R.J. Preston

The effect of cytosine arabinoside on the frequency of X-ray-induced chromosome aberrations in normal human leukocytes ☆

The yield of X-ray-induced chromosome aberrations in unstimulated human lymphocytes is greatly enhanced when the cells are incubated with cytosine arabinoside (ara-C) after irradiation. There is an increase in aberration yield with increasing time of incubation in ara-C (1, 2 or 3 h). When G2 cells are X-irradiated and incubated with ara-C until fixation, the deletion yields are considerably increased, but no interchanges are observed. In the absence of ara-C, 0.29 interchanges per cell were observed. The G2 results suggest that ara-C inhibits the repair of damage that leads to the formation of exchange aberrations. The increase in yield in unstimulated lymphocytes is interpreted to be due to an accumulation of strand breaks caused by ara-C inhibition of repair; when the inhibition is reversed with deoxycytidine, these breaks can interact to form aberrations. A possible mechanism for the induction of chromosome aberrations by X-rays is offered on the basis of these results.

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.

6-mercaptopurine, An inducer of cytogenetic and dominant-lethal effects in premeiotic and early meiotic germ cells of male mice.

Dominant-lethal effects of 6-mercaptopurine on male mice were studied using eight doses, ranging from 150 to 482 mg/kg. Effects of the 150-mg/kg dose were studied over the entire spermatogenic cycle, and those of the higher doses for matings made between days 28.5 and 41.5 after treatment. It was found that, with low doses, there was only one period in which clearcut increases in induced dominant-lethal mutations were detected, namely in matings that occurred 32.5 to 35.5 days after treatment. With higher doses, effects could be detected beyond that period through day 39.5. Spermatozoa utilized for matings during the period of greatest response were presumably derived from germ cells that were in late differentiating spermatogonial and early meiotic spermatocyte stages at the time of treatment. These results are similar to those of Ray and Hyneck. To date, 6-mercaptopurine is unique in inducing dominant lethality only at these particular stages. A study of chromatid aberration induction in the treated males themselves was carried out for 150 and 250 mg/kg doses of 6-mercaptopurine over the period of 9 to 16 days after treatment. A considerable increase in ischromatid and chromatid deletions was observed in diakinesis-metaphase-I spermatocytes on days 14 and 15 after treatment. For reasons discussed, the cells sampled at this may be assumed to have been in early meiosis (preleptotene), with some in late differentiating spermatogonial stages, at the time of treatment. The rough agreement in sensitive cell type for dominant lethality and chromatid aberration induction suggests that chromatid deletions are the cause of dominant lethality in this study. Conservative estimates of the frequency of dominant lethality expected from the chromatid aberration frequencies tend to substantiate this suggestion.

Sister-chromatid exchanges and gene mutations are induced by the replication of 5-bromo- and 5-chloro-deoxyuridine substituted DNA.

The thymidine (dT) analogue 5-chlorodeoxyuridine (CldU) induces 7-8-fold more sister-chromatid exchanges (SCE) than does 5-bromodeoxyuridine (BrdU) at equal substitution for dT in Chinese hamster ovary cells in culture. This difference facilitates study of the mechanism of induction of SCE by these analogues. Cultures were incubated with either BrdU or CldU for one cell cycle, followed by incubation in the presence of dT alone or BrdU or CldU for the second cell cycle and the SCE frequency determined in M2 cells. The results suggest that the induction of SCE is dependent only on the replication of the analogue-substituted DNA during the second cell cycle. Additional studies employed cultures grown in the presence of BrdU or CldU for 7 days to obtain mainly bifilarly substituted DNA, followed by 2 rounds of replication in the presence of dT alone. The SCE frequencies were approximately twice those found in cultures which had undergone the usual 2 rounds in the presence of the analogue; this is consistent with the replication of twice the amount of analogue-substituted DNA. Furthermore, such long-term growth in the presence of BrdU or CldU also results in concentration-dependent increases in the frequency of 6-thioguanine-resistant mutants, suggesting that gene mutations also result from the replication of analogue-substituted DNA.

The induction of specific-locus mutations and sister-chromatid exchanges by 5-bromo- and 5-chloro-deoxyuridine ☆

The induction of 6-thioguanine-resistant (TGr) mutants and sister-chromatid exchanges (SCE) by 5-bromo- and 5-chloro-deoxyuridine (BrdU and CldU) was determined with Chinese hamster ovary cells in culture. Over a concentration range of 0.5-200 microM, CldU treatment caused 3-5 times the number of SCEs found with BrdU; for example, with 10 microM CldU, 60.0 +/- 14.2 SCEs/metaphase (mean +/- S.D.) were induced, while with 10 microM BrdU, only 14.6 +/- 4.1 SCEs/metaphase were found. Despite this difference in SCE induction, both compounds were mutagenic only at concentrations greater than 50 microM, and BrdU was more mutagenic than CldU. These results indicate that SCEs and gene mutations are induced by these thymidine analogs through 2 different mechanisms.

Cytogenetic effects of environmental mutagens in mammalian cells and the extrapolation to man

Summary In order to extrapolate from cytogenetic data obtained with laboratory animals, with the intention of making an estimate of the genetic hazard of ionizing radiation, and possibly chemical mutagens, to man, several factors have to be taken into consideration. This paper represents an attempt to account for such factors and to make such an estimate. It is shown, for the mouse, Chinese hamster, and marmoset, that the yield of chromosome aberrations induced in peripheral leukocytes by X-rays was the same whether the cells were exposed in vivo or in vitro . It is also demonstrated that the yield of dicentric aberrations (asymmetrical translocations) varied from species to species, and that the yield was proportional to the number of chromosome arms available for translocation production, such that man, with 81 chromosome arms, was twice as sensitive as the mouse, which has 40. The yield of deletions did not vary from species to species. The yield of reciprocal translocations induced in spermatogonial stem cells by X-rays, and observed in primary spermatocytes, was measured in the mouse, Chinese hamster, guinea pig, and marmoset. It was found that the yield of reciprocal translocations was 0.25–0.30 of that assumed to be induced in peripheral leukocytes. It was also noted that the yield was here also related to the effective chromosome arm number. Using these and other data, it is proposed that the dicentric yield in human leukocytes can be corrected by a factor of 0.03–0.04 in order to derive an estimate of the recovered reciprocal translocations in the F 1 of irradiated males.

SCE induction is proportional to substitution in DNA for thymidine by CldU and BrdU

The incorporation of 5-bromo- and 5-chlorodeoxyuridine into cellular DNA and the induction of sister-chromatid exchanges (SCE) was examined with Chinese hamster ovary cells in culture. Although CldU caused 3-5 times more SCE than BrdU at equal extracellular concentrations, the 2 analogs were incorporated into DNA in place of thymidine similarly over a concentration range of 0.5-20 microM. The results show that SCE induction is linearly proportional to substitution for thymidine by both BrdU and CldU. Finally, based on linear extrapolation, a spontaneous level of approximately 6 SCEs per cell is estimated.

X-ray-induced chromosome aberrations in mouse dictyate oocytes. I. Time and dose relationships

Structural chromosome aberrations were analyzed in superovulated metaphase-I oocytes of the mouse, Mus musculus, at various times after a single acute dose of 200 R of X-rays. The aberrations seen were of the chromatid type, i.e., chromatid interchanges, isochromatid deletions and chromatid deletions. The aberration frequency was low during the interval 24 h to 5 days between irradiation and ovulation; peak frequency was reached when irradiation was given 14 days prior to ovulation. A dose-response study was made 14 days prior to ovulation at doses of 50, 100, 200, 300 and 400 R. A curve of these data indicated that a significant two-track component was present for both interchanges and deletions. Centromere staining revealed that symmetrical and asymmetrical interchanges occurred at approximately equal frequency and also that the asymmetrical equivalent of crossing-over was induced at a measurable frequency.

The effect of 3-aminobenzamide on the frequency of X-ray- or neutron-induced chromosome aberrations in cycling or non-cycling human lymphocytes.

3-Aminobenzamide (3-AB) is a potent inhibitor of poly(ADP-ribose) synthesis in mammalian cells. It was found to cause a 2-fold increase in dicentric frequency following X-irradiation of 9-18 h PHA-stimulated human lymphocytes, while 3-AB by itself had no effect on aberration frequency. In contrast to previously reported data, however, our results indicate that 3-AB does not increase the frequency of aberrations following either neutron or X-ray exposure of unstimulated (G0) human lymphocytes. Although 3-AB incubation after X-ray exposure in G1 caused a large increase in dicentrics, there was no effect of 3-AB incubation following neutron exposure in G1. The implications of these experiments are presently uncertain, but they do, however, suggest the importance of cycling cells for 3-AB to exert its enhancement effect, presumably on some step of DNA repair. Furthermore, these data support the hypothesis that there are different mechanisms of chromosome aberration induction with fission neutrons and X-rays (at X-ray doses above 50 rad).

X-ray-induced translocations in spermatoginia II. Fractionation responses in mice ☆

Abstract The dose-response curve for reciprocal translocations induced by X-rays in spermatognial stem cells, and observed in primary spermatocytes of mice, is “hymp-shaped”, with a maximum yield at about 600 R. To test the hypothesis that the decrease in yield with increasing dose above 600 R is a consequence of the different sensitivities of cells in different stages of the cell cycle to both cell killing and chromosome aberration induction, several fractionation experiments were carried out. A total dose of 2800 R was given in repeated doses of 400 R, separated by 8-week intervals. The yield of translocations is that expected for additivity; for example, the yield at 1600 R is approximately equal to that for four separate 400-R doses. When the total dose (500 R) which gives a translocation yield on the ascending part of the dose-response curve is given as two equal fractions separated by intervals of 30, 90, or 150 min, the translocation yield decreases with increasing interval. However, when a total dose (1000 R) which would give a translocation yield on the descending part of the dose-response curve is given in two equal fractions separated by intervals of from 30 min to 6 weeks, the response is different; the translocation yield increases with intervals up to 18 h, then decreases with intervals up to 4 weeks, and finally increases again to a yield equal to additivity with an interval of 6 weeks. These changes in translocation yield with changes in interval between the two doses are explained in terms of the differential sensitivity of cells to killing and aberration induction in the different phases of the cell cycle, and by assuming that the cells surviving the first dose and repopulating the testis different cycle characteristics from normal cells.