HHS Public Access
`Author manuscript
`Psychoneuroendocrinology. Author manuscript; available in PMC 2019 March 11.
`
`Published in final edited form as:
`Psychoneuroendocrinology. 2018 August ; 94: 72–82. doi:10.1016/j.psyneuen.2018.05.007.
`
`Sex differences in the ACTH and cortisol response to
`pharmacological probes are stressor-specific and occur
`regardless of alcohol dependence history
`
`Robert M. Anthenellia,*, Jaimee L. Heffnerb, Thomas J. Blomc, Belinda E. Daniela, Benjamin
`S. McKennaa, and Gary S. Wandd
`aDepartment of Psychiatry, University of California, San Diego, Health Sciences, La Jolla, CA,
`United States
`
`bFred Hutchinson Cancer Research Center, Seattle, WA, United States
`
`cDepartment of Psychiatry and Behavioral Neuroscience, University of Cincinnati College of
`Medicine, Cincinnati, OH, United States
`
`dThe Johns Hopkins University School of Medicine, Baltimore, MD, United States
`
`Abstract
`Women and men differ in their risk for developing stress-related conditions such as alcohol use
`and anxiety disorders and there are gender differences in the typical sequence in which these
`disorders co-occur. However, the neural systems underlying these gender-biased
`psychopathologies and clinical course modifiers in humans are poorly understood and may involve
`both central and peripheral mechanisms regulating the limbic-hypothalamic-pituitary-adrenal axis.
`In the present randomized, double blind, placebo-controlled, triple-dummy crossover study, we
`juxtaposed a centrally-acting, citalopram (2 mg/unit BMI) neuroendocrine stimulation test with a
`peripherally-acting, dexamethasone (Dex) (1.5 mg)/corticotropin-releasing factor (CRF) (1 μg/kg)
`test in euthymic women (N = 38) and men (N = 44) with (54%) and without histories of alcohol
`dependence to determine whether sex, alcohol dependence or both influenced the
`adrenocorticotropic hormone (ACTH) and cortisol responses to the pharmacological challenges
`and to identify the loci of these effects. We found that central serotonergic mechanisms, along with
`differences in pituitary and adrenal sensitivity, mediated sexually- diergic ACTH and cortisol
`responses in a stressor-specific manner regardless of a personal history of alcohol dependence.
`Specifically, women exhibited a greater response to the Dex/CRF test than they did the citalopram
`
`*Corresponding author at: Pacific Treatment and Research Center, Department of Psychiatry (0603), University of California, San
`Diego, Health Sciences, 9500 Gilman Drive, La Jolla, CA, 92093-0603 United States. ranthenelli@ucsd.edu (R.M. Anthenelli).
`Disclosures
`Dr. Anthenelli provides consulting and/or advisory services to Pfizer and US World Meds. The Pacific Treatment and Research Center
`has received grant support from Alkermes and Pfizer. Drs. Heffner, Daniel, McKenna, Wand and Mr. Blom have no competing
`interests to disclose.
`Declaration of interest
`Dr. Anthenelli provides consulting and/or advisory services to Pfizer and US World Meds. The Pacific Treatment and Research Center
`has received grant support from Alkermes and Pfizer. Drs. Heffner, Daniel, McKenna, Wand and Mr. Blom have no competing
`interests to disclose.
`Appendix A. Supplementary data
`Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.10167j.psyneuen.2018.05.007.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`SPRUCE - EXHIBIT 2001
`Neurocrine Biosciences, Inc. v. Spruce Biosciences, Inc.
`PGR2021-00088
`
`

`

`Anthenelli et al.
`
`Page 2
`
`test while men exhibited the opposite pattern of results. Women also had more robust ACTH,
`cortisol and body temperature responses to Dex/CRF than men, and exhibited a shift in their
`adrenal glands’ sensitivity to ACTH as measured by the cortisol/log (ACTH) ratio during that
`session in contrast to the other test days. Our findings indicate that central serotonergic and
`peripheral mechanisms both play roles in mediating sexually dimorphic, stressor-specific
`endocrine responses in humans regardless of alcohol dependence history.
`
`Keywords
`Dex/CRF test; Alcohol use disorder; Sex differences; Serotonin (5-HT); Citalopram stimulation
`test; Cortisol
`
`1.
`
`Introduction
`
`Women and men differ in their risk for developing stress-related mental health conditions
`such as alcohol dependence (AD) (Keyes et al., 2008), post-traumatic stress disorder (PTSD)
`(Kessler et al., 1995) and major depression (Kessler et al., 1994). For example, men develop
`AD at roughly twice the rate of women; while for PTSD, the female to male prevalence rate
`is 2:1 in the opposite direction. The role of stress in the development and maintenance of
`AD also differs between the sexes. Thus, in contrast to AD men where alcohol problems
`usually antedate the onset of anxiety and depression, AD women typically suffer from
`stress-related anxiety or mood disorders prior to the onset of AD (Hesselbrock et al., 1985;
`Kessler et al., 1997), and experience increased post-abstinence depressive symptoms
`(Hatsukami and Pickens, 1982) or frank depressive episodes (Hasin and Grant, 2002) when
`they quit drinking. While the disparity between women and men is widely known, the neural
`mechanisms underlying these gender- biased psychopathologies and clinical course
`modifiers are poorly understood.
`
`Sexual diergisms in the limbic-hypothalamic-pituitary-adrenal (LHPA) axis that regulates
`the neuroendocrine limb of the stress response have been proposed as important mediators
`of sex-specific disease risk (Bangasser and Valentino, 2014). These dimorphisms are found
`at all levels of the axis - from the cortical and limbic structures (e.g., prefrontal cortex,
`hippocampus and amygdala) that send inputs to neurons in the paraventricular nucleus
`(PVN) of the hypothalamus which secrete corticotropin-releasing factor (CRF) and arginine
`vasopressin (AVP) - to the peripheral target of these secretagogues, the pituitary, which
`secretes adrenocorticotropic hormone (ACTH) and stimulates the adrenal gland to release
`cortisol (Goel et al., 2014; Solomon and Herman, 2009). The PVN also receives stimulatory
`and inhibitory signals from monoaminergic afferents originating in the midbrain which
`themselves are influenced by sex steroids (Barth et al.,2015).
`
`The serotonin (5-hydroxytryptamine or 5-HT) system has been implicated as playing an
`important role in mediating sex-biased psychopathologies such as AD (Anthenelli et al.,
`2001; Marcinkiewcz et al.,2016) and PTSD (Ravindran and Stein, 2009). Studies in non-
`human primates and humans have found sex differences or gonadal steroid effects in 5-HT
`synthesis (Sakai et al., 2006; Sanchez et al., 2005), presynaptic autoreceptor (e.g., 5-HT1A
`receptor) activity (Pecins- Thompson and Bethea, 1999), postsynaptic receptor function
`
`Psychoneuroendocrinology. Author manuscript; available in PMC 2019 March 11.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Anthenelli et al.
`
`Page 3
`
`(Centeno et al., 2007), reuptake (Pecins-Thompson et al., 1998), and degradation (Gundlah
`et al., 2002). 5-HTergic neurons originating in the brainstem raphe nuclei innervate the
`LHPA axis centrally at the levels of the prefrontal cortex, amygdala and hippocampus, and
`also send branches to the PVN of the hypothalamus to form bidirectional feedback loops
`between the CRF and 5-HT systems (Barth et al., 2015). Thus, the 5-HT- CRF system is
`uniquely poised to regulate ACTH and cortisol release in a sex-sensitive manner.
`
`Prior work from our group implicated the 5-HT-CRF system as a potential mediator of sex-
`specific ACTH and cortisol reactivity in longterm abstinent AD men and women and
`controls (Anthenelli and Maxwell, 2002; Anthenelli et al., 2001). We found that AD men
`and women had an exaggerated and prolonged endocrine response to the 5- HT-releaser,
`fenfluramine, compared with non-AD controls, and that the ACTH response to fenfluramine
`was increased to a larger extent in AD women compared with all other groups. However, our
`earlier experiments had too few women to confirm whether there was a reproducible
`disparity in 5-HT-induced ACTH and cortisol release, and were not designed to identify the
`locus of the disturbance at either a suprapituitary (i.e., 5-HT impacting the hypothalamus or
`limbic structures regulating the PVN) or peripheral (pituitary and/or adrenal) level. This
`latter distinction is important because sex differences have also been found at the levels of
`the pituitary corticotrophs (Gallucci et al., 1993), adrenal cortex (Figueiredo et al., 2007),
`and gonadal steroids influence the glucocorticoid-dependent negative feedback loop that
`terminates the stress response (Weiser and Handa, 2009).
`
`In order to begin disentangling the effects of sex and AD on stress circuit function we
`conducted the first double-blind, placebo-controlled crossover study targeting different loci
`in the LHPA axis. Using a pharmacological stressor approach, we performed a citalopram
`stimulation test in women and men with and without histories of AD to examine whether
`sex, AD, or both affected the individual’s response to this selective 5-HTergic probe. We
`also examined glucocorticoid-dependent negative feedback, along with pituitary-adrenal
`sensitivity in these same women and men by administering the combined dex-
`amethasone/CRF stimulation test to determine sex and AD effects on those parameters.
`Based on our preliminary results, we hypothesized that: 1) women would have greater
`ACTH and cortisol responses than men to both the citalopram and dexamethasone/CRF
`stimulation tests; and 2) AD women would have substantially greater ACTH responses to
`both challenges than the other three groups.
`
`2. Material and methods
`
`2.1. Participants
`
`Adult premenopausal women and men with (n = 165) and without (n = 112) remitted AD
`(early or sustained) were recruited from treatment facilities or the general population,
`respectively. One aim of the study was to determine whether longer-term abstinent AD
`participants differed from non-AD controls in endocrine (ACTH and cortisol),
`cardiovascular (blood pressure and heart rate), and subjective responses to the citalopram
`stimulation test, thus, AD participants had been abstinent from alcohol and all other drugs
`(except nicotine) for a minimum of 60 days prior to neuroendocrine testing. The study was
`approved by the University of Cincinnati Institutional Review Board and Cincinnati VA
`
`Psychoneuroendocrinology. Author manuscript; available in PMC 2019 March 11.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Anthenelli et al.
`
`Page 4
`
`Research and Development Committee, and all participants provided written informed
`consent and were compensated for their time and effort.
`
`Participants were excluded if they were ≥ 56 years of age, or evidenced an independent (i.e.,
`non-substance-induced) mood, anxiety, psychotic or eating disorder within the past 12
`months. Psychotropic and other systemic medications that could influence the stress
`response were not allowed, and all participants were in generally good health as evidenced
`by normal physical examinations, laboratory testing and electrocardiogram. Female
`participants were excluded if they were pregnant, lactating, or using any form of hormonal
`contraception that could alter corticosteroid-binding globulin levels and, thus, affect
`measurement of plasma cortisol concentrations (Kirschbaum et al., 1999).
`
`2.2. Rationale for the double-blind, crossover study and targets of the pharmacological
`probes
`
`The study was designed to assess the central effects of the citalopram stimulation test (CST)
`in juxtaposition to the peripheral effects of the combined dexamethasone/CRF test (Dex/
`CRF) while controlling for any potential placebo effects as determined by a third test
`administering triple-dummy placebos. Acute increases in brain 5-HT levels reliably
`produced by a single dose of the selective serotonin-reuptake inhibitor (SSRI), citalopram
`(Nadeem et al., 2004), stimulate neurons in the PVN of the hypothalamus via 5-HT1A and/or
`5-HT2A/2C receptors (Hanley and Van de Kar, 2003; Heisler et al., 2007) to secrete CRF and/
`or AVP (Jørgensen et al., 2003) to initiate a stress response. Whereas our prior work in AD
`men and women demonstrated sex-specific, exaggerated ACTH/cortisol responses to
`serotonergic stimulation compared with non-AD controls (Anthenelli and Maxwell, 2002;
`Anthenelli et al., 2001), and since these increased hormonal responses might have been of
`either central or peripheral origin, in the present experiment we also administered the
`combined Dex/CRF test. In that paradigm, administration of the synthetic glucocorticoid
`inhibits the pituitary release of ACTH which, in turn, suppresses cortisol secretion. The dex-
`amethasone suppression portion of the challenge allows one to assess differences in the
`glucocorticoid-dependent negative feedback loop. The CRF portion of the test is believed to
`measure pituitary-adrenal sensitivity as well as CRF/AVP feed forward drive of the axis in
`the setting of low endogenous cortisol (Heuser et al., 1994a). Thus, differential patterns of
`activation across the CST and Dex/CRF test days has the potential to distinguish a
`suprapituitary versus pituitary-adrenal locus of response.
`
`2.3. Procedures
`
`This was a randomized, double-blind, triple-dummy crossover study wherein participants
`received each of 3 separate challenge tests (i.e., placebo, CST, Dex/CRF) in counterbalanced
`order. Effort was made to conduct neuroendocrine tests in female participants at roughly
`once- monthly intervals (mean = 44.2 ± 32.3 days between sessions) and timed to
`correspond with the follicular phase of the menstrual cycle as determined by a calendar
`method (Mortola et al., 1990) and by measurements of plasma estradiol and progesterone
`concentrations. Testing in men occurred at shorter between-session intervals (mean = 26.0
`± 43.0 days; p < 0.01; Wilcoxon rank sum test). The neuroendocrine stimulation tests
`
`Psychoneuroendocrinology. Author manuscript; available in PMC 2019 March 11.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Anthenelli et al.
`
`Page 5
`
`comprised visits 3–5 of an overall 6-session paradigm as illustrated in Fig. 1 and as
`described below.
`
`Details of Visits 1, 2 and 6 have been described previously (Heffneret al., 2011). Briefly,
`after a telephone screening procedure (n = 1199), 277 eligible participants were interviewed
`by a trained research assistant with the Semi-Structured Assessment for the Genetics of
`Alcoholism - Version II (SSAGA-II) (Bucholz et al., 1994) to determine in- clusionary and
`exclusionary diagnoses using the Diagnostic and Statistical Manual for Mental Disorders,
`4th Edition, Text-Revised (DSM-IV-TR) criteria (American Psychiatric Association, 2000).
`Recency, frequencies and quantities of alcohol, nicotine and other drug use were codified in
`the relevant sections of the SSAGA-II and monitored throughout the protocol using the
`calendar-based TimeLine Follow-Back (TLFB) technique (Sobell and Sobell, 1992). Self-
`reported abstinence from drinking and drug use was verified by repeated alcohol
`breathalyzer testing; urine drug dipstick and confirmatory urine toxicology screening; and
`evaluations of state markers of heavy drinking including liver function tests (LFTs) (e.g.,
`gamma glutamyltransferase [GGT]), mean corpuscular volume (MCV), and %
`carbohydrate-deficient transferrin. Most AD participants were residing in a controlled sober
`living environment during their participation in the protocol. This also served as another
`safeguard of AD participants’ abstinence during testing.
`
`The triple-dummy design consisted of participants ingesting a capsule at 2100 h the night
`prior to each day of neuroendocrine testing. On two of the three nights, this capsule
`contained placebo, except for the one evening that preceded the CRF stimulation test when
`participants ingested 1.5 mg of dexamethasone as part of the combined Dex/CRF test. All
`capsules contained riboflavin as a tracer which was used along with ultraviolet detection,
`reminder notices, and phone calls as a means of monitoring adherence with taking the
`capsules. After reporting to the laboratory the next morning at 0715 h and following an
`overnight fast, a heparicath attached to a 3-way stopcock was placed in the antecubital
`region of the non-dominant arm. On two of the testing days, 3cc of saline was injected
`intravenously at approximately 0830 hrs (i.e., on the placebo and CST days), except for the
`Dex/CRF day, when ovine corticotropin-releasing hormone (Ferring Laboratories) (1 μg/kg)
`diluted in saline was injected intravenously by slow push (over 10 min). Also at 0830 hrs,
`participants ingested a second capsule containing either placebo (i.e., on the placebo and
`Dex/CRF days) or citalopram (2 mg/ unit BMI: 37 mg minimum to 60 mg maximum
`dosage) with 8 ounces of water.
`
`During each laboratory session, participants reclined in a chair and were asked to stay calm
`throughout the session. Venous blood samples for baseline measures of cortisol, ACTH,
`electrolytes, LFTs and sex steroid concentrations (women only) were obtained at −45 and
`− 5 min pre-ingestion/-infusion of the capsule/IV solution. A Dinamap Pro 100 V2 (GE
`Medical Systems, Tampa, FL) and digital thermometer (Welch Allyn Sure Temp,
`Skaneateles Falls, NY) were used to measure blood pressure, heart rate and body
`temperature, respectively, at these baseline timepoints. Following administration of the study
`drugs, blood samples and vital sign measurements were obtained every 15 min for the first
`hour, and then every 30 min for the next 4 h, to monitor the endocrine and cardiovascular
`effects of the various challenge agents.
`
`Psychoneuroendocrinology. Author manuscript; available in PMC 2019 March 11.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Anthenelli et al.
`
`Page 6
`
`Subjective measures of drug and mood effects were also obtained at these same timepoints.
`These included the Subjective High Assessment Scale (SHAS) (Schuckit, 1984) which
`measures an individual’s subjective experience of a drug across 13 domains (e.g., feeling
`high, sleepy, etc.) using visual analog scales; and the Profile of Mood States (POMS),
`another self-report assessment of an individual’s emotional state that uses a Likert rating
`scale (McNair et al., 1971).
`
`2.4. Stress hormone assays
`
`Venous blood samples were collected in ice-chilled vacutainer tubes containing 0.1 ml of
`15% EDTA. Plasma ACTH concentrations were measured using an immunoradiometric
`assay (DiaSorin; Stillwater, MN) with an assay sensitivity of ~ 1.0 pg/ml and intra- and
`interassay coefficients of variance < 10%. Plasma cortisol levels were measured by
`radioimmunoassay (Siemens Health Care Diagnostics; Los Angeles, CA). The assay has a
`sensitivity of ~0.3 μg/dl across a standard range from 0.5 to 50 μg/dl. The intra-and
`interassay coefficients of variance were < 10%
`
`2.5. Data analyses
`
`Demographic and clinical characteristics were analyzed using analysis of variance
`(ANOVA) with Tukey-adjusted post hoc tests, chi- square tests or non-parametric median
`tests. ANOVA models were used to analyze baseline measures, peak changes from baseline
`measures, and hormone area-under-the-curve (AUC) values. Mixed linear models were used
`to analyze the repeated measures hormonal ratio data across the 13 time points on each of
`the test days. Log- or square root-transformations were used on the AUC and peak change
`from baseline hormonal data to correct for non-normality. For clearer interpretation of the
`results, however, all data presented are raw data. Significance levels for all hypothesis testing
`were 0.05 and test were two-sided. All analyses were performed using SAS version 9.2
`(Cary, N.C.).
`
`3. Results
`
`3.1. Demographics and clinical characteristics
`
`Table 1 presents the demographic and clinical characteristics of the sample demonstrating
`the expected main effects of a diagnosis of AD on marital status, household income,
`educational attainment level, alcoholism severity score, drinking parameters and lifetime use
`of other drugs. AD men were significantly older than men and women in the other three
`groups, and AD women had significantly higher BMIs than participants in the other groups.
`AD women had been abstinent longer than AD men, and reported the highest rates of
`lifetime cocaine/stimulant abuse or dependence compared with the other three groups.
`
`In order to examine which of the demographic and clinical variables should be considered
`for inclusion as covariates in our subsequent models of stress responsivity, Spearman
`correlations were examined among all variables found to significantly differ across the four
`groups (e.g., age, BMI, days abstinent) with the baseline and area under the curve (AUC)
`hormonal measures on each test day. If any demographic or clinical variable was
`significantly correlated with the hormonal measures, then it was included in data analyses as
`
`Psychoneuroendocrinology. Author manuscript; available in PMC 2019 March 11.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Anthenelli et al.
`
`Page 7
`
`a covariate. Based on this analysis, three variables - age, family history of alcoholism, and
`any history of illicit drug abuse or dependence - were included as covariates in subsequent
`analyses. We also included order in the models to control for the effects that receiving the
`various stimulation agents in different sequences may have had on the study results.
`
`3.2. Baseline resting AM hormonal concentrations
`
`Fig. 2 illustrates the baseline ACTH and cortisol concentrations across the three test days.
`As depicted in the upper panel (Fig. 2A), women had lower resting AM ACTH levels than
`men on the placebo test day (main sex effect: F = 7.69; df = 1; p < 0. 007), but there were no
`significant sex or alcohol group effects for ACTH on either the Dex/CRF or citalopram test
`days. Baseline cortisol concentrations, however, did not significantly differ across the four
`groups on any of the test days (Fig. 2B).
`
`3.3. Placebo day differences in hormonal concentrations
`
`Serum ACTH and cortisol levels on the placebo day are depicted in Fig. 3. After controlling
`for significant baseline ACTH differences (F = 520.0; df = 75; p < 0.001), results of a
`repeated measures ANOVA revealed a significant time effect (F = 3.4; df = 11; p < 0.001)
`and a sex x time interaction for ACTH (F = 2.7; df = 11; p < 0.006) indicating that women’s
`ACTH levels increased over time while men’s did not (see upper panel, Fig. 3A). There
`were no significant main effects of alcohol group (p = .11), sex (p = .25), or their interaction
`(p = .67).
`
`After adjusting for baseline cortisol concentrations (F = 419.4; df = 75; p < 0.0001), an
`identical analysis for the cortisol levels on the placebo day revealed a main effect for sex (F
`= 4.6; df = 1; p < 0.04), time (F = 3.2; df = 11; p < 0.002), and a significant sex x time
`interaction (F = 2.2; df = 11; p < 0.03; see Fig. 3B). Women had lower cortisol levels than
`men throughout most of the morning test hours. There was no alcohol group effect (p = .64)
`or group x sex interaction (p = .81), but there was a significant effect of a family history of
`alcoholism (F = 7.0; df = 1; p < 0.01) with family history positive (FHP) individuals having
`lower cortisol concentrations over time compared with family history negative (FHN)
`participants.
`
`Given these findings of sex differences at rest and in response to a placebo challenge, the
`placebo day AUC response was used as a covariate in all subsequent analyses.
`
`3.4. Sex- and stressor-specific hormonal responses
`
`The upper panel of Fig. 4 portrays the net integrated serum ACTH (left, Fig. 4A) and
`cortisol (right, Fig. 4B) responses to the three neuroendocrine stimulation tests using AUC
`values as the dependent variable. Regarding the ACTH response, after controlling for
`significant baseline ACTH (F = 102.5; df = 1,68; p < 0.001), family history of alcoholism
`(FHP > FHN: F = 4.1; df = 1,68; p < 0.05), and order (F = 2.5; df = 5,68; p < 0.05) effects,
`ANCOVA results revealed a significant sex x treatment (test day) interaction effect (F = 6.2;
`df = 1,68; p < 0.02) without any main sex (p = .78), AD group (p = .92) or treatment (p = .
`85) effects. Post hoc analyses demonstrated a trend (p < 0.1) for women to have greater
`ACTH release in response to the Dex/CRF test than with the citalopram test.
`
`Psychoneuroendocrinology. Author manuscript; available in PMC 2019 March 11.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Anthenelli et al.
`
`Page 8
`
`Evidence for pharmacological stressor-specificity was even more apparent upon examination
`of the cortisol AUC measures (upper right panel, 4.B). Women had significantly greater
`cortisol responses to the Dex/CRF test as compared to the citalopram test (treatment x sex
`interaction): t = −5.09; df = 68; p < 0.0001) even after controlling for significant placebo (F
`= 9.0); df = 1,68; p < 0.004) and order (F = 4.20; df = 5,68; p < 0.003) effects. There was
`also evidence of a sexually-dimorphic response to the combined Dex/CRF test: following
`dexamethasone suppression, women had significantly greater cortisol responses to
`exogenous CRF than men (treatment x sex interaction: t = 2.49; df = 68; p < 0.02).
`
`A similar pattern of findings emerged when the peak change from baseline hormonal
`measures served as the dependent variables in the ANCOVA models (lower panels, Fig. 4C
`and D). However, when this metric of HPA axis stimulation was used, the corollary of our
`previous finding was revealed with men demonstrating stressor-specific response patterns
`across the testing days. Regarding the ACTH response (lower left panel, Fig. 4C), after
`controlling for significant baseline ACTH (F = 43.1; df = 1,68; p < 0.0001), placebo day (F
`= 21.9; df = 1,68; p < 0.0001), and order (F = 3.1; df = 5,68; p < 0.02) effects, ANCOVA
`results revealed significant treatment (test day) (F = 7.3; df = 1,68; p < 0.009) and sex x
`treatment interaction effects (F = 6.9; df = 1,68; p < 0.015) without any AD group (p = .97)
`or AD group interaction effects (e.g., group x sex: p = .84). Thus, men had significantly
`greater ACTH responses on the citalopram test day than they did on the Dex/CRF day
`(treatment x sex: t = 3.8; df = 68; p = .0003). Moreover, men had significantly lower ACTH
`responses than women in response to the Dex/CRF test (treatment x sex: t = 2.7; df = 68; p <
`0.008) after controlling for the aforementioned variables.
`
`A nearly identical pattern of responses was observed for the peak change in cortisol
`responses (lower right panel, Fig. 4D), where again, after controlling for significant baseline
`cortisol (F = 19.2; df = 1,68; p < 0.0001), placebo day (F = 37.8; df = 1,68; p < 0.0001), age
`(F = 4.4; df = 1,68; p < 0.04) and order effects (F = 4.1; df = 5,68; p < 0.003), a treatment x
`sex interaction emerged (F = 8.9; df = 1,68; p < 0.004). Consistent with the ACTH results,
`men had significantly greater peak cortisol responses to citalopram than Dex/CRF (treatment
`x sex: t = 3.0; df = 68; p < 0.003). The sexual diergism in the response to the Dex/CRF test
`was again apparent with women having significantly greater peak change in cortisol
`responses compared with men (treatment x sex effect: t = 3.09; df = 68; p < 0.003).
`
`3.5. Sex- and pharmacological stressor-specific differences in adrenal sensitivity
`
`To determine whether there were sex or alcohol group effects on adrenal sensitivity, we
`calculated the cortisol to log (ACTH) ratios for each of the three neuroendocrine stimulation
`tests. This ratio has been used by others (Ulrich-Lai and Engeland, 2002) as an indirect
`measure of the adrenal’s response to pituitary ACTH. In the resting non-stimu- lated
`baseline state, cortisol to log (ACTH) ratios were similar across all four groups on each test
`day, albeit markedly smaller in magnitude on the Dex/CRF day when cortisol and ACTH
`concentrations were suppressed by the presence of the synthetic glucocorticoid (data not
`shown).
`
`However, examination of these ratios over time across each of the three test sessions
`revealed further evidence that this indirect marker of adrenal sensitivity appears to vary by
`
`Psychoneuroendocrinology. Author manuscript; available in PMC 2019 March 11.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Anthenelli et al.
`
`Page 9
`
`sex and fluctuate in response to different pharmacological stressors. Fig. 5 portrays the plots
`of the cortisol to log (ACTH) ratios on the citalopram (upper panel, Fig. 5A), Dex/CRF
`(middle panel, Fig. 5B), and placebo (lower panel, Fig.5C) test days, respectively. After
`controlling for significant baseline differences and accounting for the effects of age, illicit
`drug use, and family history of alcoholism, men exhibited greater degrees of adrenal
`sensitivity (i.e., greater cortisol to log (ACTH) ratios) than women on both the citalopram
`and placebo test days. That is, results of separate repeated measures ANCOVAs revealed
`significant main effects for sex (citalopram: F = 4.3; df = 72, p < 0.05; placebo: F = 7.2, df =
`72, p < 0.01) as well as significant sex x time interactions (citalopram: F = 2.0, df = 72, p <
`0.04; placebo: F = 1.9, df = 72, p = .05) on each of these test days.
`
`A different pattern of results emerged, however, when we examined these ratios on the
`Dex/CRF day (see Fig. 5B). After controlling for significant baseline (F = 203.3; df = 1,72;
`p < 0.0001) and illicit drug abuse effects (i.e., participants who had abused drugs in the past
`had higher ratios; p < 0.03), there was a main sex effect (F = 4.7; df = 72; p < 0.04) without
`any significant sex x time interaction (p = .2). Thus, under the conditions of the Dex/CRF
`test day, women had greater cortisol to log (ACTH) ratios than men and this sex difference
`did not fluctuate as a function of time.
`
`3.6. Cardiovascular and body temperature response
`
`Men had higher baseline systolic (p-values < 0.003) and diastolic (p-values < 0.02) blood
`pressure levels, respectively, compared with women on all three test days (see Supplemental
`Table 1). Peak changes in diastolic blood pressure were not significantly different across the
`four groups on any of the test days; however, the elevated systolic and diastolic blood
`pressures observed in men at baseline remained higher than women’s throughout each of the
`test sessions (data not shown). There were no significant differences in either baseline or
`peak change in heart rates across the groups on any of the test days.
`
`Regarding the temperature responses, there were no significant sex or alcohol group effects
`on any of the test days (data not shown). However, there was a pharmacological stressor-
`specific elevation in peak change in body temperature: women exhibited significantly
`increased body temperatures compared with men on the Dex/CRF day (main sex effect: F =
`11.8; df = 1; p < 0 .001) without any alcohol group (p = .9) or sex x group (p = .9)
`interaction effects. Body temperature changes were similar across the four groups on the
`citalopram and placebo test days.
`
`3.7. Subjective drug and mood responses
`
`Baseline total SHAS scores and peak Δ from baseline SHAS scores were examined on each
`test day. Since SHAS results were not normally distributed, this variable was dichotomized
`(i.e., no SHAS response vs. any response) and logistic regression analysis was used. There
`were no significant sex effects across the three test days, nor were there any significant
`alcohol group or group x sex interaction effects on the Dex/ CRF and placebo days (data
`now shown). On the citalopram day at baseline, a significant group effect (p < 0.04) was
`observed, and there was a trend for a group x sex interaction (p < 0.07). Peak change in
`
`Psychoneuroendocrinology. Author manuscript; available in PMC 2019 March 11.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Anthenelli et al.
`
`Page 10
`
`SHAS scores did not significantly differ as a function of sex or alcohol group status on any
`of the test days (data not shown).
`
`Supplemental Fig. 1 depicts the POMS total score at baseline across the four groups on each
`of the neuroendocrine challenge test days. Separate ANCOVAs controlling for age, family
`history of alcoholism and history of illicit drug ab