Screening of endocrine disrupting chemicals with MELN cells, an ER-transactivation assay combined with cytotoxicity assessment.

There is growing concern that some chemicals can cause endocrine disrupting effects to wild animals and humans. Therefore a rapid and reliable screening assay to assess the activity of endocrine disrupting chemicals (EDCs) is required. These EDCs can act at multiple sites. Most studied mechanism is direct interaction with the hormone receptors, e.g. estrogen receptor. In this study the luciferase reporter gene assay using transgenic human MELN cells was used. Since cytotoxicity of the chemicals can decrease the luminescent signal in the transactivation assays, a cytotoxicity assay must be implemented. Mostly the neutral red (NR) assay is performed in parallel with the estrogenicity assay. To increase the reliability and cost-efficiency of the test, a method to measure estrogenicity and cytotoxicity in the same cell culture plate instead of in parallel plates was developed and evaluated. Therefore the NR-assay was compared with the CytoTox-ONE homogeneous membrane integrity assay. The latter measures LDH (lactate dehydrogenase) leakage based on a fluorometric method. For all compounds tested, the CytoTox-ONE test showed comparable curves and EC50-values to those obtained by the NR-assay. So the CytoTox-ONE kit, which seemed more sensitive than measurements of LDH-leakage based on a colorimetric method, is recommended to test cytotoxicity to MELN cells, with the advantage to use the same cells for ER-transactivation measurements. The chemicals tested in the optimised MELN assay showed estrogenic potencies comparable to those reported for several other transactivation assays.

Stromal cells in long-term cultures of liver, spleen, and bone marrow at different developmental ages have different capacities to maintain GM-CFC proliferation.

Measurements were made of the granulocyte-macrophage colony-forming cell (GM-CFC) yield in long-term cultures established from different combinations of stroma and hemopoietic recharge inocula derived from hemopoietic organs at different stages of their embryological development. Results indicated differences in the supporting capacity of the stroma, related to the hemopoietic activity of the organ of origin. Stroma derived from hemopoietically active organs (adult bone marrow, neonatal spleen, fetal liver) supported the proliferation of GM-CFC to a larger extent than stroma derived from organs with a low hemopoietic activity (neonatal bone marrow liver at 2 days; spleen at 3 weeks). Regardless of the origin of the hemopoietic cells, stroma from adult bone marrow displayed the highest ability to support GM-CFC proliferation. The capacity of GM-CFC from hemopoietic recharge cell populations to proliferate on stroma was not related to the hemopoietic activity of their organ of origin. Regardless of their organ of origin the GM-CFC present in each of the different hemopoietic recharge populations were able to proliferate provided that they were seeded on an appropriate stroma. These experiments showed that stromal cells cultured from hemopoietic organs at different developmental ages determine the hemopoietic activity of long-term cultures as measured via GM-CFC recovery.

Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and long-term cultures.

The adherent stromal layer in long-term marrow cultures is essential to the proliferation and differentiation of hemopoietic cells. Adhering cells are heterogeneous and morphologically not adequately characterized. Comparative morphological studies were conducted on adherent cells in short-term clonal assays and long-term cultures derived from liver and bone marrow. Liver and bone marrow at different developmental ages have different hemopoietic activities in vivo and in vitro, as tested via CFU-GM recovery in long-term cultures. Adherent cells from each organ were recovered at an age with high hemopoietic activity (fetal liver and adult bone marrow) and at an age with low hemopoietic activity (neonatal liver and bone marrow). The presence of macrophages, alkaline phosphatase, acid phosphatase, myeloperoxidase, sulfated and non-sulfated glycosaminoglycans (GAGs) and fibronectin was compared. For a given organ, CFU-f colonies showed characteristics similar to those of the confluent adherent stromal layer in long-term cultures. The presence of macrophages and GAGs (sulfated and non-sulfated) in the adherent layer were directly related to the hemopoietic activity. The amount of alkaline phosphatase-positive cells and the amount of fibronectin showed no correlation with the hemopoietic activity of the cultures.

Haemopoietic long-term bone marrow cultures from adult mice show osteogenic capacity in vitro on 3-dimensional collagen sponges.

Adult murine bone marrow cells, cultured under conditions for long-term haemopoietic marrow cultures, produce bone matrix proteins and mineralized tissue in vitro, but only after the adherent stromal cells were loaded on a 3-dimensional collagen sponge. Provided more than 8 x 10(6) cells are loaded, mineralization as measured by 85Sr uptake from the culture medium, occurred in this 3-dimensional configuration (3-D) within 6 days. In contrast if undisrupted marrow fragments (containing more than 10(7) cells) are placed directly on a collagen sponge, then it requires more than 10 days before significant mineralization can similarly be detected. The 2-dimensional (2-D) long-term marrow culture system allows prior expansion of the stromal cells and some differentiation in an osteogenic direction within the adherent stromal layer. This is suggested by the presence of type I collagen and alkaline phosphatase positive cells. However; synthesis of osteonectin and a bone specific protein, osteocalcin, as well as calcification are only observed in 3-D cultures. Electron microscopy demonstrated hydroxyapatite mineral on collagen fibres, osteoblast-like cells, fibroblasts, cells which accumulated lipids, and macrophages which were retained on the collagen matrices. Irradiation of confluent long-term bone marrow cultures, prior to their loading on the collagen sponge showed that haemopoietic stem cells are not necessary for the mineralization.

Distribution of 241Am in offspring from BALB/c mice injected with 241Am at 14 days of gestation: relation to calcium and iron metabolism and comparison with distribution of 241Am after injection of adults.

Pregnant mice were given intravenous injections of 241Am citrate at 14 days of gestation. The fetal skeleton had a higher or similar uptake of 241Am per gram of fresh tissue than the liver. In comparison, the liver in adults concentrated 5 to 20 times more 241Am per gram of fresh tissue than the bones. Measurement of changes in calcium and iron content and concentration with time, showed that in the developing mice intensive calcification of bones determined the uptake of 241Am. The 241Am uptake was related to the calcium concentration of the fetal bones, which was greater at 14 days of gestation in the anterior bones, the mandibles and calvaria, than in the ribs and femurs. Transfer of 241Am to pups via milk resulted in further accumulation of 241Am in the skeleton and liver. The incorporation in the skeleton persisted after weaning and contributed to the lifetime body burden. The 241Am concentration decreased rapidly with time after injection in relation to the growth of the organs. Radiation dose rates and cumulative radiation doses were calculated for liver and bones of contaminated offspring.

Long-term effects on tumour incidence and survival from 241Am exposure of the BALB/c mouse in utero and during adulthood.

BALB/c mice were given 100, 500 or 1500 Bq/g 241Am at day 14 of pregnancy. The offspring were separated from the mothers at birth and followed until death. In addition, adult females and one group of males were also studied for the effects of 241Am following treatment with 45-213 Bq/g. Adults treated with 241Am showed significantly shortened survival and increased incidence of osteosarcoma (to 40 - 50%). The data also suggest that the female mouse is more susceptible to induction of osteosarcoma than the male. There was also a significant increase in osteosarcoma, all bone tumours, all sarcomas, and all leukaemias in the offspring from the contaminated mothers, although this appeared to occur independently of dose. Calculations of the number of osteosarcomas induced per Gy varied for contamination of adult mice between 0.2 and 0.01 and for the offspring between 6 and 0.6. Thus, offspring seemed to be about 10 times more at risk if osteosarcomas induced per mouse Gy are compared. Surprisingly, offspring from mothers treated with 241Am displayed a longer survival time than controls, possibly due to fewer deterministic lung diseases appearing early in life.

Haemopoietic cell proliferation in murine bone marrow cells exposed to extreme low frequency (ELF) electromagnetic fields.

As leukemia is one of the health hazards that is sometimes associated with exposure to extreme low frequency fields, we studied the in vitro effects of ELF fields on haemopoietic cell proliferation. First, the cytotoxic effect of 80 microT, 50 Hz magnetic fields on 3T3 cell proliferation was investigated using the neutral red test. Many chemicals are believed to cause damage because they interfere with basal or "housekeeping" cell functions. The basal cell functions are present in every cell. Non-specialized, actively dividing cells are suitable for measuring cytotoxic effects. Cytotoxic doses can be identified by exposing actively dividing cells in vitro and measuring growth inhibition caused by interference with these basal cell functions. 80 microT, 50 Hz magnetic fields caused no cytotoxicity: we were not able to demonstrate any interference with essential cell functions in the non-differentiated 3T3 cell line. Furthermore, the in vitro effects of ELF fields on murine haemopoietic and stromal stem cell proliferation were studied. Haemopoiesis is a continuous process, where mature blood cells are replaced by the proliferation and differentiation of more primitive progenitor and stem cells. Blood formation is tightly regulated by the stromal micro-environment. Exposure of murine bone marrow cells, from male and female mice, to 80 microT (50 Hz) magnetic fields showed a reduction in the proliferation and differentiation of the granulocyte-macrophage progenitor (CFU-GM) compared to non-exposed bone marrow cells. The results on the effect of the ELF-field on stromal stem cell proliferation (CFU-f) are somewhat equivocal at the moment. CFU-f from female mice showed a reduction while CFU-f from male mice were not decreased.

Haematotoxicity testing in vitro using human cord blood haemopoietic cells.

Exposure to benzene and lead results in human and experimental animals in myelotoxicity. Haemopoietic progenitor cells are considered the target cells for these effects. Currently, experimental animals are in widespread use to predict haematotoxicity. In vitro techniques allow use of human cells. This will highly improve the predictive power of myelotoxicity testing compared with experiments on syngeneic animals (mainly rat and mouse). Furthermore the number of animals required for product development and approval will be reduced. In vitro assays for studying haemopoietic progenitor cells are well developed. We examined the effect of two benzene metabolites and lead nitrate on the in vitro growth potential of human haemopoietic progenitor cells derived from human cord blood. The effects of lead nitrate and benzene metabolites such as catechol and hydroquinone on colony formation of cord blood haemopoietic progenitors were investigated in semi-solid (agar) assays (CFU-GM = Colony Forming Unit of granulocytes and/or macrophages). Toxic agents were added as single agents at the start of the cultures. Cells were exposed during the 14-day culture period. The in vitro exposure of the haemopoietic cells resulted in a dose-dependent depression of the CFU-GM numbers. The dose that caused a 50% decrease in colony formation (IC(50)) was 5 mum for catechol and 20 mum for hydroquinone. An interindividual variability was seen in the response to Pb exposure. In some cord blood samples Pb inhibited colony growth only at higher doses (IC(50) > 100 mum). In a large number of samples, haemopoietic cells were affected at much lower concentrations of Pb exposure (IC(50) < 10 mum). Effects in vitro are seen at concentration levels that are found in human blood in situ.

Response of murine stromal bone marrow cultures to alpha-particle irradiation in vivo and in vitro at osteosarcomogenic alpha-radiation doses.

Stromal cells belonging to the bone marrow microenvironment are altered in mice at risk of bone tumour development after (241)Am injection. This can be observed with selective cell culture techniques long before the tumours become manifest. Colony forming assays in vitro showed increases in the number of stromal stem cells at lower dose levels but a decrease in cell number at higher dose levels. The in vitro osteogenic capacity of marrow, which is attributed to stromal cells of the osteogenic lineage, was significantly reduced. The changes were persistent until at least 1 yr after (241)Am injection. In vitro alpha-irradiation of bone marrow also reduced the osteogenic capacity of the bone marrow at dose levels that were comparable with those producing an effect in vivo. This suggests a direct effect of alpha-particle irradiation on stromal bone marrow cells. These data and previous results on the regulatory role of the stroma in haemopoiesis show that stromal bone marrow cells should be considered as sensitive targets for chronic low level alpha-particle irradiation.

Haemopoietic and osteogenic toxicity testing in vitro using murine bone marrow cultures.

Bone marrow and the surrounding bone with its high storage capacity for inorganic compounds may accumulate various lipophilic and electrophilic substances that enter the bloodstream. In bone marrow a few stem cells are responsible for the continuous production of blood cells and bone cells during the entire life of the organism. Damage to these cells may result in haemopoietic failure, blood disorders or bone diseases. Therefore bone marrow needs to be considered as one of the major targets of chemicals that enter the circulation. A battery of different in vitro bone marrow assays is established in which interference of chemicals with proliferation and differentiation of marrow cells with haemopoietic, stromal or bone forming marrow commitment may be screened routinely. Stromal cells form the network of extracellular matrix and growth factors that is needed by the haemopoietic cells to proliferate and differentiate. If stromal marrow cells are cultured in the presence of ascorbic acid and beta-glycerophosphate, bone-specific proteins and an extracellular matrix are produced, which calcifies within 3 wk. To evaluate the specificity of the effects on marrow cells, a general cytotoxicity assay is included using 3T3-fibroblasts. Various concentrations of xenobiotics were added over the course of 3 days to the different asssays. Lead nitrate inhibited proliferation of stromal stem cells and their calcification in the bone-forming assay at much lower concentrations than those which were inhibitory to the proliferation of 3T3 cells. The benzene metabolite hydroquinone was equally inhibitory in all the marrow assays, but 3T3 cells needed 10 times more hydroquinone to reach the same degree of inhibition. Catechol, which is another benzene metabolite, was highly toxic but was equally effective in all the assays and showed no specific effects on the marrow cells. As in vivo, benzene itself and phenol showed hardly any effect in the in vitro assays. Not only pollutants but also cytokines may be screened with these assays. Differential effects on marrow cells could be demonstrated for interleukin 10.

Haemopoietic Lineage Sensitivity and Individual Susceptibility to PCB126: Relation to Glutathione S-transferase mu.

The effect of PCB126 on the in vitro growth potential of human myeloid (CFU-GM) and erythroid (BFU-E) progenitor cells derived from human cord blood haemopoietic cells was examined. The possible link between individual variability in susceptibility and the presence of the xenobiotic metabolizing enzyme glutathione S-transferase class mu (GSTmu) was studied. After density centrifugation separation, mononuclear cord blood cells were cultured in the presence of PCB126 (10(-6)m and 10(-8)m). Several cord blood samples were simultaneously tested for: (1) colony formation and (2) the presence of the GSTmu gene measured by PCR. An interindividual variability in the response to PCB126 was present. At 10(-8)m and 10(-6)m PCB126, respectively 8 (40%) and 10 (50%) out of 20 cord blood samples showed a significant dose-dependent decrease in CFU-GM numbers. Erythroid progenitors were less affected by PCB126. At 10(-8)m and 10(-6)m PCB126, respectively only two (12%) and three (18%) out of 17 cord blood samples showed a significant decrease in BFU-E numbers. The presence or absence of the GSTmu gene was determined using PCR. The GSTmu gene was present in 52% (14 out of 27 samples tested) of the cord blood samples. Damage to the myeloid and erythroid haemopoietic progenitor cells at either PCB126 concentrations was not correlated with the presence of the GSTmu gene.

Use of in vitro assays to assess hematotoxic effects of environmental compounds.

The number of chemicals being introduced into the environment increases and many of these substances may pose a health risk to exposed individuals. Many environmental toxicants with a potential toxicity to the hematopoietic system have been identified by animal experiments. Owing to the risks of severe chronic hematopoietic disorders, it is important to screen chemicals for their hematotoxicity. The aim of this work was to identify, through the use of in vitro techniques, targets for hematotoxic effects. Our study focused on myeloid and erythroid hematopoietic progenitors and stromal stem cells as possible targets. The in vitro assays showed that various hematotoxic compounds exert different effects on these cell populations. In vitro exposure of murine bone marrow cells to various inorganic (cadmium, lead) and organic (benzene metabolites, lindane. benzo-[a]-pyrene (BaP), PCB (polychlorinated biphenyl) congeners) environmental chemicals indicated that hematopoietic or stromal bone marrow cells were targets for most of the chemicals. Stromal cells were more affected by lead, cadmium, and BaP compared to myeloid cells. Benzene and phenol gave no response, but the metabolites catechol and hydroquinone were equally toxic to the stromal and the myeloid progenitor cells. Among the PCBs tested, PCB126 was most toxic. Human progenitor cells derived from cord blood were exposed in vitro to catechol, hydroquinone, lead nitrate, and PCBs. Human hematopoietic cells were sensitive to the tested compounds. Human erythroid progenitors are more susceptible to lead exposure than are myeloid progenitors. Based on the in vitro tests, humans are more sensitive to lead, catechol, and PCB126 than are mice. In contrast to the murine data, humans responded with individual differences to lead and PCB126.

Lead and catechol hematotoxicity in vitro using human and murine hematopoietic progenitor cells.

In vitro cloning assays for hematopoietic myeloid and erythroid precursor cells have been used as screening systems to investigate the hematotoxic potential of environmental chemicals in humans and mice. Granulocyte-monocyte progenitors (CFU-GM) from human umbilical cord blood and from mouse bone marrow (Balb/c and B6C3F1) were cultured in the presence of lead and the benzene metabolite catechol. Erythroid precursors (BFU-E) from human umbilical cord blood were cultured in the presence of lead. The in vitro exposure of the human and murine cells resulted in a dose-dependent depression of the colony numbers. The concentration effect relationship was studied. Results showed that: (1) Based on calculated IC50 values, human progenitors are more sensitive to lead and catechol than are murine progenitors. The dose that caused a 50% decrease in colony formation after catechol exposure was 6 times higher for murine cells (IC50 = 24 micromol/L) than for human cord blood cells (IC50 = 4 micromol/L). Lead was 10-15 times more toxic to human hematopoietic cells (IC50 = 61 micromol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 micromol/L; B6C3F1, IC50 = 536 micromol/L). (2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 micromol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 micromol/L). (3) Individual differences in the susceptibility to the harmful effect of lead were seen among cord blood samples. (4) Toxicity of lead to progenitor cells occurred at environmentally relevant concentrations.