Specific locus mutation rates after repeated small radiation doses to mouse oocytes

When female mice were given a dose of 20 × 10 rad X-rays, the specific locus mutation rate among offspring conceived up to 7 weeks after the end of treatment was 1/39887 or 0.18·10⁻⁷/rad/locus, whereas when the same total dose of 200 rad was given in a single exposure the mutation rate was 9/34813 or 1.85·10¹⁰⁻⁷/rad/locus. The lower mutation rate after the 20 × 10 rad dose was obtained whether the total of 200 rad was given over a period of 5 days or 4 weeks, and if only young conceived in the first 20 days, rather than 7 weeks, were considered. It is suggested that each 10 rad fraction had the same small effect, and hence that these results confirm and extend Russell's previous finding that the dose-response relationship for specific locus mutations in females is curved.     

The mutagenic effect of repeated small radiation doses to mouse spermatogonia: III. Does repeated irradiation reduce translocation yield from a large radiation dose?

Previous results had suggested that daily repeated doses of 10 rad X- or γ-rays to mouse spermatogonia decreased their sensitivity to translocation yield. This was tested by comparing the effects of a dose of 300 rad γ-rays given before, 24 h after, or 8 days after, 30 daily doses of 10 rad γ-rays to male mice. The yield of translocations per cell from those receiving the 300 rad dose 24 h after the repeated ones (7.3%) was significantly lower than that from the other two regimes (9.4 and 9.7% respectively). This was consistent with the explanation that the repeated irradiation had temporarily increased the resistance of the spermatogonial cell population to translocation yield. However, there must remain some doubt about this interpretation since the absolute values of translocations found were too high, and the yield from those which had received the 300-rad dose 24 h after the repeated ones was not significantly below the sum of those from a single dose and from repeated doses given separately.     

Cytogenetic effects of cyclophosphamide on mouse spermatogonia.

NMRI mice were treated with single doses of cyclophosphamide (Cytoxan) and spermatogonia were analysed for chromosome aberrations at various time intervals after treatment. The maxima of aberrations were found 24 hrs p.i. Chromatid type aberrations were observed exclusively. About half of the aberrations consisted of chromatid interchanges, 92% of which exchanging short arm fragments close to the centromeric region. The lack of meiotic multivalents in diakinesis-metaphase I after treatment of spermatogonia stem cells with cyclophosphamide in the study of Leonard and Linden (1972) is discussed.     

Specific-locus mutation rates in the mouse following inhalation of ethylene oxide, and application of the results to estimation of human genetic risk

Male (101 × C3H)F₁ mice were exposed in an inhalation chamber to ethylene oxide (EtO) in air at a concentration of (generally) 255 ppm. After accumulating total exposures of 101 000 or 150 000 ppm.h in 16–23 weeks, the males were mated to T-stock females for a standard specific-locus mutation-rate study in which 71 387 offspring were observed. The spermatogonial stem-cell mutation rate at each exposure level, as well as the combined result, does not differ significantly from the historical control frequency. At the lower and higher exposure levels, the results rule out (at the 5% significance level) an induced frequency that is, respectively, 0.97 and 6.33 times the spontaneous rate; the combined results rule out a multiple of 1.64.

The relationship between mouse spermatogonial stem-cell mutation rates and EtO-induced testis ethylations was compared with the relationship between Drosophila post-stem-cell mutation rates and sperm ethylations (Lee, 1980). The comparison does not rule out equal mutability per ethylation; but it cannot prove parallelism. An assessment of the mouse-Drosophila relationship will require a more efficient alkylator than EtO and the use of comparable germ-cell stages.

More meaningful conclusions may be drawn by utilizing the data for direct estimation of human risk by expressing the induced mutation frequency that is ruled out (at the 5% significance level) as a multiple of control rate and extrapolating to human exposure levels. The probable absence of major stem-cell killing (and thus, possibly, cell selection) by EtO indicates that such extrapolation probably does not produce an underestimate. For a human exposure concentration of 0.1 ppm on working days during the reproductive lifespan, the mouse experimental results rule out (at the 5% significance level) an induced spermatogonial stem-cell gene mutation rate greater than 8% of the spontaneous rate; for 1.0 ppm, they rule out an induced rate roughly equal to the spontaneous rate. The induced rate for any one poststem-cell stage would have to be about 3 orders of magnitude higher than that for stem cells to constitute an equivalent risk.     

Translocations in mouse spermatogonia after exposure to unequally fractionated doses of x-rays

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.     

Characteristic cytogenetic effects of small doses of ionizing radiation

A study was made of the frequency of chromosome aberrations in human lymphocyte culture after gamma-irradiation (60Co) with doses ranging from 0.05 to 1.0 Gy at dose--rates of 0,005, 0.05 and 0.5 Gy/min. The frequency of structural changes in chromosomes at low doses was higher than it was expected in the case of extrapolating the effect produced by high to low doses of radiation; within the dose range from 0.1 to 0.5 Gy a plateau was registered for aberrations of the exchange type (dicentrics and rings). The abnormal character of the dose dependence of the yield of chromosome aberrations persisted with all three dose - rates under study.     

Cytogenetic effects of cyclophosphamide on mouse spermatogonia

Summary NMRI mice were treated with single doses of cyclophosphamide (Cytoxan) and spermatogonia were analysed for chromosome aberrations at various time intervals after treatment.The maxima of aberrations were found 24 hrs p.i. Chromatid type aberrations were observed exclusively. About half of the aberrations consisted of chromatid interchanges, 92% of which exchanging short arm fragments close to the centromeric region.The lack of meiotic multivalents in diakinesis-metaphase I after treatment of spermatogonia stem cells with cyclophosphamide in the study of leonard and Linden (1972) is discussed.

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Zusammentassung NMRI-Mäuse erhielten einmalig eine Applikation von Endoxan in verschieden hoher Dosierung. Zu verschiedenen Zeiten nach der Behandlung wurden Tiere getötet und die Spermatogonien analysiert.Das Maximum der induzierten Effekte lag bei 24 Std p.i. Es gelangten ausschließlich Chromatidentypaberrationen zur Beobachtung, die annähernd zur Hälfte aus Chromatidenaustauschen bestanden. Dabei handelte es sich überwiegend (92%) um Segmentaustausche im zentromernahen Bereich.Der fehlende Nachweis von Multivalenten in der Diakinese-Metaphase I nach Behandlung spermatogonialer Stammzellen mit Cyclophosphamid (Leonard u. Linden, 1972) wird diskutiert.

     

Mutagenic adaptive response to high-LET radiation in human lymphoblastoid cells exposed to low doses of heavy-ion radiation

Adaptive response (AR) and bystander effect are two important phenomena involved in biological responses to low doses of ionizing radiation (IR). Furthermore, there is a strong interest in better understanding the biological effects of high-LET radiation. We previously demonstrated the ability of low doses of X-rays to induce an AR to challenging heavy-ion radiation [8]. In this study, we assessed in vitro the ability of priming low doses (0.01Gy) of heavy-ion radiation to induce a similar AR to a subsequent challenging dose (1-4Gy) of high-LET IR (carbon-ion: 20 and 40keV/μm, neon-ion: 150keV/μm) in TK6, AHH-1 and NH32 cells. Our results showed that low doses of high-LET radiation can induce an AR characterized by lower mutation frequencies at hypoxanthine-guanine phosphoribosyl transferase locus and faster DNA repair kinetics, in cells expressing p53.     

Comparison of the genetic effects of equimolar doses of ENU and MNU: while the chemicals differ dramatically in their mutagenicity in stem-cell spermatogonia, both elicit very high mutation rates in differentiating spermatogonia

Mutagenic, reproductive, and toxicity effects of two closely related chemicals, ethylnitrosourea (ENU) and methylnitrosourea (MNU), were compared at equimolar and near-equimolar doses in the mouse specific-locus test in a screen of all stages of spermatogenesis and spermiogenesis. In stem-cell spermatogonia (SG), ENU is more than an order of magnitude more mutagenic than MNU. During post-SG stages, both chemicals exhibit high peaks in mutation yield when differentiating spermatogonia (DG) and preleptotene spermatocytes are exposed. The mutation frequency induced by 75mgMNU/kg during this peak interval is, to date, the highest induced by any single-exposure mutagenic treatment - chemical or radiation - that allows survival of the exposed animal and its germ cells, producing an estimated 10 new mutations per genome. There is thus a vast difference between stem cell and differentiating spermatogonia in their sensitivity to MNU, but little difference between these stages in their sensitivity to ENU. During stages following meiotic metaphase, the highest mutation yield is obtained from exposed spermatids, but for both chemicals, that yield is less than one-quarter that obtained from the peak interval. Large-lesion (LL) mutations were induced only in spermatids. Although only a few of the remaining mutations were analyzed molecularly, there is considerable evidence from recent molecular characterizations of the marker genes and their flanking chromosomal regions that most, if not all, mutations induced during the peak-sensitive period did not involve lesions outside the marked loci. Both ENU and MNU treatments of post-SG stages yielded significant numbers of mutants that were recovered as mosaics, with the proportion being higher for ENU than for MNU. Comparing the chemicals for the endpoints studied and additional ones (e.g., chromosome aberrations, toxicity to germ cells and to animals, teratogenicity) revealed that while MNU is generally more effective, the opposite is true when the target cells are SG.     

Cytogenetic effects of fractionated or unfractionated X-ray exposures to mouse spermatogonia

The induction of chromosomal aberrations in mouse spermatogonia was studied, after single (50 and 100 rad) and fractionated doses (50 + 50 rad spaced 24 h apart), a short time after irradiation, by analysis of mitotic stages. From 17 to 21 h after the second fraction of the dose, the recorded frequencies of chromosomal deletions and exchanges were fully additive when compared with single “control” doses. Thus there was no suggestion of any sensitization effect of the first exposure. Possible reasons for this are discussed.     

Estimation of mutation induction rates in AT-rich sequences using a genome scanning approach after X irradiation of mouse spermatogonia

We have previously used NotI as the marker enzyme (recognizing GCGGCCGC) in a genome scanning approach for detection of mutations induced in mouse spermatogonia and estimated the mutation induction rate as about 0.7 x 10(-5) per locus per Gy. To see whether different parts of the genome have different sensitivities for mutation induction, we used AflII (recognizing CTTAAG) as the marker enzyme in the present study. After the screening of 1,120 spots in each mouse offspring, we found five mutations among 92,655 spots from the unirradiated paternal genome, five mutations among 218,411 spots from the unirradiated maternal genome, and 13 mutations among 92,789 spots from 5 Gy-exposed paternal genome. Among the 23 mutations, 11 involved mouse satellite DNA sequences (AT-rich), and the remaining 12 mutations also involved AT-rich but non-satellite sequences. Both types of sequences were found as multiple, similar-sequence blocks in the genome. Counting each member of cluster mutations separately and excluding results on one hypermutable spot, the spontaneous mutation rates were estimated as 3.2 (+/- 1.9) x 10(-5) and 2.3 (+/- 1.0) x 10(-5) per locus per generation in the male and female genomes, respectively, and the mutation induction rate as 1.1 (+/- 1.2) x 10(-5) per locus per Gy. The induction rate would be reduced to 0.9 x 10(-5) per locus per Gy if satellite sequence mutations were excluded from this analysis. The results indicate that mutation induction rates do not largely differ between GC-rich and AT-rich regions: 1 x 10(-5) per locus per Gy or less, which is close to 1.08 x 10(-5) per locus per Gy, the current estimate for the mean mutation induction rate in mice.     

The induction of reciprocal translocations in rhesus monkey stem-cell spermatogonia: effects of low doses and low dose rates

The induction of reciprocal translocation in rhesus monkey spermatogonial stem cells was studied following exposure to low doses of acute X rays (0.25 Gy, 300 mGy/min) or to low-dose-rate X rays (1 Gy, 2 mGy/min) and gamma rays (1 Gy, 0.2 mGy/min). The results obtained at 0.25 Gy of X rays fitted exactly the linear extrapolation down from the 0.5 and 1.0 Gy points obtained earlier. Extension of X-ray exposure reduced the yield of translocations similar to that in the mouse by about 50%. The reduction to 40% of translocation rate after chronic gamma exposure was clearly less than the value of about 80% reported for the mouse over the same range of dose rates. Differential cell killing with ensuing differential elimination of aberration-carrying cells is the most likely explanation for the differences between mouse and monkey.     

Hypersensitivity of the human gonadotrophs to the repeated administration of small doses of LH-RH.

The functional capacity of the gonadotrophs was assessed by repeated stimulation with small doses of LH-RH (5 microgram intravenously at 2-hour intervals for 3 injections) in normal women during the early and late follicular phases of the menstrual cycle. The results were compared to those obtained when a single dose (100 microgram) of the neurohormone was administered. During the early follicular phase, the release of LH and FSH remained about equal after the 3 successive injections of the small and after the large dose of LH-RH. During the late follicular phase, the release of LH and fsh increased progressively after the repeated administration of the 5 microgram of the neurohormone while the large dose induced a more pronounced and a more sustained pituitary response. This hypersensitivity of the gonadotrophs is observed when the E2 concentrations are higher than in the early follicular phase of the menstrual cycle.     

Reactions of mammalian nerve cells to small doses of radiation

In experiments with rat brain slices it was shown that a radiation-induced short-term increase in spontaneous neuron activity was mainly a function of dose rate. Pulsed X-radiation (pulse length of 2 X 10(-8) s, doses of 3 X 10(-5) to 6 X 10(-4) Gy) caused the most pronounced reactions that were almost completely prevented by caffeine, euphylline, and norepinephrine (10(-4) to 10(-3) M).