Psychophysiology, 51 (2014), 319–326. Wiley Periodicals, Inc. Printed in the USA. Copyright © 2014 Society for Psychophysiological Research DOI: 10.1111/psyp.12179

Temporal features of elevated hair cortisol among earthquake survivors

WEI GAO, PING ZHONG, QIAOZHEN XIE, HAIYANG WANG, JING JIN, HUIHUA DENG, and ZUHONG LU Research Center for Learning Science, Southeast University, Nanjing, China

Abstract This study aimed to determine the effect on hair cortisol level of a chronic stress response from the Wenchuan earthquake, and to explore the temporal features of elevated hair cortisol. We recruited two cohorts of earthquake survivors: cohort A consisted of 12 male adults and 8 females and cohort B of 20 male adolescents, with 23 and 29 participants as controls, respectively. Their hair samples closest to the scalp were assayed with mass spectrometry to determine cortisol content. Results revealed that hair cortisol content in survivors of cohort A was significantly higher than in the control. For survivors of cohort B, hair cortisol levels increased 6 and 22 weeks after the earthquake and decreased 43 weeks after the outburst. In conclusion, the chronic stress response elicited by the earthquake resulted in elevated hair cortisol. Timing since the earthquake outburst played an important role in the long-term response of the HPA axis to a major acute stressor. Descriptors: Hair cortisol, Hypercortisolism, Earthquake, Temporal feature reliably represent long-term activity of HPA axis in cumulative exposure to chronic stress (Russell, Koren, Rieder, & Van Uum, 2012). Empirical studies consistently demonstrate that individuals exposed to chronic stress showed significantly higher hair cortisol levels than controls (Davenport, Tiefenbacher, Lutz, Novak, & Meyer, 2006; Dettenborn, Tietze, Bruckner, & Kirschbaum, 2010; Luo et al., 2012; Skoluda, Dettenborn, Stalder, & Kirschbaum, 2012; Steudte et al., 2011; Van Uum et al., 2008; Yamada et al., 2007). Moreover, hair cortisol is primarily biologically endogenous; that is, hair cortisol is significantly and positively correlated with salivary cortisol (Bennett, & Hayssen, 2010; Davenport et al., 2006; Vanaelst et al., 2012; Xie et al., 2011) and with urinary cortisol (Sauvé, Koren, Walsh, Tokmakejian, & Van Uum, 2007). Therefore, hair cortisol as a biomarker of chronic stress could overcome the methodological limitations of point sampling plasma or salivary cortisol. On the other hand, Miller et al. (2007) emphasize that the time elapsed since chronic stressor onset and core negative emotions (depression, fear, shame, etc.), possibly elicited by chronic stressors, might produce significant impacts on the inconsistency in the association between chronic stress and cortisol response. Actually, individuals exposed to chronic stress show the parallel increase of the scores of perceived stress with the increase of hair cortisol concentrations compared to controls (e.g., Dettenborn et al., 2010; Kalra, Einarson, Karaskov, Van Uum, & Koren, 2007; Van Uum et al., 2008), and they often perceive higher anxiety, depression, and other core negative emotions possibly elicited by chronic stressors, such as chronic pain (Van Uum et al., 2008) and long-term unemployment (Dettenborn et al., 2010). However, most of the extant studies on hair cortisol utilized a cross-sectional design and focused on different types of chronic stressors. These cross-sectional studies might present different time points relative

Cortisol has been known to be a stress biomarker in psychobiological research. It is well documented that an acute stressor (i.e., physical and mental or psychosocial stress) activates the hypothalamus-pituitary-adrenal (HPA) axis and elicits a delayed increase in cortisol secretion, followed by a slow return to basal level with the offset of the stressor (Dickerson & Kemeny, 2004). With regard to chronic stress, a similar association with activation and regulation of the HPA axis has been articulated in many theories and has been summarized in a recent meta-analysis based on 107 independent studies including 8,521 individuals (Miller, Chen, & Zhou, 2007). However, the association between chronic stress and cortisol secretion was less consistent in previous empirical studies, with studies demonstrating an increase, no change, or a decrease in cortisol content (e.g., Gunnar & Vazquez, 2001; Miller et al., 2007; Wolf, Nicholls, & Chen, 2008; Yehuda et al., 1995). In the past, most studies utilized plasma or salivary cortisol as a biomarker of chronic stress. One of the main reasons for the inconsistency might be methodological limitations in an assessment of chronic stress because plasma and salivary cortisol are sensitive to acute stress and reflect an assessment at a specific time point (Wolf et al., 2008). Recently, cortisol levels in hair have been regarded to

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The authors would like to sincerely thank the participants for their contribution of time and effort into this research. The research work was supported by the Humanities and Social Science Foundation (11YJAZH019); Program for New Century Excellent Talents in University (NCET-08-0122), Ministry of Education, China; Jiangsu Provincial Social Sciences Key Project (09JYA002); and National Nature Science Foundation (60771023), China. Wei Gao thanks the Innovation Research Foundation of Southeast University for Doctoral Students (BC0905), China. Address correspondence to: Dr. Huihua Deng, Research Center for Learning Science, Southeast University, Nanjing 210096, China. E-mail: [email protected] 319

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to the stressor onsets. In other words, these studies provided no temporal features in the association between chronic stress, hair cortisol, or negative emotions. Furthermore, the meta-analysis of Miller et al. (2007) on the impact of timing was mainly based on cross-sectional studies because of a dearth of longitudinal studies in the literature. Therefore, a longitudinal study is needed to explore temporal features of association between chronic stress, hair cortisol, and core negative emotions (e.g., anxiety and depression). In the present study, we utilized hair cortisol as a retrospective biomarker to reflect long-term HPA activity of the survivors from the Wenchuan earthquake (8.1 R) that hit and destroyed Wenchuan district, Sichuan Province, China, on May 12, 2008. Epidemiogical studies have shown that a sudden earthquake could result in psychological consequences on the affected population, such as massive acute stress reactions, posttraumatic stress disorder (PTSD), and an accumulation of depressive and anxiety symptoms (e.g., Bergiannaki, Psarros, Varsou, Paparrigopoulos, & Soldatos, 2003; Kolaitis et al., 2003; Michael & Koukia, 2010). The resulting stress response would last for a considerably long period, from 2 days to several months or even several years after the earthquake outburst (e.g., Michael & Koukia, 2010). A recent cross-sectional study reports that the non-PTSD female adolescents traumatized in the Wenchuan earthquake show significantly higher hair cortisol level than controls 2–4 months after the Wenchuan earthquake (Luo et al., 2012). The main aim of this study was to investigate temporal features of the earthquake-induced increases of hair cortisol, anxiety, and depression symptoms by examining whether the association between hair cortisol, anxiety, and depression symptoms changed as time passed since the earthquake happened. The other aim was to examine whether there would be a parallel increase of earthquake-elicited negative emotions (i.e., anxiety and depression symptoms) with elevated hair cortisol among the earthquake survivors compared to controls. In addition, we aimed to extend the earthquake-induced increase of hair cortisol to adults and male adolescents exposed to the Wenchuan earthquake. Based on the literature summarized above, the present study predicts that the earthquake survivors will show significantly higher hair cortisol and symptoms of anxiety and depression than controls, and that the earthquake-induced increases in hair cortisol and symptoms of anxiety and depression will disappear with time passing after the earthquake’s outburst. Materials and Method Study Design In order to test these hypotheses, we conducted a cross-sectional design among adult survivors 4 weeks after the Wenchuan earthquake and a longitudinal design among adolescent survivors during the period from 6 weeks to 43 weeks after the earthquake. Measures The 20-item Self-Rating Anxiety Scale (SAS, Zung, 1971) and Self-Rating Depression Scale (SDS, Zung, 1976) were utilized to measure the perceived symptoms of anxiety and depression. Symptom Check List-90 (SCL-90) is a self-report questionnaire to assess distinct facets of psychopathology (Derogatis, Lipman, & Covi, 1973). The present study used SCL-90 as a brief screening instrument to find a psychiatric case. Additionally, a 5-point Likert

clinician-administered post-trauma stress disorder (CAPS) scale (Blake et al., 1995) was also used to monitor the psychiatric state of participants in the longitudinal design after the Wenchuan earthquake. These measures showed good validity and reliability in Chinese samples. In the present study, internal consistencies (i.e., Cronbach’s α coefficients) of SAS and SDS scales were .778 and .785 for adult survivors and .737 and .697 for their controls, respectively. Internal consistencies of SAS and SDS scales were .735–.766 and .665– .822 across four time points for adolescent survivors and .742–.875 and .609–.825 across three time points for their controls. Internal consistencies of SCL-90 and CAPS scales were in the range of .700–.849 and .837–.852 across three time points. Additionally, Zung recommended that raw SDS scores above 40 showed depression symptoms (Zung, 1976) and that raw SAS scores above 36 showed clinically significant anxiety (Zung, 1971). In this investigation, we used a raw SDS score of 40 and a raw SAS score of 36 as the cutoff scores in determining the prevalence of depression and anxiety. Study Participants Participants in the present study were two cohorts of healthy survivors exposed to the Wenchuan earthquake. Cohorts A and B consisted of 20 adult survivors (male/female: 12/8, wounded/ woundless: 12/8) and 20 male adolescent survivors (wounded/ woundless: 11/9), respectively. Cohort A was recruited from a resettlement center of earthquake survivors at the earthquake district. Twenty-three age- and sex-matched healthy participants previously not exposed to the earthquake were recruited as controls. Cohort B was recruited from a technical school in Wenchuan district. The participants in cohort B were Grade 11 students at a technical school in the earthquake district whose whole class was moved to a technical school in Nanjing by the Chinese government 3 weeks after the earthquake. Twenty-nine age-matched healthy male adolescents previously not exposed to the earthquake were recruited as controls from the same technical school in Nanjing. The sociodemographic data of cohorts A and B and their controls are listed in Table 1. All participants provided written informed consent before inclusion. This study was approved by the Health Science Research Ethics Board of Southeast University and followed the Helsinki Declaration. Exclusion criteria for all participants, from both cohorts and controls, were smoking, presence of dyed hair, hair length 25.0 or < 17.6 kg/m2, or total score of SCL-90 > 200, average score of psychiatric factor in SCL-90 ≥ 2, or average score of any other SCL-90 factors ≥ 3. Because SCL-90 is a 5-point Likert scale and a rating of 3 means moderate mental symptoms (Derogatis et al., 1973), the average score of 3 in certain dimensions was recommended as the cutoff score for mental symptoms in the Chinese population (Hu, 2006; Jin, Wu, & Zhang, 1986; Liu & Zhang, 2004). It was reported that the total score for 84 clinical patients with mental disorders was 229.41 ± 59.01 (M ± SD) where M is mean and SD is standard deviation (Wang, 2004). The present study utilized the more strict criteria in total score and average score of its psychiatric factor because the psychiatric factor focuses on the specific psychiatric symptom. Additionally, participants were excluded if the CAPS raw score was more than 39 (Weather, Keane, & Davidson, 2001). All participants self-reported no pre-existing mental diseases or physiological diseases and had received no medical treatment within the latest 1-month period.

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Table 1. Demographic Data, Death of Relatives, House Damage, and Prevalence of Anxiety and Depression in Cohorts A and B and Their Controls General population Variable

Cohort A (n = 20)

Control (n = 23)

Gender: male/female 12/8 14/9 Age M ± SD (years) 45.0 ± 14.2 41.5 ± 12.8 Range (years) 22∼65 23∼64 Education level Elementary school and below 60% 30% Junior high school 20% 26% Senior high school 15% 17% College and university and over 5% 26% Marital status Single/from a single-parent family 5% – Married/from a double-parent family 95% – Death of relatives (parent, spouse, sibling, offspring, grandparent) in the earthquake Yes 5% – No 95% – House damage degree Completely destroyed 65% – Heavily destroyed 15% – Partly destroyed 15% – No damage 5% – Prevalence of depressionb 40∼47 45% 22% 48∼55 20% 0% > 56 10% 0% Prevalence of anxietyc 36∼47 40% 35% 48∼55 20% 0% > 56 0% 0%

Adolescents Statistical value

Cohort B (n = 20)

Control (n = 29)

χ = 0.003

20/0

29/0

t = 1.10

16.8 ± 0.8 16∼20

16.7 ± 0.6 16∼18

100%

100%

20% 80%

– –

15% 85%

– –

65% 30% 5% 0%

– – – –

25%d 15%d 45%d

– – –

40%d 5%d 0%d

– – –

a

2

Z = 3.23*

M = mean; SD = standard deviation. *p < .01. a 2 χ is statistical value of Chi-square test, t is of t test, and Z is of U test for comparison between earthquake survivors in cohort A and their controls. b 40 in raw SDS score is used as the depression’s cutoff score. c 36 in raw SAS score is used as the anxiety’s cutoff score. d The data were collected 3 weeks after the earthquake and before the earthquake group was moved to Nanjing.

Survey and Hair Collection All participants in cohorts A, B, and the controls filled out questionnaires to report their demographic information, frequency and manner of hair washing, and perceived depression and anxiety and psychiatric states over a period of about 1 month. Thereafter, all participants provided hair samples longer than 1 cm closest to the scalp in the posterior vertex region. Given that average hair growth rate is 1 cm per month (Pragst & Balikova, 2006), the 1-cm hair segments closest to the scalp presented cortisol status within the past 1-month period for which the corresponding mental status was reported by the questionnaire. Actually, 1–3 mm hair strands were deeply embedded in the skin, and 1–2 mm hair strands closest to the scalp could not be completely cut with scissors, although hair samples were cut as close as possible to the scalp. Therefore, hair collection was done 2 weeks after the questionnaire for 1-cm hair strands and 7 weeks later for 1.5-cm hair strands in order to match the corresponding period that the questionnaires represented. Specifically, for participants in cohort A and its control, the survey was performed 4 weeks after the Wenchuan earthquake, and 2 weeks later the hair samples were collected. As shown in Figure 1, participants in cohort B reported their mental status 3 weeks after the earthquake before moving to Nanjing (Time 0). Thereafter, for participants in cohort B and its control, three sequential waves of survey collections were done 6

weeks (Time 1), 22 weeks (Time 2), and 43 weeks (Time 3) after the earthquake. Accordingly, in order to cover the survey period represented, the first wave of 1.5-cm hair samples were collected 13 weeks after the earthquake, and the second and third waves of 1-cm hair samples were collected 24 and 45 weeks after the earthquake. Three time points for hair collections corresponded to three waves of survey collections, that is, Times 1, 2, and 3, respectively. Because some of the participants in cohort A and its control had received lower education (e.g., primary school and below), a welltrained experimenter explained the literal meaning of items that they didn’t understand in their local language and helped them to finish the surveys. Hair samples closest to the scalp in the posterior vertex region were cut with iron scissors and stored in dry tubes at room temperature for cortisol analysis. Additionally, most participants washed their hair with shampoo and water 3–4 times per week in early summer (Time 1), 2–3 times per week in autumn (Time 2), and 1–2 times per week in early spring (Time 3). There was no difference in the frequency of hair washing between experimental cohorts and their controls.

Cortisol Analysis of Hair Samples Cortisol concentrations of hair segments from all subjects were analyzed by liquid chromatography tandem mass spectrometry

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W. Gao et al. Wenchuan Earthquake

Time 0

Time 1

Time 2

Time 3

occurred on

3 weeks later

6 weeks later

22 weeks later

43 weeks later

May 12, 2008

survey

survey

survey

survey

3 weeks later

13 weeks later

moving

1.5-cm hair

1-cm hair

1-cm hair

collecting

collecting

collecting

24 weeks later

45 weeks later

Figure 1. Schematic sequences of questionnaire survey (circle) and hair collection (square) after the Wenchuan earthquake outburst for cohort B (n = 20) and its control (n = 29). Questionnaire survey and hair collection in the control are performed at Times 1–3. The diamond symbol represents the move date.

(LC-MS/MS) (ABI 3200 QTRAP, USA). For the LC-MS/MS analysis, the hair strands were washed twice in 1 ml methanol for 2 min at room temperature, dried in N2, and then pulverized with a ball mill (MM440, Retsch, Germany). The washing procedures were repeated twice to completely remove any contamination and nonblood-borne cortisol coated on the outer surface of the hair stands. Then, 50 mg of powdered hair was incubated in 1 ml methanol at 40°C for 24 h. After that, the incubation medium was separated by centrifuge at 9,000 g for 10 min, then the supernatant was transferred to a dry tube and evaporated to dryness under pure N2. The extraction was resuspended with 50 μl methanol and 1 ml water, and then transferred to a C18 solid phase extraction (SPE) column, which was activated with 3 ml methanol and rinsed with 3 ml deionized water prior to use. The deposit on the activated SPE column was rinsed with a sequence of 1 ml 20 : 80 (v/v) acetone/deionized water, 1 ml deionized water, and 1 ml hexane, then dried for 30 min and eluted successively with 0.5 ml methanol three times. The eluate finally obtained was evaporated to dryness and resuspended with 50 μl mobile phase for LC-MS/MS analysis. Intraday precision was 7.6% (n = 5) at 50 ng/ml, and interday precision was 8.3% (n = 5). The detection limit was 1 pg/mg at the signal-to-noise ratio of 3.

Results Sociodemographic Data As listed in Table 1, there was no difference in age and gender between cohort A (n = 20) and its control (n = 23), but there was a significant difference in education level. There was no significant difference in age, t(47) = 0.13, p = .894, between cohort B (n = 20) and its control (n = 29).

Elevated Hair Cortisol Among Earthquake Survivors Compared to Controls Hair cortisol content (median: 31.94 pg/mg, range: 9.22– 180.11 pg/mg) in cohort A was significantly higher than that in its control (median: 7.00 pg/mg, range: 2.85–46.78 pg/mg), t(41) = 5.82, p < .001. Similarly, as shown in Figure 2, hair cortisol content in cohort B at Time 1 (M ± SD: 25.35 ± 17.08 pg/mg) was significantly higher than that in its control (median: 11.69 pg/mg,

Statistical Methods Data were processed and analyzed using the statistical package SPSS-16.0 for Windows, and statistical significance was set at p < .05. The data distribution normality was examined with a onesample Shapiro-Wilk test. All data were presented as median (range) for non-normally distributed data and for normally distributed data as M ± SD. A Mann-Whitney U test for two independent samples was conducted for comparison of SAS and SDS scores between experimental cohort and control, and a t test for two independent samples was performed for comparison of the log-transformed data of hair cortisol between the experimental cohort and control because log transformations effectively reduced the kurtosis and skewness. Wilcoxon signed-rank test for two related samples was conducted for comparison of SAS and SDS scores between Time 0 and Time 1. Repeated measures analysis of variance (ANOVA) with Greenhouse-Geisser correction was conducted for comparison of SAS and SDS scores and the log-transformed data of hair cortisol across Time 1 to Time 3.

Figure 2. Comparison of log-transformed hair cortisol content between cohort B (n = 20) and control (n = 29) at three time points. Mean values (±SEM) are presented in pg/mg. A t test for two independent samples is conducted for comparison of the log-transformed hair cortisol between cohort B and control. Repeated measures ANOVA with GreenhouseGeisser correction compares the three time points in cohort B and control, respectively. ns = nonsignificant.

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Table 2. Comparison in SAS and SDS Scores Between Cohort B (n = 20) and Its Control (n = 29) at Times 1–3a

SDS score M ± SD Median range Statistical valuec SAS score M ± SD Median range Statistical valuec

Time 0b

Time 1

Time 2

Time 3

44.87 ± 9.55 – –

40.35 ± 7.68 (38.27 ± 6.02) Z = −1.10

34.75 ± 6.36 (36.70 ± 8.52) Z = −0.65

33.05 ± 6.94 (36.85 ± 8.60) Z = −1.52

35.84 ± 7.62 – –

29.95 ± 5.32 (33.67 ± 6.40) Z = −1.98*

30.75 ± 5.72 (32.75 ± 7.50) Z = −0.87

30.40 ± 5.63 (32, 21–57) Z = −1.36

M = mean; SD = standard deviation. *p < .05. a Value in parenthesis is for subjects in the control group. b Data collected 3 weeks after the earthquake and before cohort B was moved to Nanjing. c Mann-Whitney U test for two independent samples is conducted, and Z is its statistical value.

range: 1.77–43.35 pg/mg), t(47) = 2.20, p < .05, and at Time 2 (median: 56.18 pg/mg, range: 29.94–120.62 pg/mg) was higher than that in the control (M ± SD: 25.35 ± 17.08 pg/mg), t(40.38) = 2.52, p < .05. Additionally, there was no significant difference between wounded and woundless subgroups in the logtransformed hair cortisol for cohort A and cohort B at Time 1, t(18) = 0.32, p = .756, and t(18) = −0.72, p = .484. Time-Dependent Pattern of the Elevated Hair Cortisol Among Earthquake Survivors Interestingly, the hair cortisol level in cohort B that resulted from the initially activated HPA axis at the earthquake outburst remained significantly higher than that in its control until 22 weeks (Time 2) passed since the earthquake outburst, but not significantly higher than that in the control (M ± SD: 42.40 ± 21.65 pg/mg vs. median: 31.10 pg/mg, range: 4.30–97.00 pg/mg; t(47) = 1.32, p > .05) 43 weeks after the earthquake (Time 3) as shown in Figure 2. Additionally, there was a significant difference across Time 1 to Time 3 in hair cortisol content for both cohort B and its control, F(1.42,21.33) = 10.86, p < .01 and F(2,56) = 12.60, p < .01. As shown in Figure 2, hair cortisol content was highest at Time 2 among three waves of hair samples for both cohort B and its control. There was no significant difference between cohort B and its control in the increase of hair cortisol from Time 1 to Time 2 (Z = −0.36, p = .722 for the absolute value and Z = −0.63, p = .528 for the relative value) and in the decrease of hair cortisol from Time 2 and Time 3 (Z = −0.86, p = .388 for the absolute value and Z = −1.06, p = .290 for the relative value). These results indicated that cohort B and its control might experience the same variation of hair cortisol content with the different collected times. Elevated Symptoms of Anxiety and Depression Among Earthquake Survivors Compared to Controls Cohort A showed significantly higher SAS and SDS scores than control (38.72 ± 7.31 vs. median: 30, range: 23–47, Z = −3.01, p < .01 for SAS and 43.70 ± 8.52 vs. 36.61 ± 4.90, Z = −2.84, p < .01 for SDS), and showed higher prevalence of depression and anxiety as listed in Table 1. Additionally, there was no significant difference between wounded and woundless subgroups in SDS and SAS scores, Z = 0.03, p = .977 and Z = −1.42, p = .155. As listed in Table 2, there was no significant difference between cohort B and its control in the SAS and SDS scores at Times 1, 2,

and 3, except that SAS score at Time 1 was significantly lower than that in the control, Z = −1.98, p = .048. There was no significant difference between wounded and woundless subgroups in SDS and SAS scores at Time 1, Z = −1.10, p = .270 and Z = −1.80, p = .072. Additionally, for participants in cohort B, their SAS and SDS scores at Time 0 were significantly higher than those at Time 1, Z = −3.68, p < .001 and Z = −3.83, p < .001, respectively. Their SDS score showed a significant decrease across Time 1 to Time 3, F(2,38) = 16.31, p < .001, and their SAS score showed no significant decrease, F(2,38) = 0.21, p = .812. For participants in its control, their SDS and SAS scores showed no significant decreases across Time 1 to Time 3, F(2,56) = 2.65, p = .081, and F(1.60,36.76) = 3.24, p = .061, respectively. Discussion A sudden earthquake results in massive acute stress for the affected population, and the resulting stress response lasts from 2 days to several months or even years after the earthquake occurred (Bergiannaki et al., 1990, 2003; Kolaitis et al., 2003; Metaxas, Balli, Triantafyllou, & Kalevras, 1979; Michael & Koukia, 2010; Papadatos, Nikou, & Potanianos, 1990; Soldatos et al., 1989). The present study focused on the period from the first month to the tenth month after the Wenchuan earthquake. In the investigated period, most earthquake survivors likely continued to suffer from a chronic stress response resulting from the earthquake. The present study found that survivors exposed to the Wenchuan earthquake showed significantly higher hair cortisol than their controls, as shown in cohort A and cohort B at Times 1 and 2. To our knowledge, this is the first observation of the earthquake-induced increase of hair cortisol among adults and male adolescents exposed to the Wenchuan earthquake. A recent cross-sectional study reported that the non-PTSD female adolescents traumatized in the earthquake also showed a significantly higher hair cortisol level than the controls (Luo et al., 2012). The consistent results from the two studies indicated that an earthquake is an intensive stressor, and it could activate individuals’ HPA axis, which consequently leads to increase of hair cortisol levels among individuals exposed. The present finding is inconsistent with that in the study of Goenjian et al. where lower salivary cortisol levels were observed for adolescents exposed to the 1988 earthquake in Armenia (Goenjian et al., 1996). The inconsistency between the two studies might be attributed to the limitation of salivary cortisol in assessing long-term basal cortisol level of the HPA axis. In fact,

324 salivary cortisol together with plasma and urinary cortisol are biomarkers to reflect short-term basal activity of the HPA system. However, the cortisol level of plasma/saliva/urine was utilized as a biomarker to assess long-term HPA activity in some previous research. Such biomarkers might result in inconsistent relationships between cortisol levels and chronic stresses (e.g., the occurrence of traumatic events or PTSD) with studies finding the increase of salivary cortisol (Pfeffer, Altemus, Heo, & Jiang, 2007; Young, Tolman, Witkowski, & Kaplan, 2004), lower salivary, plasma, or urinary cortisol (Boscarino, 1996; Goenjian et al., 1996; Roth, Ekblad, & Ågren, 2006; Yehuda et al, 1995), and no change (Bonne et al., 2003). In contrast, using hair cortisol as a biomarker of chronic stress, Luo et al. (2012) and Steudte et al. (2011) consistently found higher hair cortisol level among PTSD subjects traumatized in war and earthquake. The increase in hair cortisol was also observed in the systemic exposure to chronic stressors, such as relocation (Davenport et al., 2006), hospitalization (Yamada et al., 2007), severe chronic pain (Van Uum et al., 2008), long-term unemployment (Dettenborn et al., 2010), and long-term endurance (Skoluda et al., 2012). Moreover, Thomson et al. (2010) found that variation of hair cortisol concentration in Cushing’s patients was in accordance with clinical course. Thus, hair cortisol could be a new retrospective biomarker to reflect more reliably long-term basal activity of the HPA system in cumulative exposure to chronic stress. The present study confirmed that there was a time-dependent pattern of the earthquake-induced increase in hair cortisol among earthquake survivors relative to their controls, which was observed in the initial stages after the earthquake, such as 4, 6, and even 22 weeks, rather than 43 weeks after the earthquake (Figure 2). To our knowledge, this investigation is the first longitudinal study on the time-dependent pattern of an earthquake-induced increase in hair cortisol. This finding gives an important indication that the HPA system is initially activated at the stressor onset, which is persistent even when the initial stimulus is removed, then is regulated as time passes, and finally recovers to the basal level when the chronic stress truly disappears. The time-dependent pattern was also observed in previous empirical studies on relocation stress (Davenport et al., 2006) and earthquake-induced chronic stress (Luo et al., 2012). Davenport et al. (2006) reported that hair cortisol of rhesus monkeys increased 14 weeks after the mandatory relocation, and showed no difference 1 year after the relocation relative to before the relocation. Luo et al. (2012) reported that the increase of hair cortisol among traumatized non-PTSD females relative to nontraumatized controls 2–4 months after the earthquake disappeared 5–7 months after the earthquake. A metaanalysis also concluded that there might be a time-dependent pattern for cortisol increase induced by chronic stress (Miller et al., 2007). However, there was no control in the longitudinal study of Davenport et al., and no information at 1 month after the earthquake in the cross-sectional study of Luo et al. The meta-analysis conclusion of Miller et al. was based on cross-sectional results in the literature. Therefore, our finding in the present longitudinal study provides strong support for the importance of timing in the chronic response of the HPA system to a major acute stressor like an earthquake. On the other hand, a chronic stressor possibly elicits higher stress and other negative core emotions, such as fear, shame, anxiety, and depression. For instance, anxiety or depression often accompanied the increase of the perceived stress among chronic pain patients (Van Uum et al., 2008) and unemployed individuals (Dettenborn et al., 2010). The previous epidemiogical studies

W. Gao et al. showed that an accumulation of depression and anxiety symptoms resulting from a sudden earthquake could be persistent from 2 days to several months or even several years after the earthquake (Bergiannaki et al., 1990, 2003; Kolaitis et al., 2003; Metaxas et al., 1979; Michael & Koukia, 2010; Papadatos et al., 1990; Soldatos et al., 1989). The present study also found that there was a considerably high prevalence of depression and anxiety 3 and 4 weeks after the Wenchuan earthquake (Table 1). Notably, we also found that there was a parallel increase of anxiety and depression symptoms with the increase of hair cortisol among earthquake survivors in cohort A relative to its control. This implies that depression and anxiety symptoms elicited by the earthquake may be the important emotional factors that are closely associated with the activation of the HPA axis in exposure to the chronic stress response resulting from the earthquake. The present finding is similar to that in the study of long-term unemployment where unemployed individuals showed higher scores in chronic stress and psychological distress, accompanying the increase of hair cortisol (Dettenborn et al., 2010). Contrary to our predictions, the adolescents in cohort B showed no significantly higher depression and anxiety than controls from Time 1 to Time 3 (Table 2), although their depression showed a significant decrease from Time 1 to Time 3. This may be because they received major social support from the Chinese government and society after being moved from the earthquake district to a technical college in Nanjing 3 weeks after the earthquake. Such social support could possibly greatly suppress their depressive and anxiety symptoms elicited by the earthquake, as found in previous studies (e.g., Bergiannaki et al., 2003; Michael & Koukia, 2010). As a result, their anxiety and depression showed significant decreases from Time 0 (before moving to Nanjing) to Times 1–3 (after moving to Nanjing), and were not significantly higher than the control’s across Time 1 to Time 3. Lastly, the adolescents from both cohort B and its control showed a variation of hair cortisol content with the collection times. A similar variation with collection times was also observed in adults, showing a significant difference of hair cortisol level across three time points from June to November at a 2-month interval (Stalder et al., 2012). The first possible reason for the seasonal variation of hair cortisol is seasonal variation in cortisol secretion resulting from the organism’s adaptation to seasonal changes of natural environmental factors (e.g., temperature, humidity, and sunshine duration). Previous research suggested that cortisol secretion was attenuated during summer and elevated during winter (Hansen, Garde, Skovgaard, & Christensen, 2001; Persson et al., 2008; Walker, Best, Noon, Watt, & Webb, 1997). The second reason may be that loss of hair cortisol possibly varies with seasons. In fact, there was a reduction of cortisol due to hair washing (Dettenborn et al., 2010; Dettenborn, Tietze, Kirschbaum, & Stalder, 2012; Gao et al., 2010; Kirschbaum, Tietze, Skoluda, & Dettenborn, 2009; Xie et al., 2011). Such loss could be caused by various natural and artificial factors, such as ultraviolet irradiation and higher temperature (Li et al., 2012), cosmetic treatments with dyes and perms (Manenschijn, Koper, Lamberts, & Rossum, 2011; Sauvé et al., 2007), and frequent washing with shampoos and water (Hamel et al., 2011; Li et al., 2012). Thus, seasonal variations of natural factors and life habits may result in seasonal variations of hair cortisol loss. For instance, the variation of hair washing frequency with the seasons, as found in the present study, might have resulted in the variation of hair cortisol content at Times 1, 2, and 3 for both cohort B and its control. But, a recent study showed that there was no influence of hair washing frequency on hair cortisol

Temporal features of elevated hair cortisol

325

level (Dettenborn, Tietze, Kirschbaum, & Stalder, 2012). So hair washing may be a confounder, but the extent is still in debate. Effects of other external factors, such as hair humidity, room temperature, and ultraviolet irradiation duration, need to be explored in future studies. Nevertheless, there are some limitations in the present study. First, for the adolescents in cohort B, their psychological and biological stress responses might result not only from the earthquake, but also from the additional stresses of relocation and school transition because they were moved from a local technical school in the earthquake district to a technical college in Nanjing 3 weeks after the earthquake. Notably, they had been Grade 11 students and already had a 1-year independent school life from their family before their entire class was moved to Nanjing. Thus, the relocation and school transition might produce a small and ignorable impact on their psychological and biological stress responses in a short period. Additionally, the significant social support from the Chinese government and society might greatly suppress the impact of the earthquake and relocation on their psychological and biological responses. Second, the present study did not check pregnancy, the use of oral contraceptives, alcohol use, or chronic somatic diseases in cohort A, and alcohol use or chronic somatic diseases in cohort B. These factors might be closely associated with cortisol secretion (e.g., Dettenborn et al., 2012; Kirschbaum et al.,

2009; Stalder et al., 2012; Van Uum et al., 2008). Third, this investigation did not use a diagnostic interview to assess the psychiatric state of participants in cohorts A and B, and had no information about their previous experiences of psychiatric disorders or traumas. A previous study found that early adverse traumatic experiences might influence HPA-axis activity (Steudte et al., 2013). Fourth, there are other limitations including the small sample size per group and the absence of a regression analysis controlling the influence of covariates. In summary, the present study utilized the earthquake-induced chronic stress response as a model of chronic stress response and presented the first longitudinal design to explore the temporal feature of the earthquake-elicited increase in hair cortisol. It discovered that elevated hair cortisol among earthquake survivors relative to their controls was observed in the initial stages of the cumulative exposure to the earthquake-induced chronic stress, for example, 4, 6, and even 22 weeks, rather than 43 weeks after the earthquake. There was a parallel increase of anxiety and depression symptoms with the increase of hair cortisol among adult earthquake survivors relative to controls. These results might provide evidence for the importance of timing and core emotions in the long-term response of the HPA system to major acute stressors. The present findings may be helpful for understanding the complicated relationship between cortisol response and chronic stress.

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