Workshop on the validation and regulatory acceptance of innovative 3R approaches in regulatory toxicology - Evolution versus revolution.

At a joint workshop organized by RIVM and BfR, international experts from governmental institutes, regulatory agencies, industry, academia and animal welfare organizations discussed and provided recommendations for the development, validation and implementation of innovative 3R approaches in regulatory toxicology. In particular, an evolutionary improvement of our current approach of test method validation in the context of defined approaches or integrated testing strategies was discussed together with a revolutionary approach based on a comprehensive description of the physiological responses of the human body to chemical exposure and the subsequent definition of relevant and predictive in vitro, in chemico or in silico methods. A more comprehensive evaluation of biological relevance, scientific validity and regulatory purpose of new test methods and assessment strategies together with case studies that provide practical experience with new approaches were discussed as essential steps to build up the necessary confidence to facilitate regulatory acceptance.

Workshop on acceleration of the validation and regulatory acceptance of alternative methods and implementation of testing strategies.

This report describes the proceedings of the BfR-RIVM workshop on validation of alternative methods which was held 23 and 24 March 2017 in Berlin, Germany. Stakeholders from governmental agencies, regulatory authorities, universities, industry and the OECD were invited to discuss current problems concerning the regulatory acceptance and implementation of alternative test methods and testing strategies, with the aim to develop feasible solutions. Classical validation of alternative methods usually involves one to one comparison with the gold standard animal study. This approach suffers from the reductionist nature of an alternative test as compared to the animal study as well as from the animal study being considered as the gold standard. Modern approaches combine individual alternatives into testing strategies, for which integrated and defined approaches are emerging at OECD. Furthermore, progress in mechanistic toxicology, e.g. through the adverse outcome pathway approach, and in computational systems toxicology allows integration of alternative test battery results into toxicity predictions that are more fine-tuned to the human situation. The road towards transition to a mechanistically-based human-focused hazard and risk assessment of chemicals requires an open mind towards stepping away from the animal study as the gold standard and defining human biologically based regulatory requirements for human hazard and risk assessment.

Conversion of mature human ß-cells into glucagon-producing α-cells.

Conversion of one terminally differentiated cell type into another (or transdifferentiation) usually requires the forced expression of key transcription factors. We examined the plasticity of human insulin-producing ß-cells in a model of islet cell aggregate formation. Here, we show that primary human ß-cells can undergo a conversion into glucagon-producing α-cells without introduction of any genetic modification. The process occurs within days as revealed by lentivirus-mediated ß-cell lineage tracing. Converted cells are indistinguishable from native α-cells based on ultrastructural morphology and maintain their α-cell phenotype after transplantation in vivo. Transition of ß-cells into α-cells occurs after ß-cell degranulation and is characterized by the presence of ß-cell-specific transcription factors Pdx1 and Nkx6.1 in glucagon(+) cells. Finally, we show that lentivirus-mediated knockdown of Arx, a determinant of the α-cell lineage, inhibits the conversion. Our findings reveal an unknown plasticity of human adult endocrine cells that can be modulated. This endocrine cell plasticity could have implications for islet development, (patho)physiology, and regeneration.

A sandwich-cultured rat hepatocyte system with increased metabolic competence evaluated by gene expression profiling.

A rapid decline of cytochrome P450 (CYP450) enzyme activities remains a drawback of rat hepatocyte-based in vitro cultures. Consequently, judgment of the toxic potential of compounds that need bioactivation by CYP450s may not be adequate using this model. In the present study, an improved hepatocyte-based in vitro system was developed with special focus on metabolic competence. Therefore, a mixture of CYP450 inducers, phenobarbital, dexamethasone and beta-naphthoflavone, was added to culture medium of sandwich-cultured rat hepatocytes. The resulting modified model was evaluated by comparing its genome-wide expression profiles with liver and a standard model without the inducer mixture. Metabolic capacity for CYP450 enzymes showed that the modified model resembled more closely the in vivo situation. Gene expression results revealed large differences between in vivo and both in vitro models. The slight differences between the two sandwich models were predominantly represented by gene expression changes in CYP450s. Importantly, in the modified model, expression ratios of the phase I and the majority of phase II genes more closely resembled liver in vivo. The CYP450 enzyme activities corresponded with gene expression data. In conclusion, for toxicological applications using sandwich-cultured hepatocytes, the modified model may be preferred.

Separation of neural and surface ectoderm after closure of the rostral neuropore.

Separation of neural and surface ectoderm after closure of the rostral neuropore in the head region has been described by investigating the integrity of the basement membranes of these epithelia in 11- to 27-somite rat embryos. The basement membranes were visualized with polyclonal antibodies against laminin. Furthermore, cell degeneration has been investigated in relation to neural crest activity, and discontinuities of the basement membrane in 9- to 30-somite mouse embryos. The separation of the basement membranes of neural and surface ectoderm in the midline is a final phase during the fusion of the neural folds, which takes place from the closure of the rostral neuropore, at the 19-somite stage, until the 27-somite stage (rat embryos), and which occurs focally with variation in the midsagittal and the transverse planes. In the prosencephalon, neural crest activity is absent during the separation phase of both epithelia, but cell degeneration may contribute to the separation of the initially connected basement membranes. A disturbance in the separation of the neural and surface ectoderm may be the pathogenetic basis of midline skull defects, and of the fronto-ethmoidal encephalocele in particular.

Low density lipoprotein receptor expression on keratinocytes in normal and psoriatic epidermis.

Biochemical and morphologic studies on the interaction of low density lipoprotein (LDL) with cultured normal keratinocytes and squamous carcinoma cells have shown a negative correlation between LDL receptor activity and terminal differentiation of the epidermal cells [Ponec M et al, J Invest Dermatol 83:436-440, 1984 and Vermeer, BJ et al, J Invest Dermatol 86:195-200, 1986]. Whether such in vitro studies pertain to the epidermis in vivo is not known. To obtain information on the distribution of LDL receptors in the epidermis in situ, morphologic studies were performed using LDL-gold as an ultrastructural marker. When freshly isolated mouse and human epidermal cells were incubated with LDL-gold complexes, only keratinocytes with the morphologic characteristics of basal cells showed binding and uptake of LDL-gold. No LDL receptor activity was found on Langerhans cells, melanocytes or highly differentiated keratinocytes. Since cell separation techniques can destroy receptors, the staphylococcal epidermolytic toxin was utilized to produce intercellular and intra-epithelial splitting of the epidermis. In preparations of both normal mouse and human epidermis, LDL-gold binding was restricted to basal cells and a few suprabasal keratinocytes. In contrast, in psoriatic epidermis, and to a lesser extent, essential fatty acid-deficient mouse epidermis, cells in the stratum spinosum showed abundant LDL-gold binding. Thus LDL-gold may be a useful marker for epidermal differentiation.

Free sterol metabolism and low density lipoprotein receptor expression as differentiation markers of cultured human keratinocytes.

In contrast to most tissues, epidermis and its derivatives appear to lack low density lipoprotein (LDL) receptors and exhibit sterologenesis rates unaffected by circulating lipoprotein (LP) cholesterol content. Since LDL receptors have been demonstrated in both cultured squamous cell carcinoma cells and human foreskin keratinocytes, when maintained in low-calcium media, LDL receptor expression may be related to keratinocyte differentiation. We compared receptor binding and internalization of LDL-gold in normal keratinocytes at different stages of growth at physiological calcium concentrations (early, 3-5 days; preconfluent, 6-10 days; postconfluent, 12-17 days), and correlated receptor expression with sterologenesis in LP-replete vs.-depleted media. Whereas in early cultures about 60% of sterologenesis was LP dependent, this fraction declined in preconfluent and confluent cultures despite continued culture growth and little decline in total sterologenesis. Accordingly, LDL receptors were most evident in early cultures, declining in preconfluent cultures in parallel with the decrease in LP-dependent sterol synthesis. In contrast, sterologenesis in human foreskin fibroblasts was profoundly influenced by exogenous LP at all stages of confluence; total and LP-dependent sterologenesis declined in parallel with growth cessation. These studies represent the first demonstration that normal keratinocytes express functional LDL receptors at physiologic calcium concentrations. Moreover, they demonstrate that LDL receptor expression in keratinocytes, in contrast to fibroblasts, can only in part be attributed to growth requirements. Instead, loss of LDL receptor expression serves as a distinctive marker of keratinocyte differentiation and may reflect the specific functional requirements of the epidermis in vivo.

Propagation and morphologic phenotypes of human umbilical cord artery endothelial cells.

Human umbilical cord artery endothelial cells can be propagated on fibronectin-coated dishes for approximately 50 cumulative population doublings in the presence of crude preparations of endothelial cell growth factor (ECGF) and serum. The cells were characterized by immunofluorescent staining and their ultrastructure. Different morphologic phenotypes could be demonstrated: closely attached cell monolayers, atypical cells, giant cells, tube-like structures. The formation of tube-like structures can be induced by proteolytic modification of fibronectin. Our data demonstrate that umbilical arteries may provide an excellent source for the routine serial cultivation of human arterial endothelial cells.

Binding and internalization of low-density lipoproteins in SCC25 cells and SV40 transformed keratinocytes. A morphologic study.

Binding of low-density lipoproteins (LDL) to the plasma membrane and internalization of low-density lipoprotein receptor complexes were investigated in an epithelial tumor cell derived from the tongue (SCC25) and in SV40-transformed keratinocytes (SVK14 cells). For light microscopic studies an immunofluorescence technique with antiapoprotein B as well as conjugation procedure by which a fluorochrome 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanide (DIL) was conjugated with LDL (LDL-DIL) was used. Binding of LDL to the plasma membrane at 4 degrees C was observed in most SCC25 cells but not in SVK14 cells. The internalization of LDL-DIL was absent in SVK14 cells and was excessive in SCC25 cells. In SCC25 cells, internalization of the LDL-DIL particles was heterogeneously distributed over various cells. When a pulse-chase experiment was performed with LDL-DIL, less LDL was internalized into the SCC25 cells in comparison with a continuous label experiment. For the ultrastructural studies LDL conjugated with colloidal gold was used. In the binding experiments at 4 degrees C most LDL-gold particles were attached to the plasma membrane outside coated pits. During internalization experiments with LDL-gold particles it was observed that within 5-15 min at 37 degrees C several LDL-gold particles were seen in electron-dense structures near the plasma membrane. The electron-dense structures containing LDL-gold, as observed after an internalization period of 5-15 min, may represent the first endosomal compartment as described for transferrin receptors in A431 cells. After a period of 30 min at 37 degrees C the LDL-gold particles were observed in electron-lucent vesicles (multivesicular bodies) and dense bodies. However coated vesicles containing LDL-gold particles were seen sporadically. It is concluded that the route of internalization of LDL into the SCC25 cells differs from that of other cell types. No internalization of LDL gold was found in SVK14 cells, thus, in this respect, the SVK14 cells resemble normal keratinocytes. The morphologic data are in good agreement with biochemical studies published earlier (Ponec M et al, J Invest Dermatol 83:436-440, 1984). Both investigations suggest that LDL receptor activity is modulated during the process of terminal differentiation.

Modulation of low density lipoprotein receptor activity in squamous carcinoma cells by variation in cell density.

Morphological and biochemical studies on low density lipoprotein (LDL) receptor metabolism were performed in squamous carcinoma cells (SCC-15 and SCC-12F2). Modulation of terminal differentiation was achieved by culturing these cells at different cell densities. Studies on these cells cultured at low density (hardly any terminal differentiation) showed the following results: High affinity binding of LDL was excessive; LDL binding to SCC-15 cells was twice as high as that in SCC-12F2 cells and in fibroblasts. The distribution of the LDL binding visualized by LDL receptor antibodies was non-linear. There was no contact inhibition of LDL binding. LDL-gold particles were mainly bound to the plasma membrane outside coated pits. LDL-gold particles were internalized and delivered to dense bodies (= lysosomes). Degradation of LDL took place after a lag period of 10 min. Dissociation of LDL from the plasma membrane was substantial (more than 40% after a 120 min chase period). The same experiments on the cells cultured at high density (terminal differentiation present) showed several differences: A sharp decrease in high affinity LDL binding in both cell types. The internalization of surface bound LDL was defective in most of the squamous carcinoma cells. Dissociation of LDL from the plasma membrane was substantial, and after a chase period of 120 min at 37 degrees C still more than 20% of LDL remained intracellular and was not degraded. We postulate that LDL receptor-mediated endocytosis and degradation take place in squamous carcinoma cells but that during the process of terminal differentiation modulation of LDL-receptor metabolism occurs.(ABSTRACT TRUNCATED AT 250 WORDS)

Visualization of the interaction of native and modified low density lipoproteins with isolated rat liver cells.

Morphological characteristics of the interaction of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) with rat liver cells are described. These liver cell types are mainly responsible for the catabolism of these lipoproteins in vivo. Isolated rat liver Kupffer, endothelial, and parenchymal cells were incubated with LDL or AcLDL conjugated to 20 nm colloidal gold. LDL was mainly internalized by Kupffer cells, whereas AcLDL was predominantly found in endothelial cells. Kupffer and endothelial cells displayed different morphological characteristics in the processing of these lipoproteins. Kupffer cells bound LDL at uncoated regions of the plasma membrane often at the base of pseudopodia, and internalized the particles via small smooth vesicles. These uptake characteristics differ from the classical LDL uptake pathway, as described for other cell types, and may be related to the unique recognition properties of the receptor of Kupffer cells as observed in biochemical studies. Liver endothelial cells bound AcLDL in coated pits, followed by rapid uptake. Uptake proceeded through small coated vesicles, and after 5 min of incubation large (600-1200 nm) electron-lucent vacuoles (endosomes) with AcLDL-gold particles arranged along the membrane region were present. The endosomes were often associated closely with the cell membrane which might enable direct recycling of AcLDL receptors. These observations might explain the high efficiency of these cells in the processing of modified LDL in vivo.

High-affinity uptake and degradation of acetylated low density lipoprotein by confluent human vascular endothelial cells.

Confluent human vascular endothelial cells take up and degrade acetylated low density lipoproteins (Ac-LDL) via a high-affinity binding process, comparable to that for native LDL. The degradation of 125I-labelled Ac-LDL by endothelial cells was inhibited by the addition of unlabelled Ac-LDL but not by the addition of unlabelled LDL. Similarly, the degradation of 125I-labelled LDL could be inhibited by unlabelled LDL but not by unlabelled Ac-LDL. Unlabelled apolipoprotein E-free HDL did not compete with the degradation of either 125I-labelled LDL or Ac-LDL. Electron microscopical studies, using gold-labelled LDL and gold-labelled Ac-LDL, showed that at 4 degrees C both LDL and Ac-LDL bind to indented regions of the endothelial cell plasma membrane. At 37 degrees C both LDL and Ac-LDL were taken up and associated with lysosomes. Although morphologically identical, we conclude that the binding of LDL and Ac-LDL to human endothelial cells proceeds via 2 different high-affinity receptors. The uptake and degradation of Ac-LDL by human endothelial cells was about 25 and 15%, respectively, of that of native LDL. The uptake and degradation of either LDL or Ac-LDL did not lead to a massive increase in cellular cholesterol content.

Visualization of binding and receptor-mediated uptake of low density lipoproteins by human endothelial cells.

Low density lipoproteins (LDL) were conjugated to colloidal gold for investigation of the ultrastructural aspects of binding and receptor-mediated internalization of LDL by cultured endothelial cells from the human umbilical artery and vein. The number of LDL receptors was increased by preincubation in lipoprotein-depleted serum. When the cells were incubated with LDL-gold particles for 2 h at 4 degrees C, the complexes were found in coated pits as well as in clusters attached to the plasma membrane. Small vesicles containing a few LDL-gold complexes appeared in the cytoplasm close to the plasma membrane when the cells were incubated with the conjugate for 5 min at 37 degrees C. After 15 min at 37 degrees C, larger vesicles with a pale matrix and membrane-orientated LDL-gold complexes were seen. After incubation for 30 min at 37 degrees C, colloidal gold particles were present in dense bodies. Quantification of the binding of LDL-gold complexes to the plasma membrane at 4 degrees C showed no differences between arterial and venous endothelial cells.

Conjugates of colloidal gold with native and acetylated low density lipoproteins for ultrastructural investigations on receptor-mediated endocytosis by cultured human monocyte-derived macrophages.

The morphological aspects of the binding and internalization of low density lipoproteins (LDL) and acetylated low density lipoproteins (AcLDL) by cultured human monocyte-derived macrophages were investigated. For this purpose, LDL and AcLDL were conjugated to 20 nm colloidal gold particles. After incubation of the cells with the conjugated lipoproteins at 4 degrees C some LDL- or AcLDL-gold complexes were found to be attached to the cell surface, but without characteristic localization. However, after incubation of the cells at 8 degrees C with either LDL-gold or AcLDL-gold, lipoprotein-gold complexes were present in clusters on the plasma membrane, often in coated pits. Cells incubated at 37 degrees C for various time periods showed internalization of both LDL- and AcLDL-gold complexes via small coated and non-coated vesicles and processing of the complexes in smooth-walled endosomes. When the cells were pulse-chased with LDL- or AcLDL-gold for 30 min at 37 degrees C, the gold conjugates occurred in dense bodies, probably lysosomes. The results suggest that although native and modified LDL are reported to be metabolized differently by macrophages, the morphological aspects of the endocytosis of LDL and AcLDL by cultured human monocyte-derived macrophages are similar.

Development of the granule population in neutrophil granulocytes from human bone marrow.

Bone marrow from hematologically healthy adults was exposed to a number of fixation procedures for investigation of the heterogeneity of the granule population in neutrophil granulocytes at the ultrastructural level. Four main cell stages were distinguished: early promyelocyte, late promyelocyte, myelocyte, and mature neutrophil granulocyte and described separately; metamyelocytes and band-form or stab cells are described together. The characteristic changes in the cytoplasm during myelopoiesis were analysed quantitatively. Special attention was given to the development of the granule population. Three types of granule arise in successive cell stages: granules which develop a sub-structure in the matrix (nucleated granules) are formed in early promyelocytes, granules with a homogeneous electron-dense matrix (azurophil granules) in late promyelocytes, and granules with a less electron-dense matrix (specific granules) in myelocytes. The three types of granule remain present during myelopoiesis. The best results in distinguishing the granule types were obtained by prefixation either in 0.1% glutaraldehyde or in 1.5% glutaraldehyde followed by washing in phosphate-buffered Ringer solution to which aminotriazole had been added. Granule counts revealed for the mature neutrophil a total number of granules of about 220 per ultrathin section. This population of granules is composed of about 12% nucleated, 11% azurophil, and 77% specific granules. When our previous findings are taken into account, the existence of three successively formed and morphologically distinguishable types of granule in heterophil (neutrophil) granulocytes has been demonstrated for three mammalian species: the guinea pig, the rat, and man. A separate term for the early promyelocyte stage is proposed: eomyelocyte.