Fridman, A.
Citation
Fridman, A. (2011, March 2). Surviving early childhood trauma : effects of the holocaust on survivors' psychological and physiolocial well-being.
Retrieved from https://hdl.handle.net/1887/16555
Version: Not Applicable (or Unknown)
License: Licence agreement concerning inclusion of doctoral thesis in the Institutional Repository of the University of Leiden
Downloaded from: https://hdl.handle.net/1887/16555
Note: To cite this publication please use the final published version (if applicable).
CHAPTER 5
Genetic Moderation of Cortisol Secretion
in Holocaust Survivors:
The Role of ADRA2B
Fridman, A., Bakermans-Kranenburg, M.J., Sagi-Schwartz, A., Van IJzendoorn, M.H.
Manuscript submitted for publication
Abstract
In the current study we tested whether ADRA2B moderates stress regulation of Holocaust survivors as indexed by their diurnal cortisol secretion and cortisol reactivity to a stressor. Salivary cortisol levels of 54 female Holocaust survivors and comparisons were assessed during a routine day and in response to a stress-evoking procedure. Holocaust survivors with the wildtype ADRA2B displayed higher cortisol levels than did comparisons with the same genotype, whereas no difference was found between these groups in carriers of the deletion variant, previously associated with more re-experiencing of traumatic events.
Carriers of the deletion variant might be driven in the long run to resolve their vividly remembered experiences and therefore show less stress dysregulation as evident from their cortisol levels.
Introduction
Holocaust survivors show alterations of diurnal cortisol secretion as well as in response to stress more than half a century after their traumatic experiences (Van der Hal-Van Raalte, Bakermans-Kranenburg, & Van IJzendoorn, 2008; Van IJzendoorn, Fridman, Bakermans-Kranenburg, & Sagi-Schwartz, in preparation).
The deletion variant of the ADRA2B gene has been associated with more vividly re-experiencing traumatic events in Rwandan civil war refugees (de Quervain et al., 2007). Here we examine the potential moderating role of ADRA2B on the effects of the Holocaust on HPA-axis functioning of survivors.
The Holocaust is the most widely studied example of a genocide with traumatic consequences for its survivors, and its study may help gain better understanding of the adaptation of victims of other, more recent genocides that took place in countries like Cambodia, Nigeria, Rwanda, Sudan, and former Yugoslavia. In a recent meta-analysis involving 12,746 participants (from 71 samples), Holocaust survivors were found to be less well-adjusted than their age mates who did not experience the Holocaust. In particular, they showed substantially more posttraumatic stress symptoms. At the same time, they also seemed rather well-adapted in the areas of physical health and cognitive functioning, suggesting remarkable resilience (Barel, Van IJzendoorn, Sagi- Schwartz, & Bakermans-Kranenburg, 2010).
However, traumatization may be present 'under the skin', and be manifested in neurobiological functioning. Stress regulation, as assessed by the HPA-axis functioning is one possible candidate for indexing such covert long-term effects of trauma. The development of the HPA axis has been shown to be modified by early environmental stressors (Liu et al., 1997; Meaney et al., 1991; Tarullo & Gunnar, 2006). For example, atypical patterns of daily cortisol secretion were found among Ukrainian children in institutional care (Dobrova-Krol, Van IJzendoorn, Bakermans-Kranenburg, Cyr, & Juffer, 2008; Dozier et al., 2006). Such dysregulation has been observed even more than six years after adoption (Gunnar, Morison, Chisholm, & Scheuder, 2001).
In the short run, cortisol reactivity to acute danger promotes survival.
However, if activated chronically, the HPA axis may exert detrimental effects on individuals‟ health, with cardiovascular and metabolic consequences, and immune suppression (Denson, Spanovic, & Miller, 2009; Sapolsky 2003). Post traumatic stress may continue to burden HPA-axis functioning if traumatic memories remain intrusive and persistent, and are not set aside as belonging to a past that will never return (McGaugh, 2003).
In healthy young adults the deletion variant of the ADRA2B gene has been shown to be associated with individual differences in emotional memory (de
Quervain et al., 2007). Rwandan civil war refugees were more likely to re- experience traumatic events when they were deletion variant carriers rather than wildtype carriers (de Quervain et al., 2007). These findings suggest that variants of the ADRA2B gene moderate the processing of traumatic memories, and may also be implicated in long-term effects of traumatic experiences on stress regulation.
Here we test whether ADRA2B moderates stress regulation of Holocaust survivors as indexed by their diurnal cortisol secretion and cortisol responses to a stressor.
Method
Participants
Participants were recruited from a population-wide demographic information base provided by the population registry administered by Israeli Ministry of Interior (Sagi-Schwartz et al., 2003). Two groups were compared:
female Holocaust survivors who had experienced the Holocaust in their childhood and were selected if they had suffered at least the loss of both parents due to the Holocaust, and a matched comparison group of females who were born in Europe and immigrated with their parents to the British Mandate of Palestine (pre-state of Israel) just before the Second World War. For the purpose of the current study, we contacted 106 participants. However, by time of the current data collection, 10 participants of the earlier phase of the study had passed away, and for reasons of health and personal problems, only 79 (75.4%) women were eligible for participation. Of them, 65 (82.3%) agreed to take part in the study (32 Holocaust survivors, and 33 comparison respondents). Their age ranged from 71 to 84 years (M = 76.98, SD = 2.99).
Procedure
Data were collected at the participants' home to their convenience. Cheek cells for genotyping were collected first, followed by the stress task and the completion of some questionnaires. The research assistant was available to answer the participants‟ questions and to offer emotional support throughout the entire session. She could be also reached by phone at any time after the data collection session. The study was approved by the ethics committee of the Institute for Education and Child Studies, University of Leiden, the Netherlands, and participants signed a written informed consent.
Measures
Physical Health. Physical health status was assessed by a questionnaire developed by Herczeg Institute on Aging (Tel Aviv University). This questionnaire
is widely used for socio-demographic research in Israel and was used in previous Holocaust studies (e.g., Van der Hal-Van Raalte et al., 2008). Participants were asked to rate their health on a 5-point Likert scale ranging from 1 = very unhealthy to 5 = very healthy. They were also asked to indicate which of 19 listed health problems they suffered. Participants could add more health conditions if they wanted, resulting in an overall list of 40 problems. The total number of health problems ranged from 0 (no health problems) to 15. The correlation coefficient between the two health measures was r = .62 (p < .01).
Trier Social Stress Test (TSST). The TSST (Kirschbaum, Pirke, & Hellhammer, 1993) is a procedure that includes both social-evaluative threat (speaking in the presence of a critical audience) and lack of control (an arithmetic task that is impossible to complete within the time constraints). The combination of the public speaking task and the impossible arithmetic task has indeed been shown to be one of the most effective psychosocial stressors (Buske-Kirschbaum et al., 1997;
Dickerson & Kemeny, 2004).
According to standardized instructions, participants were asked to convince university experts that their grandchild would deserve a scholarship. They were told that after a short preparation period they should introduce themselves in free speech to the video camera that was placed in the room and that the video cassette would be seen by University of Haifa experts who were trained to assess both verbal and non-verbal behavior. Following these instructions, participants were given five minutes to prepare for their talk without written notes. After five minutes they were asked to sit in front of the camera and give their speech.
Whenever the participant finished her speech in less than five minutes, the research assistant responded in a standardized way: "You still have some time left. Please continue!" If the participant still finished the talk before the allotted time was over, the research assistant became quiet for 20 sec and then asked some standard questions such as "why do you think you/ your grandchild is the best candidate for this job/ scholarship?" .
After the five-minute speech participants were asked to serially subtract the number 13 from 2083 as fast and accurately as possible, and they were told that their results would be evaluated by university experts. In order to increase stress, a metronome was turned on with 40 beats per minute, and every minute the research assistant made a comment, regardless of the participant‟s performance (e. g.,
"Please be more accurate"; "Please try to be more focused"). On every failure the participant was asked to start all over again. After three failures, participants were given the option to start from 1022, instead of 2083. The task was stopped after five minutes, and the research assistant debriefed the participants that no video analysis would in fact be conducted and that the comments were standard
regardless of their performance. For the purpose of relaxation and recovery after the TSST participants watched 25 minutes of the nature movie: "Flying Birds".
Cortisol levels. Cortisol levels were measured on two separate occasions: once during the home visit around the TSST, and the second time during a regular day.
Cortisol reactivity to stress was measured three times following the stressful procedure: 20, 30 and 40 minutes after the onset of the TSST, respectively.
Participants were not allowed to eat or drink anything but water until all saliva samples were collected. Mean cortisol sampling times were 12:50 PM (SD = 3:29, baseline), 13:14 PM (SD = 3:29), 13:25 (SD = 3:28), and 13:35 PM (SD = 3:28).
Since time of baseline sample varied, it was used as covariate in the analyses. Only participants with all four cortisol samples were included in the analysis (n = 38).
Participants‟ daily cortisol secretion was measured by assessing their salivary cortisol levels at six points of time during a regular day. During the home visit participants received sampling kits including the material needed for collection and detailed written instruction how to obtain the samples, which were also elaborated face to face. Participants were asked to collect saliva six times: immediately after awakening, half an hour after awakening, an hour after awakening, before lunch time, before dinner, and before bedtime. Research staff telephoned the participants the day before the observation day to remind them of collection. Mean cortisol sampling times were 6:58 AM (SD = 0:51), 7:31 AM (SD = 0:55), 8:01 AM (SD = 0:57), 13:01 PM (SD = 1:03), 19:13 PM (SD = 0:40), and 22:44 PM (SD = 1:03).
Participants were not allowed to eat or drink anything but water at least 30 minutes before sampling. Missing samples were only imputed for participants who had maximally two missing samples per day. Samples were stored at -18°C until being assayed by the Research Center for Psychobiology at the University of Trier.
ADRA2B. Buccal swabs collected from individuals were incubated in lysis buffer (100 mM NaCl, 10 mM EDTA, 10 mM Tris pH 8, 0,1 mg/ml proteinase K, and 0,5% w/v SDS) until further processing. Genomic DNA was isolated from the samples using the Chemagic buccal swab kit on a chemagen Module I workstation (Chemagen Biopolymer-Technologie AG, Baesweiler, Germany). DNA concentrations were quantified by OD260 measurement and by agarose gel electrophoresis. The average yield was approximately 4 ug of genomic DNA per buccal swab sample.
Typical PCR reactions contained between 10 and 100ng genomic template DNA, 10 pmol of forward and reverse primers, 100 uM dNTP, 3,3% DMSO, 10x buffer supplied with the enzyme, 2,5 mM Mg2+ and 0,25 amplitaq gold (5U/μl) in a total volume of 30 ul. For amplification of the ADRA2B fragment, primers 5‟- AGAAGGAGGGTGTTTGTGGGG-3‟ (5‟ labeled with FAM) and 5‟- ACCTATAGCACCCACGCCCCT-3‟ were used. The fragment was amplified by
an initial denaturation step of 10 min at 95oC, followed by 40 cycles of 30 sec 95oC, 30 sec 58oC, 1 min 72oC, and a final extension step of 10 min 72oC.
The length of the PCR amplification product was determined by size fractionating the fragments on an ABI-3100 automated sequencer and the size data was analyzed using GeneMarker software. Based on the length of the amplified fragments, the difference between the deletion and wildtype variant was readily visible with a resolution of +/- 5 basepairs. Genotypes were in Hardy-Weinberg equilibrium, χ²(1, N = 61) = 0.11, p = .74.
Statistical Analyses
ADRA2B gene was dichotomized to wildtype carriers and deletion variant carriers because of the relative small number of deletion homozygous carriers. Both cortisol measures were inspected for outliers, which were defined as values with SD greater than 3.29 above the mean (Tabachnick & Fidell, 2007). Because the distributions of cortisol measurements were positively skewed, log10
transformations were used for analysis. Outliers in the daily distribution were made fit in the specific curve of the individual respondent. No outliers were detected in the stress reactivity measures. Cortisol measures were analyzed by multivariate analyses of variance with repeated measures (the participants‟ cortisol levels at six or four time points, respectively) with group (Holocaust survivors versus comparisons) as between-subject factor, and time of baseline sample as a covariate in the analyses of cortisol reactivity.
Results
Table 1 presents the background variables, genetics polymorphism, and transformed cortisol levels for Holocaust survivors and comparisons. Because they had spent most of their formative years in hiding or camps, Holocaust survivors had significantly fewer years of education than the comparison group, t (52) = - 4.66, p < .01. No differences were found for health issues or medication use between two groups.
To test for differences in diurnal cortisol secretion a 2 (Holocaust versus comparisons) x 6 (time of sampling) repeated measure ANOVA was conducted with ADRA2B (wildtype versus deletion) as between-subjects factor. A significant main effect was found for the time of sampling F (5, 160) = 42.87, p <.01, which confirmed the expected decrease of cortisol level from morning until evening. No significant main effect was found for group F (1, 32) = 3.47, p <.07 or for genotype F (1, 32) = 0.02, p <.90. A significant two-way interaction was found between genotype and Holocaust versus comparison group, F (1, 32) = 4.98, p <.05. In the
Table 1. Background Variables and Cortisol Values over the Day, and in Reaction to Stress
Holocaust (n = 25)
Comparison (n = 29)
M SD M SD
Age 77.48 3.54 76.59 2.50
Years of education 4.64** 3.65 8.72 2.79
Number of children 3.16 0.99 3.45 1.33
Married / widowed 15 / 10 21/ 8
Perceived health 3.48 1.01 3.62 0.98
Total number of health problems 6.12 3.00 4.86 3.03
Medication use 24 27
ADRA2B: Wildtype/ Deletion 13/ 12 12/ 17
cortisol time 1 (awakening)1 0.91a 0.44 0.85b 0.37
cortisol time 2 0.97 c 0.32 0.91 d 0.33
cortisol time 3 0.99 e 0.31 0.84 b 0.39
cortisol time 4 0.62 c 0.36 0.48 b 0.27
cortisol time 5 0.43 a 0.41 0.27 f 0.39
cortisol time 6 (bed time) 0.45 g 0.39 0.25 b 0.35
Pre- stress cortisol (baseline) 0.61 g 0.37 0.53 f 0.32
First post stress cortisol 0.65 h 0.35 0.46 f 0.33
Second post stress cortisol 0.66 e 0.35 0.49 d 0.45
Third post stress cortisol 0.60 h 0.34 0.50 b 0.38
Note. Family status was not significantly different, p = .25; ADRA2B Polymorphism was not significantly different, p = .54. 1Cortisol values logtransformed; a n = 21; b n = 25; c
n = 20; d n = 23; e n = 22; f n = 24; g n = 17; h n = 21.
* p < .05, ** p < .01
wildtype ADRA2B group, Holocaust survivors showed higher daily cortisol levels than comparisons, whereas no difference was found between Holocaust survivors and their comparisons in the deletion group (Figures 1a and 1b).
Figures 1. Diurnal Cortisol levels of Holocaust Survivors and Comparisons with the ADRA2B Wildtype and Deletion Variants
Figure 1a. ADRA2B Wildtype Carriers
Figure 1b. ADRA2B Deletion Carriers
Holocaust Comparison
Holocaust Comparison
To test for stress reactivity differences between Holocaust survivors and comparisons, an analysis of 2 (Holocaust versus comparisons) x 4 (time of sampling) repeated measures ANCOVA with ADRA2B (wildtype versus deletion) as between-subjects factor was conducted, with baseline time of sampling as covariate. As expected, time of baseline sampling had a significant effect on cortisol levels, F (1, 28) = 16.23, p <. 01, with earlier assessments related to higher cortisol production. No significant effects were found for group F (1, 28) = 0.66, p <.42 and for genotype, F (1. 28) = 0.03, p <.87. A significant two-way interaction effect of group and genotype was found F (1, 28) = 5.79, p < .05. Specifically, Holocaust survivors in the wildtype group showed higher cortisol levels than comparisons, whereas no difference was found between Holocaust survivors and their comparisons in the deletion group (Figures 2a and 2b).
Figures 2. Cortisol Reactivity of Holocaust Survivors and Comparisons with the ADRA2B Wildtype and Deletion Variants
Figure 2a. ADRA2B Wildtype Carriers
Figure 2b. ADRA2B Deletion Carriers
Holocaust Comparison
Discussion
In wildtype ADRA2B carriers, Holocaust child survivors who immigrated to Israel as orphans after the Holocaust showed higher diurnal cortisol levels than comparisons, whereas no difference was found between Holocaust survivors and their comparisons if they were deletion carriers. ADRA2B variants were also found to moderate cortisol reactivity to stress; Holocaust survivors in the wildtype group showed higher cortisol levels than comparisons, and no difference was found between Holocaust survivors and comparisons in the deletion group.
Previous research showed that the deletion variant was associated with more vivid traumatic memories in survivors of the Rwandan civil war, and may lead to elevated levels of post-traumatic stress relatively shortly after the traumatic events (de Quervain et al., 2007). However, in the long run survivors who carry the deletion variant may not be able to live with repression of their vivid memories.
More than the carriers of the wildtype variant they may be driven to work through the traumatic events over the long time-span of several decades. As a consequence they may end up with a less dysregulated HPA axis.
The current study is limited because of its small sample size. Replication in larger samples is badly needed and the current study should be considered exploratory. In future studies the mechanism of the moderating role of ADRA2B should be studied more carefully. For example, the hypothesized link between the deletion variant, more vivid memories following the trauma, and the urgency to work through these traumatic memories needs to be examined as a cascade leading to a better regulated HPA system in the long run.
Here we propose that the ADRA2B gene moderates the long-term effects of a traumatizing genocide on its survivors, even after more than 60 years because carriers of the deletion variant are driven to resolve their vividly remembered experiences in the long run and therefore show less stress dysregulation as evident from their cortisol levels. Wildtype carriers might be protected against the devastating effects of genocide in the short term but they become at risk for permanently altered HPA axis functioning in the long run.
“It is the storyteller who makes us what we are, who creates history. The storyteller creates the memory that the survivors must have –otherwise their surviving would have no meaning.” (Chinua Achebe, 1996, pp.10). Having vivid memories might be facilitative for survivors who are allowed or even urged to make meaning, whereas repressed memories might haunt survivors forever. Resolution might be advantageous in the long run, whereas repressing incomprehensible memories might be adaptable in the short term.
References
Achebe, C. (1996). Things fall apart. London: Penguin classics.
Barel, E., Van IJzendoorn, M.H., Sagi-Schwartz, A., & Bakermans-Kranenburg, M.J. (2010). Surviving the Holocaust: A meta-analysis of the long-term sequelae of genocide. Psychological Bulletin, 136, 677-698.
Buske-Kirschbaum, A., Jobst, S., Psych, D., Wustmans, A., Psych, D., Kirrschbaum, C., Rauh, W., & Hellhammer, D. (1997). Attenuated free cortisol response to psychological stress in children with atopic dermatitis.
Psychosomatic Medicine, 59, 419-426.
de Quervain, D. J. F., Kolassa, I. T., Ertl, V., Onyut, P. L., Neuner, F., Elbert, T., &
Papassotiropoulos, A. (2007). A deletion variant of the α2b-adrenoceptor is related to emotional memory in Europeans and Africans. Nature Neuroscience 10, 1137 – 1139.
Denson, T. F., Spanovic, M., & Miller, N. (2009). Cognitive appraisals and emotions predict cortisol and immune responses: A meta-analysis of acute laboratory social stressors and emotion inductions. Psychological Bulletin, 135, 823-853.
Dickerson, S.S., & Kemeny, M.E. (2004). Acute Stressors and cortisol responses: A theoretical integration and synthesis of laboratory research. Psychological Bulletin, 13, 355-391.
Dobrova-Krol, N. A., Van IJzendoorn, M. H., Bakermans-Kranenburg, M. J. Cyr, C., & Juffer, F. (2008). Physical growth delays and stress dysregulation in stunted and non-stunted institution-reared children. Infant Behavior and Development, 31, 539-553.
Dozier, M., Manni, M., Gordon, M. K., Peloso, E., Gunnar, M. R., Stovall- McClough, K. C., Eldreth, D., & Levine, S. (2006). Foster children's diurnal production of cortisol: An exploratory study. Child Maltreatment, 11, 189-197.
Fridman, A., Bakermans-Kranenburg, M. J., Sagi-Schwartz, A., & Van IJzendooen, M. H. (under review). Stress reactivity and trauma: Deviant in Holocaust survivors, but not in their daughters.
Gunnar, M. R., Morison, S. J., Chisholm, K., & Scheuder, M. (2001). Salivary cortisol levels in children adopted from Romanian orphanages. Development and Psychopathology, 13, 611-628.
Kirschbaum,C., Pirke, K. M., & Hellhammer, D. H. (1993). The "Trier Social Stress Test"- a tool for investigating psychobiological stress responses in a laboratory setting. Neuropsychobiology, 28, 76-81.
Liu D., Diorio, J., Tannenbaum, B., Cladji, C., Francis, D., Freednman, A., Sharma, S., Pearson, D., Plotsky, P, M., & Meaney, M. J. (1997). Maternal care, hippocampal glucocoticoid receptors, and the hypothalamic-pituitary-adrenal responses to stress. Science, 277, 1659-1662.
McGaugh, J. L. (2003). Memory and emotion: The making of lasting memories. New York:
Columbia University Press
Meaney, M. J., Mitchell, J. B., Aitken, D. H., Bhatnagar, S., Bondoff, S.R., Iny, L. J.,
& Sarrieau, A. (1991). The effects of neonatal handling on the development of the adrenocortical response to stress: Implications for neuropathology and cognitive deficits in later life. Psychoneuroendocrinology, 16, 85-103.
Pen a, E. D. (2007). Lost in translation: Methodological consideration in cross- cultural research. Child Development, 78, 1255-1264.
Sagi-Schwartz, A., Van IJzendoorn, M. H., Grossmann, K. E., Joels, T., Grossmann, K., Scharf, M., Koren-Karie, N., & Alkalay, S. (2003).
Attachment and traumatic stress in female Holocaust child survivors and their daughters. American Journal of Psychiatry, 160, 1086–1092.
Sapolsky, R. M. (2003). Stress and plasticity in the limbic system. Neurochemical Research, 28, 1735-1742.
Tabachnick, B. G., & Fidell, L. S. (2007). Using multivariate statistics. Boston: Pearson Education.
Tarullo, A., & Gunnar, M. R. (2006). Child maltreatment and the developing HPA axis. Hormones and Behavior, 50, 632-639.
Van der Hal-Van Raalte, E. A. M., Bakermans-Kranenburg, M. J.,& Van IJzendoorn, M. H. (2008). Diurnal cortisol patterns and stress reactivity in child Holocaust survivors reaching old age. Aging & Mental Health, 12, 631- 639.
Van IJzendoorn, M. H., Fridman, A., Bakermans-Kranenburg, M. J., & Sagi- Schwartz, A. (under review). Aftermath of genocide: Elevated levels of diurnal cortisol in Holocaust survivors but not in their adult offspring?
