Immunotoxicity In Vitro Assays for Environmental Pollutants under Paradigm Shift in Toxicity Tests

Abstract

With the outbreak of COVID-19, increasingly more attention has been paid to the effects of environmental factors on the immune system of organisms, because environmental pollutants may act in synergy with viruses by affecting the immunity of organisms. The immune system is a developing defense system formed by all metazoans in the course of struggling with various internal and external factors, whose damage may lead to increased susceptibility to pathogens and diseases. Due to a greater vulnerability of the immune system, immunotoxicity has the potential to be the early event of other toxic effects, and should be incorporated into environmental risk assessment. However, compared with other toxicity endpoints, e.g., genotoxicity, endocrine toxicity, or developmental toxicity, there are many challenges for the immunotoxicity test of environmental pollutants; this is due to the lack of detailed mechanisms of action and reliable assay methods. In addition, with the strong appeal for animal-free experiments, there has been a significant shift in the toxicity test paradigm, from traditional animal experiments to high-throughput in vitro assays that rely on cell lines. Therefore, there is an urgent need to build high-though put immunotoxicity test methods to screen massive environmental pollutants. This paper reviews the common methods of immunotoxicity assays, including assays for direct immunotoxicity and skin sensitization. Direct immunotoxicity mainly refers to immunosuppression, for which the assays mostly use mixed immune cells or isolated single cells from animals with obvious problems, such as high cost, complex experimental operation, strong variability and so on. Meanwhile, there have been no stable and standard cell lines targeting immune functions developed for high-throughput tests. Compared with direct immunotoxicity, skin sensitizer screening has developed relatively mature in vitro assay methods based on an adverse outcome pathway (AOP), which points out the way forward for the paradigm shift in toxicity tests. According to the experience of skin sensitizer screening, this paper proposes that we also should seek appropriate nodes and establish more complete AOPs for immunosuppression and other immune-mediated diseases. Then, effective in vitro immunotoxicity assay methods can be developed targeting key events, simultaneously coordinating the studies of the chemical immunotoxicity mechanism, and further promoting the paradigm shift in the immunotoxicity test.

New approach methodologies to enhance human health risk assessment of immunotoxic properties of chemicals — a PARC (Partnership for the Assessment of Risk from Chemicals) project

“…2) Building AOPs in immunotoxicology will help in identifying the most relevant pathways and key events that NAMs should evaluate. This has been well illustrated in the case of skin sensitization (Clouet et al, 2019;Wang et al, 2023) and food allergy (Bilsen et al, 2017). Developing new AOPs in immunotoxicology, as described above, is also being carried out in PARC and will feed into NAMs development (De Castelbajac et al, 2023).…”

“…Multiple chemical agents, including pesticides and other organic compounds, metals, and specific immunosuppressive medicines, have been demonstrated to exhibit immunosuppressive properties ( Bou Zerdan et al, 2021 ). However, there exist no widely accepted chemical-agnostic in vitro tests specifically designed for assessing the immunosuppressive properties of chemicals ( Wang et al, 2023 ). Therefore, there is a growing demand for developing NAMs for evaluating immunosuppression.…”

REACH out-numbered! The future of REACH and animal numbers

“…In the US, the endocrine disruptor screening program advanced earlier to a first round (Juberg et al, 2014): Notably, this first screening (tier 1, not a definitive assessment) cost about $5 million per substance; this is about the cost calculated for a full Annex X assessment, illustrating the possible dimensions. The increasing availability of in vitro and in silico approaches for developmental neurotoxicity (Smirnova et al, 2014;Fritsche et al, 2015), including most recently developed OECD in vitro guidance 35 , neurotoxicity (Schmidt et al, 2017), immunotoxicity (Wang et al, 2022), endocrine disruption (Manibusan and Touart, 2017), etc. offers opportunities to use NAMs.…”

A comprehensive battery of flow cytometric immunoassays for the in vitro testing of chemical effects in human blood cells

“…This technology allows simultaneous analysis of the majority of immune cell populations based on their precise determination of phenotype as well as response to stimuli, which is possible by careful selection of lineage and activation markers ( 10 ). The use of flow cytometry for single cell analysis allows high-throughput screening of chemicals ( 11 ), which promises to identify affected cells and determine which signaling pathway may have been affected ( 12 ). Here, we propose a battery of in vitro assays to address the diversity of immune responses in human peripheral blood.…”