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In order to evaluate the pUR288-plasmid transgenic mouse model, utilizing the bacterial lacZ gene as the mutational target, radiation-induced mutagenesis was primarily analyzed in spermatogonial stem cells. A combined hydroxyurea (HU)–X-ray treatment protocol was used, known to sensitize dramatically the induction of mutations in endogenous genes. In the testes of untreated animals, a mutant frequency of 6.7 ± 4.4 × 10–5 was found. In animals treated with HU or X ray alone, moderate elevations were seen (factors of about 4 and 2 over untreated animal values). In testes of mice having received the HU + X-ray combination treatment, a mutant frequency of 63.0 ± 36.1 × 10–5 was found. The results obtained showed a good quantitative correlation between endogenous genes and the transgene, indicating the suitability of pUR288 transgenic mice for also efficiently recording radiation-induced genetic damage. Radiosensitization, seen in spermatogonial stem cells, was not observed in other studied organs such as spleen, brain, or lung. Environ. Mol. Mutagen. 34:216–220, 1999 © 1999 Wiley-Liss, Inc.

To further characterize the radiation response of the scid mutation.X-ray induced chromosomal aberrations and cell killing were analysed using various in vivo or in vitro cell systems.Using low LET X-irradiation a reverse dose-rate effect was found for killing of differentiated and differentiating spermatogonia and the chromosomal hyperradiosensitivity of scid mice was extended to the meiotic prophase. Most striking was the observation made in vitro with synchronized established cell lines that, contrary to the situation in wild-type cells, scid cells display high levels of both chromatid- and chromosome type aberrations when irradiated during the G1-phase of the cell cycle. A time-course for induction of micronucleated polychromatic erythrocytes (MPCE) was determined for scid mice using flow analysis. No significant differences with wild-type mice were recorded. The chromosomal radiosensitivity at the G1 stage in scid cells was 4.3 times higher than in control CB-17 cells whereas G2 sensitivity differed only by a factor of 1.3.The reportedly normal radiosensitivity for MPCE in scid mice together with previous findings of hypo- or normal radiation sensitivity of scid cells could be explained by the induction of highly lethal chromatid-type damage at the G1 stage of the cell cycle leading to selective elimination of aberration-carrying cells. The differences in chromosomal radiosensitivity between wild-type and scid for the G1 and G2 stage of the cell cycle correlate with variation in the rates of DNA double-strand break (dsb) repair in scid cells during the cell cycle found by others.

The protective effects of misoprostol (MP), an analogue of prostaglandin E1, on X-ray-induced chromosomal aberrations, were studied in normal or mutant Chinese hamster cell lines grown as spheroids in vitro and on cell-killing in stem-cell spermatogonia of a mutant (acid) mouse strain or its wild-type. The mutant hamster cell lines chosen for this purpose are known to be either hypersensitive to the killing effects of X-rays and/or deficient in the repair of DNA double-strand breaks. The scid mice are deficient in the repair of DNA double-strand breaks. The results show that MP manifests varying degrees of radioprotection in all these systems, but the magnitude of these effects in the mutants is markedly reduced compared to their respective wild-type counterparts. These findings suggest a link between ionizing radiation sensitivity, DNA double-strand break repair capability and MP-mediated radioprotection.

Radiation induced chromosomal aberrations in bone marrow cells of scid and normal mice were studied at different sampling times. Fluorescence in situ hybridization (FISH) with DNA libraries specific for chromosomes 1, 11 and 13 was applied to identify the stable types of chromosomal aberrations in addition to the unstable ones. The results obtained confirm earlier observations on stem cell spermatogonia in that, contrary to the situation in normal mice, only very low levels of translocations could be recovered from scid mice at relatively long sampling times (3 weeks). However, studies at a 24 h sampling period demonstrated substantial induction of translocations in scid mice. This suggests enhanced elimination of translocation carrying cells in scid mice during successive cell proliferation, possibly via falling apart of the translocation at the original points of exchange or due to lethal damage at the translocation break points.

The radioprotective effects of prostaglandins (PGE2, PGE1 and its analogue misoprostol (MP) were investigated in cultures of V79 Chinese hamster (CHO) cells grown as spheroids and as monolayers, CHO cells grown as monolayers, and in bone marrow polychromatic erythrocytes and spermatogonial stem cells in mouse. The X-ray doses were 0.75 Gy (hamster cells) and 5, 8 and 10 Gy (mouse experiments). Prostaglandin pre-irradiation treatment resulted in a marked reduction in the frequencies of chromosomal aberrations in V79 spheroids and of reciprocal translocations in mouse stem cell spermatogonia. The amount of mouse spermatogonial stem cell killing was likewise significantly reduced. No radioprotective effects of prostaglandins could be demonstrated, however, for chromosomal aberrations in hamster cells grown as monolayers, for survival of V79 cells grown as spheroids, and for the induction of micronuclei in bone marrow polychromatic erythrocytes of mouse.

The kinetics of spermatocyte progression through meiotic prophase in cells with or without induced translocations were studied in mice that had been exposed to x-rays. Pulse-labeling experiments using 3H-thymidine, followed by autoradiographic analysis, indicated that at higher x-ray doses (6 and 7 Gy), translocation-carrying cells tend to spend more time in meiotic prophase than do normal cells. At 2 Gy, no such delay seemed to be present. The observed delay may explain the reduction in transmission of translocations to the next generation reported by others.

In order to develop mouse models for human mutagen-sensitive syndromes, we carried out cytogenetic characterization of several mouse mutants and MS/Ae mice showing enhanced radiosensitivities. The applied cytogenetic techniques include chromosomal analysis of in vitro cell cultures and lymphocyte cultures as well as in vivo UDS in hepatocytes, induction of micronuclei in polychromatic erythrocytes and translocation induction in spermatogonial stem cells. Among the mutations studied, namely the contrasted allele of steel ( Sl con ), viable dominant spotting ( W υ ), wasted ( wst ), varitint-waddler ( Va ) and dystonia musculorum ( dt ) as well as MS/Ae mice, various iso-, hyper- or hypo-sensitive conditions were recorded. Only Va and dt appear to be associated with some deficiency in DNA repair.

To shed light on the mechanism underlying the cellular response to the radiomimetic agents calicheamicin Y(1)(1) (CAL) and neocarzinostatin (NCS), several hamster cell mutants defective in different DNA repair pathways were used. Two X-ray sensitive Chinese hamster V79 mutant cell lines, XR-V9B and V-E5 were studied for their response to the induction of cell killing, micronuclei, and G2-chromosomal aberrations relative to that of parental wild-type cells. In addition, effects of CAL and NCS on bleomycin sensitive BL-V40 cells and on UV sensitive V-H1 cells were analyzed. In general, the radiosensitive cell lines showed the highest sensitivities to CAL and NCS, but also the other mutants demonstrated differences in their responses compared to wild-type cells. With respect to cell killing, expressed as D(10)-value, enhanced sensitivities of mutants with factors up to 4.4 were recorded. For the induction of micronuclei (MN) and chromosomal aberrations (CA) all cell lines, including the parental cells, show a steep increase in the frequencies at the lowest tested doses and a leveling off at higher concentrations. Probably toxic effects at the higher exposure levels are responsible for these biphasic dose effect curves. Enhanced sensitivities of the various mutants were primarily observed at the higher exposure levels. With respect to the induction of MN increased sensitivities up to a factor of 18.1 were observed for the radiosensitive mutants, whereas for CA the mutant cell lines showed a variation from resistance (0.3) of VH-1 cells up to a 3.8-fold higher sensitivity to the radiomimetic agents. However, at the lowest tested concentrations for both MN and CA, the differences between the sensitive mutants and wild-type clearly diminished, suggesting the existence of residual and/or alternative DNA repair pathways in these mutants.

The radioprotective effects of misoprostol, a synthetic stable analogue of prostaglandin E1, on spermatogonial stem cells of C3H/HeH×101/F1 hybrid mice (3H1) were analysed by establishing dose–response relationships for stem cell killing by X-rays in mice that were pretreated with misoprostol. Spermatogonial stem cell killing was studied through determination of the percentage of tubular cross-sections showing repopulation at 10 days after irradiation. In control mice, the D0 values ranged between 1·7 and 3·6 Gy, dependent on the stage of the cycle of the seminiferous epithelium the cells were in. As found previously, proliferating spermatogonial stem cells were much more radioresistant than quiescent stem cells. In the misoprostol-pretreated animals the spermatogonial stem cells were more radioresistant, the D0 values ranging from 3·6 to 5·0 Gy. Both proliferating and quiescent spermatogonial stem cells were protected by misoprostol. As the dose–response curves in control and misoprostol-pretreated mice showed about the same extrapolation number to the y-axis it was concluded that the misoprostol pretreatment did not alter the kinetics of the repopulation process.

To characterize further the radiosensitivity of severe combined immunodeficiency (scid) mice, the induction of micronuclei (MN) in polychromatic erythrocytes as well as cell killing and translocation induction in stem cell spermatogonia was studied. Scid mice turned out to be clearly hypersensitive for X-ray-induced cell killing of both bonemarrow cells and spermatogonial stem cells. The frequencies of recorded micronuclei in polychromatic erythrocytes were comparable with that reported for the normal mouse, whereas the recovery of translocations was extremely low in the scid mouse. The dose-response relationship for induced translocations was bell shaped with a maximum of about 0.5% around doses of 0.5-1.5 Gy X-rays.

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.

In a coordinated research programme sponsored by the Internation Atomic Energy Agency, the frequencies of chromocomal aberrations induced in peripheral blood lymphocytes (in vitro) by 250 kV X-rays at low doses (0.4, 1, 2, 3, 5, 10 and 30 rad) were determined. Blood from 2 donors was used to conduct one master experiment at these dose levels. The culture time used was 48 h and all samples including the controls were processed according to a standard protocol. The coded slides were scored by investigators from 10 participating laboratories. The main results are the following: (1) the frequencies of all types of chromosome aberrations at 0.4 rad are significantly lower than the control values; (2) there is no increase in the frequencies of dicentrics up to 2 rad and in those of terminal deletions up to 5 rad; (3) the mean frequencies of all aberrations considered together are not significantly different from one another at 1,2 and 3 rad ( P = 0.05); and (4) over the entire dose range the dose-effect relationship is clearly non-linear. A fit of these data to a linear quadratic model ( E ( D ) = c + αD + βD 2 ) showed that the observed total aberration frequencies at doses 1,2,3 and 5 rad are below the curve defined by the model. The deviations can be explained by an altered kinetics of aberration production at very low doses probably due to DNA repair mechanisms operating in these cells.

The influence of various X-ray fractionation regimes on translocation induction in mouse spermatogonia was studied. Splitting a single exposure of 600 R into two fractions of 300 R separated by 24 h did not influence the translocation yield (7.58% vs. 8.35%). When the dose was given in the unequal fractions,100 R+500 R and 500 R+100 R, with the same 24 h between the fractions, the translocation frequency was significantly altered (10,19 and 4.94%, respectively). As a possible explanation of these findings it is assumed that the relatively resistant cells surviving the first fraction of the dose are sensitized towards translocation induction when receiving the second fraction of the dose.

The effect of different small conditioning doses of X-rays on the production of reciprocal translocations in stem-cell spermatogonia of the mouse (scored in spermatocytes) by a second larger dose have been examined. Fractionation regimes of 25 + 975 R, 50 + 950 R, 75 + 925 R and 100 + 900 R, all with 24 h between the fractions, were applied. The size of the first fraction strongly affected the frequency of induced translocations by the second one, and a kind of threshold dose, somewhere between 75 and 100 R existed for conditioning the spermatogonial population: the translocation yield after 25 + 975 R was 3.3 %, after 50 + 950 R it was 5.0%, and after 75 + 925 R it was 5.1%; whereas 100 + 900 R resulted in 16.1% translocations. It is difficult to explain this observed threshold effect by known biological processes so far held responsible for the conditioning effect.

The results of an IAEA coordinated programme on radiation induced chromosomal aberrations in human peripheral blood lymphocytes in vitro are presented. In a master experiment, a whole blood sample from one donor was irradiated with 200 R of X-rays. Different fixation times from 46 to 82 h were used. The progression of cells into mitosis was monitored by BrdUrd incorporation. 14 investigators took part in the scoring of chromosomal aberrations. The main conclusions of this study are: (1) The mean frequencies of aberrations changed with fixation time. (2) The number of cells scored as aberrant by different laboratories was very similar, but there was variability in the number of aberrations scored per aberrant cell. (3) The differences in the frequencies of aberrations between laboratories were minimal when the scoring was restricted to the first major peak of mitotic activity and sufficient cells were scored. It is concluded that using controlled experimental conditions, human peripheral blood lymphocytes can effectively be used as a reliable biological dosimeter for absorbed radiation dose.

Summary The frequencies were studied of X-ray-induced dicentric chromosomes and deletions in peripheral blood lymphocytes of mouse and man, cultured in vitro. After doses of 100 and 200 rad, (a) the mouse was equally sensitive as man to the induction of dicentrics, and (b) the frequency of deletions was higher in the mouse, reaching statistical significance at the 200 rad level only. At the 200 rad level, the mouse with normal karyotype was compared with the T(1 13 )70H translocation heterozygote and the Ts(l13)70H tertiary trisomie of normal appearance. No differences were found either with respect to dicentrics or to deletions. At the 100 rad level, the normal mouse was compared with the tertiary trisomie mouse of the affected phenotype and with the tobacco mouse. The frequency of dicentrics was significantly higher in the phenotypically abnormal trisomies, whereas the deletion frequency was higher in the tobacco mice. C-banding of the slides enabled the locating of breaks in constitutive hetero-chromatin and euchromatin. When exchanges were classified into three categories, i.e. those between eu- and euchromatin, eu- and hetero-, and hetero- and heterochromatin, there was a preference for the first and the last whereas only few occurred between chromatins of contrasting type. Differences between previous determinations of the chromosomal radiosen-sitivity of mouse and man, using peripheral blood lymphocytes (i.e. man is twice as sensitive as mouse), and the one presented here could be attributed to differences in harvest time of the mouse peripheral blood lymphocytes. Thus the so-called “arm number” hypothesis of Brewen et al. [5] is not confirmed by the present results.

Treatment of Snell dwarf mice with high concentrations of human growth hormone from pituitaries as well as of bacterial origin, significantly increased the frequencies of chromosomal aberrations in bone-marrow cells, as measured by the micronucleus test. In vitro treatment of Chinese hamster ovary (CHO) cells with the two types of hormone likewise induced structural chromosomal aberrations.

The effect of co-cultivation of Bloom's syndrome fibroblasts with Chinese hamster ovary cells (CHO) on the incidence of sister chromatid exchanges (SCEs) was studied. The results show that suppression of the frequency of SCEs in Bloom's syndrome cells occurs only if cell to cell contact is present with CHO cells, without any effect on the SCE frequency in the latter. It is suggested that possible genetic heterogeneity between different Bloom's syndrome patients can be studied using the method of co-cultivation.

A rat-liver microsomal system in vitro has been used to activate two indirectly acting carcinogens, DMN and DEN. On activation, both compounds were extremely potent in inducing chromosomal aberrations as well as sister chromatid exchanges in Chinese hamster cells. The implications of these findings and the potential utility of this technique to detect mutagens/carcinogens are discussed.

Unstable chromosome aberrations induced by in vitro irradiation with zero plus seven low doses of 14.8 MeV D-T neutrons in the range 3.55-244 mGy have been analysed in human peripheral blood lymphocytes. In order to obtain the required large numbers of scored cells for such low doses, fourteen laboratories participated in the experiment. The dose responses for dicentrics, excess acentrics and total aberrations, fitted well to the Y = alpha D model. The alpha coefficient of yield for dicentrics, 1.60 +/- 0.07 X 10(-2) Gy-1, compares well with the values obtained in previous studies with D-T neutrons at somewhat higher doses. Results from a previous collaborative study using 250 kVp X-rays over a comparable dose range indicated the possible existence of a threshold below 50 mGy. In the present study there is no clear evidence for neutrons for such a threshold. However, the data were insufficient to permit the rejection of a possible threshold below approximately 10 mGy.

The symmetry of radiation-induced chromatid exchanges was studied in relation to the cell cycle in Chinese hamster cells in vivo and in vitro. Both in vivo and in vitro, the ratio between symmetrical and asymmetrical chromatid exchanges was about 1 to 1 during G2 and S phase of the cell cycle.

Treatment of dwarf mice with growth hormone, insulin and testosterone had no effect on the spontaneous frequencies of micronuclei (MN) in bone-marrow cells, whereas thyroxine decreased these frequencies. The induction of MN by X-rays and mitomycin C was significantly lower in dwarf mice than in normal mice. Treatment with thyroxine plus growth hormone restored normal radiosensitivity in dwarfs.

Radiation-induced stable chromosome aberrations have been studied in the testes and bone-marrow of the mouse (Mus musculus). 60 days after whole-body irradiation with a dose of 400 rad X-rays, the frequency of visible chromosome aberrations in bone-marrow cells was 19.8%. The frequency of chromosome aberrations in spermatogonia of the same mice, scored as multivalents in spermatocytes, was considerably lower — only 4.7%. The possible mechanisms underlying this marked difference in translocation yield are discussed.