This has been considered additional confirmation of the concept that depletion of any kind of spermatogonia is sufficient to modify the genetic response of stem cells. However, the subadditive and additive responses found could be considered evidence that common biological mechanisms can modulate the response to combined treatments of chemicals and ionizing radiations.

Dose—response relationship for translocation induction in spermatogonia of the crab-eating monkey ( Macaca fascicularis) by chronic γ-ray-irradiation

The induction of reciprocal translocations in spermatogonia of the crab-eating monkey ( Macaca fascicularis) by chronic γ-irradiation (1.8·10 −5 Gy/min, about 0.024 Gy/22 h/day) was examined. The frequencies of translocation per cell were 0.15% at 0.3 Gy, 0.27% at 1.0 Gy and 0.33% at 1.5 Gy. The dose—response relationship for translocation yield was a linear one with a regression coefficient ( b) of 0.16 · 10 −2. When the slope ( b) of the regression line was compared with that at a high dose rate ( (0.25 Gy/min, b = 1.79 · 10 −2), it was clear that the induction rate of translocations after chronic γ-irradiation was only about one-tenth of that after high-dose-rate irradiation. Thus, there was evidence for a pronounced dose-rate effect in the crab-eating monkey.

Reciprocal translocations in germ cells of male mice receiving external γ-irradiation

Data reported in the literature up to 1985 on reciprocal translocation induction in male mouse germ cells by external γ-ray doses ranging from 0.5 to 6.0 Gy delivered at fixed dose rates were analyzed. On the assumption of a non-threshold linear dose response, zero effect at zero dose, and a center of distribution lying on an approximately straight line, calculations were made of linear regression coefficients. These coefficients ( b), as a function of the dose rate ( P), were well fitted by two straight lines: b = (3.15 + 0.59 log P) × 10 −6 for dose rates from 0.01 to 0.1 mGy/min; and b = (7.52 + 3.86 log P) × 10 −6 for dose rates ranging from 0.06 to 1.2 × 10 3 mGy/min. The intersection point of these two lines determined the so-called threshold level of the dose rate, namely, 4.6 × 10 −2 mGy/min, at which the effectiveness of external γ-irradiation is not expected to exceed 2.36 × 10 −6/mGy. In addition, experiments were undertaken in which yields were recorded of reciprocal translocations in germ cells of male mice exposed to 0.9 Gy of γ-radiation at dose rates ranging from 6.14 × 10 −3 to 6.14 × 10 2 mGy/min (6 levels); comparisons were made with data published up to 1985 from similar studies using other fixed doses. To do this, translocation yields were expressed as relative yields ( F) and their relationship to the dose rate ( P) for the individual fixed doses was represented by an equation of the type: F = α + β log P. For most of the equations, the regression coefficients were in good agreement and a single relationship was obtained to represent them. From the analysis performed it follows that, within the 0.6–6.0 Gy dose range, the pattern of the F vs. P relationship is unaffected by the dose. This supports the initial assumption that for the dose range up to 6.0 Gy the dose response for the reciprocal translocation yield is a non-threshold straight-line relationship.

Enhanced spermatogonial stem cell killing and reduced translocation yield from X-irradiated 101/H mice

The spermatogonial stem cells of 101/H mice have been found to be more sensitive to killing by acute X-ray doses than those of the “standard” C3H/HeH × 101/H F 1 hybrid. Duration of the sterile period was longer throughout the 0.5–8.0-Gy dose range tested and “recovered” testis weights, taken after recovery of fertility, were more severely reduced. The shapes of the sterile period dose-response curves were similar, but with the 101/H mice the plateau occured at 3–5 Gy, rather than at 6 Gy. An equivalent observation was made with the testis weight data. The translocation dose-response curve was bell-shaped, as previously found with the hybrid, but yields were lower at all but the lowest doses. Notably, peak yields occured at 3–5 Gy, rather than at 6 Gy. The altered stem cell killing and genetic responses may be explained either by a higher proportion of radiosensitive cells in the heterogeneous stem cell population or by a higher ratio of cell killing to recoverable chromosome damage which might imply a reduced repair capacity. The latter finds some support in other data. The pattern of genetic response obtained when an X-ray dose was given in two fractions at various intervals was similar in 101/H and the hybrid mice, suggesting that their kinetics of stem cell repopulation following depletion differ little.

Studies on mutagen-sensitive strains of Drosophila melanogaster: IX. Modification of genetic damage induced by X-irradiation of spermatozoa in N 2, air or O 2 by 4 autosomal repair-deficient mutants

The influence of defects in DNA repair on the recovery of X-ray-induced genetic damage in spermatozoa of Drosophila melanogaster was studied. Basc males were irradiated in N 2, air or O 2 and mated to females of 4 repair-deficient mutant types: mus(2)201 D1 (excision repair deficient), mus(3)306 D1 (excision repair deficient), mus(3)302 D1 and mus(3)308 D2 (both excision repair and post-replication repair deficient). The frequencies of sex-lined recessive lethals and of autosomal translocations in the F 1 progeny were determined following standard genetic procedures. The responses in the different crosses with repair-deficient females were compared to those with repair-proficient mei + females (maternal effects). The main findings are the following: (1) with excision repair-deficient females the frequencies of spontaneous recessive lethals tend to be higher than with mei + females; (2) with excision repair-deficient females the frequencies of recessive lethals induced in N 2 and air and often in O 2 are higher than with mei + females; (3) with post-replication repair-deficient mutants a maternal effect is found for X-ray-induced translocations - both increases and decreases occur depending on the specific mutant type. The data are explained as follows: excision repair deficiencies cause the processing of primary lesions to be diverted from the error-free excision repair to the error-prone post-replication repair pathways. This results in enhanced mutational yields. After irradiation in O 2 secondary effects cause selective elimination of potential recessive lethals in those mutants that exhibit lowered fertility ( mei-9, mus-302). Therefore those mutants have no differential maternal effect on the recovery of recessive lethals after irradiation in O 2. The changes in translocation yield with post-replication repair deficiencies are thought to be the result of defects in the repair of DNA breaks. These defects might cause the post-replication repair deficiency too. The group of post-replication repair mutants is heterogenous. The mutants with low fertility seem to cause decreased translocation frequencies by selective elimination through dominant lethality. The other mutants increase the frequencies.

Dose-response relationship of γ-ray-induced reciprocal translocations at low doses in spermatogonia of the crab-eating monkey ( Macaca fascicularis)

The yield of translocations induced by acute γ-irradiation at low doses (0.25 and 0.50 Gy) in the crab-eating monkey's ( Macaca fascicularis) spermatogonia was examined. The frequencies of translocations per cell were 0.53% at 0.25 Gy and 1.07% at 0.50 Gy. Over the low dose range from 0 to 1 Gy, the dose-response relationship for translocation yield was a linear one with a regression coefficient of 1.79 · 10 −2. To estimate the sensitivity to the induction of translocations in the crab-eating monkey's spermatogonia, the slope of the regression line was compared with those in other mammalian species. Consequently, over the low dose range below 1 Gy, the sensitivity of the crab-eating monkey's spermatogonia to translocation induction was similar to several mammalian species, the mouse. Chinese hamster, and the rabbit, but significantly higher than that of the rhesus monkey and lower than that of the marmoset.

Effects of post-irradiation interval on translocation frequency in male mice

Hybrid male mice were given 5 Gy + 5 Gy acute X-rays 24 h apart, with cytological examination of testes 16-19, 39-42 and 64-66 weeks later. Mean testis weights were significantly lower in the youngest group than in the other two. However, translocation frequencies in spermatocytes of the youngest group (mean of 0.57 per cell) were significantly higher than in either of the other two groups, which gave similar values averaging 0.36 translocations per cell. There was highly significant heterogeneity in translocation yields within the youngest group. The decline in translocation yield with time after irradiation is in line with that reported by Léonard and Deknudt (1970) in inbred strain C57BL males. Analysis of all available data suggests that high translocation yields are found during late stages in the process of germ-cell repopulation of the testis after high radiation doses and may be connected with changing frequencies of radiosensitive and radioresistant stem cell populations as repopulation proceeds.

γ-Ray-induced reciprocal translocations in spermatogonia of the crab-eating monkey ( Macaca fascicularis)

The yield of translocations induced by γ-rays in the crab-eating monkey ( Macaca fascicularis) spermatogonia were studied by cytological analysis in spermatocytes derived from them. The frequencies of translocations were 0.09% at 0 Gy, 1.9% at 1 Gy, 2.5% at 2 Gy and 1.3% at 3 Gy, showing a humped dose-response curve with a peak yield around 2 Gy. No remarkable inter-seasonal or inter-animal variations in the induction of translocation were observed. The frequencies in the crab-eating monkey were significantly higher than those in the same Macaca genus, the rhesus monkey ( Macaca mulatta) (van Buul, 1976, 1980). This inter-species difference in radiosensitivity might be affected by the condition of spermatogonial stem cells at the time of exposure to radiation, depending on the seasonal change in spermatogenetic activity.

Problems of iron deficiency in different crop plants in India: Causative factors and control measures

Abstract The problem of Fe‐deficiency chlorosis in different parts of India, particularly in Maharashtra and Tamil Nadu States is described. The most affected crops are sugarcane, sorghum, groundnut and legumes. The soils in these affected areas are calcareous with pH above 8. The general method for correcting chlorosis has been the application of Fe salts through the soil or as foliar sprays. In Tamil Nadu, an “organically complexed Fe”; has been tested and is found to increase Fe translocation and increase crop yield in sorghum. Several cultivars of sorghum and chickpea were tested for Fe‐deficiency stress tolerance in solution culture, in the last few years and tolerant ones have been identified. It is suggested that Fe‐deficiency stress tolerance should be included as a criterion along with traits like high yields, in plant breeding and agronomy program.

Studies on chemical induction of chromosomal aberrations in postcopulation germ cells and zygotes of female mice. II. Induction of heritable translocations.

The induction of heritable translocations with chemicals in postcopulation germ cells of female mice was investigated. In the methyl methanesulfonate (MMS)-treated group (100mg/kg), 14 partially sterile and 5 sterile males out of 118 F1 male progeny were found. In contrast, in the mitomycin C (MC)- treated group (3mg/kg and 4mg/kg) the frequencies were low. These results indicate that the yield of heritable translocations parallels the formation of chromosome-type aberrations at the first cleavage reported previously.

Metaphase I delay as a factor influencing translocation yield from spermatogonial irradiation in mice carrying Robertsonian translocations

Metaphase I delay as a factor influencing translocation yield from spermatogonial irradiation in mice carrying Robertsonian translocations

Induction by X-rays of chromosomal aberrations in somatic and germ cells of mice with different karyotypes

Stem-cell spermatogonia of +/+, T70H translocation heterozygous and Ts(l 13)70H tertiary trisomic mice were irradiated with various doses of X-rays. Blood lymphocytes of +/+ and Ts(l 13)70H males were irradiated with 150 rad X-rays. Contrary to earlier findings (De Boer et al., 1977), tertiary trisomic mice with morphological abnormalities did not show an increased sensitivity for radiation-induced chromosome breaks. The yields of radiation-induced reciprocal translocations, scored in the primary spermatocytes, were equal for the +/+ and tertiary trisomic mice but were lower for the T70/+ males. Thus, no parallel could be established with the sensitivity of peripheral blood lymphocytes for the induction of dicentric chromosomes, where T70H/+ did not differ from +/+ mice (De Boer et al., 1977). Induced reciprocal translocations occured randomly over the late diplotene-early diakinesis and late diakinesis-metaphase I stages. Among the T70H/+ males (2 × 500 rad) there was no relation between the chiasma frequency per animal and the yield of induced translocations, though the translocation induction was positively correlated with the chiasma frequency on a per-cell basis. When all descendants of irradiated T70H spermatogonia were pooled, the induced translocation-originated multivalent contained more chiasmata in cells that had a higher chiasma score themselves. No relation could be found between the yield of induced translocations and the recovery of spermatogenesis after irradiation as expressed by the epididymal sperm count after 2 × 500 rad.

An analytical approach to the induction of translocations in the spermatogonia of the mouse

• An analysis of a series of data providing a comprehensive impression of the effect of radiation on the induction of translocations in the spermatogonia of the mouse is presented. It is assumed that the spermatogonial stem-cell population is made up of a sensitive and resistant compartment, tha the same type of basic lesion can lead to either translocation induction or cell inactivation, and that the basic lesion has a linear-quadratic dose relationship. • The same set of parameter values is used to provide a quantitative description of the acute dose-response relationship and the effects of dose-rate and short-term, 24-h and long-term fractionation. The unusual effect of 24-h fractionation can be explained by proposing that the first dose blocks the progression of sensitive cells into the resistant compartment whilst the progression of the resistant cells into the sensitive compartment is unaffected. • The analysis indicates that (1) the majority of the spermatogonial stem cells in the mouse are in the sensitive compartment; (2) that the yield of translocations is proportional to accumulated dose for chronic radiation schedules; (3) the biology of spermatogenesis should be taken into account when extrapolations are made from one animal species to another.