An Increased Capacity for Adrenal DHEA Release Is Associated with Decreased Avoidance and Negative Mood Symptoms in Women with PTSD

Home , ACTH stimulation test, Antiglucocorticoid, Glutamine, Mineralocorticoid

An Increased Capacity for Adrenal DHEA Release is Associated with Decreased Avoidance and Negative Mood Symptoms in Women with PTSD

,1 2 1 1 2 Ann M Rasmusson* , Jitka Vasek , Deborah S Lipschitz , Dolores Vojvoda , Mary Ellen Mustone , 3 2 1 2 4 Quihu Shi , Gretchen Gudmundsen , Charles A Morgan , Jessica Wolfe and Dennis S Charney

1 Department of Psychiatry, Yale University School of Medicine & VA National Center for PTSD, Clinical Neuroscience Division, VA Boston 2 Healthcare System, West Haven, CT, USA; Department of Psychiatry, Boston University School of Medicine & VA National Center for PTSD, 3 Womens’ Health Sciences Division, VA Boston Healthcare System, Boston, MA, USA; School of Public Health, New York Medical College,

The Learning Center, Valhalla, NY, USA; 4Mood and Anxiety Disorders Branch, National Institute of Mental Health, Bethesda, MD, USA

We recently found increased adrenal cortisol responses to adrenocorticotropic hormone (ACTH)1–24 and increased pituitary ACTH and

adrenal cortisol responses to corticotropin-releasing factor in premenopausal women with chronic post-traumatic stress disorder (PTSD)

compared to healthy nontraumatized subjects. This pattern of hypothalamic–pituitary–adrenal axis (HPA) hyper-reactivity has been

previously seen in healthy individuals treated with the antiglucocorticoid mifepristone. We therefore investigated whether endogenous

plasma levels of antiglucocorticoids such as dehydroepiandrosteroine (DHEA) and progesterone were increased in premenopausal

women with PTSD at baseline or in response to adrenal activation by ACTH . The study revealed that DHEA responses to 250 mg 1À24 ACTH were increased in 13 PTSD subjects compared to 13 healthy nontraumatized subjects, while DHEA levels were generally 1À24

increased in the PTSD subjects compared to seven healthy traumatized subjects. Cortisol responses to ACTH1À24 were also higher in

the women with PTSD, while progesterone levels and responses were not different among the three groups. In addition, among the

PTSD subjects, the peak change in DHEA in response to ACTH1À24 was negatively correlated with the total Clinician Administered

PTSD Scale score, while the peak DHEA to cortisol ratio was inversely associated with negative mood symptoms measured by the

Profile of Mood States scale. This work suggests that an increased capacity for DHEA release in response to extreme adrenal activation

may influence the pattern of HPA axis adaptation to extreme stress, as well as mitigate the severity of PTSD and negative mood

symptoms in premenopausal women with PTSD.

Neuropsychopharmacology (2004) 29, 1546–1557, advance online publication, 16 June 2004; doi:10.1038/sj.npp.1300432

Keywords: post-traumatic stress disorder; PTSD; cortisol; DHEA; premenopausal women; HPA axis

INTRODUCTION ovine corticotropin-releasing factor (CRF) in the PTSD group. While these findings are inconsistent with some We recently reported increased adrenal cortisol responses previous studies showing low (Mason et al, 1986, Yehuda to intravenously administered adrenocorticotropic hor- et al, 1990, 1993, 1995) or the same cortisol output (Kosten mone (ACTH)1À24 in premenopausal women with chronic et al, 1990; Baker et al, 1999; Mason et al, 2002) in PTSD post-traumatic stress disorder (PTSD) compared to healthy compared to healthy subjects, they are consistent with other nontraumatized subjects (Rasmusson et al, 2001). In studies finding relatively increased urinary free cortisol addition, we observed increased pituitary ACTH and output in premenopausal women (Rasmusson et al, 2001; adrenal cortisol responses to intravenously administered Lemieux and Coe, 1995; Friedman et al, 2001), mixed-sex samples of children (De Bellis et al, 1999) or adults (Maes *Correspondence: Dr A M Rasmusson, Psychiatry Service/116A, VA et al, 1998), and male combat veterans (Pitman and Orr, Connecticut Healthcare System, 950 Campbell Avenue, West Haven, 1990) with PTSD. Since high levels of cortisol may CT 06516, USA, Tel: 203-932-5711, ext. 2483 or 2464, Fax: 203-937- 3886, Page 203-867-3132, E-mail: [email protected] contribute to alterations in the structure and function of Received 22 September 2003; revised 8 January 2004; accepted 27 brain areas and neurotransmitter systems thought to be January 2004 involved in the mediation of PTSD symptoms (Sapolsky, Online publication: 13 February 2004 at http://www.acnp.org/citations/ 1985, 1986; McEwen, 2000; Schulkin et al, 1998; Grunde- Npp02130403432/default.pdf mann et al, 1998; Lupien et al, 1998, 1999; Newcomer et al, Increased DHEA release in women with PTSD AM Rasmusson et al 1547 1999; Gold et al, 2002), we decided it would be important METHODS to investigate the possible mechanisms underlying increased cortisol reactivity in premenopausal women with Subjects PTSD. Increased ACTH and cortisol responses to CRF admin- In all, 41 subjects were recruited via community and clinic istration have been observed in animals and humans treated advertisements in Connecticut and Boston to participate in with the antiglucocorticoid RU486 (Van Haarst et al, 1996; the study. The protocol was approved by the VA Connecti- Bertagna et al, 1994). Facilitation of HPA axis reactivity by cut and VA Boston Healthcare System Human Studies RU486 is thought to be mediated by antagonism of Subcommittees; written informed consent was obtained glucocorticoid negative feedback in the brain and pituitary, from each subject. a resulting increase in ACTH release, and trophic effects of Subjects were screened for current or significant past ACTH on the adrenal gland. Thus, we wondered whether medical illness and illicit drug use via medical history, endogenous, peripherally released steroids with antigluco- physical examination, and laboratory tests. None had corticoid activity might play a role in enhancing pituitary identifiable endocrine syndromes and only one healthy and adrenal responsiveness in our female subjects with subject reported irregular menses. All subjects were free PTSD. from psychotropic medications for months to years or had The endogenous steroids, dehydroepiandrosterone never been treated with such. Subjects abstained from other (DHEA) and progesterone, have been shown to have medications, alcohol, and illicit drugs for at least 4 weeks antiglucocorticoid activity in several tissues, including before testing, except for two PTSD and two TC subjects the brain (Chader and Reif-Leher, 1972; Kaiser et al, who used steroid hormone contraception. Two PTSD, one 1979; Bohus and DeKloet, 1981; Giesen and Beck, TC, and two NTC subjects smoked 1/2–1 pack of cigarettes 1982; Daynes et al, 1990; Blauer et al, 1991; Browne et al, per day. 1992; Araneo et al, 1993). DHEA and progesterone DSMIV PTSD Criterion A1/A2 trauma (American Psy- exert antiglucocorticoid effects at the glucocorticoid type chiatric Association, 1994) exposure was assessed by use of II receptor via mechanisms distinct from classical general and early trauma inventories (ETI) (Bremner et al, receptor antagonism (Chou and Luttge, 1998; Morfin and 2000) (Table 1). The A1 criterion requires that a person Starka, 2001), while progesterone acts as a classical experiences, witnesses or confronts an event or events antagonist at mineralocorticoid (type I glucocorticoid) involving actual or threatened death or serious injury or receptors (reviewed in Turner, 1997). Consistent with the threat to the physical integrity of self or others. The A2 antiglucocorticoid characteristics of these steroids, ACTH criterion requires that a person’s response involve intense responses to psychological stress have been shown to fear, helplessness, or horror. To qualify as a Criterion A2 increase after DHEA treatment in postmenopausal women trauma, we required that physical or sexual trauma (Kudielka et al, 1998) and dexamethasone suppression of ascertained by the ETI be scored as ‘very’ or ‘extremely’ plasma cortisol levels has been shown to be reduced during distressing and disruptive in at least two of three domains of the luteal phase of the menstrual cycle when progesterone function (emotional, interpersonal, or school performance). levels are high (Altemus et al, 1997). DHEA and its sulfated Healthy NTC subjects were evaluated psychiatrically metabolite, DHEAS, also positively modulate NMDA using the non-patient edition of the Structured Clinical receptor function and antagonize GABAA receptor- Interview for DSM-IV (SCID-NP) (First et al, 1995a); mediated chloride ion flux (Baulieu and Robel, 1998)Fef- subjects with a current Axis I diagnosis were excluded, fects that may contribute to increased HPA axis responses except for one subject with a simple phobia. PTSD and TC (Barbaccio et al, 2001). subjects were evaluated using the SCID-P (First et al, DHEA is secreted episodically and synchronously with 1995b); subjects with current or past psychotic disorders or cortisol in response to fluctuating ACTH levels (Rosenfeld bipolar disorder were excluded from participation. Current et al, 1971). In addition, the administration of CRF and co-morbid Axis I diagnoses are presented in Table 1. ACTH has been shown to increase plasma DHEA and Past psychiatric diagnoses among the PTSD subjects progesterone levels (Parker et al, 1985a, b; Genazzani et al, included major depression (n ¼ 11), panic disorder (n ¼ 2), 1998). We therefore hypothesized that premenopausal substance abuse or dependence (46 years before participa- women with chronic PTSD would have increased DHEA tion, n ¼ 6), simple phobia (n ¼ 2), eating disorder (410 and/or progesterone levels at baseline or in response to years before participation, n ¼ 1), dysthymia (n ¼ 1), OCD adrenal activation. This would be of interest not only from a (washing and cleaning only, n ¼ 1), and generalized anxiety neuroendocrine point of view, but also because DHEA(S) disorder (n ¼ 1). Two TC subjects had remote histories of has been shown protect the brain from the negative impact single episode major depression. One NTC subject had one of stress (Kimonides et al, 1998; Bastianetto et al, 1999; episode of past major depression and one a remote history Kaminska et al, 2000; Karishma and Herbert, 2002; Zhang of bulimia. et al, 2002), while the pharmacological properties of these The PTSD, TC, and NTC subjects did not differ neuroactive steroids or their metabolites might allow them significantly by age: F(2, 38) ¼ 1.59, p ¼ 0.22 (PTSD: to modulate PTSD symptoms (Rupprecht and Holsboer, 36.1 þ 2.2 years, TC: 33.1 þ 4.3 years, NTC: 30.5 þ 2.1 years) 1999). We therefore measured plasma DHEA and proges- or weight: F(2, 38) ¼ 0.65, p ¼ 0.53 (PTSD: 162.7 þ 10.4 lbs, terone levels in premenopausal women with chronic PTSD TC: 150.9 þ 10.2 lbs, NTC: 150.6 þ 6.2 lbs). The PTSD compared to healthy nontraumatized (NTC), and trauma- subjects were of mixed Caucasian (n ¼ 13), African Amer- exposed (TC) subjects before and after the intravenous ican (n ¼ 1), and Hispanic (n ¼ 1) ethnicity. TC subjects administration of ACTH1À24. were of mixed Caucasian (n ¼ 4), African American (n ¼ 2),

Neuropsychopharmacology Increased DHEA release in women with PTSD AM Rasmusson et al 1548 Table 1 Current Psychiatric Diagnoses and DSMIV Criterion A1, Table 1 (Continued) A2 Traumas Group Group (CAPS Co-morbid a (CAPS Co-morbid score ) diagnoses Trauma (age in years) scorea) diagnoses Trauma (age in years) TC (14)c Simple phobia bMotor accident (31) PTSD (74) MDD Physical abuse (2–10)b Sexual abuse (4–5) TC (12) None Physical abuse (?–17) Rape (13, 14) bRape (34) bDomestic violence (23–25) TC (0) None bWitnessed domestic violence (9) PTSD (60) None Sexual assault (6) Sexual abuse (12) bWitnessed atrocities (7)

TC (3) None bWitnessed murder (15) PTSD (59)c None Physical abuse (2–14) TC( 0) None bCombat trauma (25) Sexual abuse (1–14) TC (0) None bWitnessed shooting (16) bDomestic violence (18–34) TC (0) None bNear fatal allergic reaction (18) Rape (28) CAPS: Clinician Administered PTSD Scale; GAD: generalized anxiety disorder; PTSD (21) None bRaped twice (12) MDD: major depressive disorder; OCD: obsessive compulsive disorder; MJ: marijuana; PTSD: post-traumatic stress disorder subject; TC: healthy trauma-exposed subject; NTC: healthy nontraumatized subject. PTSD (18) GAD Physical abuse (8–13) aOne-week CAPS (Clinician Administered PTSD Scale) scores were ascertained bRape (20) at the time of the ACTH stimulation test. Subjects were instructed to report PTSD symptoms related to any of the reported A1/A2 traumas. b c Indicates DSMIV A1, A2 traumas identified as most disturbing currently. PTSD (33) GAD Physical abuse (12–17) cIndicates subjects (three PTSD, one TC) for whom childhood trauma was Eating disorder NOS Rape (13, 17) assessed using only the general trauma inventory that contained general B Attempted rape (19) questions regarding exposure to childhood physical or sexual abuse. Indicates that trauma was verified by state records. bPhysical assault (29)

PTSD (35) None bBPhysical abuse (2–14) Sexual abuse (11–13) and Asian (n ¼ 1) descent. NTC subjects were of mixed PTSD (33) None bPhysical abuse (8–18) Caucasian (n ¼ 12), African American (n ¼ 1), Asian (1), bSexual abuse (15–18) Askenazi Jewish (n ¼ 1), native Greek (n ¼ 1), Hispanic (n ¼ 1), native Russian (n ¼ 1), and subcontinental Indian (n ¼ 1) ethnicity. Years of education differed significantly, PTSD (47) Dysthymia Rape (8) b but not substantially among groups: F(2, 38) ¼ 4.46, po0.02 Domestic violence (33) (PTSD: 14.2 þ 0.54, TC: 14.9 þ 0.99, NTC: 16.5 þ 0.52 years). All subjects but one with PTSD had completed high school. PTSD (64) None Sexual abuse (1–7) b Parent’s death (7) Menstrual Cycle Monitoring

PTSD (70)c None bGang rape (16) Menstrual cycle phase at the time of the ACTH stimulation Sexual abuse (2–16) test (Table 2) was determined by use of a urine test kit to detect the mid-cycle luteinizing hormone (LH) surge (Clear Plan Easy, Whitehall Laboratories, Madison, NJ). Ovulation PTSD (20) None Physical abuse (13–15) was confirmed by measurement of plasma progesterone b Rape (14) levels 7–14 days later (Leibenluft et al, 1994). In the data analyses, subjects on oral contraceptives were considered to PTSD (36) MDD bWitnessed parent’s sudden death (10) be in the follicular phase. bRape (19), near-drowning (21) ACTH Stimulation Test PTSD (31) None Rape (15) Fifteen PTSD, 19 NTC, and seven TC subjects completed the bAbduction and rape (22) ACTH stimulation test. Subjects were asked to fast for 4 h before testing. While many subjects fasted longer, the mean PTSD (74) Dysthymia Physical abuse (2–12) fasting times were not significantly different among the Sexual abuse (2–18) groups: F(2, 27) ¼ 1.98, p ¼ 0.16 (PTSD: 8.3 þ 1.7 h, TC: bGang rape (22) 12.9 þ 1.1 h, NTC: 11.4 þ 1.5 h). Subjects also refrained from

Neuropsychopharmacology Increased DHEA release in women with PTSD AM Rasmusson et al 1549 Table 2 Menstrual Phase During the ACTH1–24 Stimulation Test DHEA was determined with a sensitivity (detectability) of 0.02 ng/ml, a cross-reactivity with DHEA-S of 0.02%, and Follicular Steroid contraception Luteal intra- and inter-assay CVs of 8 and 11%, respectively. Plasma DHEA-S was measured with a sensitivity of 2 mg/dl *Number of subjects in DHEA(S) analyses and intra- and inter-assay CVs of 11 and 15%, respectively. PTSD 8 3 2 Progesterone and estrogen were measured by solid-phase NTC 7 0 6 RIA (Coat-A-Count, Diagnostic Products Corporation, Los TC 4 2 1 Angeles, CA) with a sensitivity of 0.5 ng/ml. The inter-assay CV varied from 5 to 12%; the intra-assay CV was o5%. Number of subjects in cortisol analyses As indicated above, the subject numbers vary among the PTSD 8 3 2 hormone analyses. The cortisol assays were run first. Plasma was not available for two NTC subjects when the NTC 8 0 7 DHEA(S) assays were run. Additional NTC subjects were TC 4 2 1 completed by the time the plasma progesterone and estrogen assays were run, but we no longer had access to Number of subjects in progesterone and estrogen analyses the lab that ran the cortisol and DHEA(S) analyses. Data PTSD 8 3 2 from all subjects meeting the inclusion criteria and NTC 10 0 7 completing the ACTH study and hormone measurements TC 4 2 0 were included in the data analyses.

Statistical Analyses smoking for 4–6 h prior to ACTH administration. A random effects model was used to analyze repeated ACTH1À24 (Cosyntropin, Organon Inc.) was administered hormone measurements from the ACTH stimulation test. intravenously 1 h after IV placement between 0800 and Diagnostic group, time, and menstrual phase were included 1630 h, at a maximally stimulating dose of 250 mg. The as independent variables and a compound symmetric average time of administration did not differ among groups: variance structure (CS) was used in the mixed model F(2, 38) ¼ 0.41, p ¼ 0.66 (PTSD: 1125 h730 min, range: (Diggle et al, 1994). A high-order interaction term was 0915–1615 h; TC: 1053 h737 min, range: 0930–1315 h; removed from the model if the p-value for this term was NTC: 1144 h732 min, range: 0815–1345 h). Most of these larger than 0.1. Although Dickstein et al (1991) showed that subjects also participated in other tests designed to examine cortisol levels measured at 300 and 600 after a maximally the HPA axis at different regulatory points. We thus allowed stimulating 250 mg ACTH dose are not affected by the time subjects to schedule their ACTH tests at their convenience of testing, baseline cortisol and DHEA levels (Erosheva et al, since previous work by Dickstein et al (1991) had shown 2000) are known to progressively decrease from early adrenal capacity to be unaffected by the timing of morning until late afternoon. Baseline DHEA(S) levels are ACTH1À24 administration. also known to decrease over the age range of subjects in the Blood samples were collected at À15, 0, þ 30, and study (Hornsby, 1995). Therefore, the time of the ACTH1–24 þ 60 min relative to the ACTH1À24 infusion. They were injection and subject age were used as covariates. If there spun immediately in a refrigerated centrifuge and plasma was a statistically significant difference in the overall model, was stored at À701C until hormone measurements were pair-wise comparisons were conducted for the PTSD vs made. The Clinician Administered PTSD Scale (Blake et al, NTC, and PTSD vs TC groups; a closed testing procedure 1993) (CAPS-One Week Version; PTSD and TC subjects was applied to the pair-wise models. only) and Profile of Mood States scale (rated for the past A one-way analysis of variance (ANOVA) was used to week) (POMS; Educational and Industrial Testing Service, compare the baseline or peak cortisol and DHEA hormone San Diego, CA) were obtained during the 30 min period values among groups. The time of the ACTH1–24 injection after IV placement. was used as a covariate for the baseline but not peak steroid level analyses (Dickstein et al, 1991); age was also used as a Plasma Hormone Measurements covariate for the DHEA(S) analyses. Pearson correlations were performed between age and Plasma cortisol was measured in 35 subjects, while DHEA DHEA(S) levels. Since the symptom ratings scores were not and DHEA-S were measured in 33 subjects in the Yale distributed normally, Spearman correlations were per- Mental Health Clinical Research Laboratory. Plasma pro- formed between DHEA or cortisol measures and symptom gesterone and estrogen were measured in 36 subjects in the ratings. Due to the high degree of colinearity among rated Laboratory of Molecular Pathophysiology at the Mood and psychiatric symptoms, as well as among the hormone Anxiety Disorders Program, National Institute of Mental responses to ACTH stimulation, Bonferroni corrections Health. Plasma cortisol was measured using an antibody- may impose a high risk of Type II error. Therefore, coated tube radioimmunoassay (RIA) with an inter-assay significance was set at po0.05 for the main correlations coefficient of variation (CV) of 8.2% and intra-assay CV of between DHEA or cortisol measures in response to ACTH o5% (DiaSorin, Inc., Stillwater, MN). Plasma DHEA and and the total CAPS or total POMS scores. Bonferroni DHEA-S were measured using RIA kits supplied by corrections were used in correlations between DHEA or Diagnostic Systems Laboratories (Webster, TX). Plasma cortisol reactivity to ACTH and the CAPS subscales or

Neuropsychopharmacology Increased DHEA release in women with PTSD AM Rasmusson et al 1550 component symptoms. Descriptive statistics are expressed a as group means with standard errors of the mean. 35 It should be noted that this is not a replication study; the 30 sample of subjects in the cortisol analysis is not independent from that in our previously published paper (Rasmus- 25

son et al, 2001). Two healthy NTC subjects and the subjects PTSD (n=13) in the TC group were studied subsequent to completion of 20 the previous study. Their data were added to the cortisol NTC (n=13) 15 data from the previous group, and the data from this larger TC (n=7) sample were reanalyzed. 10 Plasma DHEA (ng/ml) 5 RESULTS 0 Plasma DHEA and DHEAS Responses to ACTH b Administration 35

Analysis of the ACTH stimulation test results indicated that 30 age had a significant effect on plasma DHEA levels:

F(1, 28) ¼ 14.32, po0.0007, and correlated negatively with 25 baseline plasma DHEA levels: r ¼À0.58, po0.0005. There PTSD (n=13)

was no effect of menstrual phase on absolute DHEA levels 20 NTC (n=15) or responses to ACTH1À24; therefore, menstrual phase TC (n=7) was removed from the model. As expected, plasma DHEA 15 levels increased in response to ACTH1À24 administration:

F(2, 59) ¼ 39.04, po0.0001 (Figure 1a). In addition, there Plasma Cortisol (ng/dl) 10 was an effect of diagnosis: F(2, 28) ¼ 7.21, po0.004, as well as a diagnosis by time interaction: F(4, 59) ¼ 2.96, po0.03. 5 Post hoc testing indicated that plasma DHEA levels were c higher across all time points: F(1, 22) ¼ 12.87, po0.002, and 1.2 increased more in response to ACTH1À24: F(2, 47) ¼ 4.93, po0.012, in the PTSD group compared to the NTC group. 1 Plasma DHEA levels were higher across all time points in the PTSD group compared to the TC group: F(1, 16) ¼ 7.82, PTSD (n=13) 0.8 po0.013, but there was no significant difference between NTC (n=12) these two groups in the DHEA response to ACTH1À24: TC (n=7) F(2, 36) ¼ 1.40, p ¼ 0.26. Baseline plasma DHEA levels were 0.6 not significantly different among the PTSD, TC, and NTC groups: F(2, 27) ¼ 1.66, p ¼ 0.21. There were, however, 0.4

differences in peak DHEA levels among the three groups: Plasma DHEA/Cortisol Ratio F(2, 27) ¼ 9.34, po0.0009. Peak DHEA levels after ACTH were higher in the PTSD group than in either 0.2 1À24 Baseline +30 +60 the NTC group: F(1, 27) ¼ 16.67, po0.0005, or the TC µ group: F(1, 27) ¼ 9.51, po0.005. Minutes From ACTH1-24 Injection (250 g IV) We also measured the effects of ACTH on the sulfated Figure 1 Plasma DHEA (a), cortisol (b), and DHEA/cortisol ratios (c) in metabolite of DHEA. As expected, age influenced plasma response to a maximally stimulating 250 mg dose of adrenocorticotropic DHEAS levels during the ACTH test: F(1, 28) ¼ 11.46, hormone (ACTH)1–24. PTSD: post-traumatic stress disorder; NTC: po0.003, and correlated negatively with baseline DHEAS nontraumatized comparison; TC: trauma-exposed comparison subjects. levels: r ¼À0.52, po0.003. While DHEAS levels increased in response to ACTH1À24 administration: F(2, 60) ¼ 7.08, po0.002, DHEAS responses to ACTH were similar among was also a significant diagnosis by time interaction: the three groups: F(4, 60) ¼ 0.46, p ¼ 0.77. We did not find a F(4, 63) ¼ 3.01, po0.025. Post hoc pair-wise testing indi- significant difference among the three groups in DHEAS cated that cortisol increased more in the PTSD than the TC levels either: F(2, 28) ¼ 1.43, p ¼ 0.26; however, the study is group after ACTH1À24: F(2, 36) ¼ 4.30, po0.03. Consistent underpowered and we cannot rule out Type II error. with our previous report (Rasmusson et al, 2001), plasma cortisol levels were higher across all time points in the PTSD Plasma Cortisol Responses to ACTH Administration compared to the NTC group: F(1, 24) ¼ 4.38, po0.05, but there was no significant difference between these groups Consistent with previous work by Kirschbaum et al (1999), in the slope of the cortisol response to ACTH1À24: menstrual phase had no effect on plasma cortisol levels or F(2, 51) ¼ 2.11, p ¼ 0.13. Baseline cortisol levels measured responses to ACTH, and was removed from the model. As prior to ACTH1À24 administration were not significantly expected, plasma cortisol increased in response to different among the PTSD, TC, and NTC groups: ACTH1À24: F(2, 63) ¼ 222.41, po0.0001 (Figure 1b). There F(2, 27) ¼ 1.63, p ¼ 0.22.

Neuropsychopharmacology Increased DHEA release in women with PTSD AM Rasmusson et al 1551 The Ratio of Plasma DHEA to Cortisol 50 There was a significant diagnosis by time effect on the ratio 45 of DHEA to cortisol: F(2, 60) ¼ 5.95, po0.005 (Figure 1c). 40 Post hoc analyses indicated that the DHEA/cortisol ratio 35 increased similarly in response to ACTH1À24 administration in the PTSD and TC groups (effect of time: F(1, 36) ¼ 8.11, 30 po0.008; diagnosis by time effect: F(1, 36) ¼ 0.43, p ¼ 0.51). 25 The NTC group ratio behaved differently than the TC group (diagnosis by time effect: F(1, 37) ¼ 13.21, po0.0009) and 20 the PTSD group (diagnosis by time effect: F(1, 47) ¼ 6.33, 15 po0.02). There were no group differences in the baseline Peak Change in DHEA (ng/ml) (F(2, 27) ¼ 1.84, p ¼ 0.18) or 30 min (F(2, 27) ¼ 1.10, 10 p ¼ 0.35) DHEA/cortisol ratio. Group differences in the 5 DHEA/cortisol ratio emerged 60 min after ACTH1À24 10 20 30 40 50 60 70 80 administration: F(2, 27) ¼ 6.91, po0.004, at which time the Total CAPS Score PTSD group ratio was significantly greater than the NTC group ratio (po0.04), while the TC group ratio fell between 50 these groups. 45

Progesterone and Estrogen Responses to ACTH 40 Administration 35 As expected, progesterone levels varied across the menstru- 30 al cycle and were about an order of magnitude higher in the 25 luteal compared to the follicular phase in all groups: F(1, 29) ¼ 68.24, po0.0001. Progesterone levels appeared to 20 change in response to ACTH1À24: F(2, 58) ¼ 5.14, po0.009, 15 but there was a significant menstrual phase by time Peak Change in DHEA (ng/ml) 10 interaction: F(2, 58) ¼ 12.39, po0.0001. Consistent with the work by Kruyt and Rolland (1982), increases in 5 progesterone after ACTH stimulation were detectable 0 5 10 15 20 25 30 35 during the follicular phase: F(1, 45) ¼ 132.93, po0.0001, CAPS C Avoidance Symptoms but not during the luteal phase: F(1, 15) ¼ 0.10, p ¼ 0.76. Figure 2 Scattergrams of the correlations between the peak change in There were no differences among the three diagnostic plasma DHEA levels in response to 250 mg ACTH1À24 and: (a) total CAPS groups in baseline progesterone levels: F(2, 26) ¼ 1.38, symptoms (Spearman r ¼À0.57, po0.04), (b) CAPS C avoidance p ¼ 0.27, or responses to ACTH1À24: F(4, 56) ¼ 0.69, symptoms (Spearman r ¼À0.70, po0.008), and (c) CAPS D hyperarousal p ¼ 0.61. symptoms (Spearman r ¼À0.53, p ¼ 0.06). Plasma estrogen levels were not different between menstrual phases: F(1, 34) ¼ 0.8, p ¼ 0.38, and did not change in response to ACTH1À24 administration: F(2, 67) ¼ 1.51, p ¼ 0.23. There were also no differences DHEA reactivity and Criterion D hyperarousal symptoms: among the three diagnostic groups in estrogen levels: r ¼À0.53, p ¼ 0.06 (Figure 2c), or Criterion B re-experien- F(2, 31) ¼ 0.17, p ¼ 0.84, or reactivity to ACTH1À24: cing symptoms: r ¼À0.19, p ¼ 0.53, were not significant. Of F(4, 65) ¼ 0.76, p ¼ 0.55. note, the peak change in DHEA correlated negatively, though not necessarily significantly, with each PTSD Correlation of Clinical Symptoms with Plasma Hormone symptom comprising the Criterion B, C, and D clusters, Responses except for ‘difficulty falling or staying asleep’ (r ¼ 0.52, po0.08). Without inclusion of the sleep disturbance item in As expected, there was a positive correlation between the the total Criterion D symptom score, the correlation peak change in cortisol and peak change in DHEA after between Criterion D symptoms and the peak change in ACTH1À24 administration among all subjects: r ¼ 0.48, DHEA was strong and significant: r ¼À0.81, po0.0009. po0.006. The peak change in cortisol in response to In addition, within the PTSD group, there was a negative ACTH1À24 correlated positively and significantly with total correlation between the mean DHEA/cortisol ratio after POMS score: r ¼ 0.41, po0.03, whereas the peak change in ACTH1À24 and the POMS scores: r ¼À0.63, po0.04. There DHEA did not: r ¼ 0.22, p ¼ 0.26. were no significant correlations between the peak change in Within the PTSD group, the peak change in DHEA cortisol in response to ACTH and total CAPS scores, correlated negatively with the total CAPS score: r ¼À0.57, summed Criterion B, C, or D scores, or total POMS scores in po0.04 (Figure 2a). This relationship appears to have been the PTSD subjects. However, the peak change in cortisol did carried largely by a significant negative correlation between correlate negatively, strongly, and significantly with ‘de- DHEA reactivity and Criterion C avoidance symptoms: creased interest and participation in everyday activities’: r ¼À0.70, po0.008 (Figure 2b). Relationships between r ¼À0.80, p ¼ 0.001.

Neuropsychopharmacology Increased DHEA release in women with PTSD AM Rasmusson et al 1552 DISCUSSION by studies that have shown: (a) increased ACTH reactivity to psychosocial stress in post-menopausal women In this study, we used a standard ACTH1À24 stimulation test treated for 2 weeks with DHEA (Kudielka et al, 1998), (b) to assess the maximum capacity of the adrenal gland for increased ACTH and cortisol reactivity in heterozygote cortisol, DHEA, and progesterone release in premenopausal carriers of 21-hydroxylase deficiency, wherein cortisol women with PTSD compared to healthy nontraumatized precursors are shunted into the androgenic steroid path- and trauma-exposed comparison subjects. This dose of ways (Witchel et al, 1997; Merke et al, 1999; Witchel and ACTH elicits cortisol responses that equal or exceed those Lee, 1998), and (c) increased CRF-induced ACTH release in associated with extreme naturalistic stressors with the depressed post-adrenarchal, pre-pubertal children with potential to cause PTSD (reviewed in Morgan et al, 2000). PTSD living in adverse environments (Kaufman et al, We found adrenal DHEA release to be increased in the 1997). In addition, DHEA(S) positively modulates NMDA women with PTSD compared to the healthy nontraumatized receptor function and antagonizes GABAA receptors (Bau- subjects, while the PTSD subjects had higher DHEA levels lieu and Robel, 1998; Rupprecht and Holsboer, 1999), effects across all time points compared to the healthy trauma- that may contribute to increased HPA axis responses exposed subjects. These findings are consistent with those (Barbaccio et al, 2001). of Lemieux and Coe (1995) showing an increase in 24-h The current study does not support a role for adrenally urinary 17-ketosteroid metabolites of DHEA and DHEAS in derived progesterone in mediating differences in HPA axis premenopausal women with PTSD. They also may be responsivity between women with and without PTSD. consistent with two studies showing increased morning However, it is important to note that the current study ‘baseline’ DHEA(S) levels in PTSD, since morning DHEA was not designed to determine whether progesterone and cortisol levels reflect the reactivity of the adrenal gland derived from the ovary might play such a role. This will to large pulses of endogenous ACTH. Spivak et al (2000) be important to assess in the future, since studies in healthy found high morning plasma DHEA and DHEAS levels, as women have demonstrated increased ACTH and cortisol well as a trend for an increase in plasma cortisol levels in reactivity during the luteal compared to the follicular phase Israeli males with combat-related PTSD. Sondergaard et al of the menstrual cycle (Kirschbaum et al, 1999; Altemus (2002) observed increased DHEAS levels among Kosovo et al, 2001; Roca et al, 2003; Schmidt et al, 2002), effects that refugees that developed PTSD but decreased DHEAS levels may be attributable to progesterone (Quinkler et al, 2003; in refugees that developed PTSD and co-morbid depression. Schmidt et al, 2002). It is also possible that antiglucocorti- In that study, DHEAS levels were observed to increase over coid effects of progesterone play a role in differentiating a 9-month period in association with increasing PTSD patterns of HPA axis adaptation to stress between symptoms, particularly sleep disturbance. While we did not premenopausal women and other groups such as men, observe significant differences in plasma DHEAS post-menopausal women, and pre-pubertal children. In levels between the traumatized subjects that did and did addition, the conversion of DHEA to DHEAS, a metabolite not develop PTSD in the current study, we cannot rule out that lacks antiglucocorticoid activity but that has more Type II error due to the relatively small number of TC potent activity at NMDA and GABAA receptors, appears to subjects. be inhibited by estrogen and facilitated by testosterone In the current study, we also found increased (Neslter, 1996). Thus, sex- as well as reproductive cortisol responses to ACTH in the PTSD compared status-related roles for DHEA and progesterone in regulat- to healthy traumatized subjects, and higher cortisol ing HPA axis responsivity may help explain why the only levels in general in the PTSD compared to nontraumatized studies finding low or similar urinary cortisol output in subjects. These findings are consistent with previous PTSD subjects have been those containing men alone 24-h urinary free cortisol studies in premenopausal or post-menopausal women (Mason et al, 1986, 2002; women and post-adrenarchal girls with PTSD, all of Yehuda et al, 1990, 1993, 1995; Kosten et al, 1990; Baker which have found increased cortisol output in the subjects et al, 1999). with PTSD (Lemieux and Coe, 1995; Maes et al, 1998; De Bellis et al, 1999; Rasmusson et al, 2001; Friedman et al, Negative Association between DHEA Responses and 2001). PTSD Symptoms and Negative Mood in PTSD Our findings lead us to suggest that adrenally derived DHEA may contribute to an increase in the reactivity Interestingly, despite the association between PTSD and of the HPA axis in premenopausal women with PTSD. high-stimulated DHEA levels in the current study, there was The antiglucocorticoid properties of DHEA may antagonize a negative correlation between DHEA reactivity and severity glucocorticoid negative feedback within the brain of PTSD symptoms within the PTSD group, suggesting that and pituitary, thereby facilitating CRF and/or ACTH DHEA may protect against the development of avoidance as release with a resulting increase in the adrenal capacity well as hyperarousal symptoms. A role for DHEA as a for cortisol release (Keller-Wood et al, 1984; Legradi resilience factor in PTSD is thus consistent with recent et al, 1997; Heim et al, 2000; Rasmusson et al, 2001). observations in military personnel of a negative correlation This possibility is supported by studies in healthy between DHEAS/cortisol ratios and dissociation and a volunteers of the glucocorticoid receptor antagonist positive correlation between DHEAS/cortisol ratios and mifepristone (RU486), which produces a pattern of military performance during extreme training stress pituitary and adrenal reactivity similar to that observed in (Morgan et al, 2004). There are also several other clinical our female subjects with PTSD (Van Haarst et al, 1996; studies showing a negative association between plasma Bertagna et al, 1994). This possibility is also supported DHEA(S) levels and depressive symptoms, and/or a positive

Neuropsychopharmacology Increased DHEA release in women with PTSD AM Rasmusson et al 1553 correlation between DHEA(S) levels and vigor or general Increased DHEA(S) responses to extreme stress, could well-being (Yaffe et al, 1998; Goodyer et al, 1998; Cruess thereby enhance the development of traumatic memories et al, 1999; Heinz et al, 1999; Michael et al, 2000; Young et al, as well as contribute to their extinction during naturalistic 2002). In addition, DHEA administration has been shown to or treatment-based trauma re-exposure (Jaycox et al, treat depressive symptoms in individuals with major 1998). depression or adrenal insufficiency (Wolkowitz et al, 1999; Arlt et al, 1999), to reduce anxiety, depression, and Factors that Influence DHEA Production and Release by negative symptoms in individuals with schizophrenia the Adrenal Gland (Strous et al, 2003), and to mitigate depressive behavioral responses in genetically bred high- but not low-anxiety rats Production of DHEA by the adrenal reticularis is a species- (Prasad et al, 1997). specific phenomenon; rodents have little circulating DHEA and the pattern of DHEA secretion differs between human Molecular and Neuropharmacological Effects of DHEA and non-human primates (Neslter, 1996). In humans, with Relevance to PTSD plasma DHEA levels are high at birth due to the presence of the adrenal fetal zone, drop shortly thereafter, and In this context, it is important to appreciate that remain low until adrenarche (age 6–8 years). DHEA levels peripherally derived DHEA(S) is thought to be the only then rapidly increase, continue to climb through adoles- source of brain DHEA(S), since the biosynthetic pathway cence, peak in the second to third decade, and then previously thought responsible for brain DHEA produc- progressively decline with further aging (Hornsby, 1995). tion functions only under nonphysiological conditions Thus, blunted DHEA responses to traumatic stress prior to (Compagnone and Mellon, 2000). Within the brain, adrenarche or later in life may limit the degree to which the region-specific metabolism of DHEA may ultimately HPA axis can upregulate in response to stress. A lack of control the nature of DHEA effects on cognition and neuroprotection by DHEA during periods of life when behavior (Rose et al, 1997). For instance, 7-hydroxylated DHEA levels are low may contribute to toxic effects of stress metabolites of DHEA have been shown to interfere with the on the brain. nuclear uptake of activated glucocorticoid receptors In the current study, disparities in the frequency, severity, in neurons of the hippocampus (Morfin and Starka, duration, and developmental timing of DSMIV criterion A 2001). This mechanism accords with the proposed anti- trauma exposure between the traumatized groups with and glucocorticoid properties of DHEA and may confer without PTSD lead one to hypothesize that increased DHEA neuroprotection in this brain region. For example, and cortisol reactivity may simply result from cumulative DHEA protects against excitatory amino acid- and oxidative stress exposure or exposure to extreme stress earlier in life stress-induced damage, restores cortisol-induced decre (eg, see Heim et al, 2002; Rinne et al, 2003). However, a ments in long-term potentiation, regulates programmed simple effect of trauma severity or developmental timing of cell death, and promotes neurogenesis in the hippocampus trauma on DHEA reactivity is not consistent with our (Kimonides et al, 1998; Bastianetto et al, 1999; Kaminska observation of a negative correlation between DHEA et al, 2000; Karishma and Herbert, 2002; Zhang et al, 2002). reactivity and PTSD symptom severity. Other factors must Further research will be required to specifically determine also be involved. whether and how DHEA may exert antiglucocorticoid Other proposed modulators of DHEA(S) levels during effects in other brain regions of relevance to PTSD, such extreme stress include insulin (Bernton et al, 1995), as the pituitary, hypothalamus, amygdala, and cortex. This interleukin 6 (Path et al, 1997; Judd et al, 2000), and B- is important since such effects may translate into behavioral adrenergic receptor activation (Oberbeck et al, 1998). In alterations: genetic downregulation of brain glucocorticoid addition, the current study and the work of Oberbeck et al receptors has been shown to reduce anxiety-related (1998) suggest that extreme activation of the adrenal gland behaviors in mice (Kellendonk et al, 2002). may result in saturation of the adrenal glucocorticoid It is also important to note that DHEA is a precursor for synthetic pathway, with shunting of steroid precursors into testosterone and estrogen, steroids with independent potent androgen pathways. Such an effect may be magnified in effects on brain, the HPA axis, and behavior (McEwen, 2002; individuals with functional polymorphisms of genes that Viau, 2002). In addition, pharmacological effects of DHEA code for adrenal enzymes involved in cortisol synthesis or DHEAS may influence the PTSD phenotype. As Spivak (Witchel et al, 1997; Jacobs et al, 1999). et al (2000) suggested, DHEA(S) may potentiate activation F of monoamine systems during stress and thereby con- Study Limitations tribute to the consistently observed phenomenon of noradrenergic hyper-reactivity in PTSD (Southwick et al, Due to the relatively low number of subjects in the trauma 1999). In addition, DHEA may antagonize the capacity of control group, there is inadequate statistical power to rule cortisol to contain some aspects of the stress response out significant differences in DHEA reactivity between the (Munck et al, 1984), perhaps explaining how noradrenergic TC and PTSD group. We also might have expected high hyperreactivity can coexist with high as well as low cortisol morning ‘baseline’ DHEA levels in the PTSD group; output in PTSD. Facilitation of NMDA receptor function by however, only about half of the subjects were studied in DHEA(S) may also influence the development of PTSD. the morning, possibly limiting our power to detect such an Activation of NMDA receptors in the amygdala has been effect. Variability in the time of the ACTH test does not, shown to facilitate the formation as well as extinction of however, alter the confidence with which we can interpret conditioned fear responses (Walker and Davis, 2002). the differences in DHEA and cortisol reactivity among

Neuropsychopharmacology Increased DHEA release in women with PTSD AM Rasmusson et al 1554 groups. Not only did we match and covary for the time of glucocorticoid negative feedback at higher levels of the ACTH1À24 administration across groups, but Dickstein et al central nervous system. (1991) demonstrated that 300 and 600 cortisol responses to a maximally stimulating ACTH dose do not differ between morning and late afternoon. There was also variability in CONCLUSION the length of fasting among participants. However, there is no evidence in the literature to suggest that baseline cortisol The current study suggests that an increased capacity for levels vary with fasting over the interval spanned in this DHEA release in response to extreme adrenal activation study. In addition, glucose effects on plasma cortisol may be protective and/or mitigate PTSD symptoms and reactivity are thought to be mediated centrally via CRF negative mood in persons with PTSD. The results of this (Gonzalez-Bono et al, 2002; Heptulla et al, 2001). Adrenal study should therefore spur us to examine how a shift steroid responses to ACTH1À24 administration do not toward androgen production, possibly influenced by genetic involve CRF. predisposition, as well as other biological and develop- Another limitation, as well as strength, of the current mental factors, influences the pattern of neuropsychiatric study is the inclusion of only two PTSD subjects with and behavioral sequelae, as well as the neuroendocrine concurrent major depression. Epidemiological data suggest profile that develops in response to extreme stress. In that major depression co-morbid with PTSD tends to addition, it will be critical to more fully understand the constitute more severe PTSD (Breslau et al, 2000), in part adaptive as well as possible maladaptive features of this because several avoidance and hyperarousal symptoms of pattern of neuroendocrine system adaptation (McEwen, PTSD (poor concentration, irritability, poor sleep, de- 2000; Fabian et al, 2001). Hopefully, such work will allow us creased interest and participation in activities, and re- to develop new treatments for PTSD as well as new methods stricted range of affect) overlap with symptoms of major to identify and protect individuals at greater risk for PTSD depression. The current findings as well as those of in advance of anticipated extreme stress exposure. Sondergaard et al (2002) suggest that a group of more severely affected PTSD subjects with co-morbid major depression might show lower DHEA levels or ACKNOWLEDGEMENTS reactivity. It is also not clear from this cross-sectional study whether This work was supported by the Veterans Affairs National individual patterns of DHEA and cortisol output in Center for PTSD, Clinical Neuroscience Division in West response to adrenal activation are trait or state character- Haven, CT, and Womens Health Science Division in Boston, istics. Indeed, 11 PTSD subjects in the study had past major MA, NIH Grant # MH30929, the Laboratory of Molecular depressive episodes. It will be important to learn whether Pathophysiology, Mood and Anxiety Disorders Program, increased cortisol and DHEA reactivity in persons with National Institute of Mental Health, and an ORWH/NIDA PTSD predisposes to such episodes and/or whether this Grant #1K12DA14038-01 (BIRWCH Award) to Dr Rasmus- pattern is maintained or changes during depressive son. We thank Valinda Fox, RN, Willie Ford, Jill Panuzio, episodes and subsequent treatment (eg, see Heuser et al, Angela Genovese, RNC, MBA, Elizabeth O’Donnell,RN, 1998). The current study also does not allow us to discern Kathy West, and George Anderson, PhD, for their profes- whether acute and brief or more prolonged surges in DHEA sional and technical support, as well as Selma Witchel, MD, are needed to affect glucocorticoid receptor function or for her scientific input. exert effects at GABA and NMDA receptors. Our data suggest that cortisol and DHEA increases after extreme REFERENCES activation of the adrenal gland may persist for some time in PTSD subjectsFthus research looking at the time course of Altemus M, Redwine L, Leong Y-M, Yoshikawa T, Yehuda R, these responses will be important. Detera-Wadleigh S et al (1997). 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