Previous studies demonstrate that the effects of one chemical stressor on selected immunological parameters can be predicted on the basis of the area under the corticosterone concentration vs. time curve. However, it is not clear if this is applicable to other chemical stressors. The present study was conducted to determine if the stress-induced immunological effects of atrazine and ethanol could be predicted, and if it is feasible to use one immunological parameter as a biomarker of stress to predict the quantity of changes expected in other immunological parameters. The area under the corticosterone concentration-versus-time curve (AUC) was measured in mice treated with ethanol (EtOH, 4, 5, 6, or 7 g/kg by oral gavage) or atrazine (ATZ, 100, 200, or 300 mg/kg, ip). The effects of the same dosages of these chemicals on thymus and spleen cellularity, lymphocyte subpopulations in the thymus and spleen, expression of MHC class II protein on splenocytes, antibody responses to keyhole limpet hemocyanin, and natural killer-cell activity were determined. Models were derived describing the relationship between corticosterone AUC and immunological changes induced by these chemicals. The results for these chemical stressors were more similar to results obtained from mice subjected to restraint stress than from mice treated with exogenous corticosterone. Some effects were greater than predicted on the basis of the stress response alone, indicating other mechanisms of immunotoxicity. One of the parameters (MHC class II expression) was evaluated as a predictive biomarker for stress-related immunosuppression, and the results suggest it could be suitable for that purpose.

Toxicological testing of compounds often is conducted at the maximum tolerated dose to identify potential target organs. Toxicities observed at these high doses may result in decreased body weight gain, food consumption and activity. These clinical signs are often associated with a generalized stress response. It has been known that stress may cause increased levels of corticosterone, which causes changes in circulating leukocyte profiles, decreases in thymus and spleen weights and changes in the microscopic structure of lymphoid organs. This makes it difficult to differentiate between stress-related changes and direct toxicity to the immune system in standard non-clinical toxicity testing in rats. In mice, MHC Class II expression was found to be a very sensitive biomarker of stress and maybe useful for the rat. Therefore, the objective of studies presented was to further characterize the effects of corticosterone and stressors on the immune system and identify potential biomarkers of stress in rats. Rats were treated with exogenous corticosterone (20 or 30 mg/kg BID) or ethanol (5 g/kg) for either 1 or 4 days. Restraint stress was also evaluated for a 3-day period. Blood and urine samples were collected during the treatment period for corticosterone measurements. At necropsy, blood samples for leukocyte differentials were collected. Spleen and thymus weights, cellularity, lymphocyte subpopulations and histopathology were also evaluated. Urine corticosterone levels were also investigated as a surrogate to measuring serum corticosterone. The results demonstrate that the pattern of responses to corticosterone or the stressors is different in mice and rats. Although, decreases in MHC Class II were found to be a sensitive indicator of stress in mice, only slight decreases were observed in rats with similar serum corticosterone AUC levels. Decreases in thymus weight were greater than spleen weight with corticosterone or ethanol or restraint stressor. No other single parameter or combination of parameters tested were obvious candidates as sensitive biomarkers of stress in rats. However, the good correlation between urine and serum corticosterone levels suggest that urine corticosterone may be a potential biomarker of stress induced changes to the immune response.

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Many chemicals and drugs can induce a neuroendocrine stress response that can be immunosuppressive. Mathematical models have been developed that allow prediction of the immunological impact of such stress responses in mice on the basis of exposure to the important stress-related mediator corticosterone. The area under the corticosterone concentration vs. time curve (AUC) has been used as an indicator of cumulative corticosterone exposure in these modeling studies. In the present study, an immunotoxicant known to induce a stress response, 3,4-dichloropropionanilide (propanil), was evaluated to determine if corticosterone AUC values are related to suppression of immunological parameters in mice treated with this chemical. Linear relationships between corticosterone AUC values and suppression of the following parameters were noted in B6C3F1 female mice: thymus cellularity and thymus subpopulation percentages, splenic subpopulation percentages, natural killer cell activity, MHC class II protein expression, and IgG1 and IgG2a antibody responses to antigen. Linear models derived in previous studies using mice treated with exogenous corticosterone or with restraint stress effectively predicted the immunological effects of 3, 4-dichloropropionanilide on the basis of corticosterone AUC values. The models derived using immobilization stress were more effective (r(2) for observed vs. predicted = 0.90) than the models derived using mice treated with exogenous corticosterone (r(2) for observed vs. predicted = 0.65). This was expected, because most stressors induce a variety of immunomodulatory mediators, not just corticosterone. These findings have implications for risk assessment in immunotoxicology.

Cell culture methods can allow investigation of the mechanisms responsible for immunotoxicity. Unfortunately, natural killer (NK) cells in rodent splenic cultures rapidly lose their cytolytic function. It is not known if death of NK cells or loss of function in viable NK cells is primarily responsible for this loss. Flow cytometry and an assay of NK cell lytic function were used to address this issue and to determine if NK cell viability could be maintained by adding selected cytokines or a caspase inhibitor to the cultures. Total cells and NK cells in untreated 18 h cultures were 79 +/- 1% and 25 +/- 2% viable, respectively, and these cultured splenocytes caused only 4 +/- 1% specific release of (51)Cr from YAC-1 target cells. Cultures including polyinosinic:polycytidylic acid (poly I:C) or IL-2 had increased NK cell viability (43 +/- 2%, 47 +/- 1%) and function (58 +/- 2 and 43 +/- 1% specific release). IL-15 significantly increased NK cell viability, but not function. Previous studies demonstrated that treatment of mice with immunotoxicants such as ethanol or corticosterone diminishes NK cell activation in vitro in response to poly I:C. To determine if alterations in viability are responsible for this decreased NK cell activity, lytic function and NK activity were measured in cultures of splenocytes treated in vivo or in vitro with ethanol and/or corticosterone. Some treatments reduced IL-2 or poly I:C-enhanced lytic activity in vitro, but there was no clear relationship between these changes in function and changes in the percentage of viable NK cells. Thus, immunotoxicants that suppress NK cell activation can be investigated in vitro because commonly used activating stimuli also permit NK cell survival. However, no agents were identified that could maintain NK cell viability and function in culture (without activation) to allow investigation of the direct effects of immunotoxicants on basal NK activity in vitro.