H. Leppens

Problems and challenges in the development and validation of human cell-based assays to determine nanoparticle-induced immunomodulatory effects

BackgroundWith the increasing use of nanomaterials, the need for methods and assays to examine their immunosafety is becoming urgent, in particular for nanomaterials that are deliberately administered to human subjects (as in the case of nanomedicines). To obtain reliable results, standardised in vitro immunotoxicological tests should be used to determine the effects of engineered nanoparticles on human immune responses. However, before assays can be standardised, it is important that suitable methods are established and validated.ResultsIn a collaborative work between European laboratories, existing immunological and toxicological in vitro assays were tested and compared for their suitability to test effects of nanoparticles on immune responses. The prototypical nanoparticles used were metal (oxide) particles, either custom-generated by wet synthesis or commercially available as powders. Several problems and challenges were encountered during assay validation, ranging from particle agglomeration in biological media and optical interference with assay systems, to chemical immunotoxicity of solvents and contamination with endotoxin.ConclusionThe problems that were encountered in the immunological assay systems used in this study, such as chemical or endotoxin contamination and optical interference caused by the dense material, significantly affected the data obtained. These problems have to be solved to enable the development of reliable assays for the assessment of nano-immunosafety.

The suitability of different cellular in vitro immunotoxicity and genotoxicity methods for the analysis of nanoparticle-induced events

Abstract Suitable assays and test strategies are needed to analyze potential genotoxic and immunotoxic health effects caused by nanoparticle exposure. The development and validation of such methods is challenging because nanoparticles may show unexpected behavior, like aggregation or interference with optical measurements, when routine in vitro assays are performed. In our interdisciplinary study, the effects of inorganic gold (4.5 nm) and iron oxide (7.3 nm) nanoparticles with a narrow size distribution were tested on human cells using different assay systems. The results show that cytotoxicity as well as immunotoxicity and genotoxicity induced by these two inorganic nanoparticles was low or absent when using a panel of cell-based tests in different laboratories. However, several technical issues had to be tackled that were specific for working with nanoparticles. The methods used, their suitability for nanotoxicity testing, and the technical problems encountered are carefully described and discussed in this paper.

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

AbstractThe 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 ofin vitro techniques, targets for hematotoxic effects. Our study focused on myeloid and erythroid hematopoietic progenitors and stromal stem cells as possible targets. Thein 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 exposedin 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 thein 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.

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

Abstract. 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 × 106 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 107 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 phos‐phatase 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.

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 μmol/L) than for human cord blood cells (IC50 = 4 μmol/L). Lead was 10–15 times more toxic to human hematopoietic cells (IC50 = 61 μmol/L) than to murine bone marrow cells from both mice strains tested (Balb/c, IC50 = 1060 μmol/L; B6C3F1, IC50 = 536 μmol/L).(2) A lineage specificity was observed after exposure to lead. Human erythroid progenitors (hBFU-E) (IC50 = 3.31 μmol/L) were found to be 20 times more sensitive to the inhibitory effect of lead than were myeloid precursors (hCFU-GM) (IC50 = 63.58 μmol/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.

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.

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.

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 Lineage Sensitivity and Individual Susceptibility to PCB126: Relation to Glutathione S-transferase μ

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.

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