The Elbmarsch leukemia cluster: are there conceptual limitations in controlling immission from nuclear establishments in Germany?

The childhood leukemia cluster in the proximity of the German nuclear establishments of Geesthacht is unique in its spatial and temporal concentration. After a steep increase in cases in 1990, the cluster continues to show a significant increase up to the present. Early investigations of blood samples from a casual sample of local residents showed an increase in dicentric chromosomes in lymphocytes, indicating exposure exceeding dose limits. Analyses of the immission data revealed several unexpected deliveries of fission and activation products in the environment but provided no explanation of the source. Because of the observed overdispersion of dicentric chromosomes in cells, the idea of a contribution by densely ionizing emitters was compelling. The routine programs, however, do not include alpha emitters. These were measured in specific studies that proved contamination by transuranic nuclides. As shown in the present investigation, routine environmental surveillance programs support the occurrence of an accidental event near Geesthacht in September 1986. Until now, neither the cause nor the complete scenario of the activity release could be established. The ongoing discussion highlights limitations in the immission-control concept, which is predominantly based on gamma-radiation monitoring.

Electromagnetic exposure effects the hippocampal dentate cell proliferation in gerbils (Meriones unguiculatus).

The chronic effect on hippocampal neurogenesis after exposure (30 min/day for 14 days) to a high frequency (35,53 kHz) electromagnetic field, double modulated at extremely low frequencies (ELF; 1, 8, 12, 29 and 50 Hz), was studied in young adult gerbils. Immediately after the last exposure proliferation of dentate granule cells was identified by in vivo labeling with 5-bromo-2-desoxyuridine (BrdU). Exposure to 1, 29 and 50 Hz resulted in a statistically significant reduction of cell proliferation rates, but only the 50 Hz-group manifested the effect highly significantly (-29,3 %). On the other hand, gerbils exposed to 8 and 12 Hz showed no significant change of postmitotic cell proliferation as compared with the sham treated controls. The results suggest that the effects of ELF on the granule cell proliferation are mediated by neurotransmitters and hormones which regulate hippocampal neurogenesis.

90Sr-90Y biokinetics at incorporation determine the radiation burden to bone marrow.

The decay characteristics of 90Sr-90Y ensure that the mother and daughter nuclides exist in radioactive equilibrium, unless they get discriminated on the basis of their chemical properties, as it happens during metabolism. Although bone is the ultimate organ of deposition, the two nuclides arrive at this target organ over different biokinetic pathways. As 90Y is not excreted, it goes through transient deposition in the liver before being secondarily deposited in bone. This leads to a temporary radioactive excess of 90Y in bone. Since the decay energy of 90Y is by a factor of about 4 higher than that of 90Sr, the initial radiation burden to the bone marrow is primarily due to 90Y. This was estimated in rats by implanting LiF thermoluminescence dosimeters (TLD) in the marrow cavity of the femur. By calibrating the TLD against a known source of 90Sr-90Y, the absorbed dose rates and cumulative doses were determined as a function of time after incorporation. Two routes of administration were employed and their influence on the radiation burden is also shown.

Fertilising capacity of human sperms: a simple predictive assay.

Male subfertility is a growing reason for assisted reproduction. A limiting factor in male subfertility is asthenospermia. Motility is a cardinal indication of sperm vitality. Thus prognostic assays are aimed at quantitative determination of progression to assess the fertilising potential. However, a method permitting reliable prognosis of the fertilising capacity has yet to be developed. The assay presented here is the outcome of empirical data based on 590 IVF (in vitro fertilisation) trials. It is essentially a further exploitation of the Swim Up procedure, the selected sperms being maintained in culture under identical conditions employed in IVF. Semi-quantitative daily recordings of linear progression until complete extinction provided an index on vitality which is directly related to the fertilising potential. The findings indicated that a threshold of 50% linear motility after 24 hr culture was required to initiate fertilisation. The fertilising potential was guaranteed when at least 60% linear motility was observed at 24 hr, making the assay a predictive one. Its simplicity is an attractive feature.

Human granulosa cells in vitro: characteristics of growth, morphology and influence of some cytokines on steroidogenesis.

Granulosa cells (GCs) were characterised morphologically by light and electron microscopy. The steroidogenic capability of GCs in vitro was estimated by radioimmunoassay (RIA): oestradiol (E2), progesterone (P) and androstenedione (A) secreted into the culture medium were measured. The influence of several culture media and anchorage of the cell either to plastic vessels (monolayer) or to collagen fibrils (in gel) were studied. As the various culture media were assayed with regard to their suitability for IVF, it was found that Ham's F10 is quite satisfactory (in agreement with other observations on embryo cultures). A chemically defined medium BM 86 was found to be inadequate. In addition to the two cell types which are known, a third cell type which can perform efficient aromatisation (E2 production) in vitro is characterised here. The influence of cytokines/growth factors (GF) like insulin-like GF (IGF-1), epidermal GF (EGF), platelet-derived GF (PDGF) and fibroblast GF (FGF) on steroidogenesis was tested either alone or with human chorionic gonadotrophin (hCG). Except for oestradiol (E2) from early GCs, hCG generally stimulated progesterone (P) and E2 secretion. EGF by itself enhanced the secretion of P but not of E2. EGF did not affect hCG stimulation of P, but reduced that of E2. In contrast, in pre-ovulatory GCs IGF-I reduced the stimulatory effect of hCG on both E2 and P. In early GCs IGF-I potentiated hCG stimulation of P. In early GCs, neither hCG nor IGF-I nor a combination of IGF-I with hCG had any effect on E2 production.

Cell culture of biopsied endometriomas after danazol/hormonal therapy: a study of growth features and fertility effects.

The growth features of cells from endometriomas biopsied from patients who had been treated with Danazol or with hormones have not been studied in vitro. Danazol is a versatile drug and despite its recognised efficacy in controlling endometriosis, little is known about is cytotoxicity and mechanism of action. Culture of the biopsied endometriomas permitted qualitative cytotoxic assessments by way of comparison with cultured normal uterine endometrial cells treated in vitro with Danazol. The growth characteristics were studied in monolayer and collagen gel cultures. Cytopathology was characterised by light and electron microscopy. Since endometriosis is associated with infertility in women, data from in vitro fertilisation (IVF) were analysed with respect to treatment modalities as compared with cases suffering fallopian occlusion. Danazol reduced pregnancy chances. Two factors may be responsible: (a) altered follicle development resulting in poor oocyte quality (b) reduced nidation because of long-lasting endometrial cytotoxicity. The experimental findings reported here support the latter explanation. Consequently, Danazol therapy should be conditional; patients wishing to achieve pregnancy should preferably receive hormonal therapy.

Human granulosa cells in vitro: influence of GnRH/GnRH-agonist on steroidogenesis and on IVF outcome.

Steroidogenic activities of the granulosa cells (GCs) from 84 IVF trials were evaluated with respect to a set of ovarian stimulation regimens. Oestradiol (E2) synthesis of the GCs in vitro (obtained at oocyte retrieval) was compared to the maximal serum E2 levels of the same patients at induction of ovulation. Three stimulation regimens were employed: human post-menopausal gonadotrophin (hMG) alone; hMG accompanied by daily doses of a gonadotrophin releasing hormone agonist (GnRH-a); hMG preceded by a single depot application of the GnRH-a. Plots of E2 synthesis in vitro against serum E2 levels indicated that the GnRH-a directly inhibited E2 synthesis in the granulosa cells. This was confirmed in vitro by adding the agonist to the culture medium: both progesterone (P) and E2 syntheses were reduced in the presence of GnRH-a. Despite this drawback, the success of in vitro fertilization (IVF), as gauged by pregnancies achieved, was best for the group which received the GnRH-a as a single depot dose during the previous menstrual cycle, prior to the commence of stimulation. This success is attributed to the lower incidence of cancellations because of premature leuteinizing hormone (LH) surges which happen sometimes during ovarian stimulation. The implications of a direct influence of GnRH-a on E2 synthesis need to be further investigated.

Extracellular matrix (ECM) and cytoskeletal modulation of cellular radiosensitivity.

Modulation of radiosensitivity by components of the extracellular matrix (ECM) and cytoskeletal elements has not been adequately studied. Although differences in the radiosensitivities of cells grown as monolayers, as spheroids, or grown in vitro in animal models are known, explanations have in the past neglected possible influences by the ECM and cytoskeleton. Using collagen gel cultures, it is shown that the fibrillar component of the ECM (which is responsible for cell anchorage) induces shifts in radiosensitivity. The effect is critically dependent on the affinity of the cell type towards collagen. The shifts in radiosensitivity induced by ECM alteration are manifested as changed Dq values. By applying four specific cytoskeletal poisons which either stabilize or destabilize specific cytoskeletal elements, the involvement of microfilaments and microtubuli was qualitatively appraised. Cytochalasin B, which destabilizes microfilaments (by preventing polymerization), caused a significant rise in radioresistance. This rise was due to increased D0. Although the cellular morphological change accompanying cytochalasin B treatment was essentially similar to that obtained with trypsin, the respective shifts in radioresponses were qualitatively different and opposite, suggesting differences in mechanism of action.

In vitro holding and PLD repair. I. On the contribution of mitotic non-quiescence in plateau-phase Chinese hamster V79 cells.

The Stationary or Plateau-Phase of commonly used rodent cell lines like the V79 are often assumed to be quiescent (non-mitotic). An analysis of cell turnover in V79 plateau-phase cultures through BrUdR-incorporation combined with FUdR-block and light exposure (S-phase cytocide) revealed such cultures to be in a state of kinetic equilibrium. Even when the state of maximal permissible density was acquired, at least 50% of the population of cells were cycling within the time for one population doubling. Attempts at holding the cells from cycling (through nutrient-depletion and serum-privation) were unsuccessful, although the turnover-rate was reduced. Our assays for X-irradiated clonogenic survivors after attempted holding combined with delayed plating (DP) showed differences in the survival curves for exponentially growing and confluent cultures. Elimination of cycling cells by S-phase cytocide removed these differences. Since a significant fraction of plateau-phase cells are not mitotically quiescent (Q), one must eliminate the proliferating (P) fraction if one wishes to examine the PLDR of the Q cells. For V79 cells, removal of the P cells eliminates the higher survival (usually interpreted as Q cell PLDR) of plateau-phase cells.

Trypsinization and the radiosensitivity of mitotic and log phase Chinese hamster V79 cells exposed to 250 kVp X-rays.

We studied the influence of trypsin-induced morphological changes on the radiosensitivity of cells plated at either low (4-600/cm2) or high (2 x 10(4)/cm2) density and grown overnight before treatments. Trypsin treatment induced contraction and rounding of spread cells. The radiosensitivity of cells trypsinized and plated either: (1) immediately before [(a) D0 = 1.7 Gy for cells at low-, and (b) 1.5 Gy at high-density] or (2) immediately after (D0 = 1.6 Gy, high-density cells) irradiation was higher than that of (3) cells at high density, irradiated and delayed plated [cells remained spread until the completion of potentially lethal damage repair (PLDR) and trypsinization, D0 = 2.2 Gy], and (4) cells at low density which were neither delayed plated nor trypsinized (i.e. remained spread) after irradiation (Do = 2.4 Gy). These data show that PLDR is reduced in trypsin-treated cells of both high (compare 1b and 2 with 3) and low (compare 1a with 4) density cultures; the latter comparison provides a direct measure of the trypsin effect. Since the comparison between conditions 1 and 2 vs. 3 and 4 is of round vs. spread cells, PLDR appears to be influenced by the cell's morphological state. Kinetic studies showed that when cells were incubated in growth medium to recover from trypsin-induced effects before irradiation, the radiation sensitivity of spread cells (plated in situ), but not of those remaining rounded (in suspension until plating and irradiation), decreased and became equal to that of delayed plated high-density cells. Neither irradiated cells treated with hypertonic saline, nor mitotic cells, showed the trypsin effect. From these results we suggest that: (1) trypsin-induced cell contraction affects the ability of cells to repair radiation damage, (2) spread cells are better able to repair PLD than rounded cells, (3) immediate plating survival of cells in high-density cultures may not represent their intrinsic radiosensitivity and (4) cell-to-cell contact is not necessary for log phase cells to repair PLD.

In vitro holding and PLD-repair: II. A flow cytometric and electron microscopic analysis of some mammalian cell lines.

The repair of potentially lethal damage (PLDR) in mammalian cells is expected to be better in quiescent cultures since PLD is supposedly fixed during cycle progression. Plateau phase cultures, therefore, serve as models because of assumed mitotic quiescence. Four established cell lines (V79, CHO, L5178Y and HELA) and one euploid cell strain IMR-90 have been analysed by flow cytometry and electron microscopy to address questions on quiescence in the plateau phase and the effect of holding (induction of quiescence by nutrient privation). In contrast to commonly held views, our results indicate that the quiescent fraction in cultures from transformed cells is exceedingly low (1% or less). Plateau phase cultures of transformed cells are constantly turning over. Euploid cells like the IMR-90 show true quiescence in the plateau phase. Holding causes typical cytopathological changes. These changes have been ultrastructurally++ characterised. Resistant sub-populations of cells can be selected out under holding-conditions. Such selected cells show completely different radiobiological characteristics, which raise questions on the interpretation of data on PLDR.

Fertilization through spermatozoal microinjection: significance of acrosome reaction.

Acrosome-reacted spermatozoa were microinjected into the perivitelline space of mouse oocytes. After 2 h incubation in culture medium containing lactate and albumin, spermatozoa were transferred into culture medium containing 12 mM of dibutyryl cyclic guanosine 3',5'-monophosphate (dbcGMP) and 10 mM imidazole for 20 min. One motile spermatozoon was injected into the perivitelline space of each oocyte. Fertilization was recognized by the presence of a second polar body and two pronuclei. The overall fertilization rate was 19.6% in the case of dbcGMP-treated spermatozoa as compared to 5.3% for non-treated spermatozoa. Thus, acrosome-reacted motile spermatozoa improve the fertilization rate of sperm microinjection. Sperm microinjection may be a method to foster fertility in cases of oligo-/asthenozoospermia in human in-vitro fertilization.

Two subsets of human alphoid repetitive DNA show distinct preferential localization in the pericentric regions of chromosomes 13, 18, and 21.

We have isolated and characterized two human middle repetitive alphoid DNA fragments, L1.26 and L1.84, which localize to two different sets of chromosomes. In situ hybridization revealed both repeats to have major and minor binding sites on the pericentric regions of several chromosomes. Probe L1.26 maps predominantly to chromosomes 13 and 21. Probe L1.84 locates to chromosome 18. Minor hybridization sites for both probes include chromosomes 2, 8, 9, and 20; in addition, L1.26 revealed minor sites on chromosomes 18 and 22. The binding to these sites strongly depends on hybridization conditions. In Southern blot hybridizations to total human DNA, both L1.26 and L1.84 give the same ladder pattern, with a step size of 170 bp, indicating their presence as tandem repeats, but with different band intensities for each probe. The chromosome-specific nature of particular multimers was confirmed by Southern blot analyses of a human-rodent hybrid cell panel. We conclude that L1.26 and L1.84, with their related sequences, constitute subfamilies of alphoid DNA that are specific for subsets of chromosomes and, in some cases, possibly even for single chromosomes.

Haematological studies on 90Sr-90Y-toxicity: II. Femoral CFU-s kinetics and mitogen response of spleen cells.

Ferrokinetic perturbations in peripheral blood of mice treated with 90Sr-90Y were demonstrated previously. This paper deals with the effects of 90Sr-90Y treatment (2.5, 5, and 10 microCi/mouse), on the haemopoietic stem cell compartment and on the immune-status. The frequency and kinetics of haemopoietic stem cells in femoral marrow (determined as colony forming units in spleen, CFU-s) and their haemopoietic efficiency (as gauged by 59Fe- and 125IUdR-uptake) were estimated. The responsiveness of splenic lymphocytes of the same animals to mitogens (Con A, PHA, and LPS) were also measured. In all assays striking dose-dependent changes marked either by depressions or overshoots were observed during the first week post-incorporation. These are correlated with the pattern of deposition (primary and secondary) of the radionuclides 90Sr-90Y. Towards the end of the period of observation and presumably thereafter, the dependence on dose disappeared and the values remained subnormal. An exception to this was the response of splenic lymphocytes to mitogens. Much higher reactivity was recorded up to the end. This higher reactivity is attributed to augmented cellular turnover, the newly recruited lymphocytes (in accord with thier extreme radiosensitivity), being probably more reactive.

Radioprotective and haemopoietic effects of some lipopolysaccharides from Rhodospirillaceae species in mice.

Lipopolysaccharides (LPS) from various Rhodopseudomonas and Rhodospirillum species were tested for their radioprotective efficiency against X-irradiation and for their influence on the growth of spleen colony forming units (CFU-s) in mice. The LPS from Rhodopseudomonas gelatinosa Dr2 gave a high survival rate. It also favoured CFU-s formation and erythroid differentiation.

Murine spermatogonial regeneration after exposure to either X-rays or 15 MeV neutrons.

The long-term regeneration of the seminiferous epithelium after irradiation with 15 MeV neutrons was studied in the mouse on a comparative basis; 150 kV X-rays were used as reference-radiation. The mice received total-body exposures at matched doses. The spermatogonial Regeneration Index (RI) was scored from stained paraffin-sections of testes which were obtained after periodic sacrifice. A biphasic pattern of regeneration was recorded. Dose-independent and dose-dependent effects have been indicated. The radiobiological implications are discussed on a cellular basis.

Haemopoietic recovery during radiation disease: comments on combined-injuries.

The regenerative ability of haemopoietic organs during combined radiation injuries has not been adequately investigated. Interactions among individual factors can critically influence the processes involved in haemopoietic recovery. An overview of radiation injuries is given, and a concept towards a hypothetical mode of action at the cellular level is presented. The influence which interacting factors can have on the concentration of pluripotential haemopoietic stem cells (HSC) is demonstrated by results from an initial experiment. The importance of synergistic and antagonistic reactions is emphasised and commented upon.

Senescence in vitro and ionising radiations--the human diploid fibroblast model.

The influence of ionising radiations on ageing is still controversial. Since Hayflick established the concept that diploid cells have finite lifespan in vitro, human diploid fibroblast (HDF) cultures have been recognised as a potent experimental model for cyto--gerontological investigations. In this study HDF cultures in phase II were exposed to acute irradiation with either X-rays or fast neutrons. The replicative potentials and labelling indices with [3H]thymidine were measured post irradiation until the cultures ceased growth in phase III. Cell mortality was measured by cloning. The apparent loss in replicative potential of irradiated mass cultures was wholly attributable to the loss of viable clonogenic cells. The current concept of precocious clonal senescence in vitro as a late effect of irradiation in clonogenic survivors is not supported by the present experiments. Instead, our results suggest that exposure to a single dose of ionising radiations either causes total replicative incapacitation (killing) of HDF cells and their progeny early after irradiation or leaves their replicative potentials unperturbed.