In vitro testing for direct immunotoxicity: state of the art

Immunotoxicity is defined as the toxicological effects of xenobiotics including pharmaceuticals on the functioning of the immune system and can be induced in either direct or indirect ways. Direct immunotoxicity is caused by the effects of chemicals on the immune system, leading to immunosuppression and subsequently to reduced resistance to infectious diseases or certain forms of nongenotoxic carcinogenicity.In vitro testing has several advantages over in vivo testing, such as detailed mechanistic understanding, species extrapolation (parallelogram approach), and reduction, refinement, and replacement of animal experiments. In vitro testing for direct immunotoxicity can be done in a two-tiered approach, the first tier measuring myelotoxicity. If this type of toxicity is apparent, the compound can be designated immunotoxic. If not, the compound is tested for lymphotoxicity (second tier). Several in vitro assays for lymphotoxicity exist, each comprising specific functions of the immune system (cytokine production, cell proliferation, cytotoxic T-cell activity, natural killer cell activity, antibody production, and dendritic cell maturation). A brief description of each assay is provided. Only one assay, the human whole blood cytokine release assay, has undergone formal prevalidation, while another one, the lymphocyte proliferation assay, is progressing towards that phase.Progress in in vitro testing for direct immunotoxicity includes prevalidation of existing assays and selection of the assay (or combination of assays) that performs best. To avoid inter-species extrapolation, assays should preferably use human cells. Furthermore, the use of whole blood has the advantage of comprising multiple cell types in their natural proportion and environment. The so-called "omics" techniques provide additional mechanistic understanding and hold promise for the characterization of classes of compounds and prediction of specific toxic effects. Technical innovations such as high-content screening and high-throughput analysis will greatly expand the opportunities for in vitro testing

In vitro immunotoxicity of bis(tri-n-butyltin)oxide (TBTO) studied by toxicogenomics

The biocide and environmental pollutant bis(tri-n-butyltin)oxide (TBTO) causes thymus atrophy in rodents. Whether the depletion of thymic lymphocytes by tributyltin compounds may be the result of inhibition of cell proliferation or induction of apoptosis is subject of debate. We examined gene expression profiles in primary rat thymocytes exposed to TBTO in vitro at dose levels of 0, 0.1, 0.3, 0.5, and 1.0muM. By measuring cell viability and apoptosis, exposure conditions were selected that would provide information on changes in gene expression preceding or accompanying functional effects of TBTO. Several processes related to TBTO-induced toxicity were detected at the transcriptome level. Effects on lipid metabolisms appeared to be the first indication of disruption of cellular function. Many transcriptional effects of TBTO at higher dose levels were related to apoptotic processes, which corresponded to present or subsequent thymocyte apoptosis observed phenotypically. The gene expression profile was, however, not unambiguous since expression of apoptosis-related genes was both increased and decreased. Stimulation of glucocorticoid receptor signaling appeared to be a relevant underlying mechanism of action. These findings suggest that TBTO exerts its toxic effects on the thymus primarily by affecting apoptotic processes, but the possibility is discussed that this may in fact represent an early effect that precedes inhibition of cell proliferation. At the highest dose level tested, TBTO additionally repressed mitochondrial function and immune cell activation. Our in vitro toxicogenomics approach thus identified several cellular and molecular targets of TBTO that may mediate the toxicity towards thymocytes and thereby its immunosuppressive effects. AD - Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Department of Health Risk Analysis and Toxicology (GRAT), Maastricht University, Maastricht, The Netherlands; National Institute of Public Health and the Environment (RIVM), Laboratory for Toxicology, Pathology and Genetics (TOX), Bilthoven, The Netherlands