session. These conditions were queried by a nurse before participation on each day of expo- sure, and subjects were rescheduled if necessary. Two subjects were tested at the same time. On the day of the first exposure session, a pregnancy test was given. Under typical room air conditions, a spirogram was performed in our clinical center before and after each expo- sure session. Subjects also completed the symptom questionnaire, practiced the POL, and performed baseline nasal lavage and salivary sampling for estradiol analysis in our clinical center. We escorted subjects to the CEF, where they were seated at a table, had ECG sensors attached, and were given a pair of headphones used to communicate instructions for adminis- tration of questionnaires and tasks by the research technician. We gave each subject a loose-leaf binder with dividers to separate each of the questionnaires and tasks to be com- pleted. Baseline measures before exposure were collected while the subject sat quietly with ﬁl- tered room air, after which the exposure was administered for 140 min (Figure 1). During the initial 20 min of the exposure, subjects read quietly. We administered a 10-min vigilance task to maintain a consistent level of alertness across subjects before introduction of the psy- chological stressor (Jennings et al. 1992). In this simple color detection task, we instructed subjects to count bars that appeared periodi- cally on a computer screen. After completion of the task, we asked subjects the number of bars counted. Subjects then completed either the public speaking task or simple arithmetic. While subjects performed the simple arith- metic task, they heard classical music through their headphones to screen out the other sub- ject’s voice. After this period, all subjects typed a standard text for 10 min. A different text was used for each experimental session. As indi- cated in Figure 1, subjects completed question- naires, collected salivary samples for cortisol analysis, and performed the neurobehavioral task during clean air baseline and the exposure period. Immediately after exposure we escorted subjects to our clinical center, where they per- formed postexposure spirometry, nasal lavage, the neurobehavioral task, and completed a questionnaires (Figure 1). E5 S 14 ± y it 3 r e v2 e s n1 a e M0 MCA VOCs VOCs + 03 125 185 Exposure end Statistical Analysis Symptoms. We analyzed the effects of exposure, stress, and time on both presence/absence of symptoms and symptom severity. For pres- ence/absence, if a subject reported any symp- tom at all, then a “yes” was recorded; otherwise, a “no” was recorded. For presence/absence, a hierarchical logistic regression (Davidian and Giltinan 1995; McCulloch and Searle 2000) modeled the log-odds of no symptoms being reported for assessment at minute 15 (baseline) through minute 185 (after removal from expo- sure facility). For symptom severity, data were analyzed using a hierarchical Poisson regression model. In both cases generalized estimating equations were used that accounted for correla- tions between repeated measurements on the same individual (Liang and Zeger 1986). Tests of the exposure effects were conducted using type 3 score tests (Liang and Zeger 1986) of the interaction between exposure and time. Time was entered into the model as a categorical variable. We used contrasts to test whether individual changes in symptoms from baseline (minute 15) to each subsequent time point dif- fered between exposures. The mean odds of reporting symptoms or the mean severity of symptoms at baseline was assumed to be the same for all three exposures. This analysis was first completed for the total symptoms and then for each classiﬁcation of symptoms: VOC physical, VOC cognitive, eye irritation, anxi- ety, upper respiratory, lower respiratory, and somatic control symptoms. Results were based on the 130 subjects who received all three exposures, including the MCA condition. Uncorrected α-values are reported, with the α level after Bonferroni correction noted for each group of multiple comparisons. Cortisol analyses. Cortisol is normally ele- vated during ovulation, potentially attenuating a woman’s cortisol response to a stressor at that time. Therefore, cortisol data were analyzed both with and without data from any speciﬁc exposure session in which the subject’s estra- diol level was ≥ 5 ng/mL. Based on this cutoff, 13 of the 130 (10%) subjects were ovulating during their MCA exposure, 13 of 130 (10%) were ovulating during the VOC exposure, and 15 of 130 (11.5%) were ovulating during the VOC + O3 exposure. On comparison of the cortisol data analyses with and without ovula- tion sessions, no differences in outcome were noted. Therefore, sessions in which subjects were ovulating were included in the final analyses of cortisol. Volatile organics, ozone, and stress Given the continuous response, SAS PROC MIXED (SAS Institute Inc. 1999) was used to analyze the fixed-factor effects. The cortisol response was right skewed and there- fore, for the linear models, was transformed using a square root transformation to better satisfy the normality assumption required for the mixed linear model. Neurobehavioral performance and lung function. A mixed linear model was used to test the effect of exposure on the composite score from the neurobehavioral task and on indicators of lung function. Results Hypothesis 1: Exposure Main Effect Symptoms. After controlling for baseline symp- toms (minute 15), the overall test of the expo- sure main effect on total symptom severity did not conﬁrm hypothesis 1 (Figure 2). However, marginal effects for presence/absence of the subscales of VOC physical symptoms (chi squared = 18.01; df = 10; p < 0.05) and severity of lower respiratory symptoms (chi squared = 16.92; df = 10; p < 0.08) were observed; no effects were observed for the other categories of symptoms. Neurobehavioral performance, salivary cortisol, and lung function. There was no sig- niﬁcant main effect of exposure on neurobe- havioral performance (F = 1.00, df = 6, 387; p = 0.4273), salivary cortisol (F = 0.40; df = 6, 316; p = 0.888), or lung function (data not shown for neurobehavioral or cortisol meas- ures; see Table 3 for lung function). Hypothesis 2: Exposure × Stressor Interaction Symptoms. Hypothesis 2 was not conﬁrmed for presence/absence or for total symptom severity. However, regardless of exposure condition, sub- jects who were in the stress condition reported signiﬁcantly greater severity on the anxiety sub- scale (chi squared = 22.73; df = 5; p < 0.0004) than those who were not. Speciﬁcally, relative to baseline either before (minute 15) or after exposure onset (minutes 60 and 90), symptoms of anxiety were signiﬁcantly more severe after the stressor at minutes 110 and 125 (Figure 3). No other symptom subscale was signiﬁcantly affected by stress or by the exposure × stressor interaction. Salivary cortisol. The hypothesized interac- tion effect of exposure × stress on cortisol was not signiﬁcant, but the main effect of stress on Table 3. Spirometry changes after exposures to MCA, VOCs, and VOCs + O3 (mean difference ± SD; n = 130). Change in FEV1 (mL) Change in FVC (mL) Change in FEF25–75 (L/sec) MCA –50.46 ± 174.02 –71.54 ± 189.22 –0.00 ± 0.41 VOCs 6.15 ± 176.56 –42.62 ± 184.40 0.51 ± 3.63 VOCs ± O3 –25.30 ± 210.33 –60.38 ± 237.11 0.09 ± 0.53 Difference is the post-minus preexposure score. Bonferroni correction: p < 0.02. 15 60 Exposure 90 110 Stressor/math Minutes Figure 2. Total mean symptom severity at each time point across exposures. Environmental Health Perspectives • VOLUME 113 | NUMBER 11 | November 2005 p-Value 0.05 0.52 0.12 1545