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The Effects of Psychosocial Stress, Hormonal Contraceptives, Exercise, and Alcohol on Cortisol, Perceived Stress, and Desire

for Alcohol

Matt London

San Jose State University

Submitted to Psychoneuroendocrinology for Spring 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 exert

anxiolytic effects in the presence of psychosocial stress. This project conducted 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 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.

Keywords: cortisol, corticosteroid-binding-globulins, Trier Social Stress Test,

HCs, attenuated response, psychosocial stress, ethinyl estradiol, alcohol

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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 the blood, 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 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 tasks to elicit a valid and reliable psychosocial

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stress inducing 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 after TSST; 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 Since these findings, many

studies have used the TSST and concluded that upon termination of psychosocial

stress, the salivary cortisol response becomes evident at 5-20 min and peaks at 10-30

min (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)

levels of significance for classification of cortisol responders (i.e. participants who

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

exhibit low levels of 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.

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When subjected to psychosocial stress, females who use 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, in hormonal contraception,

causing an abnormal rise in CBG’s, therefore attenuating bio-active cortisol

(Kirschbaum et al., 1995; Kumsta et al., 2007).

The study-wide effects 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).

Furthermore, HC excluded cortisol non-responders of Kimura (2013) showed a 1.07

nmol/l (44%) less baseline-to-peak cortisol decrease than the HC included cortisol non-

responders of Kunz-Ebrecht et al. (2003) and Buchanan & Tranel (2008).

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

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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).

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, Chennaoui, & Guezennec,

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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).

Study 1 hypothesized 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).

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

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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 Method

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

(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 1Task sequence and cortisol sampling

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Study 2 Method:

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

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 calculation were performed using R statistical software package.

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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).

Figure 3: Relative comparison of cortisol responders

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Study 2

Although there was 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 2.

Table 2. Mean PSQ Scores For Each Group in terms of TSST, Vigorous Exercise (Vig),

and Alcohol (Alc)

TSST

Vig

Alc

TSST

Vig

NoAlc

TSST

NoVig

Alc

TSST

NoVig

NoAlc

NoTSST

Vig

Alc

NoTSST

Vig

NoAlc

NoTSST

NoVig

Alc

NoTSST

NoVig

NoAlc

M

OC 3/3071

(n=1)

47

(n=1)

60.5

(n=2)

50

(n=1)

58.5

(n=2)

57.4

(n=7)

OC 1/2062

(n=1)

74

(n=1)

68

(n=2)

IUD

0.12/15

53

(n=1)

53

(n=1)

No HC

Female

56.5

(n=2)

64.5

(n=2)

46.5

(n=2)

65.9

(n=2)54 (n=1)

63.3

(n=4)

85

(n=1)

62

(n=2)

62.2

(n=16)

Male67

(n=2)

56.6

(n=6)

63.5

(n=3)

61.3

(n=4)

67.5

(n=2)

49

(n=1)

61

(n=1)

51.7

(n=3)

59.7

(n=22)

M64.1

(n=6)

60.6

(n=8)

52.3

(n=6)

63.6

(n=6)

60.5

(n=3)

57.6

(n=7)

64.6

(n=5)

57.4

(n=7)

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

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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

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

Figure 3: Relative comparison of alcohol desire within stress group

Outlier

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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 63 rd

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

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 blood and salivary cortisol. By

doing so, a total cortisol level will be exhibited in the blood 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

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

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

perceived stress and cortisol response to psychosocial stress. Guaging 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 5.5 mins. of 52 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 between 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.

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Acknowledgements

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

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

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