The Effects of Psychosocial Stress, Hormonal Contraceptives, Exercise,

and Alcohol on Cortisol, Perceived Stress, and Desire for Alcohol

Matt London

San Jose State University

May 12, 2016

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Abstract

Psychosocial stress, defined as aversive or demanding conditions that tax or exceed the

behavioral resources of the organism, incites a bioactive cortisol response, which is measured by

analysis of salivary cortisol produced after participant subjection to the Trier Social Stress Test, a

reliable psychosocial stress inducing paradigm. Hormonal contraceptive users, when subjected to

psychosocial stress, manifest an abnormally large rise in corticosteroid-binding-globulin levels.

Bio-active cortisol is rendered inactive by corticosteroid-binding-globulins and results in a

blunted cortisol response to psychosocial stress. Evidence suggests a direct correlation between

the responses to psychosocial stress of salivary cortisol and perceived stress. Exercise and

alcohol are often used as a stress coping mechanism and have been shown to exert anxiolytic

effects in the presence of psychosocial stress.

This project comprised two separate studies to examine the interactions and effects of

hormonal contraceptive use, psychosocial stress, exercise habits, and alcohol use on salivary

cortisol levels, perceived stress, and desire to drink alcohol. Statistical significance was found for

hormonal contraception causing a blunted salivary cortisol response. Observational analysis

revealed that both genders report similar desire to continue drinking alcohol after being subjected

to psychosocial stress and given one drink. Although, when stressed and given alcohol placebo,

men show a decrease in drinking desire and females show an increase. Implications for more

controlled research on cortisol response to psychosocial stress are discussed.

Keywords: alcohol, cortisol, exercise, hormonal contraceptives, perceived stress, psychosocial

stress, Trier Social Stress Test

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Introduction

Psychosocial stress is defined as aversive or demanding conditions that tax or exceed the

behavioral resources of the organism (Lazarus, 1966). The magnitude of its effects on an

organism can be measured by analysis of cortisol response (Balodis, Wynne-Edwards, &

Olmstead, 2010; Campbell & Ehlert, 2012; Dickerson et al., 2004; Kirschbaum et al. 1989;

Kirschbaum et al., 1999).

Cortisol, a glucocorticoid, serves as a negative feedback mechanism within the

hypothalamic-pituitary-adrenal axis. Psychosocial stress incites the hypothalamus to release

corticotropin-releasing hormone (CRH) to the anterior pituitary, which in turn releases

adrenocorticotropic hormone (ACTH) to the adrenal cortex. Within the zona fasciculata of the

adrenal cortex, cortisol is produced and released to the hypothalamus and the anterior pituitary,

within a negative feedback loop, to initiate inhibitory actions on these glands and to modulate

stress and inflammation pathways.

Only 2-15% of the released cortisol is not bound to protein carriers such as

corticosteroid-binding globulin (CBG), albumin, and erythrocytes. The unbound (“free,”

bioactive) proportion of cortisol is able to cause genomic cortisol effects in peripheral tissues and

the brain, as opposed to the bound cortisol that cannot cause such effects. Bio-active and bound

cortisol is found in serum, while only bio-active cortisol in found in the saliva. For this reason,

salivary cortisol collection is preferred as a more efficient and cost-effective method than serum

cortisol analysis, which can often prove to be counterproductive when the act of venipuncture,

itself, incites significant HPA activation and, therefore, an undesired cortisol response

(Kirschbaum & Hellhammer, 2000).

To incite a psychosocial stress induced salivary cortisol response, numerous studies have
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utilized public speaking (Bassett, Marshall, & Spillane, 1987) or mental arithmetic

(Frederickson, Tuomisto, Bergman-Losman, 1991; Jorgensen et al., 1990; Trestman et al., 1991)

or both (Kemmer et al., 1986), although they were not standardized into one reliable protocol.

Due to the insignificant cortisol responses and lack of reliability of these previous

protocols, Kirschbaum et al. (1993) created the Trier Social Stress Test (TSST), which consists

of a protocol that utilizes both public speaking and mental arithmetic tasks to elicit a valid and

reliable psychosocial stress induced salivary cortisol response. These researchers revealed a high

reproducibility across five separate studies, in which the psychosocial stress of the TSST induced

salivary cortisol levels to peak 10 min following TSST completion; Baseline: 4 to 9 nmol/l,

Response: 5.3 to 8.2 nmol/l above baseline levels. Males exhibit a mean salivary cortisol

response that is 1.5- to 2-fold higher than females. The salivary cortisol response to the TSST

becomes evident at 5- 20 min and peaks at 10-30 min following TSST completion (Kirschbaum

& Hellhammer, 2000). Typically, salivary cortisol follows a standard circadian rhythm where it

is boosted upon awaking in the morning and increases 50-100% until it peaks after 30 min.,

where after it decreases until, in response to substantial amounts of food at lunch time, it peaks at

about 150% and steadily decreases throughout the afternoon and into the evening without

another major secretory instance (Kirschbaum & Hellhammer, 2000).

Considering that a typical cortisol secretory episode initiates a 55.2 nmol/l increase of

total plasma cortisol and that salivary cortisol constitutes 2% to 5% of plasma cortisol

Kirschbaum et al. (1992) and Wust et al. (2000) established a 2.5 nmol/l salivary cortisol

increase to be valid classification criterion for a significant cortisol response.

A lack of systematic evaluation studies of the 2.5 nmol/l criterion has led many

experimenters to utilize 30% (Kimura et al., 2013) and 40% (Kunz-Ebrecht et al., 2003)
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levels of significance for classification of cortisol responders (i.e. participants who exhibit high

levels of psychosocial stress induced cortisol) and non-responders (i.e. participants who exhibit

low levels of psychosocial stress induced cortisol). Binary median splits were not utilized due to

a high number of participants who were highly indistinct in terms of their cortisol response

profile. Since its inception, the 2.5 nmol/l criterion has resulted in a high rate of 16.5% false-

negative classifications, where non- responders were classified as responders. Miller et al. (2013)

conducted a study to develop a cortisol response classification criterion that was more accurate

than the 2.5 nmol/l criterion. As a result, Miller et al. (2013) found a 15.5% baseline-to-peak

increase criterion to be an accurate appraisal of cortisol response, exhibiting 26.7% less

misclassifications.

When subjected to psychosocial stress, females who use hormonal contraception (HC)

exhibit “blunted or completely absent salivary cortisol responses,” despite significant secretion

of cortisol by the adrenal cortex (Kirschbaum et al., 1999). This phenomenon is explained by the

combination of psychosocial stress and ethinyl estradiol, the synthetic estrogen in HC, causing

an abnormal rise in CBG’s. Therefore, bio-active cortisol is attenuated. (Kirschbaum et al., 1995;

Kumsta et al., 2007).

The study-wide effect of the HC attenuated stress response is evident by the HC excluded

cortisol responders of Kimura (2013) showing an average stress induced baseline-to-peak

cortisol response at 9.38 nmol/l (38%) higher than the HC included cortisol responders of Kunz-

Ebrecht et al. (2003) and Buchanan & Tranel (2008). Non- responders of all three studies

exhibited salivary cortisol decreases following TSST.

Although, HC excluded cortisol non-responders of Kimura (2013) showed an average

1.07 nmol/l (44%) less baseline-to-peak cortisol decrease than the HC included cortisol
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non-responders of Kunz-Ebrecht et al. (2003) and Buchanan & Tranel (2008). The results of

these studies demonstrate the misleading data that may result from not excluding females who

use HCs from studies that administer psychosocial stress.

Studies have shown a direct correlation between the responses to psychosocial stress of

perceived stress and salivary cortisol. Kirschbaum, Pirke, and Hellhammer (1995) observed a

positive correlation of the post-TSST subjective rating of “having been nervous” and the salivary

cortisol response in both genders, regardless of HC. Kimura et al. (2013) showed that perceived

stress was increased by the TSST and immediately returned to baseline level; non-responders

reported less perceived stress than responders Since salivary cortisol response becomes evident

at 5-20 mins and peaks at 10-30 mins, this suggests that the salivary cortisol response to

psychosocial stress may be initiated by perceived stress.

Anxiety driven behavior (e.g. alcohol use) has been shown to be attenuated by the

interoceptive exposure provided by regular vigorous exercise (Medina et al., 2011; Smits, et al.,

2008; Vujanovic, et al., 2008). Medina, et al. (2011) examined 114 adults (58 women; Mage =

22.31 years, SD = 8.89) who reported exposure to at least one traumatic event and alcohol use in

the past 30 days. Medina, et al. (2011) utilized the Exercise Habits Questionnaire-Revised (EHQ-

R) in their study as a self-reported descriptive measure of physical activity of the participants.

The researchers discovered an inverse relationship between participants’ engagement in high-

intensity exercise and coping oriented alcohol use (t(57) = -2.01, p<.05). This effect was not

exhibited by participants who reported engagement in moderate- or low-intensity exercise. The

aversive symptoms (e.g. hyperarousal) that may provoke the coping oriented alcohol use among

trauma exposed individuals may be reduced by the interoceptive exposure introduced with high-

intensity exercise (Medina, et. al., 2011; Smits, et al., 2008; Vujanovic, et al., 2008).
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Smits, et al. (2012) utilized the EHQ-R in their study that found greater moderate-

intensity exercise to be significantly predictive of lower coping oriented marijuana use (β = −.15,

p < .05). The researchers expanded on previous studies that have investigated the links between

exercise intensity and anxiety sensitivity, such that substance use is used as a result of high

anxiety sensitivity and specific intensity levels of exercise decrease anxiety sensitivity (Buckner,

et al., 2007; Otto & Reilly-Harrington, 1999).

Changes in the basal nucleus of the stria terminalis (BNST) 5-HT receptor subtypes may

lead BNST neurons to prefer excitation and produce a pathological state of heightened anxiety

(Hammack, 2008). The interoceptive effect of exercise may down-regulate postsynaptic 5HT

2B/2C receptors, resulting in reduced anxiety.

An up-regulation of mRNA for 5-HT1A somatodendritic autoreceptors in the dorsal

raphe nucleus (DRN) is shown to result from six weeks of voluntary exercise (Greenwood et al.,

2003). If this up-regulation results in an up-regulation of receptor protein, the additional 5-HT

1A autoreceptors would attenuate DRN activity by ameliorating autoinhibition of DRN cell

firing, thus reducing 5-HT release in DRN projection areas that are known to be integral in

anxiety-related behaviors.

Voluntary exercise and forced exercise have shown neurobiological differences.

Voluntary exercise is associated with decreased stress induced elevation of 5-HT metabolite 5-

hydroxyindole acetic acid in the hippocampus and amygdala. This suggests that exercise

attenuated 5-HT function in these DRN targets, which are associated with anxiety-related

behavior (Dishman et al., 1997). Conversely, forced exercise has been shown to augment 5-HT

release in the hippocampus, frontal cortex, and spinal cord (Bequet, Gomez-Merino, Berthelot,

& Guezennec, 2001, 2002; Gerin, Legrand, & Privat, 1994; Gomez-Merino, Bequet, Berthelot,
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Chennaoui, & Guezennec, 2001; Meeusen et al., 1996). Therefore, forced exercise may also

produce substantially different effects on the 5-HT system. Much more research must be

conducted on the effects of exercise on anxiety (Berchtold, Chinn, Chou, Kesslak, & Cotman,

2005).

The hypothesis of Study 1 was that females who use HC are more likely to be classified

as cortisol non-responders, exhibiting a cortisol increase of less than 15.5% above baseline

(Miller et al. 2013), in response to the psychosocial stress induced by the TSST. It has been

shown that there are significant gender differences in salivary cortisol response to psychosocial

stress, although not to exercise induced physical stress (Kirschbaum et al., 1992). Study 2 sought

to determine the presence of gender differences of perceived stress in response to psychosocial

stress, with consideration of exercise habits, when participants were given alcohol to intoxication.

Methods

Subjects and General Experimental Outline

In two separate studies, a total of 113 healthy subjects were subjected to the TSST. Study 1

comprised of 65 females, of which 16 females (Mage = 18.9, SD = 1.4) currently used hormonal

contraception and 49 females (Mage = 18.8, SD = 1.1) currently did not use hormonal

contraception. Study 2 comprised of 48 participants, of which there were 7 females who

currently used OC that delivered 3mg drospirenone and 30mcg ethinyl estradiol per day (group

OC 3/30; Mage = 25.9, SD = 9.3), 2 females who currently used OC that delivered 1mg

norethindrone acetate and 20mcg ethinyl estradiol per day (group OC 1/20; Mage = 21, SD = 0),

one female that currently used an intrauterine device (IUD) that delivered 0.12mg etonogestrel

and 15mcg ethinyl estradiol per day (Mage = 21, SD = 0), 16 females who did not currently use

HCs day (Mage = 22.6, SD = 2.5), and 22 males (Mage = 23.6, SD = 3.4).
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All participants were recruited from undergraduate Psychology courses, at an

ethnically diverse university located in Northern California, and were given course credit

for their participation in the study. Participants were notified of the opportunity to

participate in the study by their professor. Before entering the study, all participants

provided written consent and completed a medical questionnaire that screened for and

excluded participants who had past or current health problems and who had used

prescription medication, except hormonal contraception, and/or nicotine in the past six

months.

Study 1 Methods

All cortisol samples were salivary cortisol samples collected with Salimetrics

SalivaBio Oral Swabs (SOS’s). Four salivary cortisol samples were collected at 10-20

min intervals. The first salivary cortisol sample was collected at 13 min following

participant's arrival to the laboratory, and a screening questionnaire, as a measure of

baseline salivary cortisol. The second salivary cortisol sample was collected

immediately following the TSST, 28 min post-participant arrival. The third cortisol

sample was collected 10 min post-TSST cessation (38 min) post-participant arrival as a

measure of peak salivary cortisol onset. The fourth, and final, salivary cortisol sample

was collected at 30 min post-TSST cessation (20 min post-3rd sample collection, 58

min post-participant arrival) as a measure of peak salivary cortisol cessation. Figure 1

displays the relative collection times of each of the four salivary cortisol samples. The

first cortisol sample was used to assess a salivary cortisol baseline. It should be noted

that the 3rd cortisol sample was intended to measure peak cortisol levels. Salivary

Cortisol samples were prepared with Salimetrics Cortisol Salivary Immunoassay Kit’s
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(ELISA/EIA), MTX Pipettes, Model 614L Laboratory Centrifuge by the Druker Lab, and a

Fisher Stirring Hotplate. Prepared cortisol samples were analyzed with a Finstruments

Microplate Reader by MTX Lab Systems Inc. and DeltaSoft JV Data Template.

Figure 1: Study 1 Task sequence and cortisol sampling

Study 2 Methods:

Tests and Measures

Perceived Stress Questionnaire (PSQ; Levenstein et al., 1993): a measure of subjective

trait stress comprising 30-items that the participant rates on a Likert-scale of 1-4 the

degree to which they felt the item applied to them in the past month, 4 being the highest

degree (e.g. #9. You fear you may not manage to attain your goals).

Exercise Habits Questionnaire-Revised (EHQ-R; Zvolensky, 2008), a self-report

descriptive measure of intensity and frequency of physical exertion (Medina, et. al.,

2011; Smits, et al., 2008; Vujanovic, et al., 2008), was used to determine if a participant

was a regular vigorous exerciser.

Desire to Drink Questionnaire comprised of a Likert-scale of 1-5, 5 being highest

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second drink desire, where the participant indicates desire to consume more alcohol.

Procedure

Psychosocial Stress was induced with the TSST.

Alcohol: Participants were given either alcohol or placebo (0.65mg/Kg vs. 0.00mg

alcohol/body weight).

Figure 2: Study 2 Task Sequence

Statistical Analysis

Factor weighting was used to account for missing PSQ data (Montero-Marin et al., 2014). All

statistical calculations were performed using R statistical software package.

Results

Study 1

A chi-squared test showed a significant difference in percentage of responders

depending on birth control use, (X2(1, 65) = 5.11, p = .02). Based on a 15.5% cortisol increase

from baseline to be classified as a responder, females who use HC demonstrated a lower

percentage of responders (56%) compared to females who do not use HC (84%) (see Figure 3).
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Figure 3: Relative comparison of cortisol responders

Study 2

Although there were no statistically significant interaction effects of contraceptive use,

exercise habits, psychosocial stress, and alcohol on PSQ scores (F(3, 45) = 1.515, p = 0.229).,

observational analysis reveals different PSQ scores based on progestin type in HC. Participants

who used HC that delivered norethindrone acetate as progestin reported a mean PSQ score of

68. Conversely participants who used HC that delivered drospirenone or etonogestrel as

progestin reported a mean PSQ score of 55.2 and a median of 57. Results are exhibited in

Table 1.
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Table 1. Mean PSQ Scores for each group in terms of TSST, Vigorous Exercise (Vig), and

Alcohol (Alc).OC 3/30: 3mg drospirenone/30mcg ethinyl estradiol per day, OC 1/20: 1mg

norethindrone acetate/ 20mcg ethinyl estradiol per day, IUD 0.12/15: 0.12mg

etonogestrel/15mcg ethinyl estradiol per day.

The lowest PSQ average was reported by the group that was not of vigorous

exercise and was subjected to psychosocial stress and alcohol. While the highest PSQ

average was reported by the group that was also not of vigorous exercise, although had

not been subjected to psychosocial stress and alcohol. This observation suggests that

alcohol may provide a greater magnitude anxiolytic effect than the anxiogenic effect of

psychosocial stress, sufficient to result in less perceived stress than the absence of both

stress and alcohol.

Within the TSST group, there was a significant interaction of gender and the

placebo effect on drinking desire scores. Both genders report similar desire to continue

drinking alcohol after being subjected to psychosocial stress and given one drink.

Although, when stressed and given alcohol placebo, females show a rise in drinking
 13

desire, while men show a decrease. (See Figure 4)

Figure 4: Relative comparison of alcohol desire within stress group

Outlier

Of the seven participants in group OC 3/30, six were of either age 21 or 22. One

participant was 46, participant 6. Furthermore, 47 of the 48 participants were aged

between 21 – 28. Participant 6 was a vigorous exerciser who did not receive TSST or

alcohol. She reported a PSQ of 69, second highest, to 71, in the OC 3/30 and in the 63rd

percentile overall. The case of this outlier suggests that age attenuates the effects of
 14

ethinyl estradiol on perceived stress. Future studies should examine how age,

specifically around menopause, affects the effects of ethinyl estradiol on perceived stress.

Discussion

Study 1 supported the results of other studies have found a significant negative

correlation with HC use and salivary cortisol response to psychosocial stress, induced

by the TSST. Since saliva is the only vehicle that is used for cortisol collection that

exclusively contains bioactive cortisol, studies that investigate total cortisol must consider

this factor. This study agrees with Kirschbaum et al. (1999) that future studies that

investigate cortisol responses should measure both serum and salivary cortisol. By doing

so, a total cortisol level will be exhibited in the serum and strictly bioactive cortisol will

be manifest in the saliva.

Based on these findings, it is recommended to re-analyze studies that have

investigated the cortisol response to psychosocial stress, did not exclude females who use

HC, and did not classify cortisol non-responders as those who exhibit less than a 15.5%

salivary cortisol increase from baseline. By removing data derived from females who used

HC and conducting analysis of the new dataset, results with more validity may be

garnered. Future studies that exclude HC users subjected to psychosocial stress will save

an average >$30 in supplies and >5 work hours.

In Study 2, the mean PSQ score of participants who used norethindrone acetate

as progestin (67) is closer to the PSQ median score found by Montero-Marin et al.

(2014) (70.29) than the median PSQ score of participants who used HC that delivered

drospirenone as progestin (57) and those who used etonogestrel as progestin (53). This

observation suggests that the type of progestin used in HC may have substantial effects
 15

on perceived stress and, thus, cortisol response to psychosocial stress. Future studies

should gauge the interactions and effects of different progestins and estrogens in HC on

perceived stress and cortisol response to psychosocial stress. Gauging the effects and

determining threshold levels will determine what data needs to be analyzed from

previous studies and screening measures that need to be enrolled in the future.

Study 2 revealed a placebo effect among both genders in the group that had

been subjected to the TSST. Both genders in the group that was given alcohol exhibited

a second drink desire of 3 out of 5. In contrast, in the placebo group, the mean desire

score was 1.8 for men and 4 for females. This effect may be due to a gender difference

in the placebo effect. Aslaksen et al. (2011) revealed that only males exhibit reduced

pain unpleasantness and stress following administration of 5.5 mins. of 52°C thermofoil

heat pain and placebo painkiller. When stressed and led to believe they have ingested

alcohol, females may be more prone, than men, to noticing that they are not physically

feeling the alcohol effects that they are expecting and, therefore, desire more than if

they had ingested alcohol. Conversely, men who are stressed and led to believe they

have ingested alcohol may be more satisfied with the psychological feeling of having

had alcohol (i.e. a reduction in negative emotions) and be less prone to continue

drinking than if they had ingested alcohol. The small HC sample size was likely to

contribute to the lack of significant difference second drink desire between HC users,

non-HC females, and males. Since HC users were not excluded from the drink desire

analysis of this study and Aslaksen (2011), it is advised to perform future studies that

examine the placebo effect differences between genders and HC users.

In summation, this project revealed the attenuating effect of HCs on psychosocial

 16

stress induced bio-active cortisol. Furthermore, the type and amount of each ingredient

in HCs appears to play a substantial role in perceived stress following psychosocial

stress. Alcohol and exercise habits were shown to exert moderate influence on

perceived stress sensitivity. Future studies that examine a larger size and collect

measurements for variables mentioned in both studies 1 and 2 are likely to see a more

robust direct correlation of perceived stress and cortisol, following psychosocial stress,

when HC, alcohol, and exercise habits are controlled for.

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Acknowledgements

Carlos Almeida, Jessica Ballin, Meylien Han, David Hunyh, Angela Mapanao, Michael

Namekata, Brissa Ortega, Mitzi Ochoa, Cheryl Chancellor-Freeland, Ph.D, and Mark

Van Selst, Ph.D.