A BrdU pulse double-labelling method for studying adaptive response.

We describe a pulse BrdU double-labelling method which we have used for a cohort-analysis study of a conventional adaptive response experiment in stimulated human lymphocytes. This method allows us to identify the cycle position occupied by each scored metaphase at the time of primer and at the time of challenge. Responses in cells at defined developmental stages can then be compared irrespective of mitotic perturbation. Using a primer X-ray dose of 1.0 cGy, and a challenge dose of 1.5 Gy 6 h later, we found a transitory depression in frequency of total aberrations present in cells sampled at 6 h but not at 9 h after challenge. When defined cohorts were summed for both sample times, no significant adaptive response was observed. A repeat experiment where terminal BrdU labelling was used after the challenge failed to provide any positive response at either 6 or 9 h. The technique presented not only ensures that similar cell mixtures from a heterogeneous, asynchronous population can be scored and compared, but it serves also to demonstrate the kinetic complexity of stimulated lymphocyte cultures.

The effect of X-irradiation on cell cycle progression and chromatid aberrations in stimulated human lymphocytes using cohort analysis studies.

Stage sensitivity for the production of chromatid-type aberrations and mitotic delay has been investigated in a stimulated human lymphocyte population, following an absorbed dose of 1.5 Gy 250 kVp X-rays. BrdU replication banding was used to obtain a fine analysis of the cell cycle and to permit cohort analysis. Fluctuations in yield with sample time were found for all aberration categories, but these could not be related simply to either the developmental stage of the cells at time of exposure, or to the time-to-run to metaphase. In general G2 and late S cells had higher aberration yields than early S and pre S cell populations. Mitotic delay and perturbation at this dose extends to all sub-phases of S and is as great, if not greater, in the earliest S cells as it is in G2.

A pulse BrdU method for SCE.

Using 'reverse' harlequin staining (bromouracil-substituted chromatin staining dark), it is possible to detect at metaphase a pulse of bromodeoxyuridine (BrdU) incorporated during S-phase. Provided that this pulse is of reasonable duration, fairly uniform staining along the chromatids of some S-cells is achieved, and no difficulty is encountered in observing and scoring SCE in such cells at second division. Thus, it is possible to define within an asynchronous population a narrow cohort of target cells and recover these for SCE scoring at second division irrespective of treatment induced perturbation. This serves to reduce the heterogeneity found in the usual terminal BrdU SCE protocols for such populations and should lead to more reliable and repeatable quantitative results. The method is illustrated for mitomycin C given to dividing human blood lymphocytes using both simultaneous and delayed pulse modes.

The human lymphocyte micronucleus assay. Response of cord blood lymphocytes to gamma-irradiation and bleomycin.

The induction of micronuclei in human cord blood lymphocytes by treatment with gamma-irradiation and bleomycin has been measured. Culture durations which gave peak MN frequencies were determined. The lowest tested doses, 0.1 Gy irradiation and 1.25 micrograms/ml bleomycin, produced significant increases in the frequency of micronuclei. The spontaneous frequency of micronucleated lymphocytes in 28 cord blood samples ranged between 0.5 and 9.5 per thousand lymphocytes, with a modal value of 2.5. The method is evaluated for its potential usefulness in monitoring populations for chromosome breakage.

Rapid screening for deletion mutations in the hprt gene using the polymerase chain reaction: X-ray and alpha-particle mutant spectra.

Conditions were devised for the isolation of DNA from single-mutant colonies on dishes, to give reproducible results in the polymerase chain reaction (PCR). Primers for 3 exons of the hamster hprt gene were used in a multiplex reaction to show rapidly whether the mutants carried deletions at these sites. 138 independent mutants were screened in total, some spontaneous and others induced by X-rays or by alpha-particles from plutonium-238. Few deletions were found among the spontaneous set, while 'total' gene deletions formed about half the mutants found after irradiation. At equitoxic doses, little difference in mutant spectrum was found for the X-ray set compared to the alpha-particle set. This rapid technique should be applicable to many instances of comparative mutagenesis.

Production of chromosome aberrations, micronuclei, and sister-chromatid exchanges by 24-keV epithermal neutrons in human G0 lymphocytes.

The induction of chromosome aberrations, micronuclei and sister-chromatid exchanges in human G0 lymphocytes by 24-keV epithermal neutrons has been measured. Positive linear dose responses were obtained for the 3 end points, with a tendency to saturation at higher dose for SCE production. In all cases, the responses to 24-keV neutrons were characteristic of high-LET radiations.

Lack of discernible effect of diagnostic ultrasound on the chromosomes of cord blood lymphocytes exposed in utero.

The levels of either sister chromatid exchanges or chromosome breakage (as assayed by the frequency of micronuclei) in cord blood lymphocytes from a total of 62 babies were analysed. The exposure of these babies to ultrasound during gestation was determined retrospectively from medical records and the data reclassified accordingly. No correlation was seen between the levels of chromosome breakage, as measured by these two end-points, and exposure to ultrasound.

BrdU pulse/reverse staining protocols for investigating chromosome replication.

By using a reverse Giemsa staining procedure (TT chromatin pale, TB chromatin dark) it is possible to detect replication in metaphase chromosomes with short (approximately 10 min) 5-bromodeoxyuridine (BrdU) pulses. A pulse protocol allows us to consider the question "What is replicating at this point in time?" and we have investigated replication patterns during cycle transit in stimulated human female lymphocytes. A clear-cut demarcation between R-zone early and G-zone late was not found. Instead, whilst replication commences (with a very staggered start) in R-zones, activity soon appears to transgress band boundaries and gives rise to cells with unclassifiable patterns where chromosomes take on a mottled or reticulate appearance. Replication in R-zones dies out leaving a clear G-zone pattern persisting for the remainder of S which terminates with a very staggered finish. When pulse duration is increased (approximately 1 h) the frequency of unclassifiable cells falls and occasional "mixed-pattern" cells appear which have, within the same cell, typical R- and G-zone regions. The existence of such cells indicates that if a mid-S replication pause exists (and the absence of any mid-S wave of pale stained cells suggests that it does not) it does not make exclusive separation between dark R- and G-band zones.

Induction of sister chromatid exchanges (SCE) in G0 lymphocytes by plutonium-238 alpha-particles.

Irradiation of human G0 lymphocytes with plutonium-238 alpha-particles and X-rays was performed to investigate the production of sister chromatid exchanges (SCE). Alpha-particles produce a significant increase in SCE and this elevation is more significant when separated lymphocytes are irradiated. X-ray irradiation did not induce any significant increase in SCE. Therefore the relative biological effectiveness (RBE) for the induction of SCE by alpha-particles in this system is undefined and effectively infinite.