Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, Louisiana 71130
Received February 26, 2003; accepted July 13, 2003
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ABSTRACT |
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Key Words: stress; corticosterone; atrazine; ethanol; predictive model; biomarker.
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INTRODUCTION |
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The effects of stress responses on the immune system are complex, but relatively intense responses that persist for a few h or more generally cause immunosuppression (Pruett, 2001). Because corticosterone is one of the major immunosuppressive mediators of stress responses, it was selected as a mediator that might predict the quantity of immunosuppression caused by stress responses. This does not imply that corticosterone is the only stress-induced mediator that can affect the immune system, but that it will likely increase in magnitude in parallel with other mediators and thus serve as a useful surrogate for the intensity of the overall stress response. Quantitative assessment of the increased exposure to corticosterone requires determination of the area under the corticosterone concentration-versus-time curve (AUC) for the entire period during which corticosterone levels are elevated (Pruett et al., 1999
). It was anticipated that the immunological effects of exogenous corticosterone would be less than the effects of a full stress response (for example, a restraint stress response) that produced comparable corticosterone AUC values. For most of the immunological parameters measured in our previous studies, this was the case (Pruett et al., 2000b
). Restraint stress generally caused greater suppression than exogenous corticosterone at equivalent AUC values. It was also anticipated that the effects of restraint stress would be similar to the effects of chemical stressors, since most stressors induce neuroendocrine mediators in addition to corticosterone (e.g., catecholamines) (de Boer et al., 1991
; Keim and Sigg, 1977
). This was the case for the one chemical stressor evaluated to date, the herbicide propanil (Pruett et al., 2000b
). However, it remains unclear if the linear models relating corticosterone AUC values induced by restraint stress and changes in several immunological parameters can also be used to predict the immunological effects of other chemical stressors. This was tested in the present study using two additional chemical stressors, ethanol (EtOH) and atrazine (ATZ).
An immunological parameter that could serve as a biomarker of stress would be useful in routine safety assessments. If stress-induced changes in such a parameter correlated to other parameters (e.g., spleen and thymus cellularity or the antibody response to a T-dependent antigen), this pattern of changes could serve as a useful indicator of the role of stress in chemical-induced immunosuppression. Some immune parameters that are affected by stress are either already recommended as part of routine safety assessments or could easily be included in them. A predictive biomarker approach would seem preferable to experiments requiring separate sets of animals in which pharmacological agents were used to block stress mediators to determine the degree to which observed immunological effects of chemicals are caused by these mediators.
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MATERIALS AND METHODS |
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Administration of ethanol (EtOH) and atrazine (ATZ).
ATZ was obtained from ChemService (West Chester, PA). It was administered intraperitoneally at dosages of 100-, 200-, or 300-mg/kg body weight in food-grade corn oil. In one experiment, ATZ was administered by oral gavage in corn oil at 500 mg/kg to match the high dosage used in the National Toxicology Programs study (as described on the NTP web site at http://ntp-server.niehs.nih.gov/htdocs/pub-IT0.html). EtOH was administered by oral gavage at dosages of 4, 5, 6, or 7 g/kg using a 32% solution in tissue culture-grade water. These dosages produce peak blood EtOH levels ranging from ~0.2 to 0.5% (Carson and Pruett, 1996). Previous studies demonstrated that the corticosterone response to vehicle administration subsides within 14 h. This yields a negligible corticosterone AUC value, which only serves to add an additional data point near the 0 value for corticosterone AUC to any models developed (Pruett et al., 1999
). Therefore, naive control groups, not vehicle control groups, were used for these modeling studies.
Determination of corticosterone AUC values for EtOH and ATZ-treated mice.
Six identical sets of mice were treated with ATZ at 0 (naive), 100, 200, or 300 mg/kg at 9:00 P.M. Thus, there were 4 groups of mice in each set and there were 5 mice in each group. One of these sets of mice was bled by decapitation at 1, 2, 4, 6, 8, and 12 h after ATZ administration. A similar approach was used for EtOH, with dosages of 0 (naive), 4, 5, 6, or 7 g/kg. Mice were bled one cage at a time in a separate room to prevent the induction of stress responses in the remaining mice. Serum was separated from each blood sample and the corticosterone was quantified using a radioimmunoassay kit (DPC, Los Angeles, CA) (Pruett et al., 1999). Graphs of the data were produced using DeltaGraph software (SPSS Inc., Chicago, Ill.), and the area between the naive control values and the treated values (AUC) was determined as described previously using NIH Image software (Pruett et al., 1999
).
Measurement of immunological parameters.
Immunological parameters were evaluated by routine methods exactly as described in our previous studies (Han and Pruett, 1995; Pruett et al., 1999
, 2000a
; Weiss et al., 1996
; Wu et al., 1994
). The methods are summarized in following paragraphs. The group size was 5 in all experiments. Cell suspensions were prepared by pressing the spleen or thymus between the frosted ends of glass microscope slides, and spleen and thymus cellularity were determined using an electronic cell counter (Coulter model Z1, Coulter Electronics, Hialeah, FL). Previous studies indicated that the greatest changes in spleen and thymus cellularity and in the lymphoid subpopulations in these organs occurred 24 h after the initiation of the stressor (Pruett et al., 2000a
), whereas the greatest changes in NK cell activity and major histocompatibility complex (MHC) class II expression occurred 12 h after the initiation of the stressor (Pruett et al., 1999
). Therefore, these times were used in the present study.
Labeling for flow cytometry was conducted using fluorescent-labeled antibodies specific for CD4, CD8, B220 (CD45R), and MHC class II (I-Ap,b,k,j,q,r,s) using antibodies from BD Pharmingen (San Diego, CA). Cells were labeled in V-bottom microtiter plates at a concentration of 106 cells per well, and an isotype control was used for each antibody. Gates were set using the unlabeled control cell and the isotype controls so that 2% or less of the cells labeled with the isotype control were outside the negative gate in any experiment.
Splenic NK cell activity was measured 12 h after initiation of treatment, using a standard 4 h 51Cr release assay with YAC-1 target cells (Pruett et al., 1999). Effectors (spleen cell) to target cell ratios of 100:1, 50:1, and 25:1 were used, and values were expressed as lytic units per 107 splenocytes (Bryant et al., 1992
) to provide a single value for modeling.
Mice were immunized with 100 µg/mouse keyhole limpet hemocyanin KLH (Pierce Chemical Co.) 12 h after treatment with EtOH or ATZ, as in our previous studies (Pruett and Fan 2000; Pruett et al., 2000b
). Two weeks after immunization mice were bled under methoxyflurane anesthesia. This is the time at which near maximum antibody levels occur (Kruszewska et al., 1995
), and it is the time used in our previous studies. Serum was diluted 1:25, 1:50, and 1:100, and IgG1 and IgG2a antibodies specific for KLH were measured by ELISA just as described in our previous studies (Pruett and Fan, 2000
; Pruett et al., 2000b
). The results indicated linear relationships of the antibody titration curves, so the absorbance values from the ELISA at the 1:25 antibody dilution were normalized (by dividing by the mean value of the naive control group and multiplying by 100), and these normalized values were used for subsequent regression analyses. Control serum from nonimmunized mice was included in every assay and yielded absorbance values less than 0.06 (as compared to typical values of 0.30.4 in sera from immunized mice).
Statistical and modeling methods.
In most cases, linear models best described the data obtained. The runs test was done for every linear model, and none of the models in the present study had a significant nonlinear component. In addition, linear models were used because they facilitated comparison of results between different experiments. Linear regression was performed using the Prism software package (GraphPad, San Diego, CA), as described previously (Pruett et al., 1999). The 95% confidence intervals were calculated by the program and are shown on the graphs presented here, which were generated using Prism. Statistical comparison of pairs of lines was done using Prism software, which implements the method described by Zar (1984)
. This method determines if the slope or the elevation (or Y-intercept) differ significantly between any pair of lines. To compare values at particular points on different lines, the 83.7% confidence intervals were calculated using StatView software (SPSS, Chicago, IL). Contrary to the common assumption, failure of 95% confidence intervals of two values to overlap does not indicate that the values are different at the 0.05 probability level. In fact, this indicates an even lower p value. Failure of the 83.7% confidence intervals to overlap indicates that the values are significantly different at the p
0.05 level (Barr, 1969
; Nelson, 1989
).
The significance of differences between control and treatment groups was determined by analysis of variance (ANOVA) followed by Dunnetts test, in some experiments.
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RESULTS |
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In a previous study, it was determined that handling and injection of vehicle induce a small increase in corticosterone levels (AUC, 303 ng/mlh) (Pruett et al., 1999), but this was not associated with significant changes in any of the immunological parameters measured. Therefore, only naive control groups were used in this study.
Linear Models of Corticosterone AUC vs. Thymus- and Spleen-Cell Numbers and Subpopulation Percentages in EtOH- and ATZ-Treated Mice
Previously published data (Pruett et al., 2000a) from mice treated with exogenous corticosterone or restraint are shown for comparison along with data from mice treated with ATZ or EtOH in the present study (Figs. 3
and 4
). The slopes of the regression lines were significantly different for ATZ and EtOH with regard to CD4+CD8+ cells in the thymus, with ATZ causing a greater decrease in this subpopulation than EtOH (at equivalent corticosterone AUC values). Our previous study demonstrated that the difference in slope between the line for exogenous corticosterone and the line for restraint is also significant, with exogenous corticosterone causing a greater decrease than restraint (Pruett et al., 2000a
). The regression lines for restraint and EtOH were not significantly different, whereas the lines for restraint and ATZ were significantly different. The slope was more negative for ATZ than for restraint, indicating that ATZ has a greater effect than restraint (at equivalent corticosterone AUC values). Together these results indicate that the effects of ATZ and corticosterone on the percentage of CD4+CD8+ were greater than the effects of restraint, whereas the effects of EtOH were similar to the previously reported effects of restraint (Pruett et al., 2000a
).
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The number of nucleated cells in the spleen was decreased dose-responsively by ATZ, but only slightly by EtOH (Figs. 5 and 6
). The slopes of the lines described by these results were significantly different. The cellular subpopulations analyzed were affected by ATZ, but not significantly by EtOH (as indicated by ANOVA). The percentage of B cells (B220+) was substantially decreased by ATZ, producing a strongly negative slope for the percentage of B220+ cells vs. corticosterone AUC. The other treatments caused only very small decreases, even at the highest corticosterone AUC values. As expected, the percentage of CD4+ and CD8+ T cells increased in a manner that corresponds to the decrease in B-cell percentage in response to ATZ, but not EtOH (Figs. 5
and 6
).
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Overall Correlation between Observed Values for Immunological Parameters in Mice Treated with Chemical Stressors and Values Predicted Using Linear Models Derived from Mice Treated with Restraint or Exogenous Corticosterone
The data shown in Figure 10 represent a comparison of the ability of the linear models obtained using restraint and exogenous corticosterone to predict each of the immunological parameters evaluated in this study for ATZ and EtOH, as well as the same parameters evaluated in a previous study in mice treated with propanil (Pruett et al., 2000b
). The observed value of each parameter at an ATZ dosage of 200 mg/kg, an EtOH dosage of 7 g/kg, and a propanil dosage of 100 mg/kg (which yielded similar corticosterone AUC values of 3387, 4068, and 4383, respectively) was compared to the values predicted using the corticosterone AUC and the restraint or the corticosterone linear model. The parameters used in this analysis were: MHC class II expression, NK cell activity, IgG1 and IgG2a responses to KLH, percentage of major subpopulations in the thymus (CD4-CD8-, CD4+CD8+, CD4+CD8-, CD4-CD8+), percentage of major subpopulations in the spleen (B220, CD4+, CD8+), thymus cellularity, and spleen cellularity. The results demonstrate correlation between observed and predicted values for both the restraint and the corticosterone models (Fig. 10
). However, the correlation coefficient was substantially higher for the restraint models (0.66) than for the corticosterone models (0.39). Also shown in Figure 10
are the idealized lines for perfect correlation. Both of the actual lines are above the ideal lines, as a result of clusters of data points for which the predicted value was greater than the observed value. Interestingly, there were very few points below the ideal line, which would indicate predicted values lower than observed values. Overall, this indicates that the chemicals tested suppress some of the parameters examined to a greater extent than can be accounted for by stress alone, but the effects of the chemicals on essentially all parameters were at least as great as predicted by the stress models (corticosterone or restraint). In general, restraint had greater effects than corticosterone alone and thus was more predictive of the effects of chemicals.
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DISCUSSION |
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EtOH was selected for these studies on the basis of numerous reports demonstrating that it activates the hypothalamic-pituitary-adrenal axis in rodents and humans (Mendelson and Stein, 1966; Pruett et al., 1998
). Atrazine was selected because it is the most abundantly used conventional agricultural chemical in the U.S. (according the U.S. EPA Web site at http://www.epa.gov/oppbead1/pestsales/97pestsales/table8.htm), and an immunotoxicology assessment by the National Toxicology Program indicates that a high dosage causes approximately 50% loss of thymocytes (these results are summarized on the NTP Web site at http://ntp-server.niehs.nih.gov/htdocs/IT-studies/IMM94002.html), a parameter that is particularly sensitive to glucocorticoids. We previously evaluated propanil in an identical set of experiments, because it has been reported that propanil induces a vigorous stress response that is responsible for at least some of its immunological effects (Cuff et al., 1996
). The prevalence of the induction of stress responses by immunotoxic drugs or chemicals is not known. However, there are at least 21 drugs or chemicals that have been reported to induce a stress response (listed in Pruett et al., 1999
). In addition, we have reported that sodium methyldithiocarbamate (the third most abundantly used agricultural pesticide in the U.S. induces a stress response, and that this is completely responsible for the thymic atrophy observed in mice treated with this compound (Myers and Pruett, 2001
). Because the EPA recommends the use of a high dosage, near the maximum tolerated dosage (which by definition produces some signs of overt toxicity), it seems likely that, at least at the higher dosages, stress responses will be common in immunotoxicology safety evaluation. Although the serum corticosterone AUC values for EtOH and ATZ were linearly related to the dose of each compound (Figs. 1
and 2
), it is clear that the effects of ATZ on serum corticosterone are complex. For example, corticosterone is maximal at 1 h for both the 100 and 200 mg/kg dosages, but the maximum level occurs at 6 h for the 300-mg/kg dosage. It is not clear what effects such differences may have on immune parameters, but it is likely that the differences, as well as time of day (in relation to normal circadian changes in corticosterone), have some impact on immunological outcomes.
The results reported here are applicable only to acute effects of a single dose of chemical stressor. Because immunotoxicological safety evaluations often involve dosing daily for 28 days, it will be important to determine if these relationships persist after 28 days of dosing. It is possible that some degree of tolerance to the stress-inducing effects of some drugs or chemicals will develop over 28 days. However, it seems unlikely that this will occur for all chemicals and drugs. Although it is also possible that the immune system will become tolerant to the effects of stress-induced neuroendocrine mediators, it is unlikely this will be of sufficient magnitude to substantially alter all immunological effects. For example, it is well documented that chronic stress and metabolic disorders such as Cushings syndrome, that involve increased concentration of stress hormones, cause chronic changes in the immune system (Kronfol et al., 1996; Vedhara et al., 1999
).
In addition to the implications of these findings with regard to safety evaluations, there are implications with regard to the effects of stress in general and drug and chemical-induced stress in particular. The effects of the chemical stressors are more similar to the effects of restraint than to the effects of exogenous corticosterone. This was expected, because it was assumed that immunosuppressive stress mediators in addition to corticosterone would be induced by restraint and that this would lead to greater immunosuppression. This was observed for most parameters, and greater than expected suppression was noted for NK cell activity in mice treated with EtOH and for the percentage of B cells in the spleen with ATZ. This may be due to direct action of these chemicals. Alternatively, it is possible that corticosterone is regulated differently and affects the immune system differently when it is given exogenously (presumably representing only the afferent limb of the stress response) than when it is produced endogenously (representing efferent and afferent limbs of the stress response). We initially chose to evaluate IgG1 and IgG2a instead of IgM because of reports that stress differentially affected these two immunoglobulins (Pruett and Fan, 2000). However, we did not observe meaningful differences in this study or in our previous study (Pruett and Fan, 2000
). The effects on lymphocyte subpopulations in the thymus were actually reversed, suggesting the possibility that mediators other than corticosterone might actually protect CD4+CD8+ thymocytes from the action of corticosterone. The effects of EtOH on thymocyte subpopulations were generally intermediate between the effects of stress and exogenous corticosterone, whereas the effects of ATZ were more similar to the effects of restraint (Figs. 3
and 4
). Results for mice treated with propanil were also similar to results for mice treated with restraint (Pruett et al., 2000b
). Because changes in thymic subpopulations are obviously under complex regulation and are not consistent between restraint and exogenous corticosterone or among the chemicals tested, this parameter was not included in the predictive models shown in this study.
The observation that a single stress response induced by EtOH or ATZ, lasting no more than 8 h, can cause substantial changes in several immunological parameters is interesting and has not previously been reported. The effect of EtOH on NK cell activity and thymus cell number and subpopulations has been reported previously (Han and Pruett, 1995; Han et al., 1993
; Weiss et al., 1996
; Wu and Pruett, 1996b
), but the effects of atrazine and the other effects of acute EtOH have not been previously reported in the peer-reviewed literature. The dosages of EtOH used in this study produce blood EtOH levels that are relevant with regard to at least some human binge drinkers (Carson and Pruett, 1996
), and high blood EtOH levels are associated with a stress response in humans (Pruett et al., 1998
). Thus, the models derived here have potential for application in predicting immunological changes in human alcoholics or binge drinkers. Although the dosages of ATZ used here are higher than typical for environmental exposure of humans, they are representative of dosages that would be required to meet the testing requirements of the EPA for approval of a new pesticide, which indicates that the highest dose should be near the maximum tolerated dose (U.S. EPA, http://www.epa.gov/oppts_harmonized/870_health_effects_test_guidelines/series/870-7800.pdf).
Decreased spleen and thymus weight, preferential loss of splenic B cells, and decreased resistance to B16F10 melanoma cells have been noted the day after 14 daily doses of ATZ at 500 mg/kg (by gavage) (http://ntp-server.niehs.nih.gov/htdocs/IT-studies/IMM94002.html). However, there was no effect on the primary IgM response to sheep erythrocytes. This study was conducted through the National Toxicology Program. The results are available online, but they have not been published. In any case, the effects reported were mostly similar to those noted in the present study. The difference with regard to effects on a primary antibody response could reflect differences in the antigens used or the immunoglobulin isotypes measured. It is also possible that differences could be attributable to the use of multiple doses in the NTP study, possibly allowing tolerance to develop to some effects. An earlier study did not identify any meaningful, dose-related immunosuppression using a single dose of ATZ at 27.5875 mg/kg (Fournier et al., 1992). However, immunological parameters were not evaluated until 7 days after dosing. This probably was a sufficient time to allow recovery of most immunological parameters to normal values. In any case, it should be noted that oral gavage of ATZ at the high dosage used in the NTP-sponsored evaluation of this compound (500 mg/kg) significantly increased serum corticosterone in the present study. This suggests that the stress response induced by ATZ is not entirely dependent on the route of administration and was likely involved in the immunosuppression reported in the NTP study.
The ultimate goal of this research is to complete a risk-assessment parallelogram that would allow extrapolation of immunotoxicology data obtained in mice to humans. By obtaining data similar to that described here using mouse blood (rather than spleen) as a source of immune system cells and by treating human subjects with cortisol and obtaining similar immunological data from them, it should be possible to directly relate stress-mediated immunotoxicity in mice and humans. This could then be taken further by measuring host resistance in mice under that same condition, and using these data and the relationships between immunological parameters in mice and humans to estimate the effects of stressors on host resistance in humans.
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ACKNOWLEDGMENTS |
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NOTES |
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