Evaluation and Prevalidation of an Immunotoxicity Test Based on Human Whole-blood Cytokine Release

Ingrid Langezaal, Sebastian Hoffmann, Thomas Hartung, & Sandra Coecke

Immunotoxicology is a relatively new field in toxicology, and is one of emerging importance, because immunotoxicity appears to contribute to the development of cancer, autoimmune disorders, allergies and other diseases. At present, there is a lack of human cell-based immunotoxicity assays for predicting the toxicity of xenobiotics toward the immune system in a simple, fast, economical and reliable way. Existing immunotoxicity tests are mainly performed in animals, although species differences favour humanbased testing. Whole-blood cytokine release models have attracted increasing interest, and are broadly used for pharmacological in vitro and ex vivo studies, as well as for pyrogenicity testing. We have adapted those methods for immunotoxicity testing, to permit the potency testing of immunostimulants and immunosuppressants. Following stimulation with a lipopolysaccharide or staphylococcal enterotoxin B, monocytes and lymphocytes release interleukin-1β and interleukin-4, respectively. Thirty-one pharmaceutical compounds, with known effects on the immune system, were used to optimise and standardise the method, by analysing their effects on cytokine release. The in vitro results were expressed as IC50 values for immunosuppression, and SC4 (fourfold increase) values for immunostimulation, and compared with therapeutic serum concentrations of the compounds in patients, and in vivo LD50 values from animal studies. The in vitro results correlated well with the in vivo data, so the test appears to reflect immunomodulation. Results were reproducible (CV = 20 ± 5%), and the method could be transferred to another laboratory (r2 = 0.99). We therefore propose this method for further validation and for use in immunotoxicity testing strategies.
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The Effects of Heavy Metals on Common Carp White Blood Cells In Vitro

Malgorzata Witeska and Marta Wakulska

The in vitro effects of cadmium, copper, lead and zinc, and various cadmium compounds (chloride, sulphate and nitrate) on common carp (Cyprinus carpio) lymphocyte viability and phagocyte activity, were evaluated. The percentage of dead lymphocytes was determined after Trypan blue staining, and phagocyte activity was measured by using the nitroblue tetrazolium (NBT) reduction test. Lead was the most toxic to lymphocytes — the maximum mortality exceeded 30%, and was significantly higher at 1μM of lead, compared to the control. The maximum mortality caused by cadmium was below 10%, but was significantly elevated with 5μM or more of cadmium. Zinc induced lymphocyte mortality from 10μM, whilst no effect was observed with copper. The incubation of full blood with the three cadmium compounds (at 5mg/l of cadmium) showed that cadmium nitrate and cadmium sulphate were more toxic (over 35% and 25% mortality, respectively) than cadmium chloride (about 15% mortality). This was confirmed by the results of tests on isolated cells —1mg/l of cadmium as nitrate and sulphate increased lymphocyte mortality compared to the control and cadmium chloride. Phagocytic activity was less sensitive to heavy metals than was lymphocyte viability. It was significantly reduced following exposure to 50μM and 100μM cadmium, and 100μM zinc, but no effects were observed with either lead or copper.
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