In vitro SCE discrepancy between embryonic and extraembryonic mouse tissues

In vitro sister-chromatid exchange (SCE) background levels and cytokinetics were compared in embryonic (whole embryo cell suspensions) and extraembryonic (yolk sac and amnion, placenta) cells of inbred and outbred strains at various gestational stages (days 12-17). Results indicate a tissue origin (embryonal, extraembryonal) related variation in the formation of baseline SCE frequencies and cytokinetics. The significant higher SCE levels in extraembryonic tissues (maximum increase of 2 X the background values of the embryo cells) were independent of mouse strain and gestational stage. An average of 4-5 SCEs/cell in embryo cells is contrasted by 7-9 SCEs/cell in extraembryo cells. Mitotic index was generally lower and average generation time longer (by 2-3 h) in extraembryonic tissue cells. No significant differences in SCE frequencies and no changes in cytokinetics were detected at the BrdU concentrations used (1.2-4.8 micrograms/ml). The reason for the inter-tissue differences in baseline SCE is still not clear.     

A unique human mutant B-lymphoblastoid cell line (ataxia telangiectasia) which exhibits increased siste-chromatid exchange retaining hypersensitivity to neocarzinostatin and bleomycin

Chromosome aberrations and sister-chromatid exchanges (SCEs) were examined in 4 ataxia telangiectasia (AT)-derived B-lymphoblastoid cell lines (B-LCLs) (AT-S, AT-SHI, AT-SHI B13A and AsHa) following treatments with neocarzinostatin (NCS) and bleomycin. All of these cell lines exhibited extremely high frequencies of chromosome aberrations with the NCS and bleomycin treatments. Among them, AsHa, a mutant B-LCL originating from an AT patient, showed high frequencies of SCEs under high bromodeoxyuridine (BrdU) concentrations retaining hypersensitivity to NCS and bleomycin with regard to chromosome aberrations. A clear BrdU dose-dependent increase in SCEs (9.85 SCEs/cell at 40 μg/ml, 36.65 SCEs/cell at 100 μg/ml on average) in this mutant was observed. When AsHa mutant cells were treated with NCS (0.02 μg/ml) and/or bleomycin (5.0 μg/ml) under 40 μg/ml BrdU (minimum BrdU concentration for sister-chromatid differential staining), SCE levels increased from 9.85 (baseline level) to 21.1 with NCS and 20.5 with bleomycin, in a dose-dependent manner. These observations indicate that AsHa is a unique AT-derived mutant cell clone with a high SCE character retaining the original hypersensitivity to bleomycin and NCS.     

Spontaneous and busulphan-induced sister-chromatid exchange (SCE) frequencies in haematologically normal human bone marrow and lymphocytes

In a study of 14 patients who were not treated with either chemotherapy or irradiation, 13 patients had lower siste-chromatid exchange (SCE) frequencies in their bone-marrow cells than in their lymphocytes. For both bone marrow and lymphocytes, there was significant inter-patient variability in SCE frequencies, but there was no correlation between the bone-marrow and lymphocyte values.

The effect of exposing bone-marrow cells to busulphan (BUS) in vitro was investigated using doses up to 5.0 μg/ml. The dose-response relationships between BUS and SCEs in vitro were found to be similar for bone marrow and lymphocytes.     

Bromodeoxyuridine (BrdU) template and thymodine pool effects on high frequencies of sister-chromatid exchange (SCE) in Bloom syndrome cells and a mutant cell line (AsHa) originated from ataxia telangiectasia

The effect of bromodeoxyuridine (BrdU)-substituted DNA template and thymidine (dT) pool on excess sister-chromatid exchanges (SCEs) was studied in Bloom syndrome (BS) cells and an ataxia telangiectasia (AT)-derived mutant cell line (AsHa). When BS endomitotic cells were labeled with low and high (or high and low) BrdU concentrations during S₁ and S₂, only the BrdU concentration during S₁ phase affected the observed SCE. In BS cells about a 10-fold increase in SCEs occurs during or following replication on a BrdU-substituted template (high-high and high-low BrdU labeling) relative to the normal DNA template. SCEs decreased to about half in AsHa cells labeled with various BrdU doses (40, 60, 80 and 100 μg/ml) during only S₁, compared with those labeled during S₁ and S₂. Co-cultivation of AsHa and BS cells resulted in a significant reduction in SCE level from 70 to 13–17 in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential (SCD) staining from 40 to 10 μg/ml with normal SCE level and resulted in decreased level of SCEs at high BrdU concentrations (80–100 μg/ml) 12–14 SCE) in AsHa cells, compared with the originally increased SCE level (36.65 SCE at 100 μg/ml) without co-culture. However, co-cultivation between AsHa and normal cells lowered the BrdU dose necessary for SCD staining from 40 to 30 μg/ml; the dT pool possibly balanced at this level, which is clearly higher than that at co-cultivation between AsHa and BS cells. The reason for the very high BrdU doses needed to achieve SCD would seem to be that AsHa cells have high levels of thymidylate (TMP) synthetase, which maintain a large endogenous thymidine pool. This has been confirmed by direct measurement. These findings strongly support that excess and decreased dT pools are closely related to the condition necessary for high SCE induction.     

Influence of chromosomal determinants on development of androgenetic and parthenogenetic cells

We have examined the role of germline-specific chromosomal determinants of development in the mouse. Studies were carried out using aggregation chimaeras between androgenetic----fertilized embryos and compared with similar parthenogenetic----fertilized chimaeras. Several adult chimaeras were found with parthenogenetic cells but none were found with androgenetic cells. Analysis of chimaeras at mid-gestation showed that parthenogenetic cells were detected in the embryo and yolk sac but that androgenetic cells were found only in the trophoblast and yolk sac and not in the embryo. The contribution of parthenogenetic cells to the embryo and yolk sac was increased by aggregating 2-cell parthenogenetic and 4-cell fertilized embryos but the contribution of parthenogenetic cells in extraembryonic tissues remained negligible even after aggregation of 4-cell parthenogenetic and 2-cell fertilized embryos. Furthermore, parthenogenetic cells were primarily found in the yolk sac mesoderm and not in the yolk sac endoderm. These results suggest that maternal chromosomes in parthenogenetic cells permit their participation in the primitive ectoderm lineage but these cells are presumably eliminated by selective pressure or autonomous cell lethality from the primitive endoderm and trophectoderm lineages. Conversely paternal chromosomes in androgenetic cells confer opposite properties since the embryonic cells can be detected in the trophoblast and the yolk sac but not in the embryos, presumably because they are eliminated from the primitive ectoderm lineage. The spatial distribution of cells with different parental chromosomes may occur partly because of differential expression of some genes, such as proto-oncogenes, and partly due to their ability to respond to a variety of diffusible growth factors.     

In vitro development of murine T cells from prethymic and preliver embryonic yolk sac hematopoietic stem cells.

Mature T cells are derived from prethymic stem cells, which arise at one or more extrathymic sites and enter and differentiate in the thymus. The nature of these prethymic stem cells is a critical factor for the formation of the T-cell repertoire. Although the bone marrow of adult mice can provide such stem cells, their origin during murine embryogenesis is still undetermined. Among potential sites for these progenitor cells are the fetal liver and the embryonic yolk sac. Our studies focus on the yolk sac, both because the yolk sac appears earlier than any other proposed site, and because the mammalian yolk sac is the first site of hematopoiesis. Although it has been shown that the yolk sac in midgestation contains stem cells that can enter the thymic rudiment and differentiate toward T-cell lineage, our aim was to analyze the developmental potential of cells in the yolk sac from earlier stages, prior to the formation of the liver and any other internal organ. We show here that the yolk sac from 8- and 9-day embryos (2-9 and 13-19 somites, respectively) can reconstitute alymphoid congenic fetal thymuses and acquire mature T-cell-specific characteristics. Specifically, thymocytes derived from the early embryonic yolk sac can progress to the expression of mature T lymphocyte markers including CD3/T-cell receptor (TCR), CD4 and CD8. In contrast, we have been unable to document the presence of stem cells within the embryo itself at these early stages. These results support the hypothesis that the stem cells capable of populating the thymic rudiment originate in the yolk sac, and that their presence as early as at the 2- to 9-somite stage may indicate that prethymic stem cells found elsewhere in the embryo at later times may have been derived by migration from this extra-embryonic site. Our experimental design does not exclude the possibility of multiple origins of prethymic stem cells of which the yolk sac may provide the first wave of stem cells in addition to other later waves of cells.     

CD41 expression defines the onset of primitive and definitive hematopoiesis in the murine embryo

The platelet glycoprotein IIb (alpha(IIb); CD41) constitutes the alpha subunit of a highly expressed platelet surface integrin protein. We demonstrate that CD41 serves as the earliest marker of primitive erythroid progenitor cells in the embryonic day 7 (E7.0) yolk sac and high-level expression identifies essentially all E8.25 yolk sac definitive hematopoietic progenitors. Some definitive hematopoietic progenitor cells in the fetal liver and bone marrow also express CD41. Hematopoietic stem cell competitive repopulating ability is present in CD41(dim) and CD41(lo/-) cells isolated from bone marrow and fetal liver cells, however, activity is enriched in the CD41(lo/-) cells. CD41(bright) yolk sac definitive progenitor cells co-express CD61 and bind fibrinogen, demonstrating receptor function. Thus, CD41 expression marks the onset of primitive and definitive hematopoiesis in the murine embryo and persists as a marker of some stem and progenitor cell populations in the fetal liver and adult marrow, suggesting novel roles for this integrin.     

In vivo BrdU-33258 Hoechst analysis of DNA replication kinetics and sister chromatid exchange formation in mouse somatic and meiotic cells

BrdU (5-bromodeoxyuridine)-33258 Hoechst methods have been adapted for in vivo analyses of replication kinetics, sister chromatid differentiation and sister chromatid exchange (SCE) formation in mice. Sufficient in vivo BrdU substitution for cytological detection was effected with multiple intraperitoneal injections of the analogue. The combination of centromere staining asymmetry and sister chromatid differentiation at metaphase permits unambiguous determination of the number of replications in BrdU and dT (deoxythymidine) undergone by individual cells. Late-replicating regions in marrow and spermatogonial chromosomes are highlighted by bright fluorescence after sequential incorporation of BrdU followed by dT during a single DNA synthesis period. SCEs are analyzed in marrow and spermatogonial metaphases after successive complete cycles of BrdU and dT incorporation. Significant induction of SCE was observed with both mitomycin C and cyclophosphamide; the latter drug requires host-mediated activation to be effective. In meiotic metaphase cells harvested two weeks after BrdU incorporation, satellite DNA asymmetry, sister chromatid differentiation and SCE could be detected in a few chromosomes, most frequently the X and the Y.     

Anomalous neural differentiation induced by 5-bromo-2'-deoxyuridine during organogenesis in the rat

The influence of 5-bromo-2'-deoxyuridine (BrdU) on rat embryo development and neurogenesis was investigated using a rat conceptus culture system during organogenesis (pregnancy days 10-13). The embryos and visceral yolk sacs of conceptuses cultured with BrdU were examined for overall growth, morphological anomalies, incorporation of radiolabeled BrdU into DNA, and neurotransmitter enzyme activities in embryos. In addition, neural tubes from cultured whole embryos were isolated and mechanically dissociated into fragments and cultured again to assess neural cell differentiation into neuron-like cells. BrdU was found to incorporate differentially into embryonic and visceral yolk sac DNA with simultaneous stage-specific retardation and anomalous organogenesis in proportion to the increasing concentrations used. Neural tube differentiation of cultured embryos was markedly altered, and there were morphologically distinct neural anomalies. The neurite outgrowth from neuroblast cells (type 1) of explanted spinal neural tube fragments from BrdU-treated embryos was markedly reduced in length and number compared to those from similar areas of embryos grown without BrdU. In contrast, BrdU at the same doses did not affect differentiation of a number of neural tissue-related enzymes. These results indicate that BrdU incorporation into DNA of primordial embryonic cells significantly affects neurogenesis and differentiation of neurites from neuroblasts, which is a specific neural cytodifferentiation characteristic of neuronal cells.     

Experimental yolk sac dysfunction as a model for studying nutritional disturbances in the embryo during early organogenesis

Our investigations concerning the importance of cell surface macromolecules during embryonic development led us to the discovery in 1961 that heterologous anti-rat kidney serum produced teratogenesis, growth retardation and embryonic death when injected into the pregnant rat during early organogenesis. It was established that IgG was the teratogenic agent, primarily directed against the visceral yolk sac (VYS) but not the embryo. Heterologous anti-rat VYS serum was prepared which was teratogenic localized in the VYS and served as a model for producing VYS dysfunction and embryonic malnutrition. The role of the yolk sac placenta in histiotrophic nutrition is now recognized to be critical for normal embryonic development during early organogenesis in the rodent. VYS antiserum affects embryonic development primarily by inhibiting endocytosis of proteins by the VYS endoderm, resulting in a reduction in the amino acids supplied to the embryo. Our laboratory has recently developed teratogenic monoclonal yolk sac antibodies (MCA) which can be utilized; to study VYS plasma membrane synthesis and recycling, to compare yolk sac function among different species, and to identify components of the plasma membrane involved in pinocytosis. MCA prepared against certain VYS antigens provide an opportunity to study embryonic nutrition with minimal interference with the nutritional state of the mother. Recent developments in the study of the human yolk sac along with our laboratory's ability to isolate a spectrum of yolk sac antigens, prepare monoclonal antibodies, and perform functional studies, should provide information that will increase our understanding of yolk sac function and dysfunction in the human and determine the relative importance of various amino acids to normal development during mammalian organogenesis.     

Spontaneous and mitomycin-C induced sister-chromatid exchanges : Comparison of in vivo and in vitro systems

Frequencies of sister-chromatid exchanges (SCE) were measured in vitro in mouse fibroblasts and in vivo in mouse bone-marrow cells. SCE levels in these cell systems were measured in response to varying concentrations of bromodeoxyuridine (BrdU) and mitomycin-C (MMC). Although BrdU was found to induce SCE in both cellular systems, baseline SCE levels were 2- to 3-fold higher in vitro than in vivo. SCE induction was found to be a linear function of MMC concentration in vivo and in vitro; however the slope of the in vivo curve was 5-fold higher. The interaction of BrdU substituted DNA and MMC was examined by administering a fixed dose of MMC with increasing concentrations of BrdU. The induced SCE frequencies appeared to be additive. In addition to measuring drug-induced SCE, the BrdU differential staining technique allows concomitant measurement of the inhibition of cellular replication by the test drugs.     

Proteolytic activity in the yolk sac membrane of quail eggs.

Extraembryonal degradation of yolk protein is necessary to provide the avian embryo with required free amino acids during early embryogenesis. Screening of proteolytic activity in different compartments of quail eggs revealed an increasing activity in the yolk sac membrane during the first week of embryogenesis. In this tissue, the occurrence of cathepsin B, a lysosomal cysteine proteinase, and cathepsin D, a lysosomal aspartic proteinase, has been described recently (Gerhartz et al., Comp Biochem Physiol, 118B:159-166, 1997). Determination of cathepsin B-like and cathepsin D-like proteolytic activity in the yolk sac membrane indicated a significant correlation between growth of the yolk sac membrane and proteolytic activity, shown by an almost constant specific activity. Both proteinases could be localized in the endodermal cells, which are in direct contact to the yolk. The concentration of proteinases in the endodermal cells appears to be almost unaltered in the investigated early stage of quail development, whereas the amount of endodermal cells increases rapidly, seen by a complicated folding of the yolk sac membrane. In the same cells quail cystatin, a potent inhibitor of quail cathepsin B (Ki 0.6 nM), has been localized at day 8 of embryonic development. Approximately at this stage of development, the quail embryo stops metabolizing yolk. In conclusion, it is strongly indicated that the amount of available free amino acids, produced by proteolytic degradation and supporting embryonic growth, is regulated by the growth of the yolk sac membrane.     

Mouse Wnt receptor gene Fzd5 is essential for yolk sac and placental angiogenesis

Wnts are secreted signaling molecules implicated in various developmental processes and frizzled proteins are the receptors for these Wnt ligands. To investigate the physiological roles of frizzled proteins, we isolated and characterized a novel mouse frizzled gene Fzd5. Fzd5 mRNA was expressed in the yolk sac, eye and lung bud at 9.5 days post coitum. Fzd5 specifically synergized with Wnt2, Wnt5a and Wnt10b in ectopic axis induction assays in Xenopus embryos. Using homologous recombination in embryonic stem cells, we have generated Fzd5 knockout mice. While the heterozygotes were viable, fertile and appeared normal, the homozygous embryos died in utero around 10.75 days post coitum, owing to defects in yolk sac angiogenesis. At 10.25 days post coitum, prior to any morphological changes, endothelial cell proliferation was markedly reduced in homozygous mutant yolk sacs, as measured by BrdU labeling. By 10.75 days post coitum, large vitelline vessels were poorly developed, and the capillary plexus was disorganized. At this stage, vasculogenesis in the placenta was also defective, although that in the embryo proper was normal. Because Wnt5a and Wnt10b co-localized with Fzd5 in the developing yolk sac, these two Wnts are likely physiological ligands for the Fzd5-dependent signaling for endothelial growth in the yolk sac.     

Yolk sac development in lizards (Lacertilia: Scincidae): New perspectives on the egg of amniotes

Embryos of oviparous reptiles develop on the surface of a large mass of yolk, which they metabolize to become relatively large hatchlings. Access to the yolk is provided by tissues growing outward from the embryo to cover the surface of the yolk. A key feature of yolk sac development is a dedicated blood vascular system to communicate with the embryo. The best known model for yolk sac development and function of oviparous amniotes is based on numerous studies of birds, primarily domestic chickens. In this model, the vascular yolk sac forms the perimeter of the large yolk mass and is lined by a specialized epithelium, which takes up, processes and transports yolk nutrients to the yolk sac blood vessels. Studies of lizard yolk sac development, dating to more than 100 years ago, report characteristics inconsistent with this model. We compared development of the yolk sac from oviposition to near hatching in embryonic series of three species of oviparous scincid lizards to consider congruence with the pattern described for birds. Our findings reinforce results of prior studies indicating that squamate reptiles mobilize and metabolize the large yolk reserves in their eggs through a process unknown in other amniotes. Development of the yolk sac of lizards differs from birds in four primary characteristics, migration of mesoderm, proliferation of endoderm, vascular development and cellular diversity within the yolk sac cavity. Notably, all of the yolk is incorporated into cells relatively early in development and endodermal cells within the yolk sac cavity align along blood vessels which course throughout the yolk sac cavity. The pattern of uptake of yolk by endodermal cells indicates that the mechanism of yolk metabolism differs between lizards and birds and that the evolution of a fundamental characteristic of embryonic nutrition diverged in these two lineages. Attributes of the yolk sac of squamates reveal the existence of phylogenetic diversity among amniote lineages and raise new questions concerning the evolution of the amniotic egg. J. Morphol. 278:574–591, 2017. © 2017 Wiley Periodicals, Inc.     

Blood-forming potential of vascular endothelium in the human embryo

Hematopoietic cells arise first in the third week of human ontogeny inside yolk sac developing blood vessels, then, one week later and independently, from the wall of the embryonic aorta and vitelline artery. To address the suggested derivation of emerging hematopoietic stem cells from the vessel endothelium, endothelial cells have been sorted by flow cytometry from the yolk sac and aorta and cultured in the presence of stromal cells that support human multilineage hematopoiesis. Embryonic endothelial cells were most accurately selected on CD34 or CD31 surface expression and absence of CD45, which guaranteed the absence of contaminating hematopoietic cells. Yet, rigorously selected endothelial cells yielded a progeny of myelo-lymphoid cells in culture. The frequency of hemogenic endothelial cells in the yolk sac and aorta reflected the actual blood-forming activity of these tissues, as a function of developmental age. Even less expected, a subset of endothelial cells sorted similarly from the embryonic liver and fetal bone marrow also exhibited blood-forming potential. These results suggest that a part at least of emerging hematopoietic cells in the human embryo and fetus originate in vascular walls.     

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 analoques. 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.     

Demonstration of a phagocytic cell system belonging to the hemopoietic lineage and originating from the yolk sac in the early avian embryo.

It is well established that hemopoietic cells arising from the yolk sac invade the avian embryo. To study the fate and role of these cells during the first 2.5-4.5 days of incubation, we constructed yolk sac chimeras (a chick embryo grafted on a quail yolk sac and vice versa) and immunostained them with antibodies specific to cells of quail hemangioblastic lineage (MB1 and QH1). This approach revealed that endothelial cells of the embryonic vessels are of intraembryonic origin. In contrast, numerous hemopoietic cells of yolk sac origin were seen in embryos ranging from 2.5 to 4.5 days of incubation. These cells were already present within the vessels and in the mesenchyme at the earliest developmental stages analyzed. Two hemopoietic cell types of yolk sac origin were distinguishable, undifferentiated cells and macrophage-like cells. The number of the latter cells increased progressively as development proceeded, and they showed marked acid phosphatase activity and phagocytic capacity, as revealed by the presence of numerous phagocytic inclusions in their cytoplasm. The macrophage-like cells were mostly distributed in the mesenchyme and also appeared within some organ primordia such as the neural tube, the liver anlage and the nephric rudiment. Comparison of the results in the two types of chimeras and the findings obtained with acid phosphatase/MB1 double labelling showed that some hemopoietic macrophage-like cells of intraembryonic origin were also present at the stages considered. These results support the existence in the early avian embryo of a phagocytic cell system of blood cell lineage, derived chiefly from the yolk sac. Cells belonging to this system perform phagocytosis in cell death and may also be involved in other morphogenetic processes.     

In vivo and in vivo/in vitro kinetics of cyclophosphamide-induced sister-chromatid exchanges in mouse bone marrow and spleen cells

In several acute and chronic exposures to various chemicals in vivo and in vitro, the average sister-chromatid exchange (SCE) frequencies in human, mouse, rat, and rabbit lymphocytes generally decrease with time following treatment. The rate of this decline varies, but little data have been published pertaining to the comparative kinetics of SCEs both in vivo and in vivo/in vitro (exposure of animals to the test compound and culturing of cells) simultaneously in the same tissues. In this study, a single dose of cyclophosphamide (40 mg/kg) was injected for varying periods (6-48 h) and its effects, as assessed by the induction of SCEs, were analyzed under both in vivo and in vivo/in vitro conditions in mouse bone marrow and spleen cells. In vivo, the cyclophosphamide-induced SCEs increased with increasing time up to 12 h, stayed at approximately the same level until 24 h, and then decreased with increase in post-exposure time. However, the SCE levels remained significantly higher than controls at 48 h post-exposure time in both bone marrow and spleen cells. Under in vivo/in vitro conditions, the SCEs in bone marrow decreased with increase in post-exposure time until reaching control values by 48 h post exposure. However, in spleen cells, the decrease in SCE level was gradual, and by 48 h post-exposure time, the cells still had approximately 6 times higher SCEs than the control values. These results suggest that there are pharmacokinetic differences for cyclophosphamide in mouse bone marrow and spleen. Also, there is a differential SCE response to cyclophosphamide under in vivo and in vivo/in vitro conditions.     

Analysis of baseline and BrdU-dependent SCEs at different BrdU concentrations

In the present paper we have used a rationale based on the development of theoretical equations that define sister-chromatid exchange (SCE) frequencies as a function of two variables, namely the baseline (BrdU-independent) and the BrdU-dependent SCE frequencies. The experimental design includes the estimation of SCE frequencies in second division chromosomes when both cycles occurred in the presence of BrdU and when BrdU incubation took place only during the first cycle in a wide range of BrdU concentrations. The final SCE yields in second division chromosomes could be separated into three different components: (1) The BrdU-independent, ‘spontaneous’ or baseline SCEs, whose low but biologically significant frequency was calculated to be about 0.06 SCEs per pg of DNA; this figure could be similar for most of the cell types; (2) the BrdU-dependent SCEs whose frequency increases with BrdU dose, probably as a result of BrdU substitution for thymidine; (3) the BrdU-dependent SCEs as a consequence of other cellular factors such as disturbance of nucleotide pool sizes. At high BrdU concentrations (300 μM upward) the three components appear to have a significant value in the final SCE yield, whereas at lower BrdU doses the third component seems to be negligible.