Induction of Chromosomal Aberrations after Exposure to the Auger Electron Emitter Iodine-125, the ß--emitter Tritium and Cesium-137 γ rays.

High-LET-type cell survival curves have been observed in cells that were allowed to incorporate 125I-UdR into their DNA. Incorporation of tritiated thymidine into the DNA of cells has also been shown to result in an increase in relative biological effectiveness in cell survival experiments, but the increase is smaller than observed after incorporation of 125I-UdR. These findings are explained in the literature by the overall complexity of the induced DNA damage resulting from energies of the ejected electron(s) during the decay of 3H and 125I. Chromosomal aberrations (CA) are defined as morphological or structural changes of one or more chromosomes, and can be induced by ionizing radiation. Whether the number of CA is associated with the linear energy transfer (LET) of the radiation and/or the actual complexity of the induced DNA double-strand breaks (DSB) remains elusive. In this study, we investigated whether DNA lesions induced at different cell cycle stages and by different radiation types [Auger-electrons (125I), ß- particles (3H), or γ radiation (137Cs)] have an impact on the number of CA induced after induction of the same number of DSB as determined by the γ-H2AX foci assay. Cells were synchronized and pulse-labeled in S phase with low activities of 125I-UdR or tritiated thymidine. For decay accumulation, cells were cryopreserved either after pulse-labeling in S phase or after progression to G2/M or G1 phase. Experiments with γ irradiation (137Cs) were performed with synchronized and cryopreserved cells in S, G2/M or G1 phase. After thawing, a CA assay was performed. All experiments were performed after a similar number of DSB were induced. CA induction after 125I-UdR was incorporated was 2.9-fold and 1.7-fold greater compared to exposure to γ radiation and radiation from incorporated tritiated thymidine, respectively, when measured in G2/M cells. In addition, measurement of CA in G2/M cells after incorporation of 125I-UdR was 2.5-fold greater when compared to cells in G1 phase. In contrast, no differences were observed between the three radiation qualities with respect to exposure after cryopreservation in S or G1 phase. The data indicate that the 3D organization of replicated DNA in G2/M cells seems to be more sensitive to induction of more complex DNA lesions compared to the DNA architecture in S or G1 cells. Whether this is due to the DNA organization itself or differences in DNA repair capability remains unclear.

RENEB Inter-Laboratory Comparison 2021: Inter-Assay Comparison of Eight Dosimetry Assays.

Tools for radiation exposure reconstruction are required to support the medical management of radiation victims in radiological or nuclear incidents. Different biological and physical dosimetry assays can be used for various exposure scenarios to estimate the dose of ionizing radiation a person has absorbed. Regular validation of the techniques through inter-laboratory comparisons (ILC) is essential to guarantee high quality results. In the current RENEB inter-laboratory comparison, the performance quality of established cytogenetic assays [dicentric chromosome assay (DCA), cytokinesis-block micronucleus assay (CBMN), stable chromosomal translocation assay (FISH) and premature chromosome condensation assay (PCC)] was tested in comparison to molecular biological assays [gamma-H2AX foci (gH2AX), gene expression (GE)] and physical dosimetry-based assays [electron paramagnetic resonance (EPR), optically or thermally stimulated luminescence (LUM)]. Three blinded coded samples (e.g., blood, enamel or mobiles) were exposed to 0, 1.2 or 3.5 Gy X-ray reference doses (240 kVp, 1 Gy/min). These doses roughly correspond to clinically relevant groups of unexposed to low exposed (0-1 Gy), moderately exposed (1-2 Gy, no severe acute health effects expected) and highly exposed individuals (>2 Gy, requiring early intensive medical care). In the frame of the current RENEB inter-laboratory comparison, samples were sent to 86 specialized teams in 46 organizations from 27 nations for dose estimation and identification of three clinically relevant groups. The time for sending early crude reports and more precise reports was documented for each laboratory and assay where possible. The quality of dose estimates was analyzed with three different levels of granularity, 1. by calculating the frequency of correctly reported clinically relevant dose categories, 2. by determining the number of dose estimates within the uncertainty intervals recommended for triage dosimetry (±0.5 Gy or ±1.0 Gy for doses <2.5 Gy or >2.5 Gy), and 3. by calculating the absolute difference (AD) of estimated doses relative to the reference doses. In total, 554 dose estimates were submitted within the 6-week period given before the exercise was closed. For samples processed with the highest priority, earliest dose estimates/categories were reported within 5-10 h of receipt for GE, gH2AX, LUM, EPR, 2-3 days for DCA, CBMN and within 6-7 days for the FISH assay. For the unirradiated control sample, the categorization in the correct clinically relevant group (0-1 Gy) as well as the allocation to the triage uncertainty interval was, with the exception of a few outliers, successfully performed for all assays. For the 3.5 Gy sample the percentage of correct classifications to the clinically relevant group (≥2 Gy) was between 89-100% for all assays, with the exception of gH2AX. For the 1.2 Gy sample, an exact allocation to the clinically relevant group was more difficult and 0-50% or 0-48% of the estimates were wrongly classified into the lowest or highest dose categories, respectively. For the irradiated samples, the correct allocation to the triage uncertainty intervals varied considerably between assays for the 1.2 Gy (29-76%) and 3.5 Gy (17-100%) samples. While a systematic shift towards higher doses was observed for the cytogenetic-based assays, extreme outliers exceeding the reference doses 2-6 fold were observed for EPR, FISH and GE assays. These outliers were related to a particular material examined (tooth enamel for EPR assay, reported as kerma in enamel, but when converted into the proper quantity, i.e. to kerma in air, expected dose estimates could be recalculated in most cases), the level of experience of the teams (FISH) and methodological uncertainties (GE). This was the first RENEB ILC where everything, from blood sampling to irradiation and shipment of the samples, was organized and realized at the same institution, for several biological and physical retrospective dosimetry assays. Almost all assays appeared comparably applicable for the identification of unexposed and highly exposed individuals and the allocation of medical relevant groups, with the latter requiring medical support for the acute radiation scenario simulated in this exercise. However, extreme outliers or a systematic shift of dose estimates have been observed for some assays. Possible reasons will be discussed in the assay specific papers of this special issue. In summary, this ILC clearly demonstrates the need to conduct regular exercises to identify research needs, but also to identify technical problems and to optimize the design of future ILCs.

RENEB Inter-Laboratory Comparison 2021: The Gene Expression Assay.

Early and high-throughput individual dose estimates are essential following large-scale radiation exposure events. In the context of the Running the European Network for Biodosimetry and Physical Dosimetry (RENEB) 2021 exercise, gene expression assays were conducted and their corresponding performance for dose-assessment is presented in this publication. Three blinded, coded whole blood samples from healthy donors were exposed to 0, 1.2 and 3.5 Gy X-ray doses (240 kVp, 1 Gy/min) using the X-ray source Yxlon. These exposures correspond to clinically relevant groups of unexposed, low dose (no severe acute health effects expected) and high dose exposed individuals (requiring early intensive medical health care). Samples were sent to eight teams for dose estimation and identification of clinically relevant groups. For quantitative reverse transcription polymerase chain reaction (qRT-PCR) and microarray analyses, samples were lysed, stored at 20°C and shipped on wet ice. RNA isolations and assays were run in each laboratory according to locally established protocols. The time-to-result for both rough early and more precise later reports has been documented where possible. Accuracy of dose estimates was calculated as the difference between estimated and reference doses for all doses (summed absolute difference, SAD) and by determining the number of correctly reported dose estimates that were defined as ±0.5 Gy for reference doses <2.5 Gy and ±1.0 Gy for reference doses >3 Gy, as recommended for triage dosimetry. We also examined the allocation of dose estimates to clinically/diagnostically relevant exposure groups. Altogether, 105 dose estimates were reported by the eight teams, and the earliest report times on dose categories and estimates were 5 h and 9 h, respectively. The coefficient of variation for 85% of all 436 qRT-PCR measurements did not exceed 10%. One team reported dose estimates that systematically deviated several-fold from reported dose estimates, and these outliers were excluded from further analysis. Teams employing a combination of several genes generated about two-times lower median SADs (0.8 Gy) compared to dose estimates based on single genes only (1.7 Gy). When considering the uncertainty intervals for triage dosimetry, dose estimates of all teams together were correctly reported in 100% of the 0 Gy, 50% of the 1.2 Gy and 50% of the 3.5 Gy exposed samples. The order of dose estimates (from lowest to highest) corresponding to three dose categories (unexposed, low dose and highest exposure) were correctly reported by all teams and all chosen genes or gene combinations. Furthermore, if teams reported no exposure or an exposure >3.5 Gy, it was always correctly allocated to the unexposed and the highly exposed group, while low exposed (1.2 Gy) samples sometimes could not be discriminated from highly (3.5 Gy) exposed samples. All teams used FDXR and 78.1% of correct dose estimates used FDXR as one of the predictors. Still, the accuracy of reported dose estimates based on FDXR differed considerably among teams with one team's SAD (0.5 Gy) being comparable to the dose accuracy employing a combination of genes. Using the workflow of this reference team, we performed additional experiments after the exercise on residual RNA and cDNA sent by six teams to the reference team. All samples were processed similarly with the intention to improve the accuracy of dose estimates when employing the same workflow. Re-evaluated dose estimates improved for half of the samples and worsened for the others. In conclusion, this inter-laboratory comparison exercise enabled (1) identification of technical problems and corrections in preparations for future events, (2) confirmed the early and high-throughput capabilities of gene expression, (3) emphasized different biodosimetry approaches using either only FDXR or a gene combination, (4) indicated some improvements in dose estimation with FDXR when employing a similar methodology, which requires further research for the final conclusion and (5) underlined the applicability of gene expression for identification of unexposed and highly exposed samples, supporting medical management in radiological or nuclear scenarios.

Inter-laboratory comparison of gene expression biodosimetry for protracted radiation exposures as part of the RENEB and EURADOS WG10 2019 exercise.

Large-scale radiation emergency scenarios involving protracted low dose rate radiation exposure (e.g. a hidden radioactive source in a train) necessitate the development of high throughput methods for providing rapid individual dose estimates. During the RENEB (Running the European Network of Biodosimetry) 2019 exercise, four EDTA-blood samples were exposed to an Iridium-192 source (1.36 TBq, Tech-Ops 880 Sentinal) at varying distances and geometries. This resulted in protracted doses ranging between 0.2 and 2.4 Gy using dose rates of 1.5-40 mGy/min and exposure times of 1 or 2.5 h. Blood samples were exposed in thermo bottles that maintained temperatures between 39 and 27.7 °C. After exposure, EDTA-blood samples were transferred into PAXGene tubes to preserve RNA. RNA was isolated in one laboratory and aliquots of four blinded RNA were sent to another five teams for dose estimation based on gene expression changes. Using an X-ray machine, samples for two calibration curves (first: constant dose rate of 8.3 mGy/min and 0.5-8 h varying exposure times; second: varying dose rates of 0.5-8.3 mGy/min and 4 h exposure time) were generated for distribution. Assays were run in each laboratory according to locally established protocols using either a microarray platform (one team) or quantitative real-time PCR (qRT-PCR, five teams). The qRT-PCR measurements were highly reproducible with coefficient of variation below 15% in ≥ 75% of measurements resulting in reported dose estimates ranging between 0 and 0.5 Gy in all samples and in all laboratories. Up to twofold reductions in RNA copy numbers per degree Celsius relative to 37 °C were observed. However, when irradiating independent samples equivalent to the blinded samples but increasing the combined exposure and incubation time to 4 h at 37 °C, expected gene expression changes corresponding to the absorbed doses were observed. Clearly, time and an optimal temperature of 37 °C must be allowed for the biological response to manifest as gene expression changes prior to running the gene expression assay. In conclusion, dose reconstructions based on gene expression measurements are highly reproducible across different techniques, protocols and laboratories. Even a radiation dose of 0.25 Gy protracted over 4 h (1 mGy/min) can be identified. These results demonstrate the importance of the incubation conditions and time span between radiation exposure and measurements of gene expression changes when using this method in a field exercise or real emergency situation.

Examining Radiation-Induced In Vivo and In Vitro Gene Expression Changes of the Peripheral Blood in Different Laboratories for Biodosimetry Purposes: First RENEB Gene Expression Study.

The risk of a large-scale event leading to acute radiation exposure necessitates the development of high-throughput methods for providing rapid individual dose estimates. Our work addresses three goals, which align with the directive of the European Union's Realizing the European Network of Biodosimetry project (EU-RENB): 1. To examine the suitability of different gene expression platforms for biodosimetry purposes; 2. To perform this examination using blood samples collected from prostate cancer patients (in vivo) and from healthy donors (in vitro); and 3. To compare radiation-induced gene expression changes of the in vivo with in vitro blood samples. For the in vitro part of this study, EDTA-treated whole blood was irradiated immediately after venipuncture using single X-ray doses (1 Gy/min(-1) dose rate, 100 keV). Blood samples used to generate calibration curves as well as 10 coded (blinded) samples (0-4 Gy dose range) were incubated for 24 h in vitro, lysed and shipped on wet ice. For the in vivo part of the study PAXgene tubes were used and peripheral blood (2.5 ml) was collected from prostate cancer patients before and 24 h after the first fractionated 2 Gy dose of localized radiotherapy to the pelvis [linear accelerator (LINAC), 580 MU/min, exposure 1-1.5 min]. Assays were run in each laboratory according to locally established protocols using either microarray platforms (2 laboratories) or qRT-PCR (2 laboratories). Report times on dose estimates were documented. The mean absolute difference of estimated doses relative to the true doses (Gy) were calculated. Doses were also merged into binary categories reflecting aspects of clinical/diagnostic relevance. For the in vitro part of the study, the earliest report time on dose estimates was 7 h for qRT-PCR and 35 h for microarrays. Methodological variance of gene expression measurements (CV ≤10% for technical replicates) and interindividual variance (≤twofold for all genes) were low. Dose estimates based on one gene, ferredoxin reductase (FDXR), using qRT-PCR were as precise as dose estimates based on multiple genes using microarrays, but the precision decreased at doses ≥2 Gy. Binary dose categories comprising, for example, unexposed compared with exposed samples, could be completely discriminated with most of our methods. Exposed prostate cancer blood samples (n = 4) could be completely discriminated from unexposed blood samples (n = 4, P < 0.03, two-sided Fisher's exact test) without individual controls. This could be performed by introducing an in vitro-to-in vivo correction factor of FDXR, which varied among the laboratories. After that the in vitro-constructed calibration curves could be used for dose estimation of the in vivo exposed prostate cancer blood samples within an accuracy window of ±0.5 Gy in both contributing qRT-PCR laboratories. In conclusion, early and precise dose estimates can be performed, in particular at doses ≤2 Gy in vitro. Blood samples of prostate cancer patients exposed to 0.09-0.017 Gy could be completely discriminated from pre-exposure blood samples with the doses successfully estimated using adjusted in vitro-constructed calibration curves.

Study on cell survival, induction of apoptosis and micronucleus formation in SCL-II cells after exposure to the auger electron emitter (99m)Tc.

OBJECTIVE: To study the biological effectiveness of Auger electrons emitted by (99m)Tc on cell survival, induction of apoptosis and micronucleus (MN) formation in the human squamous cell carcinoma cell line SCL-II and compare the effects observed to those observed after exposure to external 60Co gamma radiation. MATERIAL AND METHODS: Cells were either gamma(60Co)-irradiated (0.67 Gy/min) or exposed to (99m)Tc-pertechnetate (0.95-14.3 MBq/ml) for 24 h under cell culture conditions and assayed for cell survival (colony-forming assay), micronucleus formation (cytochalasin B assay) and the frequency of apoptotic cells (fluorescence microscopy). Monte Carlo based dosimetry has been applied to derive the absorbed dose corresponding to the accumulated decays of (99m)Tc under the given geometry. RESULTS: Absorbed doses up to 0.5 Gy could be achieved after 99mTc-exposure leading to no substantial cell killing in this dose range except at one dose point (0.1 Gy) resulting in an relative biological effectiveness (RBE)SF 0.9 of 0.64 when compared to the 60Co reference radiation. MN formation was described best by a linear dose response and was consistently lower after 99mTc exposure when compared to 60Co irradiated cells resulting in an RBE of 0.37. Apoptosis induction was significantly increased after 99mTc exposure at much lower doses (0.1 Gy) when compared to the reference radiation. The (99m)Tc uptake experiments revealed an activity concentration ratio cells vs. medium of 0.07 after 24 h of exposure. CONCLUSION: No overall increased biological effectiveness due to the emitted Auger electrons of (99m)Tc, applied as sodium-pertechnetate, could be observed in the investigated cell line when compared to acute external gamma radiation. The RBEs in the range of 0.37-0.64 might be well explained by dose rate effects. The significantly increased apoptotic response after (99m)Tc-exposure at very low doses has to be further investigated.

Stimulation of phagocytosis and free radical production in murine macrophages by 50 Hz electromagnetic fields.

Effects of 50 Hz electromagnetic fields on phagocytosis and free radical production were examined in mouse bone marrow-derived macrophages. Macrophages were in vitro exposed to electromagnetic fields using different magnetic field densities (0.5-1.5 mT). Short-time exposure (45 min) to electromagnetic fields resulted in significantly increased phagocytic uptake (36.3% +/- 15.1%) as quantified by measuring the internalization rate of latex beads. Stimulation with 1 nM 12-0-tetradecanoylphorbol-13-acetate (TPA) showed the same increased phagocytic activity as 1 mT electromagnetic fields. However, co-exposure to electromagnetic fields and TPA showed no further increase of bead uptake, and therefore we concluded that because of the absence of additive effects, the electromagnetic fields-induced stimulation of mouse bone marrow-derived macrophages does not involve the protein kinase C signal transduction pathway. Furthermore, a significant increased superoxide production after exposure to electromagnetic fields was detected.

Micronucleus induction in Syrian hamster embryo cells following exposure to 50 Hz magnetic fields, benzo(a)pyrene, and TPA in vitro.

Electromagnetic fields (EMFs) have been associated with increased incidence of cancer suggested by epidemiological studies. To test the carcinogenic potency of EMF, the in vitro micronucleus assay with SHE cells has been used as a screening method for genotoxicity. A 50Hz magnetic field (MF) of 1mT field strength was applied either alone or with the tumour initiator benzo(a)pyrene (BP) or the tumour promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). All three treatments were applied in single, double or triple treatment regimes. MF or TPA (1nM) alone did not affect the number of micronuclei (MN) in initiated and non-initiated SHE cells. Changing the schedule of the typical initiation protocol, namely applying the initiator (BP) during exposure to MF, results in an 1.8-fold increased MN formation compared to BP treatment alone. Combined experiment with BP, TPA and MF did not cause further MN formation. Since initiation during MF exposure caused a significant increased MN formation, our findings suggest that MFs enhance the initiation process of BP. We think that this MF-enhanced co-carcinogenic effect is caused by an indirect "cell activation" process. The resulting genomic instability is proposed to be due to free radicals and/or to the unscheduled "switching-on" of signal transduction pathways.

Apoptosis induction and micronucleus formation after exposure to the Auger electron emitter zinc-65 in a human cell line.

Induction of apoptosis and micronucleus formation has been studied in a transformed human squamous cell carcinoma cell line (SCL-II) after exposure to the Auger electron emitter Zinc-65 (65Zn) and after external low-LET radiation. Exposure to non-radioactive Zn and unirradiated cells served as controls. Studies on the cellular uptake of 65Zn2+ have been carried out in vitro and conventional dosimetric models have been applied to derive the absorbed radiation dose. Auger electrons, generated during decay of 65Zn2+, are strong inducers of micronuclei as well as of apoptosis, in comparison to external low-LET irradiation. The relative biological effectiveness has been determined and was found to be in the range of 1.2-2.7 for the two investigated biological endpoints, depending on which mathematical model for describing the dose-effect curves was used. A non-uniform distribution of intracellular Zn2 + was observed, showing a strong signal in the perinuclear region. We conclude that separate radiation weighting factors for Auger electrons should be established depending on the nuclide and its ability to interact with the DNA (e.g. 65Zn by Zinc-finger proteins).

Absence of synergistic effects on micronucleus formation after exposure to electromagnetic fields and asbestos fibers in vitro.

Exposure of human amniotic fluid (AFC) cells to horizontally applied magnetic fields (hMF) of 50 Hz and 1 mT generated in a Helmholtz-coil system leads to a significant increase in micronucleus frequency (MN), without affecting cell proliferation. To investigate whether hMF-exposure has an additive or synergistic effect on the genotoxic capacity of asbestos fibers, MN induction was investigated in hMF pre-exposed cells, treated before or after with asbestos (1 microg/cm2). Neither synergistic nor additive effects on MN induction were observed. The results indicate, that under our experimental conditions, exposure to hMF and treatment with asbestos fibers possess genotoxic capability, but no interactive effects, in AFC cells.

Delayed cytotoxic and genotoxic effects in a human cell line following X-irradiation.

BACKGROUND: In order to clarify the relationship between delayed reproductive death and radiation-induced genomic instability, the colony-forming efficiency of surviving, irradiated human squamous carcinoma cells and centromere positive as well as centromere negative micronuclei in surviving progeny were examined. MATERIALS AND METHODS: Colony-forming ability and micronucleus (MN) frequency in binucleated cells 24 h after the addition of cytochalasin B during 2 weeks of post-irradiation growth were determined in a squamous cell carcinoma cell line (SCL-II) of human origin. In addition, centromeres in micronuclei were detected using FISH. RESULTS: In the human epithelial cell line used for these experiments, delayed reproductive death was pronounced and persisted for at least 2 weeks after irradiation. Although there is evidence for an increased rate of centromere positive micronuclei, but not of centromere negative micronuclei, arising during the first week of post-irradiation proliferation, this decreases later while the rate of delayed reproductive death remains elevated. CONCLUSION: In the studied cell line, the observed delayed reproductive death is not closely related to the investigated criteria of radiation-induced genomic instability. This casts doubt on the common assumption that delayed reproductive death is a direct manifestation of radiation-induced genomic instability.

Micronucleus formation in human amnion cells after exposure to 50 Hz MF applied horizontally and vertically.

Micronucleus (MN) induction as a genotoxic effect of extremely-low-frequency electromagnetic fields (ELF-EMF, 50 Hz, 1 mT) was studied in human amniotic fluid cells (AFC) after continuous exposure to magnetic fields (MF), oriented horizontally and vertically with respect to the surface of the culture medium, at different time points. To compare the effectiveness of different exposure systems, a Helmholtz-coil system and a so-called Merritt-coil system was used. A statistically significant increase in MN frequency could be detected in exposed cells compared to controls after 72 h continuous exposure to MF applied vertically in the Merritt-coil system, while no effect was found after exposure in the Helmholtz-coil system. Furthermore, a significant increase in MN induction occurred after 24, 48 and 72 h exposure to MF applied horizontally in the Helmholtz-coil system in comparison to controls, whereas horizontally MF generated in the Merritt-coil system induced no genotoxic effects. To exclude suppression of indirect EMF-induced DNA-lesions, we studied MN formation in the presence of N-Acetyl-p-aminophenol (APAP, Paracetamol(R)), which is an inhibitor of DNA-repair mechanisms. We found a dose-dependent increase of MN formation in APAP-treated AFC cells, but no significant further increase in MN frequency after additional MF exposure. Therefore we conclude, that EMF-induced MN formation is not caused by directly or indirectly induced clastogenic mechanisms. The obtained results show that the orientation of MF with respect to the cell culture dish and the physical condition of the exposure system is of major importance for the induction of micronuclei in certain cell types. Therefore, the reason for inconsistent results published in the literature may be caused by the variability of exposure systems, the exposure conditions and the cell types used.

Effects of 50 Hz EMF exposure on micronucleus formation and apoptosis in transformed and nontransformed human cell lines.

Effects of applying extremely low-frequency electromagnetic fields (ELF-EMF) for different durations (24, 48, and 72 h) and different field intensities (0.1-1.0 mT) on micronucleus (MN) formation and induction of apoptosis were examined in a human squamous cell carcinoma cell line (SCL II) and in a human amniotic fluid cell line (AFC). A statistically significant increase of MN frequency and of induction of apoptosis in SCL II cells after 48-h and 72-h continuous exposure to 50 Hz magnetic field (MF) (0.8 and 1.0 mT) was found. However, exposure of AFC cells to EMF of different intensities and for different exposure times showed no statistically significant differences when compared with controls. These results demonstrate that different human cell types respond differently to EMF. Dose-dependent induction of apoptosis and genotoxic effects, resulting in increased micronucleus formation, could be demonstrated in the transformed cell line, whereas the nontransformed cell line did not show statistically significant effects. These findings suggest that EMF could be a promotor but not an initiator of carcinogenic effects.

An in vitro model study of BSp73 rat tumour cell invasion into endothelial monolayer.

In order to study the process of invasion in more detail we developed an in vitro model of the vessel wall. Rat tumour cells derived from an adenocarcinoma of the pancreas, BSp73 AS--of high invasive but low metastatic capacity--and BSp73 ASML--not invasive but highly metastatic--were compared for their mode of invasion into confluent monolayers of endothelial cells. Corneal as well as vascular endothelial cells were plated alternatively onto the basal lamina-like bovine lens capsule that was mounted in a combi-ring dish or reconstituted extracellular matrix (Basement Membrane Matrigel) as substrata. The endothelial monolayers were confronted with AS- and ASML-tumour cells. The interaction of the various cell types was followed by scanning and transmission electron microscopy. The invasive cell type AS was able to force the endothelial cells to retract and subsequently undermined the endothelial cell layer. In the noninvasive cell population ASML most cells remained in the typical roundish morphology and did not interact with the endothelial cell layers. Only a very minor fraction of ASML populations was able to attach to and also invade into the endothelial cell monolayer. It could be shown that AS-cells individually and as small groups penetrated the endothelial cell layer. The results of transmission and scanning electron microscopy suggest that endothelial cell retraction and underlapping of adjacent endothelial cells by tumour cells play an important role in invasion and extravasation through blood vessels. Against all expectations, the nonmetastasizing tumour cell variant (AS-cells) exhibited a dramatic invasive behaviour whereas the highly metastatic ASML-variant mostly retained its spherical shape and showed invasive activity only in exceptional cases.