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Interpreting Stress Responses during Routine Toxicity Studies: A Review of the Biology, Impact, and Assessment Nancy E. Everds, Paul W. Snyder, Keith L. Bailey, Brad Bolon, Dianne M. Creasy, George L. Foley, Thomas J. Rosol and Teresa Sellers Toxicol Pathol 2013 41: 560 originally published online 7 March 2013 DOI: 10.1177/0192623312466452 The online version of this article can be found at: http://tpx.sagepub.com/content/41/4/560

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Scientific and Regulatory Policy Committee Review Toxicologic Pathology, 41: 560-614, 2013 Copyright # 2013 by The Author(s) ISSN: 0192-6233 print / 1533-1601 online DOI: 10.1177/0192623312466452

Interpreting Stress Responses during Routine Toxicity Studies: A Review of the Biology, Impact, and Assessment NANCY E. EVERDS1, PAUL W. SNYDER2, KEITH L. BAILEY3, BRAD BOLON4, DIANNE M. CREASY5, GEORGE L. FOLEY6, THOMAS J. ROSOL7, AND TERESA SELLERS8 1

Amgen Inc., Seattle, Washington, USA Purdue University, West Lafayette, Indiana, USA 3 Oklahoma Animal Disease Diagnostic Laboratory, Oklahoma State University, Stillwater, Oklahoma, USA 4 Department of Veterinary Biosciences and the Comparative Pathology and Mouse Phenotyping Shared Resource, The Ohio State University, Columbus, Ohio, USA 5 Huntingdon Life Sciences, East Millstone, New Jersey, USA 6 Abbott Laboratories, Abbott Park, Illinois, USA 7 Department of Veterinary Biosciences, The Ohio State University, Columbus, Ohio, USA 8 GlaxoSmithKline, King Of Prussia, Pennsylvania, USA 2

ABSTRACT Stress often occurs during toxicity studies. The perception of sensory stimuli as stressful primarily results in catecholamine release and activation of the hypothalamic–pituitary–adrenal (HPA) axis to increase serum glucocorticoid concentrations. Downstream effects of these neuroendocrine signals may include decreased total body weights or body weight gain; food consumption and activity; altered organ weights (e.g., thymus, spleen, adrenal); lymphocyte depletion in thymus and spleen; altered circulating leukocyte counts (e.g., increased neutrophils with decreased lymphocytes and eosinophils); and altered reproductive functions. Typically, only some of these findings occur in a given study. Stress responses should be interpreted as secondary (indirect) rather than primary (direct) test article–related findings. Determining whether effects are the result of stress requires a weight-of-evidence approach. The evaluation and interpretation of routinely collected data (standard in-life, clinical pathology, and anatomic pathology endpoints) are appropriate and generally sufficient to assess whether or not changes are secondary to stress. The impact of possible stress-induced effects on data interpretation can partially be mitigated by toxicity study designs that use appropriate control groups (e.g., cohorts treated with vehicle and subjected to the same procedures as those dosed with test article), housing that minimizes isolation and offers environmental enrichment, and experimental procedures that minimize stress and sampling and analytical bias. This article is a comprehensive overview of the biological aspects of the stress response, beginning with a Summary (Section 1) and an Introduction (Section 2) that describes the historical and conventional methods used to characterize acute and chronic stress responses. These sections are followed by reviews of the primary systems and parameters that regulate and/or are influenced by stress, with an emphasis on parameters evaluated in toxicity studies: In-life Procedures (Section 3), Nervous System (Section 4), Endocrine System (Section 5), Reproductive System (Section 6), Clinical Pathology (Section 7), and Immune System (Section 8). The paper concludes (Section 9) with a brief discussion on Minimizing Stress-Related Effects (9.1.), and a final section explaining why Parameters routinely measured are appropriate for assessing the role of stress in toxicology studies (9.2.). Keywords:

stress; toxicity; glucocorticoid; epinephrine; immune system; hypothalamic-pituitary-adrenal (HPA) axis; endocrine system.

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. The author(s) received no financial support for the research, authorship, and/or publication of this article. Address correspondence to: Nancy E. Everds, Amgen Inc., 1201 Amgen Court West, Seattle, WA 98119, USA; e-mail: [email protected]. Abbreviations: ACTH, adrenocorticotropic hormone (corticotropin); ALP, alkaline phosphatase; ANS, autonomic nervous system; AUC, area under the curve; AVP, arginine vasopressin; 11bHSD2, 11-beta-hydroxysteroid dehydrogenase, type 2; BBB, blood–brain barrier; BDNF, brain-derived neurotrophic factor; CA, catecholamine/catecholaminergic; CA1, Cornu Ammonis, field 1; CA3, Cornu Ammonis, field 3; CBG, corticosteroid-binding protein, also known as transcortin; CCK, cholecystokinin; CFU-GM, colony forming unit–granulocyte/macrophage; CNS, central nervous system; CRH, corticotropin-releasing hormone; CRL, Charles River Laboratories; CVS, chronic variable stress; DA, dopamine; DAMPs, Danger-associated molecular patterns; DN, double negative (lymphocyte); DP, double positive (lymphocyte); EAA, excitatory amino acids; EAE, experimental allergic encephalitis; EPI, epinephrine (or adrenalin); F344, Fischer 344 (rat strain); FOB, functional observational battery; FSH, follicle-stimulating hormone; GnRH, gonadotropin-releasing hormone; hCG, human chorionic gonadotropin; HPA, hypothalamic–pituitary–adrenal axis; HPG, hypothalamic–pituitary–gonadal axis; HPT, hypothalamic–pituitary–thyroid axis; Hsp, Heat shock protein; 5HT, 5-hydroxytryptamine (i.e., serotonin); IL-1, interleukin-1; LH, luteinizing hormone; MHC, major histocompatibility complex; MZ, marginal zone; NE, norepinephrine (or noradrenalin); NGF, nerve growth factor; NHP, nonhuman primate; NK, natural killer; NO, nitric oxide; NPY, neuropeptide Y; PALS, periarteriolar lymphoid sheath; PNMT, phenylethanolamine N-methyltransferase; PNS, peripheral nervous system; POMC, pro-opiomelanocortin; PRL, prolactin; PVN, paraventricular nucleus (of the hypothalamus); RBC, red blood cell; ROS, reactive oxygen species; SAM, sympatho–adrenal–medullary (system); SD, SpragueDawley (rat strain); SER, smooth endoplasmic reticulum; SNS, sympathoneural system; STP, Society of Toxicologic Pathology; rT3, reverse T3 (tri-iodothyronine); T3, tri-iodothyronine; T4, thyroxine; TCR, T cell receptor; TEM, transmission electron microscopy; TH, tyrosine hydroxylase; TNF-a, tumor necrosis factor-alpha; TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone (thyrotropin); VIP, vasoactive intestinal peptide; ZF, zona fasciculata (of the adrenal gland); ZG, zona glomerulosa (of the adrenal gland); ZR, zona reticularis (of the adrenal gland). 560

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TABLE 1.—In-life and pathology parameters routinely evaluated in toxicity studies: Typical responses to stress. Stage of study In-life

Affected system Nutrition Circulating blood cells

Organ weightsa

Immune Endocrine Immune Reproductive

Organ histology

Digestive Lymphoid Reproductive

Endocrine

Parameter Body weight or body weight gain Food consumption Eosinophil counts Lymphocyte counts Neutrophil counts Macrophage phagocytosis Adrenal gland Thymus Spleen Testisb Epididymis Seminal vesicles Prostate Ovaries Uterus Gastric ulceration Thymic cellularity Spleen cellularity Testis Epididymis, prostate, seminal vesicles Ovary, Uterus Vagina Adrenal cortex

Potential stress effect (may also be unchanged) Decreased Decreased Decreased Increased or decreased Increased Decreased Increased Decreased Decreased Unchanged (rats) or decreased (mice) Decreased Decreased Decreased Decreased Decreased Increased Decreased Decreased Unchanged (rat) or degeneration (mice) Possible atrophy Inactive Atrophy, mucification Hypertrophy/hyperplasia

Note: Parameters affected by stress depend on the species, sex, age, and type and duration of stressor. Refer to appropriate text sections for species-specific details. a Absolute, relative to body, and/or relative to brain. b Absolute weight only.

1. SUMMARY Animal stress is an inherent feature of toxicity studies, attributable to the dose of test article and/or experimental procedures, whether these studies are conducted for the chemical or pharmaceutical industry. A primary objective of these studies is to determine the potential adverse effects of test articles when given at high doses (e.g., appropriate limiting doses depending on the purpose of the study, such as doses with large exposure multiples or that saturate exposure, or maximally tolerated or maximum feasible doses as defined in regulatory documents; HED Guidance 2003; ICH M3 (R2) 2010; Organisation for Economic Co-operation and Development [OECD] 407 2008; Rhomberg et al. 2007; U.S. Environmental Protection Agency [U.S. EPA] 1998). At high doses, primary test article– related effects may be of sufficient magnitude and/or duration to result in stress as a secondary effect. At lower doses, primary effects due to the test article are generally less pronounced and may cause little or no secondary stress. Because primary effects of test articles are generally proportional to the dose administered, the findings due to stress may also show dose proportionality. For this reason, difficulties may arise in discerning primary test article–related findings from secondary stress-related changes during toxicity studies. Nonetheless, in the majority of cases, the data set collected during toxicity studies is sufficient to distinguish test article–related findings from stress. 1.1. Hallmarks of Stress Stress is perceived by the brain and results in adaptive responses in other organ systems via neural and neuroendocrine

pathways. Stress responses are readily identifiable for many routinely evaluated parameters (e.g., leukocyte counts) and organ systems that are sensitive to stress (Table 1). The hallmarks of the stress response include decreased total body weights or body weight gain, food consumption and activity; altered organ weights for selected organs (decreased for thymus and spleen, and increased for adrenal gland); lymphocyte depletion in the thymus and spleen; altered circulating leukocyte counts (e.g., increased neutrophil counts with decreased lymphocyte and eosinophil counts); and altered reproductive functions. Although any or all of these systems may be affected by stress in a given toxicity study, a weight-of-evidence approach is required to differentiate whether these changes should be attributed directly to test article exposure or indirectly to stress. In general, circulating leukocyte counts, lymphocyte subsets, and adrenal gland and thymus weights are the most sensitive parameters that indicate stress in toxicity studies, although the relative sensitivity of parameters to stress varies depending on the species, the type of stress, the physiologic state of the animal, and the inherent interanimal variability of the data. In routine toxicity studies, effects related to acute stress may present as changes in clinical pathology parameters (e.g., a stress leukogram during the clinical pathology assessment) and/or histologic changes in sensitive cell populations (e.g., glucocorticoid-induced diffuse apoptosis of thymic lymphocytes) rather than alterations in gross organ appearance. In contrast, chronic stress may impact total body weight or organ weights, including increased adrenal weights in conjunction with decreases in total body weight and weights of thymus, accessory

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TOXICOLOGIC PATHOLOGY

TABLE 2.—Stressors associated with procedures and processes that may occur during routine toxicity studies. Procedure or process Husbandry Handling Effects of the test article Intercurrent disease

Examples Cage changing, food restriction, fasting, social structure, exercise, transportation Dosing, sample collection, evaluations, restraint End-organ toxicity, vomiting, diarrhea, seizures, lethargy Chronic progressive nephropathy (male rats), species/strain-specific neoplasia

Note: The resulting stress experienced by animals depends on the species, sex, age, and type and duration of stressor. Refer to appropriate text sections for species-specific details.

sex organs, and spleen (Table 1). Body and organ weight changes are generally greater if the stressful stimulus is more pronounced.

1.2. Weight-of-Evidence Approach To differentiate whether changes in in-life and clinical and anatomic pathology parameters are due indirectly to stress or directly to the test article, data must be evaluated in an integrated (i.e., whole body) fashion. Changes for individual animals and entire treatment groups must be assessed with respect to the severity and time course of the clinical signs and other in-life endpoints. The changes must be evaluated and interpreted with respect to their sensitivity to stress. In animals affected by stress, the more sensitive indicators of stress are present at lower doses or at greater severity and/or incidence compared to changes that are less sensitive to stress. When considering the impact of stress on outcomes in toxicity studies, it is informative to follow some simple guidelines:

Consider the biology of the test article and its intended target. If the test article has a primary effect on an organ system sensitive to stress, attributing the effect to stress can be difficult. In these instances, effects of the test article at lower doses may offer insight into whether the findings are related to stress or to the test article. Evaluate the magnitude of the response in stresssensitive parameters and organs. The magnitude of the response should be commensurate with the magnitude of the stress. For example, animals that experience mild repetitive stress (e.g., due to clinical signs associated with dosing of a test article) over a 1month period might have minimal to mild changes in adrenal weights and histology. Examine the dose–response of potential stressrelated changes versus the dose–response of probable primary test article–related changes. Stress-related changes generally do not occur at doses at which there are no test article–related effects. For example, in a study with dose–related decreases in circulating and thymic lymphocytes, increased adrenal weights, and body weight loss at the middle and high doses, but with only increased adrenal weights at the low dose, the latter effect might be a primary test article–related change.

Consider other potential interpretations. For most changes that can be attributed to stress, there are one or more alternative interpretations. These alternatives (differential diagnoses) and the evidence supporting or refuting them should be fully considered prior to making an interpretation. For example, adrenocortical hypertrophy/hyperplasia can be due to stress or a primary adrenal toxicant that inhibits the production of glucocorticoids. Adrenocortical hypertrophy/ hyperplasia in the absence of any other stressassociated changes suggests a possible primary effect on adrenocortical hormone synthesis and is generally not sufficient evidence to support the assumption of stress. Similarly, lymphocyte depletion in the spleen and lymph nodes but not the thymus (generally a more sensitive organ to stress) in animals not showing any other signs of stress may suggest primary effects of the test article on the immune system. Evaluate the strength of the weight of evidence. Generally, less sensitive indicators of stress do not occur in the absence of more sensitive indicators. For example, if there are alterations in reproductive organs without any other evidence of stress such as body weight or lymphoid organ changes, it is less likely that the reproductive organ findings are attributable to stress.

The answers to the above questions will generally provide sufficient weight of evidence to determine whether findings are due to stress. Less commonly, the data are insufficient to make a determination about which changes are primary test article effects as opposed to secondary effects of stress. In these cases, additional studies (e.g., specific immunotoxicity studies, investigative studies on adrenal gland function) may be necessary to further investigate underlying mechanisms. 2. INTRODUCTION The effects of stress may confound the interpretation of many clinical pathology and some anatomic pathology findings evaluated in toxicity studies. Understanding different types of stressors and their potential sequelae within the context of a study helps differentiate between primary test article–related effects and secondary stress-induced effects. In the current article, stress-related findings as identified by routinely evaluated parameters and organ systems are described from

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pathophysiologic, morphologic, and functional perspectives. Potential investigative studies to differentiate primary test article effects from secondary stress-related changes are described where relevant. At present, regulatory documents addressing whether findings in toxicity studies are primary (direct) test article–related changes or secondary to stress (indirect) are limited to those found in guidelines for immunotoxicity testing, specifically those promulgated by the International Committee on Harmonisation (ICH) and the Environmental Protection Agency of the United States (http://ntp-server.niehs.nih.gov/htdocs/IT-studies/ IMM94002.html; U.S. EPA 1998). Common clinical pathology and anatomic pathology changes indicative of stress have been defined in general terms within these guidelines: Interpretation of Stress-related Changes ‘‘With standard toxicity studies, doses near or at the maximum tolerated dose can result in changes to the immune system related to stress. These effects on the immune system are most likely mediated by increased corticosterone or cortisol release. Commonly observed stress-related immune changes include increases in circulating neutrophils, decreases in circulating lymphocytes, decreases in thymus weight, decreases in thymic cortical cellularity and associated histopathologic changes (‘‘starry sky’’ appearance), and changes in spleen and lymph node cellularity. Increases in adrenal gland weight can also be observed. In situations with clear clinical observations (e.g., decreased body weight gain, decreased activity), some or all of the changes to lymphoid tissue and hematology parameters might be attributable to stress rather than to a direct immunotoxic effect. The evidence of stress should be compelling (From ICH guidelines: S8 IMMUNOTOXICITY STUDIES FOR HUMAN PHARMACEUTICALS, Section 1.4).’’ These ICH guidelines stipulate that if findings in the immune system are thought to be attributable to stress, evidence to support this interpretation must be ‘‘compelling.’’ In the absence of strong evidence of stress as a likely cause of immune system lesions, additional investigations of immunotoxicity may be required (ICH S8 Immunotoxicity Testing Guidelines; http://ntp-server.niehs.nih.gov/htdocs/IT-studies/ IMM94002.html). Therefore, it is important to distinguish secondary stress-related findings from primary test article–related effects on the immune system. The ability to differentiate primary/direct immunotoxicity from secondary/indirect stress-related effects can be difficult, given the normal variability in immune parameters due to differences in health, age, and intercurrent experimental conditions. During toxicity studies, stress often affects organ systems other than the immune system, most prominently endocrine and reproductive systems, and clinical pathology (primarily hematologic) parameters. Similar to changes in the immune system, stress-related effects on these systems can be difficult

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to differentiate from direct test article–related alterations. Although stress-related effects on parameters other than those of the immune system are not specifically addressed by regulatory guidance documents, perturbations in these nonimmune organ systems can be essential in providing sufficient weightof-evidence to support the interpretation that stress is the underlying cause of findings observed in animals during toxicity studies. The objective of toxicity studies is to identify target organ toxicities caused by a test article, by design these studies are often stressful (Chida et al. 2004; Masson et al. 2010). The effects of stress are multisystemic and are indicated by a wide range of effects that include changes in behavior, activity, food consumption, body weight and organ weight changes, histology and clinical pathology parameters, and reproductive parameters (see Table 1). When some or all of these hallmarks occur, they likely are attributable to stress. The objective of this review is to provide a pathophysiologic understanding of the effects and interpretation of stress on toxicity studies as measured by standard clinical and anatomic pathology endpoints. To meet this objective, this article (1) reviews the biology of stress; (2) reviews normal structural and functional components of various organ systems that may mediate or be modified by stress; (3) describes changes to standard toxicity parameters that can result from acute or sustained stress; and (4) presents a pathophysiologic approach for determining whether findings are primary and due directly to the test article, or secondary and due indirectly to stress. 2.1. History and Definition of Stress The current understanding of stress is based on pioneering research by Walter Cannon and Hans Selye. Cannon proposed that an organism responds to changes in its environment and suggested that these physiological responses are necessary to survive, introducing the concepts of ‘‘homeostasis’’ (the tendency toward a relatively stable equilibrium between opposing but interdependent physiological processes) and the ‘‘fight or flight’’ response (Cannon 1915, 1929a, 1929b, 1932, 1939). Selye termed physiologic stress to be a ‘‘general adaptation syndrome’’ and identified several cardinal features of this biological response including adrenal enlargement, gastrointestinal bleeding, and decreased immune responsiveness (Selye 1936, 1950a, 1950b). He also introduced the terms ‘‘distress’’ and ‘‘eustress’’ to differentiate good stress (e.g., receipt of a passing grade on a difficult test; environmental enrichment) from bad stress (e.g., a series of emotionally upsetting incidents; physical pain). The physiologic differences between eustress and distress for animals are not well understood, although these two terms are increasingly used in literature concerning the welfare of research animals (Morton 2000). Another applicable concept used by stress researchers is ‘‘allostasis,’’ which is the set of responses whereby body systems respond actively and collectively to restore normal physiological conditions following exposure to various stressors (McEwen 1998, 2004). Stressors resulting in mild changes are not necessarily deleterious as some of these changes are the result of

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and immune systems that mediate stress (Guyton and Hall 2006; Latimer and Prasse 2003). 2.2. Pathways Involved in Stress Responses

FIGURE 1.—Components of the hypothalamic–pituitary–adrenal axis involved in the response to stress, including potential stressors and adaptive responses for organ systems evaluated in routine toxicity studies.

appropriate adaptive mechanisms and result in advantageous adaptations (Koolhaas et al. 2011). For the purposes of this review, the generic term ‘‘stress’’ encompasses physiological responses to perturbations of homeostasis, including those effects caused by changes in either neural (mainly catecholamine) or endocrine (mainly glucocorticoid) pathways (Figure 1). This definition of stress is broader than those used in clinical human and veterinary medicine, but it is consistent with definitions used by stress researchers and reflects the current understanding of the complex interrelationships among neurologic, endocrine,

Stressful conditions elicit adaptive physiological responses that are loosely divided into primary (mostly neurologic and/ or hormonal) and secondary responses. The major neurologic responses are the limbic system (emotional brain) and the sympathetic pathways of the brain and adrenal medulla. The major hormonal pathway is the hypothalamic–pituitary–adrenal (HPA) axis. Neurologic (see Nervous System section) and endocrine (see Endocrine System and Reproductive System sections) responses to stressors are closely interconnected because the hypothalamus is a critical component of both the limbic system and is the origin of releasing factors that affect downstream stress organs (e.g., adrenal gland, reproductive organs). Secondary responses to stressors are those that occur in other organs in response to activation of the sympathetic nervous system and/or HPA axis. These secondary responses can be abrogated by inhibiting the neurologic and/or hormonal response to stress and can be replicated to some degree by exogenous administration of compounds that simulate a stress response. Examples of secondary responses to stress include increased serum glucose concentrations in response to epinephrine (EPI), or decreased lymphocyte counts in blood in response to glucocorticoids. Specific mechanisms and outcomes that can be attributed to other major organ systems (e.g., immune system, reproductive system) that are part of stress-related secondary responses are detailed in this review. Responses to stressors are not monotypic (i.e., the same regardless of inciting cause), as originally postulated by Selye, but instead are modulated by both past experience and the current physiologic state. Therefore, seemingly minor events may have dramatic impact on stress responses in an individual animal while eliciting no detectable responses in others. This may explain why, in toxicity studies, there is often interanimal variability in endpoints relating to stress. For example, only some animals with decreased body weight and food consumption might have decreased circulating lymphocyte counts. 2.3. Animal Models of Stress Stress models, although not directly applicable to toxicity studies, are useful in dissecting pathways involved in stress. The stress literature includes experimental paradigms that vary markedly in type and duration of stress, experimental subjects, and data collected. However, animal studies that evaluate stress generally can be categorized as follows:

Stress of real-life situations: These studies evaluate the stress of typical laboratory animal procedures. Examples of such variables reported in the literature include single- versus group-housed animals, handling procedures, and various means of blood collection.

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Disease: These studies evaluate the effect of acute or chronic illness on stress-related parameters (e.g., mice with experimental allergic encephalitis [EAE]). A limited number of these studies provide information relevant to repeated dose safety studies. Experimental perturbation of environment: These models are generally divided into emotional or physical stress. Examples of emotional stress include fear, aggression, lack of control over one’s environment, and immobilization (e.g., in tube restraint). Examples of physical stress include heat, cold, electric shock, and restraint (e.g., cannot move limbs or head). These models may be acute (e.g., a single exposure to the stressful conditions for 2 hr) or chronic (e.g., 2 hr of stress repeated once daily for 5 consecutive days). Pharmacological/surgical induction of stress: These models involve modulation of the activity in neural and/or humoral (endocrine) pathways to study various aspects (usually related to chemical signaling pathways) of the stress response.

Dietary restriction is a stressor commonly used by investigators studying stress. This model of stress is particularly relevant to toxicity studies, since high doses of test articles can cause animals to consume less feed. Diet restriction studies can produce many of the stress-related changes observed in animals administered a dose of a test article that causes them to decrease food intake. 2.4. Acute and Chronic Stress Stressors experienced by animals can be acute (a single, short event) or chronic. Sources of acute stress include dosing, restraint for up to a few hours, or short-duration effects of test articles (e.g., emesis immediately after dosing). Chronic stress results from stressors that last for more than a few hours and may result from a continuously applied stressor (e.g., chronic social stress) or repeated intermittent application of acute stressors over a period of days to weeks (e.g., restraint once daily for 7 days). Intermittently applied stressors may be further classified as consistent (same stressor at each interval) or variable (unpredictable stressors). The duration of stress can markedly influence the resulting physiologic response. For example, in one model of stress in rats, acute stress results in increased plasma corticosterone concentrations whereas chronic intermittent stress does not (Bauer et al. 2001). Most research on stress, with the exception of studies of chronic disease, involves either acute or chronic intermittent stress. Although these models predominate in the stress literature, they do not emulate the pattern of stress experienced during toxicity studies. Stress observed secondary to test article–related effects is often continuous over the course of a study (e.g., lethargy, inappetance, and decreased body weight gain over 2 weeks), as opposed to stress resulting from typical stress study paradigms (e.g., restraint for 1 hr a day for 2 weeks). Stress research also differs from toxicity studies in that different types of data are collected. For example, stress experiments

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generally measure a limited number of stress-specific endpoints (e.g., only lymphocyte counts or only adrenal weights and adrenocorticotropic [ACTH] concentrations) rather than the broader but less stress-targeted set of measurements (complete clinical pathology, organ weights, and histology) collected for routine toxicity studies. A review of the literature showed that the most sensitive indicator of acute stress differed depending on the experimental protocol and the endpoints measured, and varied among circulating leukocyte counts, lymphocyte subsets, and adrenal and thymus gland weights. Research on stressors affecting humans provides some of the best information about effects of chronic stress. These studies generally rely on cognitive tests and sampling of blood or saliva to assess stress responses. Although these data are useful to understand peripheral changes as a result of stress, they lack complete data on organ weights and histologic changes that are the hallmarks of stress on toxicity studies. Habituation, as measured by the return to baseline of one or more stress-related parameters, generally occurs in response to repeated consistent stress (Dave et al. 2000; Fukuhara et al. 1996; Kant et al. 1992; Marquez, Nadal, and Armario 2004; Retana-Marquez, Bonilla-Jaime, Vazquez-Palacios, MartinezGarcia, et al. 2003; Wong, Cassano, and D’mello 2000). Habituation of the HPA axis response to stressors applied over 7 or more days is mediated by glucocorticoids acting on mineralocorticoid and glucocorticoid receptors of the posterior paraventricular thalamus in rats (Jaferi and Bhatnagar 2006). Chronic variable stressors (i.e., different stressors each day) as opposed to consistent stressors (i.e., the same stress at the same time every day) or severe stressors may result in hyperresponsiveness rather than habituation (Koolhaas et al. 2011; Marin, Cruz, and Planeta 2007; Orr et al. 1990). These observations in rats suggest that negative feedback attributable to corticosterone modulates the influence of previous stressors on an individual animal’s response to subsequent stresses. Glucocorticoids may modulate, enhance, or suppress responses to stressors (Sapolsky, Romero, and Munck 2000). Recovery from single or repeated episodes of stress varies depending on the duration and type of stressor, the consistency of the stressor, and the parameter measured as an indicator of stress (e.g., see Dhabhar et al. 1995b; Drozdowicz et al. 1990; Garcia et al. 2000; Servatius et al. 2000). Unpredictable and uncontrollable stress tends to cause more or larger effects than exposure to the same stressor in a predictable or controllable manner. Because most toxicity studies are done in a manner that is predictable (dosing at the same time of day in the same manner), it is likely that habituation to mild stressors would occur during most multiple dose toxicity studies. 2.5. Primary Test Article (direct) versus Secondary (indirect) Effects For toxicity studies, effects of test articles can be divided into primary (direct) test article–related effects or secondary (indirect) effects. Primary effects are specifically caused by

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exposure to the test article while secondary effects (which include stress) are those that occur downstream of the primary effects. Secondary effects include those related to stress (e.g., body weight loss, lymphoid depletion in the thymus); these are analogous to other downstream effects that may not be relevant for human risk assessment. The goal of toxicity studies is to identify and characterize toxicities of test articles in experimental animals. Understanding and interpreting what changes may be related to stress is an important consideration in determining whether or not certain effects observed during a given toxicity study are primary and which are secondary. Test articles that have a pharmacologic effect on pathways involved in stress may produce some changes that may be similar to those elicited by stress. For example, administration of exogenous glucocorticoids would be expected to induce leukocyte changes similar to stress, but stress-related adrenal hypertrophy would not occur because the administered glucocorticoids would decrease serum ACTH concentrations by negative feedback. 2.6. Rat as the Species of Reference Stress has been studied in many species including humans, nonhuman primates, dogs, rabbits, rats, and mice. Comparisons across studies that evaluate stress are difficult because of the variability in experimental design factors such as the species/ strains used, the strength and duration of the stressor, and the endpoints measured. An identical stressor can have different effects depending on the age of the test subject, complicating the interpretation of stress (Koenig et al. 2011; Sapolsky, Romero, and Munck 2000). Even prenatal stress can program and influence stress in postnatal life (Darnaudery and Maccari 2008). Most of the work described in this review article focuses on rodent-related stress research due to the plethora of publications and the relevance of rodents to toxicity research. Information about other species commonly used in toxicity studies is included when available. Strains of rodents differ in the magnitude of their responses to stress. Of the strains of rats used in toxicity studies, Fischer 344 (F344) rats generally have the highest basal and stimulated concentrations of corticosterone, Lewis rats have low basal and stimulated concentrations, and Sprague-Dawley (SD) rats are intermediate between F344 and Lewis rats (Dhabhar, McEwen, and Spencer 1993; Gomez, De Kloet, and Armario 1998; Griffin and Whitacre 1991; Windle et al. 1998). Wistar-Kyoto rats, compared to SD rats, have exaggerated neuroendocrine responses to stress and exhibit neurotransmitter changes consistent with a depressive state (De La Garza and Mahoney 2004; O’Mahony et al. 2011; Rittenhouse et al. 2002). Differences have also been reported in stress responses between suppliers of the same strain of rat (Turnbull and Rivier 1999) or from mice of the same strain bred by a vendor versus inhouse (Olfe et al. 2010). For instance, SD rats from Charles River Laboratories (CRL) are reported to have a significantly higher ACTH response to inflammatory stressors, but not to foot shock stress, when compared to Harlan-supplied SD rats.


Despite the higher ACTH response in CRL SD rats, the two strains have similar corticosterone concentrations for all stressors. Quantitative trait locus (QTL) analysis has identified several genetic elements that correlate with strain differences in response to stress (e.g., see Potenza et al. 2004; Solberg et al. 2006; Vendruscolo et al. 2006). Similar strain differences have been noted for mice. BALB/ c, C57BL/6, DBA/2, and NOD mice subjected to restraint, lipopolysaccharide, or interleukin-1 (IL-1) stress have differential responses as determined by plasma corticosterone and glucose concentrations regardless of the stressor (C57BL/6 NOD < DBA/2 < BALB/c; Harizi et al. 2007). Differential sensitivity to stress has also been reported by other researchers (e.g., Anisman et al. 2001). Vendor-derived mice may have altered HPA axis activity that continues into adulthood. In one study, these mice had no activation of the HPA axis in response to acute stress compared to mice raised in-house (Olfe et al. 2010). 3. IN-LIFE PROCEDURES

AND

STRESS

Factors that induce stress responses in laboratory animals during the pretest and study periods include transportation, housing conditions, handling, restraint, diet, diagnostic procedures, sample collection, and dosing (Conour, Murray, and Brown 2006, Table 2). Animals respond to these stressors by releasing a number of mediators such as catecholamines and glucocorticoids, as described in subsequent sections of this article. In general, the effects of these stress-induced mediators tend to be transient and result in habituation within 1 to 4 days (reviewed in Prager et al. 2011). Interpretation of study data from toxicity studies should be undertaken with several considerations in mind. Potential stressors associated with environmental factors, husbandry practices, and experimental procedures must be comparable for groups treated with the vehicle and test article. Effects in the control group estimate the degree of stress associated with experimental manipulations such as test article administration (gavage, inhalation, or parenteral) and physiological assessment (e.g., behavioral testing). Biases due to order of collecting samples or taking measurements must be minimized by conducting procedures on animals across groups in either a random or a round-robin (stratified) fashion (Hall and Everds 2008; Sellers et al. 2007). 3.1. Transportation The effect of transportation on laboratory animals has been studied extensively due to animal welfare concerns (Bergeron et al. 2002; Dymsza et al. 1963; Flynn, Poole, and Tyler 1971; Obernier and Baldwin 2006; Slanetz et al. 1956; Tuli, Smith, and Morton 1995; Wallace 1976; Weisbroth, Paganelli, and Salvia 1977). The extent of the stress response depends on how animals are transferred to shipping containers, the method and duration of transportation, the age of the animals, any preexisting physiological conditions (e.g., pregnancy), environmental conditions during transit, and the availability of water during shipment.

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As early as 1956 (Slanetz et al.) and later in 1963 (Dymsza et al.), studies were conducted in an attempt to characterize the ‘‘equilibration’’ period in rats, mice, and guinea pigs following transportation by truck, rail, and air. In rats, the most common alterations reported during transportation include weight loss and increased or decreased heart rate (Capdevila et al. 2007). Increased water intake and increased myocardial antioxidant enzyme activity have also been reported in Wistar and SD rats, respectively (Rowland et al. 2000; van Ruiven et al. 1998); the average time for equilibration of these alterations varied from 3 to 14 days. Transportation is reported to increase corticosterone concentrations in mice for 24 hr (Tuli, Smith, and Morton 1995b), and indicators of stress (e.g., behavioral observations such as feeding and activity level) were still present at 4 days. Stresses associated with transportation impaired reproductive performance in deer mice for several weeks following transatlantic transport (Hayssen 1997). For routine toxicity studies, these data support the modern practice of acclimating rodents for 1 week after arrival in the test facility in order to recover from the stress of shipping (Conour, Murray, and Brown 2006; Furudate et al. 2005). Larger animals experience similar responses to shipping stress (Kuhn et al. 1991). In Beagle dogs, air and ground transportation have been associated with increased heart rate, increased mean plasma and salivary cortisol concentrations, increased circulating neutrophil count, and decreased lymphocyte count (Bergeron et al. 2002). Similar parameters were altered following transportation of rabbits (Toth and January 1990) and cynomolgus monkeys (Kim et al. 2005). In addition, rabbits develop anorexia and hyperglycemia in response to the stress of transportation. Rabbits and cynomolgus monkeys recover from transportationrelated changes by 48 hr and 1 week, respectively, after arrival at a new destination (Kim et al. 2005; Toth and January 1990). 3.2. Study Procedures Husbandry and experimental procedures cause stress. Minimization of husbandry and experimental stress decreases pre-analytical effects of stress and yields more consistent experimental data for clinical and anatomic pathology, as well as other data collected on toxicity studies (Conour, Murray, and Brown 2006; Klumpp et al. 2006; Leishman et al. 2011). 3.2.1. Caging Stressors associated with caging include such routine procedures as cleaning cages, housing density, movement of cage racks, and provision of environmental enrichment activities. Stress in rats is associated with the routine act of changing cages and may contribute to transient increases in heart rate and blood pressure (Duke, Zammit, and Lawson 2001). Cage changing, animal weighing, and other routine noninvasive procedures increase heart rates in rats to a degree comparable to or greater than parenteral injections or the odor of rat blood (Sharp et al. 2003). Movement of cages of BALB/c mice from one room to another results in increased HPA axis activation and decreased circulating lymphocyte counts; these changes recover within a few days (summarized in Olfe et al. 2010).

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TABLE 3.—Changes in body weight and standard organ weight parameters potentially associated with mild or severe stress responses in routine toxicity studies. Tissue Body weight Thymus Spleen Adrenal glands Testes Seminal vesicles Prostate Ovaries Uterus

Mild stressors Unchanged or decreased Unchanged or decreased Unchanged Unchanged Unchanged Decreased Decreased Unchanged Unchanged

Severe stressors Decreased Decreased Unchanged or decreased Increased Unchanged or decreased Decreased Decreased Decreased Decreased

Note: Parameters affected by stress depend on the species, sex, age, and type and duration of stressor; some of these stress responses occur only in rodents. Refer to appropriate text sections for species-specific details.

Current federal guidelines and laws (Guide for the Care and Use of Laboratory Animals, The National Academies Press 2011, Table 3.2, pg 57 and the United States Department of Agriculture [USDA] Animal Welfare Act 9 CFR Parts 1, 2, and 3, respectively; reviewed in White, Hawk, and Vasbinder 2008) provide specific recommendations and laws for the amount of space required by experimental animals. The primary reason for these recommendations and requirements is animal welfare. The current understanding is that animals housed individually or that have no environmental enrichment will have more distress (‘‘bad’’ stress) than those that are group housed or with access to environmental stimuli (see also History and Definition of Stress section). Rats housed in enriched environments experience a state of eustress (‘‘good’’ stress) as indicated by generally higher corticosterone measurements relative to agematched counterparts in traditional caging (BenaroyaMilshtein et al. 2004; Konkle et al. 2010; Marashi et al. 2003; Moncek et al. 2004). The effects of caging conditions on weights of organs sensitive to stress (e.g., spleen, thymus, adrenal gland) are inconsistent among studies (Abou-Ismail and Mahboub 2011; Konkle et al. 2010; Moncek et al. 2004). Group-housed female rats exhibit decreased stress responses to routine caging manipulation as measured by heart rate and mean arterial pressure (Sharp et al. 2003). Male rats subjected to crowding have decreased food consumption with concurrent decreased body weight gain and increased water intake (Armario, Luna, and Balasch 1983). Isolated female rhesus monkeys have higher cortisol concentrations and a dampened circadian rhythm compared to those in a stable social environment (Gust et al. 2000), but other researchers have found no difference in singly versus co-housed monkeys (Reinhardt, Cowley, and Eisele 1991). Dogs show less stereotypical behavior when socialized (Beerda, Schilder, van Hooff, et al. 1999; Beerda, Schilder, Bernadina, et al. 1999; Dean 1999; Hetts et al. 1992; reviewed in Meunier 2006). Taken together, these data indicate that stress in animals placed on toxicity studies may be substantially lessened by sufficient attention to housing and socialization needs (Prager et al. 2011).

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With sufficient attention to study design, animals may be temporarily housed under novel conditions for sample collection procedures without causing undue stress. Isolating rats in novel cages to collect urine corticosterone transiently increases serum corticosterone concentrations approximately 8-fold over baseline; corticosterone returns to baseline after approximately 120 min in the new cage (Fluttert, Dalm, and Oitzl 2000; Prager et al. 2011). Overnight fecal corticosterone in rats is unchanged when animals are placed in novel metabolic cages compared to those individually housed in traditional solid-bottom polycarbonate cages (Eriksson et al. 2004). Body weight gain in rats after 3 days in a wire mesh metabolic cage is only slightly less than body weight gain in animals maintained in solid-bottom caging, suggesting that being housed in wire mesh cages is only slightly stressful (Eriksson et al. 2004). 3.2.2. Diet and Exercise 3.2.2.1. Fasting: Fasting prior to sample collection is stressful in rodents but not in dogs, as measured by circulating ACTH and glucocorticoid concentrations. Fasting of rats starting 1 to 2 hr before the dark period increases ACTH and corticosterone concentrations during the dark period (Akana et al. 1994; Hanson, Levin, and Dallman 1997). Corticosterone concentrations of fasted rats are approximately 3-fold higher than values from ad libitum–fed animals. A stressor applied during the beginning of the light period results in heightened ACTH but similar corticosterone responses in fasted compared to fed rats (Akana et al. 1994). Fasting of up to 36 hr causes a decrease (12 and 24 hr) or no change in cortisol concentrations of dogs (Knies, Erhard, and Ahrens 2005; Reimers, McGarrity, and Strickland 1986). For rhesus monkeys habituated to being fed once daily, one missed meal causes a transient but significant (approximately 1.4-fold) increase in cortisol beginning shortly after the usual feeding time (Helmreich, Mattern, and Cameron 1993). 3.2.2.2. Dietary Restriction: The effects of dietary restriction on mice, rats, and nonhuman primates are similar in that decreased food availability generally results in decreased body weight gain and increased concentrations of plasma glucocorticoids. In general, dietary restriction in rats experiencing stress results in more rapid decrease of body weight compared to dietary restriction in nonstressed rats (Servatius et al. 2001). Although absolute pituitary and adrenal gland weights remain unchanged in rats following 3 days of stress exposure, relative (to body weight) adrenal weights are consistently increased and pituitary weights are variably increased (Servatius et al. 2001). In contrast, a study in rats evaluating the additive effects of caloric restriction and chronic stress showed an enhanced adaptation to stress (Gursoy et al. 2001). These authors proposed a threshold-type response in weight loss with increased plasma corticosterone concentrations as well as improved tolerance upon exposure to additional stressors. Altering the normal feeding time of rodents has downstream effects on the HPA axis. The normal circadian rhythm


in circulating corticosterone concentrations is reversed in rats offered a diet in the early light period restricted to 75% of that provided to a control group fed ad libitum (Gallo and Weinberg 1981). This finding has implications for pair-feeding in toxicity studies, in which a second control group is given a restricted diet to match the food consumption of a group that has decreased food consumption as a result of the test article. In such studies, the pair-fed control group would likely consume its diet during the first few morning hours, as opposed to the test article–related group that would likely eat during the normal nocturnal time. Additionally, increased plasma corticosterone concentrations, thymic atrophy, and adrenal hypertrophy and hyperplasia have been observed in control pair-fed rats, confirming the effects of stress due to dietary manipulation (Gorski et al. 1988). 3.2.2.3. Exercise: Moderate physical exercise is thought to improve the ability to cope with stress. Mice with access to a running wheel voluntarily run approximately 4 km/day during the early dark period. This self-selected exercise period results in adaptive changes in the HPA axis, including adrenal gland hypertrophy, loss of asymmetry in adrenal gland size (i.e., an increase in right adrenal cortex and medulla dimensions), lower ACTH concentrations in the morning, greater plasma corticosterone in the early dark period, increased concentrations of circulating cortisol-binding globulin, and greater activity of tyrosine hydroxylase (TH) in the adrenal medulla (Droste et al. 2003). These changes presumably represent a eustress (‘‘good’’ stress) response. In contrast to mice, rats exercised on treadmills do not develop increased adrenal weights (Brown et al. 2007; Fediuc, Campbell, and Riddell 2006). The initial increase in corticosterone in exercised rats attenuates after 8 weeks (Campbell et al. 2009). Rats with access to voluntary exercise have dampened stress responses to noise and habituate to noise stress faster than sedentary rats (Masini et al. 2012). In contrast to voluntary or mild exercise, extreme exercise can be stressful (Ferry et al. 1993). 3.2.3. Restraint and Handling Effects of restraint and handling on specific parameters (e.g., clinical pathology parameters, HPA axis) are covered in their respective sections below. The effects of restraint and handling on clinical signs are discussed here. Restraint of laboratory animals is associated with increased heart rate and increased blood pressure (Irvine, White, and Chan 1997; McDougall et al. 2000; McDougall, Lawrence, and Widdop 2005), in addition to decreased food consumption and weight loss (Harris et al. 1998). Various restraint methods can also impact the production of stress hormones. Nonhuman primates subjected to chair-restraint, squeeze cages, or manual restraint have increases in serum and urine corticosterone concentrations (reviewed in Reinhardt, Liss, and Stevens 1995). Adaptation to daily restraint tubes for nose only exposures, as measured by normalization of body temperature and heart rate, occurs after 2 weeks in rats and mice (Narciso et al. 2003).

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The manner in which animals are handled can also induce stress that affects study results. For example, lifting rats and mice by their tails predisposes them to convulsions under certain testing conditions (Boyle and Villanueva 1976; Goldstein 1973). Environmental cues can produce a stress response even in the absence of restraint and handling. The mere appearance of individuals in medical attire can cause increased blood pressure in dogs as well as humans (White Coat Syndrome: http:// www.whitecoatsyndrome.org/, Marino et al. 2011). The presumed explanation is that the individuals remember the visual cues from prior episodes. However, additional aural and/or olfactory cues could also conceivably play a role in establishing the memory. 3.2.4. Collection of Blood The least stressful procedures for collection of blood and tissues are those that are rapid and/or minimally invasive to the living animal. Generally, circulating catecholamine concentrations increase as soon as 10 sec after a stressor, while increases in glucocorticoid concentrations are not detected until approximately 3 min after a stressor. Historically, blood collection by decapitation has been considered one of the least stressful methods for rodents. It was considered to allow the collection of samples essentially unperturbed by stress due to the speed with which the full aliquot can be acquired from a senseless subject (AVMA: http://www .avma.org/issues/animal welfare/euthanais.pdf). For aesthetic, animal welfare, and technical reasons, alternatives to blood collection by decapitation have been investigated. In rats, collection from the tail vein or from an indwelling catheter is appropriate for measurement of ACTH and corticosterone during stress studies, whereas decapitation after CO2 or pentobarbital causes stress-related effects on ACTH and corticosterone concentrations measured in trunk blood (Vahl et al. 2005). Alterations in heart rate and blood pressure are significantly lower in rats subjected to jugular bleeding when compared to tail vein or periorbital blood sampling, suggesting that jugular bleeding is less stressful (Toft et al. 2006). However, jugular or tail vein collection results in slightly prolonged elevations in body temperature when compared to periorbital bleeding. Similarly, EPI and norepinephrine (NE) concentrations in mice (based on the within-study mice and historical literature) are approximately 5- to 6-fold higher in blood samples collected by decapitation relative to those of blood collected from preestablished arterial cannulae of unanesthetized mice or from the retro-orbital plexus of mice under halothane anesthesia (Grouzmann et al. 2003). In addition, catecholamine concentrations measured after cold stress were different from nonstressed concentrations only when blood was collected by intravenous indwelling catheter or retro-orbitally, but not by decapitation (Grouzmann et al. 2003). These studies suggest that the procedures involved in decapitation are a source of acute stress for rodents and that their extent may be substantial enough to impact certain biochemical parameters.

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Accordingly, collection methods other than decapitation should be considered for measuring hormones associated with the stress response. The stress of in-life blood collection is also influenced by the volume taken, the frequency of collection, and the anesthetic used for immobilization and pain management. Collection of small volumes of blood via a previously implanted catheter in rats is considered nonstressful. Repeated collection of small blood aliquots (5–6 collections resulting in the withdrawal of approximately 10% of blood volume in total) from implanted jugular catheters or from jugular phlebotomy under anesthesia (isoflurane or CO2:O2) results in increased corticosterone concentrations in rats. Corticosterone increases associated with blood collection are slightly more pronounced in phlebotomized rats anesthetized with CO2:O2 compared to isoflurane, and slightly more pronounced in phlebotomized rats anesthetized with isoflurane compared to cannulated rats (Altholtz et al. 2006; Vachon and Moreau 2001). CO2 restraint is more stressful than isoflurane anesthesia. The increase in heart rate associated with jugular phlebotomy is partially abrogated in rats anesthetized with isoflurane compared to the rates measured in unanesthetized animals (Toft et al. 2006). Diethyl ether has been shown to be more stressful than halothane/O2/N2O anesthetic mixtures (de Haan et al. 2002; van Herck et al. 2000). Barbiturates can induce anesthesia without increasing stress-related mediators (Donnerer and Lembeck 1990). Tail bleeding leads to an increase in corticosterone in mice immediately after bleeding (Tuli, Smith, and Morton 1995a). The transient rise is reversed within 24 hr. Corticosterone concentrations in other mice housed in the same room are not affected (Tuli, Smith, and Morton 1995a). In general, blood collection in larger animals is associated with fewer stress-related changes compared to rodents. For dogs, there is no difference in cortisol, luteinizing hormone, or testosterone concentrations when blood is collected by venipuncture or pre-placed catheter, nor is there any difference in hormone measurements between blood collected from cagerestrained dogs and dogs removed from their cages and restrained in an anteroom (Knol et al. 1992). However, research dogs exposed to a novel outdoor environment prior to blood collection have a dramatic increase in cortisol of approximately 3-fold (Knies, Erhard, and Ahrens 2005). Collection of blood has a small effect on stress measurements in nonhuman primates (Capitanio, Mendoza, and McChesney 1996; Herndon et al. 1984; Lambeth et al. 2006). For rhesus monkeys, cortisol concentrations were lower when blood was collected from animals in their cage as opposed to a restraint apparatus (Reinhardt et al. 1990, reviewed in Reinhardt, Cowley, and Eisele 1991). 3.3. In-life Observations Associated with Stress The primary in-life effects of stress are decreased body weight or body weight gain and decreased food consumption, and have been reviewed extensively (Committee on

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Recognition and Alleviation of Distress in Laboratory Animals 2008). 4. NERVOUS SYSTEM

AND

STRESS

The nervous system of an organism receives input through sensory pathways. When these stimuli are interpreted collectively as a stressor, the brain responds by mobilizing effector activities mediated by various neurologic centers, endocrine organs, and the immunologic system (McEwen 2006). The stress response may be marshaled for genuine perils (e.g., a physical hazard such as disease, handling, or restraint) or for perceived threats (e.g., an emotional peril such as constant change, crowding, fear, or isolation). Neuroactive xenobiotics tend to be more effective at eliciting a stress response, although these effects tend to be modulations in the concentrations of stress hormones rather than anatomic and functional changes to the nervous system proper (DeBoer, Slangen, and van der Gugten 1992). Two primary efferent neural circuits modulate stress responses. The first is the HPA axis, a well-characterized neuroendocrine loop. The HPA axis consists of certain hypothalamic nuclei, the three lobes of the pituitary gland, and the three zones of the adrenal cortex. The second circuit is the sympathoneural system (SNS), one peripheral arm of the autonomic nervous system (ANS; Kandel, Schwartz, and Jessell 2000) and the primary source of circulating catecholamines (CA) released in response to stress (Carrasco and Van de Kar 2003). The SNS is composed of sympathetic pre-ganglionic neurons and the chromaffin cells of the adrenal medulla, as well as sympathetic nerves that supply key effector organs involved in the ‘‘flight or fight’’ response. The HPA axis and SNS interact closely in the periphery (Kvetnansky et al. 1995). The two systems are also highly intertwined with respect to central circuits that connect their respective brain centers (Table 4 and Figure 2). Detailed descriptions of the HPA axis and the related hypothalamic–pituitary–gonadal (HPG) and hypothalamic– pituitary–thyroid (HPT) axes as well as their associations with stress-induced physiological responses are discussed in the Endocrine and Reproductive System sections. Accordingly, the current section focuses on ways in which the central nervous system (CNS) and the SNS are impacted by the stress response and influence adaptive responses to stress. Numerous reports indicate that stress can alter the sensitivity to and responsiveness of various neural cells to toxicants, and vice versa. Demonstrated mechanisms include enhanced sensitization to raised glucocorticoid concentrations and heightened vulnerability to excitotoxic agents. Specific changes in neural structure and/or function may include increased neuronal death (e.g., following exposure to an excitotoxicant), decreased neuronal numbers (usually as a consequence of developmental exposure), and/or impaired cognitive function. In routine toxicity studies, the nervous system is assessed for structural (e.g., organ weights, macroscopic and histologic analysis) and functional (e.g., behavioral tests, functional observational battery [FOB]) effects. Generally, none of these endpoints is altered by stress during the course of toxicity studies.


TABLE 4.—Structural and functional responses of neuroanatomical components directly or indirectly involved in stress responses. Relationship

Anatomic structure

Limbic system

Amygdala Hippocampus

Hypothalamus

Thalamus

Major functions

Cerebrocortical centers

Cingulate gyrus

Entorhinal cortex

Medial prefrontal cortex (mainly rodent)

Sympathoneural system

Parietal cortex (mainly human)

Sympathetic ganglia and trunk

Perception of fear Processing of fear Learning Memory Spatial orientation Homeostatic control Behaviors Circadian rhythms Feeding Sleep Temperature Neuroendocrine regulation (pituitarytargeted hormones) Dispatch center for cortical input from subcortical areas Sensation Spatial orientation Information processing Regulation of consciousness Anterior part Emotional processing Selecting actions Posterior part Adaptation to trauma Emotional processing Memory Formation Optimization Retention Spatial orientation Emotional perception Emotion-driven responses Emotional collation with memory Emotional perception Emotion-driven responses Emotional collation with Memory Music Enhances muscle activities needed for ‘‘fight or flight’’ (e.g., heart rate, limb muscle readiness, vessel wall tone) Invokes pilo-erection and sweat gland secretions Represses homeostatic functions (e.g., intestinal motility)

4.1. Neurological Control of Stress 4.1.1. CNS Centers and Pathways Stress-induced sensory input may travel via either shortloop reflexes mediated at the spinal or supraspinal levels or

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FIGURE 2.—Schematic diagram of a rat brain demonstrating the approximate locations of the major neural domains that control the stress response. The major functional associations are the limbic system or ‘‘emotional brain’’ (red shapes nos. 1–4); the cerebrocortical centers that mediate attention, emotion, and memory functions (blue shapes nos. 5–8); the neuroendocrine circuitry (shapes nos. 3, 9, and 12); and the central sympathetic centers (orange shapes nos. 10 and 11). Brain regions: 1 ¼ hippocampus, 2 ¼ thalamus, 3 ¼ hypothalamus, 4 ¼ amygdala, 5 ¼ medial prefrontal cortex, 6 ¼ cingulate cortex, 7 ¼ parietal cortex, 8 ¼ entorhinal cortex, 9 ¼ pituitary gland, 10 ¼ locus coeruleus, 11 ¼ raphe nuclei, 12 ¼ adrenal gland (a peripheral endocrine effector organ). The locations, sizes, and significance of these brain regions vary among species. While projected to the midsagittal plane in this diagram, several of these domains are actually located more laterally in the 3-dimensonal brain.

by long-loop neuronal circuits that are integrated in and influenced by higher brain centers (Kvetnansky, Sabban, and Palkovits 2009). The integrated input of physical sensations and/or emotional perceptions across many brain regions defines what is threatening (stressful) to the individual. The capacity of the nervous system to control an individual’s perception and responsiveness to stress indicates that neural structures are integral contributors to the patterns of stressrelated alterations that manifest in all body systems. Pathways involved in the neural response to stress are widely distributed in the CNS. The brain regions that control primary stress responses throughout the body are the limbic system (‘‘emotional brain,’’ composed of the amygdala, hippocampus, hypothalamus, and thalamus); various cerebrocortical centers (cingulate gyrus, and the entorhinal, medial prefrontal, and parietal cortices) that serve as major sources of incoming and outgoing signals for the limbic system (reviewed in Carrasco and Van de Kar 2003); and the locus coeruleus (a major sympathetic center and the main source of catecholamines in the brain). Limbic areas exhibit sexual dimorphism, suggesting that the HPG axis can affect their structure and function (Becker et al. 2007). The master control center for integrating stressful stimuli and controlling the stress response is the hypothalamus (Fitzgerald, Gruener, and Mtui 2007; Kandel, Schwartz, and Jessell 2000). The paraventricular nucleus (PVN) of the hypothalamus serves as a coordinating center, receiving input from many brain regions (Harbuz 2002). The primary

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chemicals produced in the PVN include corticotropinreleasing hormone (CRH) and, in about 50% of cells, arginine vasopressin (AVP; Akana et al. 1992; Carrasco and Van de Kar 2003; Harbuz 2002). Release of CRH stimulates the discharge of ACTH as the first step in activating the HPA axis; CRH also acts as a neurotransmitter for various neuron populations in the limbic system and SNS-related brain centers (Preil et al. 2001). However, AVP is required for optimal ACTH activity as rats with AVP deficiency but normal CRH concentrations do not exhibit a normal stress response (Kjaer et al. 1993). Under basal conditions, expression of AVP and CRH in the PVN is very low, but levels increase substantially following stress (Imaki et al. 2001; Van de Kar and Blair 1999). Feedback inhibiting hypothalamic CRH release is provided by glucocorticoid receptors on PVN neurons (Meijer and De Kloet 1998). Hippocampal neurons also provide a major source of inhibitory feedback to CRH-expressing neurons in the PVN to dampen the response of the HPA axis (Gesing et al. 2001; Herman and Cullinan 1997). The two major neurotransmitters involved in CNS processing of stressful stimuli are catecholamines (CA such as EPI and NE), and serotonin (5-hydroxytryptamine [5HT]), an indoleamine. Brain nuclei that produce these neurotransmitters are dispersed in the pons and medulla oblongata. Ascending catecholaminergic and serotonergic pathways extend to the integrative centers of the forebrain (e.g., amygdala, hippocampus, hypothalamus, and cortical regions associated with the limbic system) to influence the activity of the HPA axis. These aminergic projections innervate distant brain regions with many collateral branches. For example, the NE-rich neurons of the locus coeruleus are directly connected to the CRH-expressing neurons in the PVN (Habib, Gold, and Chrousos 2001), while the 5HT-rich neurons of the raphe nuclei contact the hippocampal neurons that supply the PVN (Meijer and De Kloet 1998). The descending pathways expressing these transmitters supply the sympathetic pre-ganglionic neurons of the intermediolateral domains of the thoracic spinal cord. Other neurotransmitter classes have been demonstrated to be important in stress neurobiology. In particular, many neuropeptides regulate the stress response. Vasoactive intestinal peptide (VIP) is widely distributed in the limbic system and throughout the HPA axis (reviewed in Carrasco and Van de Kar 2003). In these areas, VIP often is co-localized in PVN neurons and is highly expressed in some nuclei within the amygdala. Levels of VIP increase within the median eminence (which forms the tissue bridge between the hypothalamus and pituitary gland) following chronic but not acute restraint stress (Armario et al. 1993) and in the amygdala after noise or vibration stress (Nakamura et al. 1994), suggesting that VIP is primarily a factor during sustained stress. Similarly, levels of neuropeptide Y (NPY) are increased in certain brain regions (including the PVN) and sympathetic ganglia following stress (Nankova et al. 1996; Rybkin et al. 1997), seemingly as a means of reducing negative responses to stress (Heilig and Thorsell 2002; Holden and Pakula 1999). Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide

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produced mainly by hypothalamic neurons (including those of the PVN), enhances gene transcription in the hypothalamus, pituitary gland, and adrenal medulla as well as providing trophic support to many CNS neuronal populations and to peripheral sympathetic neurons (Hashimoto et al. 2011; Stroth et al. 2011). Cholecystokinin (CCK) expression and release is increased in the hypothalamus and pre-frontal cortex during stress (Hernando, Fuentes, and Ruiz-Gayo 1996), while CCK inhibition dampens stress-related intracellular calcium concentrations in isolated pituitary cells and in rats conditioned to exhibit a fear response when placed in a novel environment (Tsutsumi et al. 2001). Leptin and neuromedin U act on CRH neurons in the PVN to regulate the flow of ACTH (Jethwa et al. 2006). Additional neurochemicals that impact the stress response include growth factors, cytokines, histamine, and nitric oxide. Various growth factors, such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), modulate stress pathways in the CNS and peripheral ANS (Joca, Ferreira, and Guimaraes 2007; Magarinos et al. 2011; Smith et al. 1995). In mice subjected to social stress, NGF is released into the blood from the submandibular salivary gland, which promotes an increase in adrenal chromaffin cell numbers; NGF is also produced locally in the hypothalamus and hippocampus (reviewed in Cirulli and Alleva 2009). Indeed, NGF has been hypothesized to be a mediator of anxiety and mood disorders in humans (reviewed in Cirulli and Alleva 2009). During exercise and inflammatory stress, cascades of cytokines such as interleukin (IL)-1, IL-6, and tumor necrosis factor (TNF)-a stimulate hypothalamic production of CRH and/or AVP as a means of inducing glucocorticoid secretion to limit the immune activity (reviewed in Mastorakos and Pavlatou 2005). In particular, IL-6 seems to be an important factor in this response as its production is stimulated by catecholamines and it results in higher elevations of plasma ACTH and cortisol concentrations in humans than does CRH. Histaminergic neurons in the caudal hypothalamus control the HPA axis during stress, mainly by elevating secretion of ACTH and CRH (reviewed in Miklos and Kovacs 2003). Nitric oxide, a gaseous neurotransmitter, has also been proposed as a player in certain CNS domains (e.g., hippocampus) that modulate the stress response (Joca, Ferreira, and Guimaraes 2007). 4.1.2. Efferent PNS Pathways The peripheral nervous system (PNS) consists of an extensive neural net sending signals to somatic (voluntary) or autonomic (involuntary) effector organs. In general, specific neural sampling for routine toxicology studies is focused on the somatic trunks (e.g., sciatic nerve and its branches, which innervate skeletal muscles). Autonomic nerves and ganglia typically are collected only as they occur within the other tissues (e.g., nerve trunks in the mesentery, ganglia within the walls of various viscera). The somatic PNS is the primary efferent neural pathway that implements ‘‘fight or flight’’ decisions in response to


immediate peril. Incoming signals from various peripheral receptors are carried by the PNS to the spinal cord, and thence to the brain via multiple spinal cord tracts located chiefly in the dorsal (posterior) and lateral white matter (Bolon 2000). These afferent signals are borne to the cerebellum, sensory cerebral cortex, and thalamus. After processing, efferent signals are sent from the motor cerebral cortex, cerebellum, and various regions of the brain stem (e.g., olivary nucleus, red nucleus) to lower motor neurons in the spinal cord ventral (anterior) horn. Efferent signals from these motor neurons course peripherally through large-diameter, thickly myelinated axons of somatic PNS nerves. The thick myelin insulation allows rapid transmission of impulses. The axons of somatic nerves synapse directly on voluntary muscle cells. The SNS can complement the acute ‘‘fight or flight’’ activities of the somatic PNS. This signaling is mediated by the sympatho–adrenal–medullary (SAM) axis, which results in the release of EPI and NE from the adrenal medulla into the blood (Wetherell et al. 2006). Central control of this process rests with various brain stem regions (e.g., locus coeruleus, raphe nuclei, reticular formation) that supply sympathetic pre-ganglionic neurons in the intermediolateral gray matter (i.e., lateral horn) of the thoracic and lumbar spinal cord segments. These neurons send SNS impulses to peripheral organs—chiefly viscera—via small-diameter nerves with thinly myelinated (unmyelinated) axons. Axons from the pre-ganglionic SNS neurons generally synapse in a peripheral sympathetic ganglion rather than following the somatic nerve pattern of directly innervating the effector organ; another small-diameter, thinly myelinated fiber leaves the ganglion as a post-ganglionic SNS axon to convey the signal to the effector organ. The ganglia of the sympathetic chain are the main peripheral autonomic ganglia involved in the stress response. The chain is present bilaterally in the thoracolumbar region as a series of ganglia connected by a long, thin nerve trunk located deep to the vertebral column. The adrenal medulla serves as a peripheral sympathetic ganglion in this system. Neuronal cell bodies localized in a given spinal cord region innervate specific peripheral ganglia and cell types. For example, sympathetic neurons that supply the adrenal medulla reside in segments T5 to T9. The SNS generally sends activating signals to effector organs during stress. Common SNSmediated responses to acute threats include catecholamine release by the adrenal medulla, increased heart rate, and pupillary dilation. The SNS also inhibits maintenance activities (e.g., digestion) that are likely to impede the stress response. Responses to stressors that are perceived to be less immediately dangerous are carried by the hormones of the HPA axis rather than unmyelinated nerves. The endocrine loops involved in these responses are described more fully in the Endocrine System section. The responses of the SNS to stress are generally countered by the reciprocal actions of the parasympathetic (PSNS) arm of the ANS to maintain normal organ function. Common outcomes of increased PSNS activity include enhanced digestive activity and slower heart rate.

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The association between stress and gastric ulceration is thought to depend chiefly on parasympathetic innervation and its excitatory signals (e.g., acetylcholine, histamine), which decrease mucosal resistance to gastric acids; sympathetic fibers act via their CA transmitters like EPI and NE to promote mucosal resistance (Hernandez 1986). Except for the cardiac and digestive systems, PSNS activation generally does not enhance stress responses and may in fact dampen them (Hamer and Steptoe 2007). Activation of the PSNS may be decreased during periods of chronic stress, such as adolescence (Hollenstein et al. 2012). 4.2. General Responses to Stress Neurologic changes as a result of stress can include various combinations of structural, functional, and neurochemical alterations in the CNS and the PNS (Kvetnansky and Sabban 1998). Such stress-induced neural responses are dependent on the nature of the inciting event. Various stressors impact different brain areas (down to distinct nuclei in specific areas), and stressful stimuli may reach these regions through diverse neuronal pathways (e.g., auditory vs. pain vs. visual sensations; reviewed in Kvetnansky, Sabban, and Palkovits 2009). Early stressful experiences modify the assembly of sympathetic circuits that regulate the stress response (Card et al. 2005). These stress-induced changes are evident in mature individuals as alterations in structural, behavioral, and neuroendocrine responses. For example, in animals, including humans, stress-related neural changes such as altered cell numbers or disrupted synthesis of brain neurotransmitters may be transient (Lucassen et al. 2006) or permanent (Card et al. 2005; Maccari et al. 2003). Alterations tend to be more pronounced and more permanent if stress is prolonged. The impact of stress on neural anatomy, chemistry, and function varies by age, sex, duration/ frequency/type of stressor, and the parameter measured (CorySlechta et al. 2008; Page, Blakely, and Kim 2005; Perez-Cruz et al. 2007; Richardson et al. 2006; Romeo et al. 2006; Zuena et al. 2008). Prior stressful events alter the neural response to subsequent episodes of stress (Akana et al. 1992; Romeo et al. 2006). Individuals may become habituated to repeated episodes of stress (Kant et al. 1985), resulting in a reduction (habituation) or quenching (extinction) of the neurological stress response over time. For neurological pathways as well as other parameters, habituation is more likely when the stress is consistent and predictable. With respect to toxicity studies, neural changes specifically related to stress typically are not observed. Total and regional brain weights as well as gross and histologic features generally are within normal limits. Changes in clinical signs may be observed but are generally nonspecific. Further investigation using specialized techniques (e.g., electron microscopy or stereology) is not warranted as such methods cannot differentiate subtle changes due indirectly to stress from any resulting directly from toxicity.

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is decreased neuronal numbers, in which two mechanisms have been implicated (Czeh et al. 2007; Fuchs, Flugge, and Czeh 2006; Lucassen et al. 2006). The first is decreased neuron production, which may occur during either initial development (Card et al. 2005; Korosi et al. 2012) or in the course of adult brain remodeling (Borcel et al. 2008; Czeh et al. 2007; Danzer 2012; Fuchs, Flugge, and Czeh 2006; Joca, Ferreira, and Guimaraes 2007; Lyons et al. 2010; Yan et al. 2011). The second mechanism is increased neuronal apoptosis (Lucassen et al. 2006). Deficits in neuronal number are permanent but may have no permanent functional consequence if suitable compensatory neural pathways are established. Stress also impacts the organization of neuronal substructures, particularly synapses (Card et al. 2005). Such effects may occur during development or adulthood. For example, two commonly reported stress-related consequences in the hippocampus and prefrontal cortex are fewer synapses and altered synaptic remodeling (Fuchs, Flugge, and Czeh 2006). Likely explanations for such effects are diminished synapse formation and/or retraction or pruning of previously formed processes during dendrite remodeling (Blugeot et al. 2011; Fuchs, Flugge, and Czeh 2006; Lucassen et al. 2006; Magarinos et al. 2011; Perez-Cruz et al. 2009). Together, these effects interrupt the normal circadian pattern of neural plasticity (Perez-Cruz et al. 2009) that is necessary to maintain the efficiency of brain circuit function in the face of constant change; increased glucocorticoid production is a likely cause of this effect as affected animals have heavier adrenal glands. Another possible mechanism contributing to decreased synaptic numbers is deficient production of neurotrophic growth factors by target cells (typically other neurons). For example, expression of BDNF is decreased in the adult rat hippocampus (CA1 and CA3 pyramidal neurons, and dentate gyrus granule neurons) within 1 hr of immobilization stress (Smith et al. 1995). In contrast, expression of neurotrophin-3 (NT-3) increased in the dentate gyrus and also the locus coeruleus only after repeated immobilization (Smith et al. 1995). These two neurotrophic factors are required to guide growing axons to their correct targets during development and to sustain their synaptic terminals in adulthood (Magarinos et al. 2011). Alterations in dendrite or synaptic ultrastructure may be partially or fully reversible once stress is alleviated. While present, these changes have functional implications, such as impaired memory (centered on the hippocampus; Borcel et al. 2008). Such functional changes are generally reversible after an acute stressor. Persistent changes in neural cytoarchitecture generally require extended periods of stress such as a single ongoing episode or repeated events over a long period. Serotonin (5HT) likely exerts a protective role in the hippocampus and attenuates the behavioral consequences of stress by activating 5HT1A receptors in this structure (Joca, Ferreira, and Guimaraes 2007; Savitz, Lucki, and Drevets 2009).

4.2.1. Structural Effects The limbic system (e.g., amygdala, hippocampus) and prefrontal cortex seem to be more sensitive to stress than are other regions. A major stress-induced outcome in these areas

4.2.2. Functional Effects Stress can cause behavioral and cognitive alterations. Changes that may be observed after stress include amplified

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aggression, attention deficits, heightened anxiety, and cognitive impairment; these have been observed in both animals and humans (Chida and Hamer 2008; Glover 2011; Kikusui and Mori 2009; Korte et al. 2005; Ma et al. 2011; McEwen 2006; Sotiropoulos et al. 2011). Additional changes include decreased associative capability, diminished memory, and distorted spatial orientation/navigation. Prolonged stress resulting in relatively higher sympathoadrenal activity and lower parasympathetic tone may have downstream consequences. Myocardial infarction in humans experiencing emotional stress is an example of ANS imbalance mediating changes in cardiovascular dynamics (Soufer, Jain, and Yoon 2009). The connections between ANS and downstream events are difficult to establish under the conditions of toxicity studies. Stress responses may be alleviated by prior exposure to intense stressors, such as parturition (Rima et al. 2009). Early positive life experiences can permanently affect subsequent neurological performance, and thus can protect against the adverse sequelae associated with stress (Cirulli et al. 2010). This protective effect may be enhanced by better maternal care during the neonatal period, as cross-fostering of pups from a more anxious (e.g., BALB/c) strain to the care of a dam from a more ‘‘relaxed’’ (C57BL/6) strain reduces the susceptibility to stress later in life (Priebe et al. 2005). During adulthood, environmental enrichment serves the same purpose. Animals living in more complex habitats exhibit enhanced neural structure and function (Beauquis et al. 2010; Goshen et al. 2009; Hu et al. 2010; Madronal et al. 2010; Schloesser et al. 2010). The neurobehavioral effects of stress are greater in immature compared to mature individuals. Age-related sensitivity to stress might be due to critical periods of neural development in brain regions that regulate stress reactivity and emotional responses (such as the integrated circuits linking the limbic system and cortex). In rats, stress experiences prior to weaning, including during the gestational period, result in HPA hyperactivity and higher brain CA turnover accompanied by an increase in defensive behaviors during adulthood (Takahashi, Turner, and Kalin 1992). Interestingly, enhanced HPA reactivity fades over time, but abnormal CA metabolism does not. For humans, the increased sensitivity of young individuals has been proposed to explain why stressful experiences during early life enhance the likelihood of developing psychological dysfunction later in life (Romeo et al. 2006). The stress response decreases with age (Cizza, Pacak et al. 1995). Stress has been linked to accelerated brain senescence (Noorlander et al. 2008; Wolkowitz et al. 2010) and cognitive decline (McDonald, Craig, and Hong 2008). The likely cause is progressive distortions in the expression patterns of many genes, including among others pro-apoptosis and proinflammatory proteins and stress-induced factors (Lukiw 2004). The outcomes produced by stress are both functional (cognitive deficits) and structural (cerebrocortical atrophy, tau protein deposition), possibly as a consequence of longterm glucocorticoid hypersecretion (Sotiropoulos et al. 2011).


4.2.3. Neurochemical Effects Stress impacts neurological function by altering the balance among neurotransmitter pathways (reviewed in Kvetnansky, Sabban, and Palkovits 2009). Acute stress leads to altered transmitter production or release by pre-synaptic neurons, aberrant removal or inactivation of transmitters from synaptic clefts, and/or modified numbers and affinities of receptor molecules; the nature of the change (increase or decrease) depends on the specific system. Such changes are typically transient. In the case of chronic or repeated stress, however, neurons remain in a highly active state for extended periods, which eventually depletes transmitter stores. When presented with novel stressors, the chronically stressed brain compensates by enhancing the expression of transmitter-synthesizing enzymes. However, this adaptive response often is only partially successful as transmitters are not restored to basal levels. The primary neurotransmitter changes caused by stress are increased CA or decreased 5HT signaling. Neurons that express CA are generally activated in experimental animals in response to stressors, resulting in accelerated CA release and turnover (reviewed in Carrasco and Van de Kar 2003; Kvetnansky, Sabban, and Palkovits 2009). The typical mechanisms are enhanced EPI and NE metabolism arising from greater release of stored transmitters as well as amplified activity of the main NE-synthesizing enzyme, TH. The NE-rich neurons of the locus coeruleus respond to most stressors by activating CRH-expressing neurons in the PVN (Habib, Gold, and Chrousos 2001). However, the activation of the HPA axis through CA innervations of PVN neurons occurs after some but not all stressors. Serotonergic neurons also have a major influence on neuroendocrine responses to stress, and stressful stimuli result in elevations in serotonin turnover in limbic centers (reviewed in Carrasco and Van de Kar 2003). The cell bodies of these 5HT neurons are located mainly in the dorsal or median raphe nuclei of the midbrain and project to the hypothalamus with collateral branches to the amygdala and possibly to other limbic forebrain regions. Serotonin influences the neurons in the PVN, which have high expression of 5HT receptors. The stress-related decrease in serotonin reserves in experimental animals mirrors the situation in humans, where depletion of brain serotonin is associated with increased anxiety (i.e., perceived stress). Stress may transiently or permanently alter gene transcription and protein synthesis in neurons (Fuchs, Flugge, and Czeh 2006). Neurons of stressed animals generally have higher levels of c-Fos, a transcription factor involved in regulating the expression of many genes in highly active neurons. The induction of c-Fos in response to acute stress is widespread in the cerebral cortex and limbic system of rats. The magnitude of c-Fos expression varies among regions of the brain and is dependent on the intensity and duration of stressful stimuli and sex (Figueiredo, Dolgas, and Herman 2002; Kvetnansky, Sabban, and Palkovits 2009). The degree to which c-Fos is expressed also depends on age; a greater number of CRH-containing PVN neurons express c-Fos after brief

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restraint in prepubertal (28-day old) male rats relative to young adult (77-day old) males (Romeo et al. 2006; Romeo 2010). This difference is functional rather than structural as immature and mature animals both have the same number of CRHproducing neurons. Intermittent social stress alters the expression of hundreds of genes in the nonhuman primate (Saimiri sciureus [squirrel monkey]) prefrontal cortex, with most of these genes being downregulated to a modest degree (Karssen et al. 2007). Affected genes are involved in cell cycle progression, nuclear receptor signaling, synaptic plasticity, and ubiquitin conjugation. Whether these changes are harmful or simply adaptive is uncertain. PNS structures also exhibit differential gene activity in response to stress. For example, stress increases expression of CA synthetic enzymes in both the adrenal medulla (HPA axis) and sympathetic ganglia (SNS), with peak expression occurring more rapidly and at higher levels in the adrenal medulla (Kvetnansky, Sabban, and Palkovits 2009). Neurochemical changes during natural disease can modulate the effects of stress. Some populations of CNS neurons can be sensitized to subsequent episodes of stress following exposure to inflammatory cytokines such as IL-1b (Johnson et al. 2004). Critical systemic illness leads to dysregulation of neuroendocrine axes involving the hypothalamus and pituitary gland, which increases the risk of morbidity and mortality in severely ill human patients. The dysregulation is driven by central deficiency of hypothalamic releasing factors (specifically CRH and TRH) that promote pituitary secretion as well as several hormones (especially GH and LH; Langouche and Van den Berghe 2006). Specific mechanisms by which hypothalamic neuronal function is decreased during systemic illness have not been identified. The lack of hypothalamic releasing factors suppresses pulsatile discharge of pituitary hormones. Rapid normalization of the circulating concentrations of hormones precedes clinical recovery. Biochemical mediators and processes of neurons other than those directly involved in signal transition may be affected by stress (reviewed in McEwen 2008). Metabolic changes in neurons linked to stress include depleted ATP and glutathione levels, increased intracellular Caþþ concentrations, increased production of reactive oxygen species (ROS), and decreased glucose uptake by cells (Anju, Jayanarayanan, and Paulose 2011; Lian and Stringer 2004; Madrigal et al. 2001; McIntosh and Sapolsky 1996b). Chronic stress induces lipid peroxidation and mitochondrial dysfunction in rat brain (Madrigal et al. 2001). Glucocorticoids have been hypothesized to disrupt energy metabolism in stressed hippocampal cells (Stein-Behrens et al. 1994) and enhance ROS-mediated neurotoxicity in vitro (McIntosh and Sapolsky 1996a). Glucocorticoids also potentiate release and accumulation of excitotoxic amino acids (EAA) in the limbic cortex (Chambers et al. 1999) and hippocampus (Bremner 1999; Danilczuk et al. 2005) following stress. While stress induces numerous neurochemical changes, not all of these alterations are undesirable. Stress-associated expression of heat shock proteins (Hsp) appears to protect the brain and certain viscera. For example, the number of mRNA transcripts for Hsp70 increases in the cerebral cortex and

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stomach of rats following acute restraint water-immersion stress (Fukudo et al. 1997). This protein, which protects cells from stress, appears to be localized to synapses (Bechtold, Rush, and Brown 2000). Various Hsp also play a role in stress-induced hyperphagia, as rats given a sucrose diet while being restrained generated more Hsp27 and Hsp70 in the cerebral cortex and cerebellum as well as more Hsp70 in the hypothalamus (Kageyama et al. 2000). Increased synthesis of Hsp72 following hyperthermia protects the integrity of the blood–brain barrier (BBB) in rats from a subsequent hyperosmotic episode (Lu et al. 2004). The distribution of both Hsp70 and glucocorticoid receptors decline in the cerebral cortex and hippocampus of adult Wistar rat males exposed to acute stress (cold, immobilization) but are essentially unchanged following chronic stress (crowding, daily swimming, social isolation; Filipovic et al. 2005). Taken together, these findings suggest that the presence of Hsp may attenuate the detrimental effects of repeated stress by helping to lessen the activity of the HPA axis.

4.3. Interactions between Neurotoxicants and Stress Neuroactive agents have been reported to be more effective at initiating a stress response relative to other xenobiotics (DeBoer et al. 1992). The more pronounced effects of neurotoxicants reflect modulations in stress hormone concentrations rather than overt structural and functional alterations defined to specific CNS or PNS domains. Histologic changes observed in neural tissues after exposure to neuroactive drugs typically are ascribed to the effects of the xenobiotic rather than the direct outcome of stress. Clinical and experimental data indicate that stress itself also is harmful to the nervous system. Neurons of animals experiencing stress are more sensitive to excess amounts of some neurotransmitters, especially EAA such as glutamate (Chambers et al. 1999). Hyperglutamatergic states may be critical in provoking both the acute and long-lasting consequences of traumatic stress (Chambers et al. 1999). Glutamate associated with traumatic stress may be derived from the adrenal medulla and may exert its effects by promoting long-term potentiation of neuronal synapses (Weiss 2007). Brain cells of animals experiencing stress have a greater sensitivity to neurotoxicants, possibly as a consequence of glucocorticoid-mediated sensitization (Cory-Slechta et al. 2008; McDonald, Craig, and Hong 2008). The converse is also true: neurotoxicants (e.g., heavy metals) can enhance the vulnerability of brain cells to stress (Cory-Slechta et al. 2008). Simultaneous exposure to a neurotoxicant and stress can cause synergistic effects on the nervous system and can be detrimental even if neither the stress nor the toxicant alone is damaging (Cory-Slechta et al. 2008, 2010). In general, the changes are evident as alterations in levels of neurotransmitters and/or hormones of the HPA axis. To our knowledge, a detailed description of anatomic and functional abnormalities in this setting has not been reported.

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Toxicants and stress can interact to affect neurologic pathways. Some neurotoxicants (e.g., exogenous corticosteroids, metals) alter the architecture of stress circuitry in the brain, acting to decrease neuronal numbers (especially in the hippocampus) by apoptosis and decreased proliferation (Bremner 1999; Cooper and Kusnecov 2007; Noorlander et al. 2008; Robinson et al. 2010). Glucocorticoids exacerbate the loss of hippocampal neurons of animals experiencing stress to the excitotoxicant kainic acid in vivo (Stein-Behrens et al. 1994) and enhance ROS-mediated, adriamycin-induced neuronal toxicity in vitro (McIntosh and Sapolsky 1996a). Endogenous corticosteroids from dams experiencing stress can also affect developing nervous systems of fetuses after having crossed the placenta, typically by decreasing neuronal numbers in certain populations (Maccari et al. 2003; Noorlander et al. 2008; Zuena et al. 2008). Neuronal loss in toxicant-treated animals experiencing stress is associated with attenuated cognitive ability (Cooper and Kusnecov 2007; Noorlander et al. 2008). In adult rats, stress in combination with voluntary self-administration of ethanol has been shown to stimulate the release of ACTH and corticosterone, thereby sensitizing the brain to a neurochemical balance favoring a prolonged stress response (Richardson et al. 2008). These experiments correlate stress-induced neurochemical changes with addictive tendencies. 4.4. Minimizing Stress-related Nervous System Effects During Toxicity Studies Despite the known effects of stress on the nervous system, specific neurological findings reflective of stress generally will be undetectable in toxicity studies. This is a result of the subtle nature of stress-induced neural alterations and the relative insensitivity of anatomic and functional methods used in toxicity studies to these changes. Evidence that stress has been a contributing factor in a test article–related effect during a toxicity study should rely on demonstration of the hallmarks of stress that can already be observed in other organ systems (Table 1). Stress-related effects in the nervous system may be observed in routine toxicity studies as subtle functional alterations (behavioral and/or clinical), but will rarely be associated with morphologic findings in the nervous system. 5. ENDOCRINE SYSTEM

AND

STRESS

The HPA axis, particularly the adrenal gland, is a sensitive indicator of stress in toxicity studies. Adrenal gland weights and the morphology of the adrenal cortex are often altered in subacute to chronic stress. In contrast, serum concentrations of glucocorticoids are not generally a sensitive indicator of stress due to interanimal variability in their response to stress and the normal diurnal cycles of glucocorticoid production. Stress leads to an increase in circulating ACTH, which results in hypertrophy of the cells of the zonae fasciculata and reticularis of the adrenal cortex. If stress is prolonged, hyperplasia of the zona fasciculata (ZF) and zona reticularis (ZR) may occur. This results in an increase in the weight of the adrenal glands due to hypertrophy of the cortex. Adrenal cortical hypertrophy


TABLE 5.—Morphologic findings for endocrine organs potentially associated with acute or chronic stress responses in routine toxicity studies. Tissue Adrenal medulla Adrenal cortex Thyroid gland

Acute stressa Chromaffin cell degranulation Decreased vacuolation No change

Chronic stressb Hypertrophy/hyperplasia/ pheochromocytoma (rats only) Hypertrophy/hyperplasia No change

Note: Morphologic effects of stress depend on the species, sex, age, and type and duration of stressor. Refer to appropriate text sections for species-specific details. a Acute: minutes to hours. b Chronic: days to weeks.

due to stress is the end result of prolonged and variable endocrine and nervous system responses to daily or periodic stressors. In toxicity studies where there is an increase in adrenal gland weights, it is important to differentiate adrenal gland hypertrophy (due to stress) from degenerative changes of the adrenal cortex that are often characterized by cellular hypertrophy and vacuolation due to disruption of steroidogenesis. To complicate the interpretation, both stress and degenerative changes can occur simultaneously in the adrenal cortex, and adrenal cortical toxicity can interfere with the normal stress response of the adrenal cortical cells. 5.1. General Responses to Stress The endocrine system responds in both an acute and a chronic manner to stress in order to maintain homeostasis (Tables 5 and 6). The stress response is mediated by the adrenal medulla through EPI and NE and the HPA axis through glucocorticoids (Figure 1). EPI and NE cause increased heart rate and blood pressure, decreased blood flow to the viscera, and an altered metabolic state. Glucocorticoids and catecholamines act in concert and complement each other to increase energy mobilization and blood pressure. Under some conditions, EPI increases blood glucose concentrations and is more potent than NE in increasing heart rate and cardiac output. The left adrenal gland in mice and other species is normally larger than the right (Droste et al. 2003). The cause of the size differential is unknown, but it may be due to greater sympathetic innervation or function in the left adrenal gland as a result of greater NE concentrations and control of the sympathetic system on the right side of the brain. Neural input to the adrenal gland is an important factor for growth of the adrenal cortices and sensitivity to ACTH concentrations (Droste et al. 2003). Chronic stress that causes adrenal cortical hypertrophy would be expected to decrease or abolish the size differential between the adrenal glands. There are important interactions between the HPA and SAM axes during normal conditions and stress. In chromaffin cells, NE is N-methylated by the enzyme phenylethanolamine N-methyltransferase (PNMT) to produce EPI; PNMT is required for EPI production. The activity of PNMT is regulated

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TABLE 6.—Hormonal parameters potentially associated with mild or severe stress responses in routine toxicity studies. Hormone CRH ACTH Catecholamines Cortisol or corticosterone T4 T3 rT3 Estrogen Progesterone Testosterone Prolactin LH FSH GnRH

Acute Stressa

Chronic Stressb

" " " " !"# !# # "#! "#! "#! "#! "#! "#! "#!

!"# !"# !" !"# !# !# !"# #! "#! #! !"# #! !# #

Note: Hormones affected by stress depend on the species, sex, age, and type and duration of stressor. Refer to appropriate text sections for species-specific details. a Acute: minutes to hours. b Chronic: days to weeks.

by both hormonal and neural stimuli, and its activity can serve as a marker of adrenergic function (Wurtman 2002). Glucocorticoids enter the adrenal medulla directly from the corticomedullary portal system of capillaries and upregulate PNMT by increasing PNMT mRNA transcription and enzyme stability. Although glucocorticoids are the primary regulator of PNMT gene expression, PNMT production is also induced by the sympathetic nervous system in response to cholinergic and depolarization stimuli (Lee et al. 1999). Knockout mice lacking CRH have decreased basal- and stress-induced concentrations of plasma corticosterone and EPI, as well as decreased PNMT activity (Jeong et al. 2000). In stress models that result in a predominantly sympathetic response, there is prolonged activation of the sympathetic nervous system resulting in chronically increased plasma concentrations of EPI and NE and increased adrenal TH activity. In some forms of stress, decreased corticosteroid-binding globulin (CBG) can contribute to increased free corticosterone concentrations (Tinnikov 1999). The increased adrenocortical response also results in hypertrophy of the adrenal gland. Bilateral adrenalectomy in rats leads to undetectable circulating concentrations of corticosterone and EPI, which demonstrates that the adrenal glands are the principal source of these hormones. However, NE is still measurable in adrenalectomized rats due to contributions from nonadrenal sources. Although PNMT is largely restricted to the adrenal medulla, the enzyme is also expressed at low levels in other tissues, such as cardiac atria, cardiac ventricles, and sympathetic ganglia (Kvetnansky et al. 2006). Stress increases extra-adrenal PNMT expression in the cardiac atria and sympathetic ganglia, but not in cardiac ventricles. Activation of the HPA axis causes an increase in circulating glucocorticoids (principally cortisol in hamsters, dogs, cats, and primates and corticosterone in birds, rodents, and rabbits; Rosol et al. 2001; Vinson,Whitehouse, and Hinson 2007).

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Rabbits are unusual in that they have significant amounts of both cortisol and corticosterone. During stress, rabbits tend to shift synthesis toward cortisol (Kolanowski et al. 1985). Glucocorticoid concentrations increase 10-fold or more in lifethreatening stress and by 3- to 5-fold in more moderate stress. Mild chronic stress may only increase trough concentrations, while mean peak concentrations may remain in the normal range. The biological effects of glucocorticoids are mediated by the mineralocorticoid and glucocorticoid receptors. Glucocorticoids can increase or decrease the numbers or activity of these receptors depending on the tissue and physiological condition, but in general chronic stress causes downregulation (Zhe, Fang, and Yuxiu 2008). Under normal conditions, glucocorticoids are secreted intermittently in a pulsatile manner, approximately once per hour. In addition, glucocorticoids exhibit a circadian pattern with peak concentrations at times of peak activity (e.g., for rodents during the first few hours of the dark period) and trough concentrations at times of reduced activity (e.g., for rodents about the first 6 hr of the light period; Lightman et al. 2002). Circadian rhythms of glucocorticoids in diurnal (i.e., non-nocturnal) animals are the opposite of those of nocturnal animals. For example, in rhesus monkeys, baseline peak cortisol concentrations (12–15 mg/dl) occur between 8 and 16 hr while trough concentrations (6.5–8.0 mg/dl) fell between 20 and 24 hr (Morrow-Tesch, McGlone, and Norman 1993). Stress during the dark period induces less ACTH and corticosterone response in rats compared to stress during the light phase (RetanaMarquez, Bonilla-Jaime, Vazquez-Palacios, DominguezSalazar, et al. 2003). Circadian secretory patterns are reduced or lost in pregnant or lactating rats. 5.2. Effect of Gender and Age on Glucocorticoids Glucocorticoid concentrations vary with many physiological factors, including sex, strain, metabolic state, and age. The majority (80–90%) of serum glucocorticoids are bound to the protein, CBG, while l0% is bound to albumin. Free glucocorticoids are 10% or less of the total amount in serum. The circulating half-lives of total and free corticosterone are 25 and 15 min, respectively, in rats (Sainio, Lehtola, and Roininen 1988). Serum CBG and corticosterone increase dramatically in rats during postnatal weeks 3 and 4 (Tinnikov 1993). Interestingly, research on the effects of stressors is often done in male rats, which have lower circulating corticosterone concentrations with less frequent secretory pulses than agematched females. Males also have a less rapid increase in corticosterone following stress compared to females (Kant et al. 1983). Human males with chronic pain syndrome had decreased cortisol compared to females with the same condition (Turner-Cobb et al. 2010). Female mice and rats have higher total corticosterone concentrations compared to males, but this is counterbalanced by their higher CBG concentrations (Tinnikov 1999). Neonatal rats (days 4–14) are less responsive to stress possibly due to inhibitory maternal factors and negative feedback from higher free corticosterone concentrations

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(Zelena et al. 2011). In addition, various functions of the HPA axis are downregulated during aging (Gust et al. 2000). For example, cerebral glucocorticoid receptors and hippocampal neurons are decreased during aging, which is consistent with an escape from glucocorticoid negative feedback in this region of the brain. The normal inhibition of the pituitary gland and hypothalamus by circulating glucocorticoids is also decreased in aging rodents. The attenuation of this negative feedback loop results in higher ACTH and corticosterone concentrations in aged compared to young rats. In contrast, aged (15–27 years old) female rhesus monkeys have lower cortisol concentrations compared to younger (7–8 years old) female monkeys (Gust et al. 2000). 5.3. Adrenal Medulla Acute stress leads to degranulation of adrenal medullary chromaffin cells and increased secretion of EPI and NE. Immobilization stress in rats increases PNMT mRNA in the adrenal medulla due to the promotion of transcription by glucocorticoids (Tai et al. 2007). Pharmacological doses of ACTH or glucocorticoids increase plasma concentrations of NE and EPI and decrease their reuptake and metabolism. In contrast to pharmacologic doses of corticosterone, endogenous concentrations of serum corticosterone have an inhibitory effect on stressinduced NE synthesis, release, reuptake, and metabolism in peripheral sympathetic nerves during immobilization or cold stress in rats (Fukuhara et al. 1996; Kvetnansky et al. 1993). This effect is abrogated in adrenalectomized rats. Chronic stress leads to adrenal medullary hypertrophy with increased catecholamine synthesis, enhanced proliferation of chromaffin cells, and an increased incidence of pheochromocytomas in some species (especially some strains of rats; Rosol et al. 2001; Tischler, Powers, and Alroy 2004; Tischler and Coupland 1994). 5.4. HPA Axis Acute stress results in activation of the HPA axis and increased synthesis of glucocorticoids. Synthesis and secretion of glucocorticoids from the adrenal cortex are regulated by ACTH secreted from chromophobic corticotrophs in the adenohypophysis (anterior pituitary gland). Secretion of ACTH is controlled by CRH and AVP secreted from the hypophysiotropic neurons in the PVN of the hypothalamus into the portal circulation of the median eminence. The CRH-producing neurons (50% in rats and 100% in mice) also express AVP during basal conditions. AVP may be a more important HPA secretogogue in neonatal rats compared to adult rats (Zelena et al. 2011). Basal as well as circadian variations of ACTH secretion are regulated by the hypothalamus. Multiple inhibitory and excitatory neural pathways also regulate the HPA axis (see Nervous System above). Expression of the early response gene c-fos in the PVN reflects the CNS response to stressful stimuli (Chan et al. 1993). Negative feedback of glucocorticoids on the HPA axis is regulated directly and indirectly by glucocorticoid receptors


in the hippocampus, hypothalamus, and adenohypophysis. The distribution and specificity of glucocorticoid receptors influence stress responses (Miller et al. 1994). There are two types of glucocorticoid receptors with different binding affinities: type I (mineralocorticoid; higher affinity) and type II (glucocorticoid; lower affinity). Low concentrations of glucocorticoids maintain basal HPA activity through high-affinity mineralocorticoid receptors (principally in the hippocampus), whereas high concentrations interact with lower-affinity glucocorticoid receptors to negatively regulate the HPA axis (principally in the PVN and the adenohypophysis). (Spencer et al. 1998; Weiser and Handa 2009). Approximately 9% of the pituitary corticotrophs in the adenohypophysis of unstressed rats contain stores of ACTH that can be detected immunohistochemically (Sasaki et al. 1990). Ultrastructurally, corticotrophs in unstressed rats typically contain a single row of secretory granules beneath the cell membrane. Acute stress increases the percentage of corticotophs that stain positively for ACTH and may result in three or more rows of cytoplasmic secretory granules near the cell membranes. Chronic stress leads to increased pituitary weight due to changes in the number and size of corticotrophs of the adenohypophysis. At the cellular level, corticotrophs exhibit hypertrophy and proliferation; at the subcellular level, cytoplasmic degranulation in conjunction with hypertrophy of the endoplasmic reticulum and Golgi apparatus are principal corticotroph features. Stress also increases the number of acidophils in the pituitary gland (Knigge 1958), and glucocorticoids suppress the secretion of growth hormone from acidophils (Pecile and Muller 1966). During chronic stress there is typically increased CRH and AVP in the PVN and increased mRNA of proopiomelanocortin (POMC, an ACTH precursor) in the pituitary corticotrophs (Zelena et al. 2003). In some chronic stress situations in rodents, AVP may be the primary secretogogue for ACTH since CRH concentrations can be downregulated by circulating glucocorticoids. The synthesis and secretion of NE in the PVN can also be inhibited by glucocorticoids (Pacak et al. 1993). Chronic stress in male rats decreases responsiveness of the HPA axis by transitory alterations in CRH expression within the PVN (Ostrander et al. 2006). 5.5. Adrenal Cortex 5.5.1. Normal Adrenal Cortex Adrenal gland weights in mice increase from 3 to 7 weeks of age. They subsequently remain stable in female mice but decrease by 25% in male mice from weeks 7 to 9 (Bielohuby et al. 2007). Accordingly, adult female mice have larger adrenal glands and greater circulating corticosterone concentrations compared to male mice. Serum corticosterone concentrations decrease from weeks 3 to 11 in male and female mice. Mouse strains may differ in the rate of postnatal adrenal gland growth (Badr, Shire, and Spickett 1968). In an unstressed state, cellular proliferation in the adrenal cortex follows a circadian rhythm controlled by ACTH.

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Proliferative cells reside predominantly in the outer, less differentiated portion of ZF (the middle adrenocortical layer that produces glucocorticoids in response to ACTH). Peak proliferation occurs in the ZF of rats at 3 to 4 hr, about 4 hr after peak blood ACTH concentrations occur. The proliferative cells in the ZF are not synthetically mature because they lack cytochrome P450 enzymes, aldosterone synthase, and 11b-hydroxysteroid dehydrogenase (Mitani et al. 1994). The proliferating cells migrate medially toward the center of the gland into the ZF and ZR (the inner layer) to populate these regions, eventually undergoing apoptosis near the ZR–medullary junction (Mitani et al. 2003) in the presence of tissue macrophages (Mitani et al. 2003). The proliferating cells are positive for the marker Ki67 and have increased DNA synthesis. Compensatory growth occurs in the contralateral adrenal cortex after unilateral adrenalectomy principally by proliferation of cells in the undifferentiated cell zone of the outer ZF (Engeland et al. 2005). In addition to cell numbers, ACTH regulates the growth and division of mitochondria in the ZF and ZR (Mazzocchi et al. 1976). Mice and rats differ from many other species in that they do not have a distinct ZR because they lack 17-alpha-hydroxylase expression and do not secrete adrenal androgens. The X-zone of the inner adrenal cortex in mice is the putative remnant of the fetal adrenal cortex. It varies in thickness depending on strain and sex (wider in females). The enzyme 20-ahydroxysteroid dehydrogenase (20aHSD), which catabolizes progesterone and 11-deoxycorticosterone, is a marker of the X-zone since it is restricted to this region of the adrenal cortex (Hershkovitz et al. 2007). Morphology of the X-zone is regulated by genetics and endogenous hormones. In male mice, the X-zone rapidly involutes during puberty due to the presence of testosterone with regression of 20aHSD activity after 3 weeks of age, while in female mice it persists until the first pregnancy. Androgens cause X-zone regression in females, and gonadectomy causes X-zone regrowth in mature males and females. In humans and nonhuman primates, fetal adrenal glands grow rapidly beginning in the middle of the first trimester due to hyperplasia and hypertrophy of the fetal cortical cells (Mesiano and Jaffe 1997). The primate fetal adrenal cortex atrophies after birth due to apoptosis, and the adult adrenal cortex forms from the outer proliferative zone. The zona glomerulosa (ZG, the outer adrenocortical layer) secretes the mineralocorticoid, aldosterone. Production of aldosterone is primarily regulated by aldosterone synthase (P450aldo), which in turn is controlled by the renin–angiotensin system. Peak proliferation in the ZG occurs at between 0600 and 0700 hr in rats (light period beginning at 0600 hr) in response to angiotensin II (Miyamoto et al. 2000). Although angiotensin II and Kþ are the principal regulators of ZG cell growth and function, ACTH can also stimulate aldosterone production and hypertrophy of ZG cells in concert with the renin– angiotensin system (Nussdorfer et al. 1977). In addition, ACTH can increase the volumes of cells, mitochondria, and smooth endoplasmic reticulum (SER) in the ZG of rats (Nussdorfer et al. 1977). The ZG atrophies in hypophysectomized rats.

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5.5.2. Acute Stress The stress response results in activation of the HPA and renin– angiotensin systems and secretion of ACTH, glucocorticoids, adrenal catecholamines, oxytocin, prolactin (PRL), and renin. Treatment of animals with single agents associated with the stress response, such as ACTH, does not effectively model stress due to the complex hormonal and neural responses and counterregulatory actions associated with stress. During acute stress, ACTH secretion is principally stimulated by CRH, a peptide hormone produced by the parvocellular neurons of the PVN. Thus, brain-derived CRH is the chief mediator of the behavioral and autonomic responses to stress. Glucocorticoids inhibit the stress response at the levels of the hypothalamus, pituitary gland, and neural circuits. Intra-neuronal repression of CRH following stress also limits the stress response (Aguilera et al. 2007). AVP is a weak agonist of ACTH and is synergistic with CRH, but is not as sensitive to negative feedback by glucocorticoids (Akil 2012). AVP may mobilize different pools of ACTH, compared to CRH, in the stress response. Brattleboro rats are deficient in AVP and have an impaired ACTH response to various stressors, supporting the concept that AVP is an important regulator of the stress response (Carrasco and Van de Kar 2003). Acute stress has variable effects on the morphology of the adrenal cortex. Acute heat stress (1 hr) in rats results in decreased adrenal mass and volume due to smaller ZF cells with fewer cytoplasmic lipid droplets (Koko et al. 2004). In addition, the cortical stroma becomes more prominent. In contrast, acute stress due to ethanol administration in rats has the opposite effects on the adrenal cortex after 30 min (Milovanovic et al. 2003). The diameter and volume of the ZF cells are increased during proestrus but not during diestrus. One hour of ACTH perfusion in rats leads to expansion of capillaries in the adrenal cortex and development of cell membrane filopodia that may be related to enhanced secretory mechanisms (Pudney et al. 1981). The effects of ACTH on adrenal cortical cells are reversed following cessation of stress and subsequent decreases in circulating ACTH concentrations. After a fall in ACTH concentrations, ZF and ZR cells undergo atrophy characterized by decreased mitochondria and SER within 48 hr. Microphagocytic vacuoles accumulate over the succeeding 3 days (Rebuffat et al. 1989). Athymic (nude) mice have an impaired HPA axis as indicated by a blunted ACTH response to stress, high basal ACTH concentrations, and hypertrophy of the ZF. These findings suggest that the immune system is necessary for normal function of HPA axis (Daneva et al. 1995). 5.5.3. Chronic Stress Chronic stress in rats generally increases adrenal gland weight and may increase basal corticosterone concentrations. If mild chronic stress increases the concentrations of corticosterone in rats, it will be most evident during the circadian trough period (Dallman et al. 2000). This response in rats is

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similar to that observed in humans. In addition, there may be a mild decrease in peak concentrations, but in general there is no change in the daily mean values of corticosterone (calculated as the average concentration from samples taken every 2 to 4 hr). It is important to recognize that chronic stress in animals and humans may not result in increased circulating corticosterone or cortisol concentrations. Rats experiencing chronic stress may have normal ACTH concentrations but an increased corticosterone response to ACTH. Chronic stress leads to histological and ultrastructural changes in the adrenal cortex including decreased neutral lipid vacuoles, cellular hypertrophy, hypertrophy of SER, increased mitochondria, cristolysis of mitochondria, and cellular proliferation. Adrenal cortical changes of hyperplasia and hypertrophy associated with stress are diffuse and bilateral, in contrast to focal adrenal cortical hypertrophy which is an incidental or background finding in many species (Harvey and Sutcliffe 2010; Kaspareit 2009; Rohr et al. 1978; Soldani et al. 1999). Chronic variable stress (CVS) in rats induces cellular hypertrophy of the ZF and adrenal medulla, hyperplasia of the outer medulla, and increased maximal responses to ACTH (Ulrich-Lai et al. 2006). Either ACTH administration for 7 days or chronic stress induces hypertrophy of rat ZF cells due to increased volumes of mitochondria and SER. After ACTH removal, ZF cells atrophy and form increased numbers of dense bodies, which are microautophagocytic bodies filled with SER, macroautophagocytic bodies with mitochondria, and residual bodies (Rebuffat et al. 1989). A CVS paradigm consisting of multiple stressors (twice daily exposure to hypoxia, 8% O2; warm swim, 28 to 31 C for 15 min; cold swim, 16 to 18 C for 5 min; cold room, 4 C for 1 hr; shaker platform, 100 rpm for 1 hr; restraint; 30 min; overnight isolation; overnight crowding, 6/cage) in SD rats results in increased adrenal weights, decreased body weight gain, hyperplasia of the outer ZF, hypertrophy of cells in the inner ZF and medulla (measured by decreased nuclear density), and atrophy of ZG cell size (Ulrich-Lai et al. 2006). In this setting, the maximal corticosterone response to ACTH is increased in proportion to the increased adrenal cortex mass. Chronic noise exposure is particularly stressful to rats and leads to a progressive increase in corticosterone concentrations (Soldani et al. 1999) and ultrastructural modifications of the adrenal cortex. Changes in the ZG related to noise stress include cellular hypertrophy and morphological changes consistent with a conversion to ZF-like cells with transformation of tubular to vesicular cristae in mitochondria. These changes are similar to those in rats following chronic treatment with ACTH, which also impairs the conversion of corticosterone to aldosterone in the ZG (Nussdorfer and Mazzocchi 1982). Prominent ultrastructural changes in the ZF include increased numbers and size of mitochondria and SER. Some mitochondria are abnormally shaped with signs of degeneration and cristolysis, and some SER are swollen. In contrast to other classic stress models, nutrient stress is not associated with increases in CRH. Instead, AVP appears to play the major role in eliciting glucocorticoid release, and


the adrenal response to ACTH is amplified by pancreatic polypeptides that are secreted during periods of hypoglycemic stress (Leakey, Seng, and Allaben 2004). The hormonal changes associated with nutrient stress (e.g., fasting, starvation, or insulin-induced hypoglycemia) in rodents are characterized by increased glucocorticoid serum concentrations in the absence of increased serum concentrations of ACTH or inflammatory cytokines (Leakey, Seng, and Allaben 2004). This hormone signature has certain similarities to the stress response elicited by many chemicals when administered at their maximum tolerated dose (Leakey et al. 1998). Mice exposed to chronic subordinate colony housing (i.e., confinement of subordinates with a dominant male) have decreased body weight, increased relative adrenal weight, and increased plasma NE concentrations. These mice have acute (but not chronic) increases in plasma corticosterone and hypothalamic CRH mRNA during the light phase and decreased plasma corticosterone and responsiveness to ACTH during the dark period (Reber et al. 2007). These changes indicate that adaptive responses occurring in the HPA axis during the chronic stress limit the ability of the adrenocortical cells to synthesize and secrete corticosterone. In addition, these mice exhibit increased inflammation in the colonic mucosa and increased pro- and anti-inflammatory cytokine secretion by mesenteric lymph nodes, supporting a role for chronic stress in the induction of inflammatory bowel syndrome. 5.6. HPT Axis Acute and chronic stress can alter thyroid homeostasis including thyroid hormone secretion and peripheral thyroid hormone concentrations. Likewise, an intact HPT axis is necessary for a normal adrenal response to stress. Hypothyroidism due to iodine deprivation in rats decreases both stressinduced and circadian corticosterone secretion in rats (Nolan et al. 2000). Two active forms of thyroid hormone exist in the circulation: 3,5,30 —Tri-iodothyronine (T3) is the biologically active form since it has higher affinity for thyroid hormone receptors than does thyroxine (T4). Peripheral tissues can convert T4 to T3 by the action of 50 -deiodinase. These two hormones are regulated by the production of thyroid-stimulating hormone (TSH [thyrotropin]), which is released from the adenohypophysis under the control of thyrotropin-releasing hormone (TRH) from the PVN of the hypothalamus. The peripheral conversion of T4 to T3 is inhibited by increased glucocorticoids. Repeated foot-shock stress in rats increases plasma corticosterone and decreases serum T3 and T4 with no change in TRH mRNA in the PVN (Helmreich et al. 2005). Inescapable foot-shock stress, but not escapable stress, decreases T3 in rats (Helmreich et al. 2006). Immobilization stress in rats increases plasma corticosterone, decreases serum T3, increases reverse T3 (rT3, an inactive isomer of T3), and reduces T4-converting 50 -deiodinase activity in the liver and kidney (Bianco et al. 1987). Administration of dexamethasone to rats reduces extrathyroidal conversion of

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T3 from T4 in rats and also reduces serum protein and cellular binding of T3, thus facilitating clearance of T3 (Cavalieri, Castle, and McMahon 1984). The inhibition of the HPT axis by stress is attenuated by aging in male F344/N rats (Cizza, Brady et al. 1995). Acute stress is associated with transiently increased T4 secretion and increased circulating T4 concentrations within a few minutes of the stressor (Langer et al. 1983). The mechanism is unknown, but it may be due to catecholamine release or sympathetic innervations of the thyroid gland. Chronic stress is often associated with decreased serum T3 and may be associated with decreased T4 in more severe cases, without altered TSH concentrations. An additional change that may be observed with chronic stress is decreased rT3. The condition of decreased peripheral T3 and T4 concentrations without concomitant alterations in TSH concentrations in the face of nonthyroidal illness is referred to as ‘‘euthyroid sick syndrome’’ or nonthyroidal illness syndrome, which can be substantial in rodents, dogs, and humans (Bello et al. 2010). The reductions in T3 and T4 are likely an adaptive response to chronic stress since they have a protein- and energysparing effect on the stressed animal. Short- and long-term experimental hyperthyroidism in rats results in increased serum corticosterone and CBG, but not increased ACTH. These changes are associated with increased adrenal weights and decreased thymus weights (Johnson et al. 2005). A dog with hyperthyroidism due to a functional thyroid tumor exhibited an increase in urinary cortisol:creatinine ratio and an exaggerated response of cortisol to a low-dose dexamethasone challenge test, which may have been due to altered cortisol metabolism or stress from the hyperthyroid state (Stassen et al. 2007). In contrast to hyperthyroidism, experimental hypothyroidism in rats is associated with decreased plasma corticosterone after stimulation with ACTH but exaggerated ACTH release in response to CRH injection (Tohei, Watanabe, and Taya 1998; Tohei 2004). Hypothyroidism is also associated with decreased adrenal weights (Tohei et al. 1998). 6. REPRODUCTIVE SYSTEM

AND

STRESS

The effects of stress on the male and female reproductive system are complex. These effects are influenced by many factors, including the nature of the stressor, duration of the stress, species and strain under investigation, and whether the animal is a continuous or seasonal breeder. Suppression of the HPG axis in response to stress results in decreased gonadotropin-releasing hormone (GnRH), gonadotropin hormones (follicle stimulating hormone [FSH] and luteinizing hormone [LH]), and gonadal sex steroids (testosterone, progesterone, and estradiol), and downstream effects on the reproductive system. In male rodents, the most sensitive reproductive endpoint to stress is a decrease in the weights of the accessory sex organs (prostate and seminal vesicles). Epididymal weights may also be decreased. Testis weights are generally unaffected in rats but may be decreased in mice. There are generally no detectable

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histologic changes in any of the reproductive organs, except in instances of repeated or more severe stress, which may result in minimal tubular degeneration in the testes of mice. The reproductive system of male dogs and nonhuman primates is generally less susceptible to the effects of stress and few structural changes are seen. Exceptions include the effects of social stress (decreased size and weights and lower spermatogenic potential) of testes in subordinate monkeys. The female reproductive system is more susceptible to stress than the male system and is particularly sensitive to the stress associated with decreased food intake and/or decreased body weights. In all species, the most sensitive reproductive parameter is disturbance of the estrous or menstrual cycle. In general, stress results in extended duration of the estrous cycle and may also result in anovulatory cycles and infertility. In addition, stress and decreased food intake may delay puberty in the female. In rodents, the downstream effects of decreased hormone concentrations may be reflected by decreased ovarian and uterine weights and a general inactive appearance of the reproductive organs, consistent with diestrus or anestrus. Also, there may be decreased numbers of ovarian follicles and corpora lutea, atrophy of the uterine endometrial glands, and atrophy and mucification of the vaginal epithelium. Most female dogs are in an inactive phase (diestrus or anestrus) for the duration of most toxicity studies, obfuscating identification of any effects of stress on the estrus cycle (Chandra and Adler 2008). In mature monkeys, disruption of menstrual cyclicity is a sensitive indicator of stress; this is commonly seen when individuals are introduced into new social groupings. Animals may have extended or anovulatory cycles, or become acyclic. Effects on the estrous cycle or menstruation in nonhuman primates are generally difficult to detect using only organ weights and histology as a result of the low number of animals and normal variability of these parameters. 6.1. HPG Axis and Stress Stress-related stimulation of the HPA axis is usually associated with inhibition of the HPG axis, but the response of the individual hormones of the HPG axis is variable, particularly during acute stress. The stress response is controlled from the PVN in the hypothalamus and involves the secretion and interaction of a variety of chemicals and neurotransmitters including CRH, AVP, glucocorticoids, b-endorphins, VIP, leptin, and CCK. GnRH, which is secreted in a pulsatile manner by the GnRH neurons located within the hypothalamus, orchestrates the endocrine regulation of male and female reproductive function by controlling gonadotropin hormone (FSH and LH) secretion from the pituitary gland, which in turn regulates secretion of the gonadal sex steroids (testosterone, progesterone, and estradiol) from the gonads. Secretion of GnRH into the hypophyseal portal tract is regulated not only by negative feedback of the gonadal sex steroids but also by many of the same pathways involved in regulation of the stress response and in the maintenance of energy status (for reviews, see Cunningham, Clifton, and Steiner 1999; Roseweir and Millar

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2009; Weinbauer et al. 2008). The critical regulatory gatekeeper integrating these various hypothalamic inputs to regulate GnRH secretion involves the kisspeptin/GPR54 ligand–receptor complex, which is present in multiple hypothalamic nuclei adjacent to the GnRH-secreting neurons. Kisspeptin/GPR54 has been shown to be pivotal at the time of pubertal onset in many species (Garcia-Galliano, Pinilla, and Tena-Sempere 2011; Roseweir and Millar 2009; Tena-Sempere 2006). Reproductive success is dependent on food and energy availability and adequate metabolic reserves. Many of the neurotransmitters involved in energy homeostasis such as leptin, ghrelin, CCK, and VIP are major regulators of the GnRH neurons in the hypothalamus, and decrease reproductive capability during times of low food availability. Female reproductive capacity is particularly sensitive to these environmental factors. Dietary restriction studies model the nonspecific toxicity (e.g., decreased body weight gains) produced by test articles administered at high doses. Reproductive endpoints are affected through stress-mediated and energy-mediated pathways acting at the level of the hypothalamus. Stress can directly inhibit FSH and LH secretion by the pituitary gland and also block gonadal sex steroidogenesis in the gonads, as well as affect GnRH secretion in the hypothalamus. Inhibition of steroidogenesis at the level of the gonad may be the result of decreased sensitivity of the testicular interstitial (Leydig) cells or ovarian follicular cells to gonadotropin stimulation or occur by inhibition of gonadal sex steroidogenic enzymes. In addition, stress may result in decreased sensitivity of peripheral target tissues to sex steroids produced by the gonads. Experiments performed to elucidate stress-induced changes in reproductive hormones have generated conflicting results, likely due to variables associated with the nature, severity, and duration of the stress models chosen. Most animal research on the effect of stress on the reproductive system has been done in rats. There is also a growing body of information on the effects of social or hierarchical stress on the reproductive system in nonhuman primates, which provides important information for the understanding of findings on toxicity studies where social housing is being used more commonly. 6.1.1. Effects on Hormones of the HPG Axis of Males Stress and CRH inhibit pituitary LH secretion in both sexes (reviewed by Leakey, Seng, and Allaben 2004). Stress or high glucocorticoid concentrations in male rats inhibit LH-mediated testosterone secretion in cultured Leydig cells. Dexamethasone treatment decreases testosterone, while adrenalectomy increases testosterone. Glucocorticoids also inhibit pituitary secretion of PRL but do not alter mean serum LH concentrations. Dietary restriction in male rodents results in decreased GnRH secretion, which is the primary cause of subsequent hormonal alterations of the HPG system (Bergendahl, Perheentupa, and Huhtaniemi 1989, 1991; Dong, Rintala, and Handelsman 1994; Dong et al. 1994). Four to six days of starvation in male rats causes a 25% loss in body weights, a 50% decrease in GnRH receptor


expression in the pituitary gland, and a 25 to 50% decrease in serum concentrations of FSH, LH, and PRL (Bergendahl, Perheentupa, and Huhtaniemi 1989). This regimen also decreases testicular and serum testosterone concentrations by 70 to 80%, testicular LH receptor expression by 26%, and PRL receptor expression by 50%. Coadministration of a GNRH agonist to fasted animals prevents the decline in serum concentrations of FSH, LH, and testosterone, and coadministration of human chorionic gonadotropin (hCG, a substitute for LH) prevents the decline in the serum testosterone concentration. These results demonstrate that the effects of dietary restriction are acting at the level of the hypothalamus to suppress GnRH secretion rather than as direct effects on hormone production at either the pituitary gland or the testis. Plasma progesterone concentrations are increased, and testosterone and androstenedione concentrations may be decreased in male rats experiencing stress (Pellegrini et al. 1998). Immobilization stress (1 hr/day for 7 consecutive days) in male rats increases plasma corticosterone and progesterone concentrations and depletes lipid stores from the ZF cells (Pellegrini et al. 1998). 6.1.2. Effects on Hormones of the HPG Axis of Females In females, glucocorticoids decrease FSH-stimulated aromatase activity and estrogen production by ovarian granulosa cells, suppress ovulation, and inhibit ovarian prostaglandin metabolism. Glucocorticoids also inhibit the pre-ovulatory pituitary LH surge in vivo as well as estradiol- and GnRHinduced LH production in cultured rat pituitary cells. Stress and CRH inhibit pituitary LH secretion in both sexes (reviewed by Leakey, Seng, and Allaben 2004). Effects of dietary restriction on female rodent reproductive hormones include decreased LH pulsatility, resulting in reduced or failed ovulation and decreased synthesis of estradiol and progesterone from the ovary. These hormonal changes also result in delayed puberty. Effects of stress on primate reproductive cycles have been studied primarily in humans. In women, amenorrhea, delayed menarche, and infertility are commonly seen in association with malnutrition, eating disorders, chronic alcoholism, and physiological stress such as intense exercise. In women, the main effects appear to be (1) suppression of GnRH secretion under the control of CRH and b-endorphin; (2) cortisol-induced inhibition of GnRH, LH, ovarian estradiol, and progesterone biosynthesis; and (3) cortisol-induced target tissue resistance to estradiol. Disruption of normal menstrual cyclicity and reproductive hormone patterns with rapid development of amenorrhea has also been demonstrated in cynomolgus monkeys subjected to strenuous exercise training (Williams et al. 2001). The effect is thought to be primarily due to elevation of CRH and glucocorticoids causing suppression at all levels of the HPG axis (reviewed by Chrousos, Torpy, and Gold 1998; Donadio et al. 2007). 6.1.3. Modulation of the HPA Axis by Reproductive Hormones The adrenal cortex is more sensitive to ACTH stimulation in the presence of high circulating concentrations of PRL, resulting

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in increased secretion of corticosterone and progesterone. Acutely stressed male rats with experimental hyperprolactinemia have increased peak ACTH, corticosterone, and progesterone concentrations (Jaroenporn et al. 2007). The increased PRL normally observed in aging SD female rats is in part responsible for increased basal cortisol in mature (12 months) and senescent (24 months) female rats (Lo, Kau, and Wang 2006). High PRL can also cause adrenocortical cell hypertrophy. Experimental hyperprolactinemia induced by the DA inhibitor domperidone (4 mg/kg SQ per day) in rats increases adrenal weight and hypertrophy of ZF cells (Silva et al. 2004). Administration of domperidone promotes PRL secretion by reducing the ability of hypothalamic DA to inhibit PRL release. Estrogen modulates the HPA axis by potentiating peripheral stress responses and suppressing central stress responses. Estrogen potentiates the HPA axis response to numerous acute stressors in female rats. Estrogen decreases the ability of glucocorticoids to exert negative feedback signals to the HPA axis by binding to the estrogen receptor-a in the hypothalamus of SD rats (Weiser and Handa 2009). In addition to potentiating the central HPA axis, estrogen promotes glucocorticoid secretion in females. Estrogen increases the corticosterone response to ACTH in dexamethasone-blocked ovariectomized females. Therefore, estrogen increases the sensitivity of the adrenal gland to ACTH. There is no effect of estrogen on corticosterone metabolism in the liver of rats (Figueiredo et al. 2007). Estrogen is also responsible for increasing CBG concentrations in female rats. In contrast to the effects of estrogen that amplify stress responses, estrogen can also have a beneficial effect in blunting some stress responses. The mechanism is unknown, but estrogen may enhance the inhibition of glucocorticoids on the HPA axis, increase the sensitivity or reduce the degradation of glucocorticoid receptors, or have direct effects on the brain (Carrasco and Van de Kar 2003). Estrogen decreases the acute stress response in rats. Estrogen inhibits the c-fos response of the PVN and ACTH response to acute stress in female SD rats (Figueiredo et al. 2007). Estrogen replacement therapy suppresses the HPA axis in postmenopausal women. 6.1.4. Dietary Restriction/Decreased Food Consumption and the Hormones of the HPG Axis Although dietary restriction/decreased food consumption and loss in body weight may be a part of the overall stress response, energy balance plays a special role in the control of reproductive function and behavior and has its own regulatory pathways that feed into the HPG axis. Similar to its role in stress, GnRH suppression in the hypothalamus is a major pathway responsible for the changes in the reproductive axis associated with energy (food) availability. The specific pathways that lead to GnRH suppression are unknown but appear to involve a number of metabolic hormones involved in energy homeostasis and appetite regulation mediated through the kisspeptin/GPR54 system (for reviews, see Cunningham, Clifton, and Steiner 1999; Hileman, Pierroz, and Flier 2000; Korner and Aronne 2003). Kisspeptin has a role in energy homeostasis, and

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the Kiss1 gene expressed in pancreas, liver, intestine, and testis is involved in the actions of leptin and ghrelin on appetite suppression and adiposity. Kisspeptin has also been shown to be involved in the onset of puberty, potentially serving as a mediator for the link between body weight/adiposity and the timing of puberty (Roseweir and Millar 2009; Tena-Sempere 2006). 6.1.5. Acute versus Chronic Effect of Stress on Reproductive Hormones Acute and chronic stress have different effects on reproductive hormones (reviewed by Rivier and Rivest 1991; Table 6). Acute stress primarily affects receptor sensitivity and steroidogenesis of the ovaries and testes, while chronic stress decreases plasma concentrations of GnRH, LH, and gonadal sex steroids (testosterone, progesterone, and estradiol). Acute stress can produce a brief and transient increase in LH and gonadal sex steroids, possibly due to stress-induced changes in plasma volume and liver clearance and/or activation of hypothalamic NE-producing neurons. However, these effects are highly variable and difficult to measure. Conversely, chronic stress generally causes a decrease in circulating LH, which is the primary cause of stress-induced inhibition of ovulation. Acute stress has different effects depending on when it occurs with respect to stages of the estrous cycle (Donadio et al. 2007). Stress applied in the morning of proestrus decreases the number of oocytes maturing to ovulation, decreases LH concentrations, and promotes progesterone and PRL surges later in the same day. These changes are not seen when restraint is applied in the afternoon of proestrus (at the time of the PRL surge). Instead, reduced sexual behavior (lordosis quotient) is seen during the evening of proestrus when stress is applied during the prior afternoon. 6.2. Male Reproductive System 6.2.1. Male Reproductive Organs (Table 7) In male rodents, reproductive organ weights and histologic changes associated with stress are due to the decreased testosterone secretion caused by suppression of GnRH. The most sensitive and frequently seen changes are decreased weights of accessory sex organs (seminal vesicles and prostate; Chapin and Creasy 2012). The secretory function of these accessory sex organs is highly dependent on androgen concentrations, and the decreased organ weights are primarily the result of decreases in the secretory products. Except in severe cases, the decreased weights are not associated with any detectable histologic changes. Absolute testis weights generally are not changed due to stress (with the exception of mice) because there are negligible effects of stress on spermatogenesis. This is due to conservation of testicular growth/weight, which occurs despite loss of body weight. Epididymal weights (absolute and relative to body weight) may be decreased due to lower testosterone concentrations; however, epididymal weights are not as sensitive an indicator as the weights of the seminal vesicles and prostate. In rats, there are generally no significant stress-

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TABLE 7.—Reproductive system parameters potentially affected by mild or severe stress responses in routine rodent toxicity studies. Sex

Degree of stress Effects

Male

Mild Severe

Female Mild Severe

Decreased prostate and seminal vesicle weights Markedly decreased prostate and seminal vesicle weights Testicular degeneration (mouse only) Irregular estrous cycle, reduced numbers of corpora lutea (CL) Persistent diestrus, reduced CL, reduced fertility

Note: Hormones affected by stress depend on the species, sex, age, and type and duration of stressor. Refer to appropriate text sections for species-specific details.

induced histologic effects on testis or epididymis unless the stress is severe. Atrophic changes (e.g., apoptosis, atrophic epithelium and decreased secretion) may be seen in the seminal vesicles and prostate as a qualitative finding, but organ weights are generally a more sensitive and quantitative endpoint. Histological testicular changes have been reported in rats restrained in inhalation tubes, but this may be due to heating of the testes rather than to restraint stress per se (Brock et al. 1996; Dodd et al. 1999; Rothenberg et al. 2000). In mice, but not rats, dietary restriction can result in mild degeneration and/or partial depletion of germ cells from the testis (Chapin, Gulati, Barnes et al. 1993; Chapin, Gulati, Fail et al. 1993). The reproductive organs of male dogs are less sensitive to the effects of stress than those of rodents. Prostate weights may be decreased with severe stress or severe body weight loss, but normal interanimal variability in prostate weights makes this measurement a relatively insensitive endpoint. Testis and epididymis weights are not affected. Dogs experiencing stress generally do not have histologic changes in the testes or epididymides but may show atrophy of the prostatic epithelium. Nonhuman primates may have decreased testicular size, weights, and spermatogenesis associated with stress. However, weights and histology of seminal vesicles and prostate are not sensitive indicators of stress in these species. 6.2.2. Specific Stressors 6.2.2.1. Dietary Restriction and Decreased Body Weight: There have been numerous studies on the effects of dietary restriction on the reproductive system of male rodents. Decreases in plasma testosterone and PRL concentrations as well as decreased epididymal, prostate, and seminal vesicle weights have been consistently demonstrated with a wide variety of experimental regimens ranging from mild dietary restriction to starvation (Bergendahl, Perheentupa, and Huhtaniemi 1989; Chapin, Gulati, Barnes et al. 1993; Chapin, Gulati, Fail et al. 1993; Glass, Herbert, and Anderson 1986; Grewal, Mickelsen, and Hafs 1971; Mulinos and Pomerantz 1941; O’Connor et al. 2000; Rehm et al. 2008; Trentacoste et al. 2001; Widdowson, Mavor, and McCance 1964). Except in situations of chronic severely decreased food intake, testicular weights and histology are generally unaffected in the rat. Mild


testicular degeneration has been reported in rats after 2 weeks of severe dietary restriction (Levin, Semler, and Ruben 1993). Using a detailed stage-aware histologic evaluation, minimal and subtle changes in the testes, characteristic of those seen with testosterone depletion (e.g., degeneration of occasional stage VII/VIII round spermatids and spermatocytes) have been reported in rats following an approximately 25% dietary restriction for 6 weeks (Rehm et al. 2008). Decreases in reproductive hormone concentrations and weights of accessory sex organs (seminal vesicles and prostate) may be substantial without histologic evidence of reproductive tract changes. For example, over 15 days of dietary restriction in rats, decreases in body weight gain of 26% (compared to controls) result in decreased absolute weights of epididymis and accessory sex organs but no change in absolute testis weights (O’Connor et al. 2000). Histology of testis and epididymis are unaffected. Significant decreases in PRL and testosterone concentrations are seen at body weight decreases of 21% (O’Connor et al. 2000). Similar results were demonstrated following graded dietary restriction of SD rats maintained at body weights that were 90%, 80%, or 70% of ad libitum–fed controls for up to 17 weeks (Chapin, Gulati, Barnes et al. 1993). Seminal vesicle and prostate weights (absolute and relative to body weight) were decreased in all groups of diet restricted rats, but there were no effects on testis or epididymal weights or histology. Dietary restriction produces a greater decrease in testosterone concentrations in younger rats (e.g., 6–8 weeks) compared with older rats (e.g., 10–12 weeks; Rehm et al. 2008). In older rats, 6 weeks of dietary restriction causes minimal degeneration of stage VII pachytene spermatocytes (a characteristic of low testosterone concentrations) in the testes. Regardless of age, seminal vesicle, prostate, and epididymal weights are decreased following 2 or 6 weeks of dietary restriction, but plasma LH is unaffected. Seminal vesicle weights are considered the most sensitive reproductive endpoint for stress in rats (Rehm et al. 2008). Mice are more sensitive than rats to dietary restriction for reproductive endpoints. When Swiss CD-1 mice are subjected to dietary restriction that caused the same decrease in body weight gains (90, 80, or 70% of ad libitum–fed controls), there are significant decreases in weights of testis (absolute) as well as epididymis, prostate, and seminal vesicles in all diet restricted groups, and also histologic evidence of altered testicular function (increased incidence of tubular degeneration) in approximately one-fourth of the food-restricted mice. Intratesticular testosterone levels were decreased in all diet restricted groups, falling to 16% of control values in mice maintained at 70% of control body weight (Chapin, Gulati, Fail, et al. 1993). Dietary restriction generally has greater effects on reproductive function in species that are seasonal breeders compared to continuous breeders. This phenomenon is presumed to result from the ability of seasonal breeders to shut down their reproductive hormones and functions in response to suboptimal environmental conditions, such as decreased food availability and diminishing light. For example, 30% dietary restriction of house mice (Mus musculus, which are continuous breeders)

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and deer mice (Peromyscus sp., which are seasonal breeders) significantly decreases sperm counts in deer mice but not house mice (Blank and Desjardins 1984). Marked interindividual variation in the response of the deer mice between those showing azoospermia and others showing no effect was also reported. Most of the species used in toxicity studies are nonseasonal breeders with the exception of hamsters and rhesus monkeys. It has been proposed that hamsters and rhesus monkeys may be more sensitive to the effects of stress, although there is currently no research to support this hypothesis. Although no published information was found on the effects of moderate dietary restriction or body weight loss on the reproductive tissues of male dogs and monkeys, these larger species appear to be relatively insensitive to developing morphological changes in the testes or accessory sex organs, based on personal observations on one author (Creasy). Published reports of dietary restriction in these species primarily describe hormonal changes. Rhesus monkeys subjected to marked caloric restriction (17% of ad lib feeding) for 20 to 34 days have decreased body weights and reductions in plasma LH and FSH concentrations (Dubey et al. 1986). Moderate caloric restriction (30%) of prepubertal rhesus monkeys for a period of 7 to 8 years had no effects on plasma LH or testosterone concentrations but showed minor changes in pituitary and testicular gene expression (Sitzmann et al. 2009). 6.2.2.2. Restraint/Immobilization-induced Stress: Protocols using immobilization in tube restraints have been developed to investigate the effects of stress on a variety of endpoints. Since restraint of rodents using nose-only inhalation tubes (4–8 hr/day for 5–7 days/week) is a common procedure during inhalation studies, the results of these restraint models are relevant to inhalation toxicity studies. Acute (2-hr) immobilization and chronic intermittent immobilization increase circulating glucocorticoid concentrations and decrease testosterone concentrations as well as increase germ cell apoptosis in the seminiferous tubules of adult rats (Charpenet et al. 1981, 1982; Kostic et al. 1997; Yazawa et al. 1999). Testicular lesions are commonly noted in control rats in nose-only inhalation studies when compared with oral gavage studies (Lee et al. 1993). In addition, testicular degeneration exhibits a dose-related trend in increased incidence and/or severity following nose-only inhalation exposure of rats to trifluoroiodomethane or trifluoroethane (Brock et al. 1996; Dodd et al. 1997). After equivalent dosing by whole body inhalation exposure, testicular changes were absent (Brock et al. 1996; Dodd et al. 1999). The effects observed in the nose-only exposure setting were attributed to stress and immobilization caused by restraint (Lee et al. 1993) and thermal stress due to restraint (Brock et al. 1996; Dodd et al. 1999). These immobilization and thermal stress effects on reproductive parameters, including changes in organ weights, histology, sperm parameters, or fertility are eliminated when rats are first acclimated to the tubes and tube restraint is performed in a cooled environment (Rothenberg et al. 2000).

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Histologic testicular changes observed in control rats used in nose-only inhalation studies of 2 weeks duration include prominent spermatid retention and eosinophilic globular bodies (degenerate round spermatids; Lee et al. 1993). In more severely affected animals, elongating spermatids were totally depleted and round spermatids were partially absent. Although these findings were not attributed to low testosterone concentrations in the original article, the profile of changes described are consistent with those resulting from decreased intratesticular testosterone concentrations (Creasy 2001). Simple restraint of male or female monkeys in primate chairs has been reported to inhibit pulsatile GnRH secretion (Chen et al. 1992; Norman and Smith 1992). However, it is unlikely that short periods of intermittent restraint, as typically occurs in toxicity studies, would result in any detectable changes in histology or reproductive function. In contrast, total immobilization of monkeys for a prolonged period (14 days) results in severe germ cell loss from seminiferous tubules (Gondos, Zemjanis, and Cockett 1970). 6.2.2.3. Social/Hierarchical Stress in Nonhuman Primates: Most nonhuman primates live in social groupings in their natural habitat, and it has become common practice to co-house monkeys during toxicity studies to reduce stress. However, initial establishment of social hierarchy in recently introduced groups of monkeys is stressful and can result in dramatic changes in reproductive parameters. Niehoff, Bergmann, and Weinbauer (2010) reported a 45% decrease in testis volume of subordinate male cynomolgus monkeys following introduction into a social grouping. Most of the decrease occurred during the first 3 months of social housing. The testis volume returned to baseline after 6 months of social interaction. Testicular volume of dominant males in the same groupings stayed constant or increased over the same period. Although histologic evaluation was not conducted, the decreased testicular volume was not associated with decreased sperm count. The dominant males generally had increased basal concentrations of cortisol and an initial decrease in body weight gain whereas the subordinate males had normal basal cortisol concentrations, but an increased response to ACTH. Dominance was closely correlated with initial body weight. Although dominant males did not always have the highest testosterone concentrations at introduction, higher testosterone concentrations were observed in dominant versus subordinate individuals, once dominance was established. 6.2.3. Male Puberty In males, there are no definitive markers of puberty to provide an accurate timing of sexual maturation. However, compared with females, stress and deceased food intake have less impact on male reproductive development. Male rats subjected to 50% dietary restriction prenatally (dams fed 50% of control intake throughout gestation and lactation) and postnatally from weaning through puberty have delayed initiation of sexual behavior and in the appearance of sperm in a penile smear (23 days and 16 days delay, respectively; Larsson et al. 1974). Graded dietary restriction (up to 33% of control intake)

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in male rats results in a weak inverse correlation (r ¼ 0.31) between growth rate and age at first conception and a weak positive correlation between growth rate and litter size. At 51 days of age, daily sperm production and spermatogenic development are similar regardless of growth rate, but testis size is slightly decreased in severely diet-restricted rats. The degree and duration of decreased food intake that has been shown to delay pubertal development in the male is unlikely to occur in toxicity studies. Effects of immobilization on reproductive parameters differ from those of dietary restriction in male rats. Intermittent immobilization stress (6 hr/day immobilization for 15 days) in prepubertal Wistar rats (40 days of age) results in decreased testicular maturation as indicated by a 16% decrease in the number of maturing spermatids per seminiferous tubule cross section, a 32% reduction in epididymal caudal sperm concentration, and a 17% decrease in seminal vesicle weights. In addition, at the end of 15 days of intermittent immobilization, increased (rather than decreased) concentrations of testosterone were recorded in association with decreased plasma LH, and increased corticosterone and PRL concentrations. When intermittent immobilization stress is applied for 60 days to 40-day-old prepubertal rats, testosterone is decreased and corticosterone and PRL are increased (Almeida et al. 1998; Almeida et al. 2000). 6.3. Female Reproductive System 6.3.1. Female Reproductive Organs and Cyclicity (Table 7) The most sensitive indicator of stress on the female reproductive system is a disturbance in the estrous or menstrual cycle. Cyclicity and ovulation are tightly regulated by the complex cascade of hormones secreted from the hypothalamus, pituitary gland, and ovaries. Minor disturbances in the amplitude, timing, and/or frequency of these hormones will alter (generally prolonging) the duration of the estrous cycle. In general, mice are more sensitive than rats in their reproductive response to various stressors, but there are also differences between strains. For example, chronic mild stress significantly disrupts the estrous cycle of Long-Evans rats but has relatively little impact on estrus stage in SD rats (Konkle et al. 2003). Potential morphological consequences of stress-induced hormonal disturbances in rodents are characterized by a generalized inactivity of the reproductive organs. The inactivity may be manifested as ovarian atrophy with inactive interstitial glands, atrophy of uterine epithelium and endometrial glands, and atrophy of the vagina (Yuan and Foley 2002). In rodents, a decrease in ovarian and uterine weights may occur but may be difficult to distinguish from the inherent normal variability and sampling of these tissues. The most common histologic changes in female rodents include decreased or absent corpora lutea and increased follicular atresia in the ovary accompanied by atrophic interstitial gland. Atrophic changes in the uterus and vagina (e.g., atrophy and mucification) and/or evidence of prolonged diestrus or anestrus are also observed (Chapin, Gulati, Barnes, et al. 1993; Chapin, Gulati, Fail, et al. 1993; Yuan and Foley 2002).


The effects of stress on the estrous cycle of dogs are unknown. However, due to the long duration of the canine estrous cycle, 70 to 80% of female dogs are in an inactive phase (diestrus or anestrus) for the duration of most toxicity studies, obfuscating the identification of any effects of stress (or test article) on progression of the cycle (Chandra and Adler 2008). In addition, since female Beagle dogs do not mature until 10 to 14 months of age, dogs used on toxicity studies may be immature and have contracted inactive uteri and ovaries without developing follicles and corpora lutea and/or with atretic follicles. These characteristics of immature dog reproductive tracts add to the difficulty of detecting changes in canine reproductive cyclicity. In monkeys, stress is most commonly reflected by prolongation of the menstrual cycle and failure of ovulation. These effects are best detected by in-life parameters, as organ weights and histology of the ovaries and uterus are not sensitive indicators in routine toxicity studies, given the normal variability due to maturation and stage in the menstrual cycle. 6.3.2. Specific Stressors 6.3.2.1. Dietary Restriction and Decreased Body Weight: In female rats, dietary restriction leading to a >16% decrease in mean body weight compared with controls has been associated with persistent diestrus, decreased corpora lutea, and decreased fertility (likely due to the decreased corpora lutea; Terry et al. 2005). On the basis of minor changes seen in estrous cyclicity at 16% decrease in body weight gain, decreases of 10 to 15% were considered nonadverse to reproduction in the female rat. Dietary and/or caloric restriction has been shown to delay the age at which reproductive senescence occurs, particularly in the Sprague-Dawley rat (reviewed by Leakey, Seng, and Allaben 2004). Reproductive parameters of mice are more sensitive to dietary restriction than are those of rats. Both mice and rats had increased lengths of the estrous cycle, decreased ovarian weights, and decreased numbers of corpora lutea following dietary restriction (Chapin, Gulati, Barnes et al. 1993; Chapin, Gulati, Fail et al. 1993). In rats there was no effect on overall fertility or number of implants per dam, but in mice these parameters were significantly adversely affected, even at relatively minor (10%) reductions in body weight gain. In rhesus monkeys, dietary restriction (animals offered 30% less food than controls) for a period of 6 years (resulting in a 16% decrease in mean body weight compared with controls) had no effect on the menstrual cycle or on circulating concentrations of reproductive hormones (Lane et al. 2001). Simple restraint of male or female monkeys in restraint chairs will inhibit pulsatile GnRH secretion (Chen et al. 1992; Norman and Smith 1992). Although short periods of intermittent restraint may not affect the menstrual cycle, longer periods of restraint may cause disturbances if used in a toxicity study.

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6.3.2.2. Social/Hierarchical Stress in Nonhuman Primates: Introduction of female cynomolgus monkeys from single housing into social groups prolongs the menstrual cycle from an average of 31 days to 46 days for about 6 months before returning back to baseline. Prolongation of the cycle correlates with a decrease or loss of the preovulatory estradiol peak and loss of the normal luteal rise in progesterone (Weinbauer et al. 2008). Once females have adapted to pairing or social groupings, menstrual cycle duration normalizes. If pairs of females with no previous contact are housed together, one assumes a socially dominant role and retains a normal cycle while the other develops a prolonged cycle. In a group of female monkeys containing a male, the dominant female cycles normally while the subordinate females demonstrate hormone suppression, irregular or arrested cycles, and infertility (reviewed by Weinbauer et al. 2008). 6.3.3. Female Puberty The timing of puberty in the female is influenced by metabolic status and is delayed by decreased food intake (reviewed by Cameron 1990, 1996). In rats, severe dietary restriction in the prepubertal female delays puberty (as defined by vaginal opening or first estrus), prevents ovulation (as shown by an absence of corpora lutea), and results in smaller ovaries (Lintern-Moore and Everitt 1978; Widdowson, Mavor, and McCance 1964). Although some follicles undergo development up to pre-ovulatory follicles in the diet-restricted rat, all antral follicles undergo atresia and ovulation does not take place. In addition, there are increased numbers of primordial follicles in diet-restricted rats due to their decreased entry into the pool of growing follicles (LinternMoore and Everitt 1978). Prepubertal rats maintained at 45% of their expected body weights by dietary restriction have no evidence of pulsatile GnRH and do not ovulate by 50 days of age. Reinstating access to food induces pulsatile GnRH within 12 hr, and ovulation occurs within 2.5 to 3.5 days (Bronson 1986). Social stress can affect timing of puberty in nonhuman primates. Subordinate females may show delayed puberty compared with females having a higher social status. 6.4. Pregnancy and the Developing Fetus Animal models have been used to study the effects of prenatal stress on the developing fetus through to adulthood. Effects have been reported in brain structure and function, sexual differentiation, and various changes in the autonomic, neuroendocrine, immune, and reproductive systems (for a general review, see Gulati and Ray 2011). Chronic stress in pregnant rats results in decreased weight gain and food consumption, and increased adrenal gland weights in dams (Mairesse et al. 2007). In addition, fetuses have decreased body weights and decreased pancreas and testis weights; decreased pancreatic b-cell mass; decreased placental expression and activity of 11b-hydroxysteroid dehydrogenase Type 2 (11b-HSD2); and decreased plasma concentrations of

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TABLE 8.—Clinical pathology parameters potentially affected by acute or chronic in routine toxicity studies. Parameter Neutrophil count Lymphocyte count Eosinophil count RBC mass parameters (RBC, HGB, HCT) Reticulocyte count Bone marrow cellularity Glucose concentration

Acutea

Chronicb

" " ! !" !" ! !"

!"# # # !# !# !# "#!

Note: Hormones affected by stress depend on the species, sex, age, and type and duration of stressor. Refer to appropriate text sections for species-specific details. a Acute: minutes to hours. b Chronic: days to weeks.

growth hormone, ACTH, and glucose (Mairesse et al. 2007). Offspring of pregnant animals (including humans) that experience chronic prenatal stress have decreased body weights and develop long-term metabolic, behavioral, and neuroendocrine changes, such as hyperglycemia and increased food consumption after fasting. 7. CLINICAL PATHOLOGY

AND

STRESS

Stress-related changes can affect several hematology and clinical chemistry parameters (Table 8). Of these, circulating leukocyte counts are most sensitive to stress. Immediately (seconds to minutes) after a stressor, total and differential leukocyte counts tend to be increased in proportion to their normal circulating numbers due to effects of EPI. Later (minutes to hours), stressors tend to cause increases in neutrophil counts and decreases in lymphocyte and eosinophil counts; these changes are mediated by glucocorticoids. With more mild stressors, only one or two of these leukocyte changes may be observed. Determining whether changes in circulating leukocyte counts are related to stress involves evaluating other endpoints (in-life, clinical pathology, histology) to determine the underlying cause of these changes. For example, increases in neutrophils and lymphocytes are typical with inflammatory processes in rodents. Evidence of inflammation (e.g., acute phase protein responses or histologic changes indicating inflammation) would make it likely that these peripheral leukocyte effects are a primary result of inflammation, rather than a secondary response to acute stress. In rats, decreased food intake and decreased weight gain suppress hematopoiesis, with the most noticeable effect being decreased reticulocyte counts. Clinical pathology parameters occasionally altered by stress include red blood cell and platelet counts, serum glucose concentrations, and bone marrow cellularity. Other routine clinical pathology parameters are rarely affected by stress, due either to their lack of sensitivity to stress and/or their inherent variability among individuals. Several endocrine hormones sometimes measured as components of clinical chemistry test panels are sensitive to acute stressors. These hormonal effects are covered in the Endocrine and Reproductive sections.

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7.1. Hematopoiesis Hematopoiesis is influenced by stress as well as administration of catecholamines or glucocorticoids. The sympathetic nervous system maintains hematopoiesis by cell retention in the bone marrow (Afan et al. 1997). Catecholamines also directly stimulate erythropoiesis in vitro through stimulation of b2-adrenergic receptors, but the in vivo relevance of this mechanism is uncertain (Afan et al. 1997; Brown and Adamson 1977). Glucocorticoids are essential for responses to erythropoietic stress in vivo (Bauer et al. 1999; Leberbauer et al. 2005; Wessely et al. 1997). However, administration of exogenous glucocorticoids has variable effects on erythropoiesis: increased (Fruhman and Gordon 1955a, 1955b; Malgor et al. 1974) or unchanged (Laakko and Fraker 2002). Glucocorticoids are also involved in expansion of all developmental stages of myeloid bone marrow cells, contributing to increased circulating neutrophil counts (Bishop et al. 1968; Trottier et al. 2008). In mice, injection of corticosterone at doses approximating short-term stress affects the bone marrow by decreasing lymphoid cell and increasing granulocytic precursors, without affecting erythroid or monocytic cell counts or total cellularity (Laakko and Fraker 2002). Additionally, granulocyte colony stimulating factor (G-CSF) has been shown to modulate increased circulating neutrophil counts due to hyperthermic stress, likely at the level of the bone marrow (Ellis et al. 2005). Glucocorticoids also promote survival of neutrophils and apoptosis in eosinophils in circulation (Meagher et al. 1996). In rats, profound stressors generally result in moderate to marked decreases in hematopoiesis for all three lineages (red and white blood cells and platelets), accompanied by decreased cellularity of the bone marrow. The stress of moderate to marked dietary restriction or decreased food consumption consistently causes a marked decrease in bone marrow hematopoiesis in rats along with decreases in circulating cell counts; decreased reticulocytes and hypocellular bone marrow are generally the most prominent changes (Levin, Semler, and Ruben 1993). Stressor-related decreased hematopoiesis is a common finding in toxicity studies in rats when the test article decreases food consumption and/or body weight gain. In studies in which rats exhibit decreased reticulocyte counts and/or bone marrow hypocellularity along with decreased dietary intake and/or body weight gain, decreased hematopoiesis is likely secondary to the inanition, rather than a primary test article–related change. The suppression of hematopoiesis due to decreased food consumption is not consistently observed in other species, although humans with anorexia nervosa occasionally exhibit bone marrow failure (Hutter, Ganepola, and Hofmann 2009). Stress also decreases the number of colony forming unitsgranulocyte/macrophage (CFU-GMs) in the bone marrow of rats (Malacrida et al. 1997). In other species of toxicologic relevance, stress effects on hematopoiesis are less prominent or not observed. Bone marrow failure may develop in humans with severe traumatic injuries that result in sustained increases in catecholamines and glucocorticoids (Fonseca


et al. 2005). Other chronic diseases in humans which result in stress are also associated with bone marrow suppression that contributes to decreased red cell mass (Cullis 2011). Psychological stress can have varying effects on erythropoiesis. Sleep deprivation decreases measures of erythropoiesis in mice (Skurikhin et al. 2005).

7.2. Red Blood Cells (RBC) 7.2.1. Effects of stressors Stressors may increase, decrease, or have no effect on red cell mass (assessed by red blood cell count, hemoglobin concentration, and hematocrit), depending on the duration and intensity of the stressor. In general, acute stressors causing primarily EPI-related effects (such as handling, exercise, or restraint) result in increased red cell mass and increased circulating reticulocytes due to release of mature and immature red blood cells from spleen and bone marrow (Hannon, Bossone, and Rodkey 1985; Joles et al. 1982; Laub et al. 1993; Szygula et al. 1985). In contrast, chronic stress is associated with decreased red blood cell mass and decreased or unchanged reticulocyte counts; for toxicity studies, these changes are most commonly observed in association with decreased food consumption and/or poor health (see above: Stress effects on hematopoiesis). Decreased red cell mass due to chronic stress is analogous to the clinical condition referred to as anemia of chronic disease, which is the most common cause of nonregenerative decreased red cell mass in humans and animals (Stockham and Scott 2012; Weiss 2002). Rats restrained for 6 hr/day and exposed to air in nose-only inhalation devices have transiently increased circulating reticulocytes after 1 episode of restraint, but not after 4 episodes (Pauluhn 2004). This transient effect indicates habituation of the reticulocyte response to a repeated stressor. 7.2.2. Effects of Exogenous Mediators of Stress Direct sympathetic stimulation or injection of EPI or NE causes increased red cell mass, primarily due to contraction of the spleen (Geiger, Song, and Groom 1976; Hannon, Bossone, and Rodkey 1985; Modin, Pernow, and Lundberg 1993; Nelson and Swan 1972; Wachtel and McCahan 1974; Wade 1973). Because the population of splenic erythrocytes is enriched for reticulocytes, sympathetic-modulated release of splenic erythrocytes increases circulating reticulocyte counts as well (Berendes 1959; Sorbie and Valberg 1970; Wade 1973). Red blood cells residing in bone marrow also contribute to stress-related reticulocytosis. Intra-marrow infusion of NE or direct stimulation of the sympathetic trunk decreases bone marrow reticulocytes and increases circulating reticulocytes via b-adrenergic receptors (O’Flynn and de Pace 1987; Webber et al. 1970). Circulating IL-6 also may mediate acute increases in circulating reticulocytes in response to stress. A single dose of IL-6 transiently increases circulating reticulocytes and late erythroid

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precursors in the bone marrow at 12 to 24 hr (Ulich and del Castillo 1991; Zhou et al. 1993). 7.2.3. White Blood Cells White blood cells, specifically neutrophils, lymphocytes, and eosinophils, are fairly sensitive indicators of stress. In general, short-term stress (i.e., minutes to an hour) results in increases in all blood cell types in proportion to circulating counts, whereas longer term stress (i.e., an hour to days) results in increased neutrophil counts and decreased lymphocyte and eosinophil counts. The timing and magnitude of such effects depends on the degree and duration of the stressor; restraint stress for 1 hr results in an approximately 50% decrease in circulating lymphocytes in rats, but the lymphocyte count recovers within 24 hr. Because leukocyte counts are so dynamic, the timing and method of blood collection can influence the magnitude of the effect observed as a result of a stressor. Both EPI and glucocorticoids affect white blood cell counts. In general, EPI increases counts of all classes of circulating leukocytes in approximate proportion to basal counts with the exception of some subsets of lymphocytes (Schedlowski et al. 1996; Tonnesen, Christensen, and Brinklov 1987; see Immune System section). In contrast, glucocorticoids increase circulating neutrophil counts while decreasing lymphocyte, monocyte (most species), and eosinophil counts (Calvano et al. 1987; Dhabhar et al. 1996; Speirs and Sullivan 1953; Stevenson and Taylor 1988; Tonnesen, Christensen, and Brinklov 1987). The decrease in lymphocyte counts as a result of glucocorticoids is more prominent than the increase in neutrophil counts in species with lymphocyte predominance such as rodents (e.g., rats and mice; Ulich and del Castillo 1991). Decreases in eosinophil counts can be seen in species with either lymphocyte predominance or neutrophil predominance (e.g., dogs and monkeys) using flow cytometric-based automated hematology analyzers with animal-specific software for differential counting. 7.2.4. Effects of Stressors Effects of stress models on leukocyte distribution and counts have been evaluated in most laboratory animal species and in humans. In general, leukocyte changes caused by stressors are similar to those observed in animals with pharmacologically induced glucocorticoid responses. Most studies of stress models measure the HPA response to the stressor (e.g., serum ACTH or serum glucocorticoid concentrations) and do not document effects on sympathetic mediators (e.g., concentrations of catecholamines). Stressors, including mild restraint of 2 hr or short periods of transportation, consistently increase circulating neutrophil counts and decrease circulating lymphocyte, eosinophil, and monocyte counts in rodents (Dhabhar et al. 1995b; Drozdowicz et al. 1990). Effects after mild stressors are similar to those observed after corticosterone administration, suggesting that corticosterone-related effects on leukocytes likely dominate

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over EPI-related effects in terms of leukocyte responses in rodents (Bowers et al. 2008; Dhabhar et al. 1995b, 1996). Social stress decreases circulating lymphocyte counts and increases granulocyte counts to a greater degree in male rats relative to female rats (Stefanski and Gruner 2006). Under social stress, monocyte counts are slightly increased in males and decreased in females. Decreases in lymphocyte counts due to stress are specifically due to glucocorticoid rather than mineralocorticoid effects and can be reproduced in corticosterone-treated adrenalectomized animals. Administration of aldosterone, a type I (mineralocorticoid) adrenoreceptor agonist, does not affect lymphocyte counts of rats (Dhabhar et al. 1996). In addition, administration of glucocorticoids or other type II (glucocorticoid) adrenoreceptor agonists has similar effects on lymphocyte counts compared to stressors (Dhabhar et al. 1996). Other mediators influence lymphocyte counts, since adrenalectomy does not completely abolish stress-related decreases in lymphocyte counts (Engler, Dawils, et al. 2004). Among the possible modulators are glucocorticoids from nonadrenal sources, endorphins, and other hormones and mediators of the HPA axis (Ader, Felten, and Cohen 1990; Altemus et al. 2001; Dhabhar et al. 1995a, 1995b, 1996; Qiu, Peng, and Wang 1996; Stojic-Vukanic et al. 2009; Vacchio, Papadopoulos, and Ashwell 1994). The stress of transportation and handling affects circulating leukocyte counts in rodents, dogs, monkeys, and other species. Transportation of dogs results in increased serum concentrations of cortisol and increased red cell mass (RBC count, hemoglobin, hematocrit), and changes in circulating leukocyte counts (increased neutrophil and decreased lymphocyte counts; Bergeron et al. 2002; Kuhn et al. 1991). Fifteen hours of transportation results in increased cortisol and circulating neutrophil counts and decreased lymphocyte counts in cynomolgus monkeys. Stress-related hematologic changes in monkeys resolve by 7 days after transportation (Kim et al. 2005). Three-hour chair restraint of rhesus monkeys is associated with increased serum cortisol concentrations, total circulating leukocyte counts, and relative neutrophil counts as well as decreases in relative percentages of lymphocytes and monocytes (Morrow-Tesch, McGlone, and Norman 1993). 7.2.5. Effects of Exogenous Catecholamines The role of catecholamines in stress-related circulating leukocyte changes has been studied by exogenous administration of catecholamines, adrenergic agonists, and adrenergic blocking agents. Catecholamines increase circulating leukocyte counts primarily by demargination of the marginal pool and mobilization from the spleen (Davis et al. 1991; Engler, Dawils, et al. 2004; Gader and Cash 1975; Iversen et al. 1994; Kradin et al. 2001; Toft, Tonnesen, Svendsen, Rasmussen, and Christensen 1992; Toft et al. 1994). Redistribution and trafficking of leukocytes are mediated through expression of adhesion receptors and their ligands on leukocytes and endothelial cells, and through the elaboration of chemokines.

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Demargination is likely a result of decreased adhesive forces and possibly increased blood flow and shear forces. Leukocytes residing in bone marrow and lymphatic vessels and organs inconsistently contribute to EPI-induced leukocytosis (Benschop, Rodriguez-Feuerhahn, and Schedlowski 1996; Iversen et al. 1994; Rogausch et al. 1999; Schedlowski et al. 1996; Toft, et al. 1992; Ulich et al. 1989). Adrenergic effects on neutrophils are thought to be modulated primarily by aadrenergic receptors, while those on lymphocytes are modulated by b2-adrenergic receptors (Benschop, RodriguezFeuerhahn, and Schedlowski 1996). Adrenergic effects on monocytes are due primarily to peripheral b-adrenergic effects, as shown by a series of experiments using various adrenergic antagonists (Engler, Dawils, et al. 2004). Circulating neutrophil and lymphocyte counts transiently increase in rabbits approximately 10 to 20 min after a bolus injection of EPI (Toft, Tonnesen, Svendsen, Rasmussen, and Christensen 1992). In humans, circulating neutrophil counts begin increasing by 15 min after EPI injection, and double at approximately 3 to 6 hr post-injection (Athens et al. 1961; Davis et al. 1991). Prolonged (12 hr) infusions of a—adrenergic agents to humans cause a glucocorticoid-type pattern in circulating leukocyte counts (i.e., increased neutrophil and decreased lymphocyte counts; Stevenson et al. 2001), presumably due to secondary stress and increased glucocorticoid concentrations as a result of persistent sympathetic stimulation. Injections of EPI increase neutrophil and lymphocyte density in the pulmonary capillary bed and decrease lymphocyte density in the spleen of mice, suggesting that organ-specific redistribution of leukocytes also occurs with EPI (Kradin et al. 2001; Morken et al. 2002). 7.2.6. Effects of Exogenous Glucocorticoids Both endogenous and exogenous glucocorticoids affect leukocyte counts. Endogenous glucocorticoids are responsible for circadian rhythms and, in part, the associated variability in leukocyte counts (Bradbury et al. 1991; Dhabhar et al. 1994; Morrow-Tesch, McGlone, and Norman 1993; Pelegri et al. 2003). In diurnal (daytime) animals, total leukocyte counts are highest in the evenings (end of the light period), while in nocturnal animals, total leukocyte counts and individual cell types peak between the 3rd and 7th hr of the light period in a 12-hr light/dark cycle (Abo et al. 1981; Dhabhar et al. 1994; Pelegri et al. 2003; see Immunology section for lymphocyte subsets). Glucocorticoid-induced increases in neutrophil counts are caused by prolongation of neutrophil half-life (possibly due to decreased apoptosis), increased release of the storage pool of neutrophils from bone marrow, and expansion of all developmental stages of myeloid bone marrow cells (Bishop et al. 1968; Cox 1995; Trottier et al. 2008; Valenzona et al. 1998). Glucocorticoid-induced decreases in lymphocyte counts result from redistribution of lymphocytes from blood, spleen, and bone marrow to other lymphoid organs (Toft, Tonnesen, Svendsen, and Rasmussen 1992) as well as promotion of apoptotic pathways in susceptible lymphocytes (Garvy, Telford et al.


1993). The magnitude of the decrease can be quite remarkable, but the changes are rapidly reversible. The approximate 70% decreases in circulating lymphocyte counts after acute mild stress in rats and humans reverse within 1 to 3 hr. Based on the rapid time course, it is likely that these alterations are due primarily to redistribution of lymphocytes (altered trafficking) rather than to cell death (Calvano et al. 1987; Dhabhar et al. 1995b, 1996). With automated hematology analyzers, it is common to see decreases in eosinophil counts in blood as a fairly consistent sign of stress in most species. The relationship between increased glucocorticoids and decreased eosinophils in bone marrow and peripheral blood is firmly established (Fruhman and Gordon 1955b). The mechanism for glucocorticoidrelated decreases in eosinophils may involve decreased mobilization from the bone marrow, decreased adherence to vascular walls, sequestration in the marginal pool and/or lymphoid tissues (predominantly the spleen), and decreased survival (Altman et al. 1981; Elsas et al. 2004; Sabag, Castrillon, and Tchernitchin 1978; Wallen et al. 1991). The importance of distribution of circulating eosinophils into the spleen after glucocorticoid administration is shown by the near abolishment of glucocorticoid-induced eosinopenia in splenectomized rats (Sabag, Castrillon, and Tchernitchin 1978). Glucocorticoids have inconsistent effects on circulating monocyte counts. The circadian rhythm of monocyte counts in rats is directly related to that of corticosterone. Accordingly, monocyte counts are lowest in the beginning of the light period (i.e., when corticosterone is lowest) and highest a few hours into the dark period (Dhabhar et al. 1994; McNulty et al. 1990). In contrast, monocyte counts decrease during infusion of cortisol in humans; the pattern of decrease parallels that of lymphocytes (Calvano et al. 1987). Dosing of humans with synthetic glucocorticoids results in decreases in monocyte counts, sometimes followed by sustained increases in the face of continued dosing (Rinehart et al. 1975; Steer, Vuong, and Joyce 1997). Decreased monocyte counts may be caused by decreased production, as sustained exposure to glucocorticoids results in decreased generation and release of monocytes from bone marrow in mice (Thompson and van Furth 1973). Effects of exogenously administered glucocorticoids on circulating white blood cell counts have been studied in humans, rats, mice, monkeys, and rabbits. A bolus injection of species-appropriate glucocorticoid (either cortisol or corticosterone) results in increased neutrophil counts and decreased lymphocyte and eosinophil counts (Davis et al. 1991; Tonnesen, Christensen, and Brinklov 1987). Similar to changes in other species, circulating neutrophil counts increase and lymphocyte counts decrease in rabbits approximately 1 to 6 hr after a single injection of the synthetic glucocorticoid dexamethasone (Toft, Tonnesen, Svendsen, and Rasmussen 1992). In humans, glucocorticoid administration causes a transient decrease in monocyte counts during the first few hours after glucocorticoid administration, after which monocyte counts increase to or rise above basal counts by approximately 24 hr (Rinehart et al. 1975). Concurrent administration of glucocorticoids and catecholamine initially causes changes in leukocyte

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counts consistent with those observed with EPI alone. At later time points, the effects of glucocorticoids become superimposed on those caused by catecholamines (Brohee et al. 1990). 7.3. Platelets Platelets are not a sensitive indicator of stress, although some research suggests that they may be increased in response to a stressor. The relative paucity of data concerning the effects of stressors on platelet counts may be due to the lack of an effect or the inability to detect stress-related changes due to the wide variation in platelet counts that occurs normally in healthy animals. Reports detailing the effects of emotional or physical stress models on platelet counts are scarce. Strenuous exercise and psychological stress both increase platelet counts in humans, and IL-6 increases platelet counts in rats (Dawson and Ogston 1969; Jongen-Lavrencic et al. 1996; Mischler et al. 2005). Administration of EPI has been reported to increase (rat, dog, and human), have no effect on (pig), or decrease (dog) platelet counts (Castro-Faria-Neto et al. 1989; Fortunato et al. 1991; Freden et al. 1978; Ljungqvist 1971; McClure, Ingram, and Jones 1965; Wachtel and McCahan 1974). Increases in platelet counts due to EPI are generally attributed to splenic contraction (Castro-Faria-Neto et al. 1989; Freden et al. 1978; Ljungqvist 1971). Decreases may be related to EPI-mediated activation of platelets and subsequent consumption/removal. 7.4. Serum/Plasma Glucose Concentrations Glucose concentration is not a sensitive indicator of stress. For example, glucose concentration in rats is less sensitive to exogenous corticosterone administration than body weight gain, thymus or adrenal weights, or serum insulin and CBG concentrations (Akana et al. 1992). Glucose concentration is also not specific for stress; numerous other factors affect glucose. Serum glucose concentrations can change rapidly in response to stress depending on the condition of the animal. Pronounced stress (e.g., moribundity or severe debilitation) in most species can result in minimally to markedly increased or decreased glucose concentrations. Stress affects glucose metabolism and homeostasis via stress-related hormones, such as catecholamines and glucocorticoids, and by glucose-modulating hormones, such as insulin and glucagon. Stress-related hormonal changes may result in increased glucose concentration and insulin intolerance (Eigler, Sacca, and Sherwin 1979). Injected EPI directly increases plasma glucose by inducing hepatic gluconeogenesis and glycogenolysis. Glucocorticoids stimulate glucose production by mobilizing amino acids for conversion to glucose, decreasing glucose utilization by cells, and decreasing sensitivity of cells to insulin. Glucagon release is potentiated by stress, thus further increasing plasma glucose concentrations. Glucagon counters the effect of insulin by directing hepatocytes to convert stored glycogen into glucose for release into the circulation. Concentrations of more than one stress-related hormone must be increased to result in relevant increases in glucose concentrations (Eigler, Sacca, and Sherwin 1979). Infusion of EPI,

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glucagon, or cortisol alone to fasted dogs has mild or no effects on plasma glucose concentrations. Infusion of EPI plus glucagon results in a transient increase in glucose and insulin. Infusion of EPI, glucagon, and cortisol causes a sustained and more prominent increase in glucose. Additionally, adrenergic blockers are unable to prevent increased glucose caused by EPI or NE injection, presumably because other mediators of hyperglycemia are also involved in causing increased glucose (Hermansen and Hyttel 1971). Stressor-related increases in glucose can be transient or sustained (Niezgoda et al. 1987; Pierzchala, Niezgoda, and Bobek 1985). For example, although repeated stress-induced increases in ACTH concentrations habituate and return toward normal during 13 daily stressors in rats, increases in glucose are more sustained and do not show the same degree of habituation (Marquez, Nadal, and Armario 2004). The effect of stressors on glucose concentration is dependent on the fasting state of the animal, and responses caused by corticosterone are synergistic with those caused by glucagon and EPI (Eigler, Sacca, and Sherwin 1979). Fasted rats have only a minimal increase in plasma glucose concentrations after immobilization stress, while fed rats have marked increases (Yamada et al. 1993). Increased glucose in stressed fed rats is accompanied by pronounced increases in plasma insulin; these changes are dependent on an intact adrenal gland (Yamada et al. 1993). The effects of stress on glucose are strain-dependent (Harizi et al. 2007). 7.5. Acute Phase Proteins and Cytokines The response to stress or inflammation causes characteristic increases in positive acute phase proteins and decreases in negative acute phase proteins primarily by altering hepatic protein synthesis. While these alterations can occur in the absence of typical inflammatory stimuli they are less sensitive indices than other markers of stress. Both IL-1 and IL-6 are critical mediators of these stress-related acute phase protein changes (Moshage 1997). Inescapable shock causes decreases in CBG (a negative acute phase protein) and increases in haptoglobin and a1-acid glycoprotein (positive acute phase proteins, Deak et al. 1997). No alterations in a panel of acute phase proteins occurred in humans during or up to 18 hr after a 6-hr combined infusion of physiologic doses of EPI and glucocorticoids. However, moderate changes in leukocyte counts were observed, suggesting that acute phase proteins were not as sensitive as leukocyte counts to pharmacologically relevant doses of these 2 mediators of stress. Acute and chronic stresses have differential effects on immune function, and some of these effects involve modulation of cytokines. An acute stressor may serve as an ‘‘endogenous adjuvant’’ that enhances the ability of the immune system to protect against pathogens and injury, while chronic stress causes immune suppression and dysregulation largely mediated through suppression of type 1 (cell-mediated) cytokine responses and enhancement of

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pro-inflammatory and type 2 (antibody-mediated) cytokine responses (Dhabhar 2009). Acute stress generally results in increases in pro-inflammatory cytokines (e.g., TNF-a, IL-6, and IL-1b). In turn, IL-1b and IL-6 activate the HPA axis at multiple levels (Kovalovsky et al. 2004; Prickett et al. 2000; Sekiyama et al. 2006; Sugama and Conti 2008). Effects of chronic stress may be mediated in part by TNF-a. During chronic stress, IL-6 helps maintain increased concentrations of IL-1b and glucocorticoids, and also antagonizes some of the effects of glucocorticoids (Nguyen et al. 1998; Shintani et al. 1995; Smith et al. 2007). In addition, the production of IL-6 during stress may be modulated in part by EPI (DeRijk et al. 1994; LeMay, Vander, and Kluger 1990; Zhou et al. 1993). IL-6 in conjunction with glucocorticoids is important for the acute phase protein responses discussed above (Hernandez et al. 2000). Both IL-6 and glucocorticoids are required for production of a nuclear regulatory protein involved in the regulation of acute phase protein synthesis (Nishio et al. 1993; Whalen, Voss, and Boyer 2004). IL-6 also directly stimulates pituitary corticotrophs to produce ACTH (Bethin, Vogt, and Muglia 2000). Studies in adrenalectomized mice show that corticosterone inhibits the stress-induced increase in IL-6 (Takaki et al. 1994; Zhou et al. 1993). In rats experiencing stress, increases in IL-6 parallel those of corticosterone and are also abrogated by adrenalectomy but not b-adrenergic antagonists (Zhou et al. 1993). 7.6. Other Clinical Chemistry Parameters Other clinical chemistry parameters are insensitive indicators of stress as it is observed during routine toxicity studies. Stressors can increase the activities of serum enzymes commonly assessed on toxicity studies. Administration of exogenous EPI or NE to rats, mice, guinea pigs, rabbits, and dogs results in increases in various serum enzymes, including alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LD), and aldolase (Highman, Altland, and Garbus 1965). These changes are diminished if animals are pretreated with a b-adrenergic blocker (Garbus, Highman, and Altland 1967). The primary clinical chemistry effect of exogenous glucocorticoids to rats or dogs is increased glucose concentrations. Dogs treated with pharmacologic doses of glucocorticoids or that have the clinical condition of hyperadrenocorticism can have increases in the serum activity of a dog-specific steroid-induced isoenzyme of alkaline phosphatase (ALP; Solter et al. 1993; Syakalima et al. 1997). Although this enzyme can be increased in dogs experiencing chronic stress, it is not considered a sensitive or specific marker of activation of the HPA axis (Center 2007; Sepesy et al. 2006). A variety of stressors consistently increase activities of serum/plasma enzymes (ALT, AST, CK, LD; up to *6-fold increases) and concentrations of other serum constituents (glucose, urea, cholesterol) in humans, rats, mice, dogs, and sheep (Apple et al. 1993; Arakawa et al. 1997; Ohta et al. 2009; Sanchez et al. 2002). The increases in these serum/plasma


constituents are thought to be due to increased sympathetic tone (i.e., increased catecholamine release) and may be differentially regulated by a- and b-adrenergic pathways (Arakawa et al. 1997). Increases in serum enzyme activities in response to stress are thought to be stressor-nonspecific since the inciting cause can be exercise, restraint, hypothermia, or emotional/social stress. Mice generally habituate to stress-related effects on serum constituents within 4 days (Sanchez et al. 2002). Dogs transported by ground for 9.5 hr had no alterations in activities of several serum enzymes (ALT, ALP, CK, aldolase, glutamate dehydrogenase, LD, and a-hydroxybutyrate dehydrogenase; Kuhn et al. 1991). In summary, the main effects of stress on clinical pathology parameters are those on circulating leukocyte counts. Other parameters such as reticulocyte counts and glucose concentrations are less frequently affected. Serum enzyme activities can be transiently affected by stress and may habituate after repeated stressors. 8. IMMUNE SYSTEM

AND

STRESS

The physiological and morphologic effects of stress on the immune system can be observed in blood, thymus, spleen, and bone marrow. Of the lymphoid organs, the thymus is the most sensitive; responses to stress can be elicited within hours. Glucocorticoids can cause redistribution of lymphocytes, alterations in cellular responses, and lymphocyte destruction. Responses of circulating lymphoid cells occur rapidly, temporally followed by histologic alterations in lymphoid organs. Only organ weight changes approximating 20% decreases are associated with histologic findings of lymphoid depletion. Interpretation of stress-induced pathologic findings in the thymus and spleen can be challenging due to the physiologic processes within the normal involuting thymus and the variability of the splenic architecture under normal physiologic conditions. Because immature lymphocytes are most sensitive to the effects of stress, the cortex of the thymus frequently is decreased in size as a result of lymphocyte depletion. The predominance of mature lymphoid cells in bone marrow and spleen compared to the thymus make the stress response in these organs less dramatic. Stress-related changes occur less consistently in other secondary lymphoid organs. 8.1. Physiological Organization of the Immune System The immune system is divided into the innate and the adaptive immune system. The innate immune system is the nonspecific arm of immunity that is responsible for the first line of defense against pathogens. It is dependent on the acute amplification of nonspecific mediators (including cytokines, complement, etc.) and the function of innate immune system cells (including granulocytes, monocytes, and natural killer [NK] cells). The ‘‘flight or fight’’ response to acute stress is directly linked through the sympathetic nervous system and the HPA axis to the innate immune system (Gadek-Michalska and Bugajski 2010; Goshen and Yirmiya 2009; Nance and Sanders 2007). The interactions between the sympathetic nervous

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system and the innate immune system are bidirectional; cytokines that are part of the innate immune system (e.g., IL-1b, IL-6, and TNF-a) activate the HPA which can then influence immunologic responses. Danger-associated molecular patterns (DAMPs) including heat shock proteins (HSP) and various nuclear components are released by stressed cells in association with the elaboration of IL-1b and IL-6 as well as activation of the HPA axis and sympathetic nervous system (Pedra, Cassel, and Sutterwala 2009; Zhou et al. 1993, 1996). These signals are associated not only with initiating immune responses but also with physiological adaptations to stress. The response of the innate immune system to antigen or tissue damage directs the response of the adaptive immune system. The adaptive (i.e., acquired) immune system is the antigenspecific arm that is largely organized into lymphoid organs consisting of lymphoid cells, accessory cells, and stromal cells. These cell populations form complex interactions involved in antigen-independent lymphocyte development in primary (central) lymphoid organs, and antigen-dependent lymphocyte activation in secondary (peripheral) lymphoid organs. Lymphocytes exit the circulation and enter peripheral lymphoid tissues through post-capillary high endothelial venules before returning to the circulation via the efferent lymphatic vessels. The trafficking of lymphocytes to and from lymphoid organs and other tissues is a highly complex and tightly regulated process dependent on chemokines and adhesion molecules. In a normal immune response, trafficking leads to ‘‘lymphocyte trapping’’ or antigen-induced sequestration that allows for the development of a specific immune response to a given antigen. The circulating population of immune cells, together with tissue-based immune cells in primary and secondary lymphoid organs, comprise the immune system. The primary lymphoid organs of most mammalian species—including the rat, mouse, dog, nonhuman primate, and human—are the thymus and bone marrow. The thymus is the source of T-lymphocytes, which control the adaptive immune responses and also participate in the cell-mediated component of this response. The bone marrow is the origin of B-lymphocvtes, which form the humoral (antibody) branch of the adaptive immune response. In the neonate and adult, lymphocytes develop from pluripotent hematopoietic stem cells in bone marrow and undergo complex developmental processes in primary lymphoid organs before emigrating as immunocompetent but naive lymphocytes to secondary lymphoid organs, including the spleen, lymph nodes, tonsil, and other lymphoid aggregates (e.g., gut-associated lymphoid tissue). The morphology of primary and secondary lymphoid organs can be quite variable under normal physiologic conditions due to the influence of the animal’s health status, age and condition, and bidirectional interactions among immune, endocrine, and neurologic systems. For these reasons, differentiation between primary (or direct) immunotoxic effects and secondary indirect (or stress-related) effects in routine toxicity studies can be challenging (Cizza, Pacak, et al. 1995; Cizza, Brady, et al. 1995; Cizza et al. 1996; Dominguez-Gerpe and Rey-Mendez 1998). Nevertheless, the morphology of thymus and spleen are an important part

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of the data set used for the weight-of-evidence approach for evaluating potential stress-related changes. 8.1.1. Normal Thymus Physiology The thymus is the site of T-lymphocyte development and maturation. During development, the cells enter the thymus from the bone marrow of as an immature population CD4- and CD8-negative (DN, double negative) cells that rapidly expand in the thymic cortex into a CD4- and CD8-positive (DP, double positive) population. Approximately 80% of developing T-lymphocytes undergo apoptosis as a result of not receiving appropriate signals, a process referred to as neglect. The remaining DP cells undergo complex developmental processes. The immature DP cells, comprising the majority of cortical lymphocytes, begin to express the T cell receptor (TCR) and undergo two selection processes before exiting the thymus as either CD4-positive or CD8-positive (i.e., single positive) effector cells. Once the TCR is expressed, the DP cells undergo positive selection in the thymus which is the process whereby only single positive T-lymphocytes with TCR of an appropriate affinity for self-peptides bound to MHC (major histocompatibility complex) molecules are positively selected for continued development. Positive selection is the basis of MHC class restriction, which is required to prevent the adaptive immune response from targeting self-antigens. Positive selection is followed by negative selection, which is the deletion of single-positive T-lymphocytes with high affinity TCRs to selfantigen MHC complexes. Negative selection is the basis of central tolerance and is essential to eliminating self-reactive T-lymphocytes. Less than 5% of DN T-lymphocytes that enter the thymus will exit as single-positive CD4 or CD8 effector cells. While the thymus is essential to establishment of the peripheral T lymphocyte repertoire, once the repertoire is established its function is less well understood particularly in mature animals. The primary mechanism regulating circulating T-lymphocyte counts is thought to be a process called homeostatic expansion (Berzins, Boyd, and Miller 1998; Marrack et al. 2000), by which the circulating T-lymphocyte population is maintained through T-lymphocyte proliferation in the secondary lymphoid organs rather than release of cells from the thymus. This may in part explain why significant morphologic changes in the thymus are not always correlated to findings in secondary lymphoid organs or in blood during routine toxicity studies. In mice, the number of circulating T-lymphocytes is largely unrelated to the number of T-lymphocytes within the thymus. Progressive thymic involution occurs near the time of sexual maturity in most species of interest for toxicity studies. Its primary morphologic feature is the marked decrease in the number of cortical lymphocytes, attributed to a decrease in proliferative capacity and an increased sensitivity to apoptosis (Kapasi and Singhal 1999; Plecas-Solarovic et al. 2006; Rinner et al. 1996). After progressive involution, the thymus consists of only adipose tissue and remnants of connective tissue and lymphoepithelial rests.

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The rate and extent of mammalian thymic involution is species-, strain-, age-, and sex-dependent (Haley 2003). The thymus attains maximal weight relative to body weight at birth and reaches maximal absolute weight approximately at sexual maturity. In the rat, thymic involution results in progressive reductions in thymic size and relative weight beginning at about 8 to 12 weeks of age (Losco 1992). In aged rats (18 months of age), thymic weight remains constant although there is a gradual decrease in the lymphoepithelial component that is offset by an increase in the volume of adipose connective tissue (Plecas-Solarovic et al. 2006). In contrast, adults of some but not all strains of mice have an age-dependent decrease in thymic size (Hsu et al. 2005; Li et al. 2003; Nabarra and Andrianarison 1996). The distinction between normal progressive involution, stress, and immunotoxicity, particularly in the NHP thymus, is often confounded because the exact age of the animal is unknown and the high degree of variability of thymic weights (Greaves 2012; Spoor, Radi, and Dunstan 2008). This is particularly true for the NHP thymus, as is evident from the evaluation of thymic weight data from a large number of control animals (Figure 3). This variability among normal animals can lead to an overinterpretation or misinterpretation of the finding of decreased thymic weights (Greaves 2012). The exact mechanisms involved in progressive thymic involution are unknown. The stimulus for progressive involution is preprogrammed and intrinsic to the thymus, and not attributable to emigration of cells from the bone marrow to the thymus (Aspinall 2000; Mackall and Gress 1997). Peripubertal increases in gonadal hormones and decreases in growth hormone promote involution (Chen et al. 2010; Sfikakis et al. 1998). Local concentrations of glucocorticoids are important for appropriate thymic development and T-lymphocyte development, differentiation, and selection in younger animals (Herold, McPherson, and Reichardt 2006; Jaffe 1924a, 1924b, 1924c; Jondal, Pazirandeh, and Okret 2004; Pazirandeh, Jondal, and Okret 2004; Stojic-Vukanic et al. 2009). Thymic glucocorticoid effects are mediated by high local expression of Type II glucocorticoid receptors. While physiologic concentrations of glucocorticoids are necessary for thymic development, supraphysiologic concentrations can be damaging and lead to increased apoptosis of cortical lymphocytes and cortical thinning.


FIGURE 3.—(A–C): Thymus weight parameters of control cynomolgus monkeys from 53 studies collected at a single CRO between 2002 and 2007. Values are presented as absolute weights (3A) and relative to body (3B) or brain (3C) weights. A: Absolute thymus weights (gm) of control cynomolgus monkeys from 53 studies collected at a single CRO between 2002 and 2007. Each column depicts data from a single control group. Each symbol is an individual animal. B: Thymus weights relative to final body weight (%) of control cynomolgus monkeys from 53 studies collected at a single CRO between 2002 and 2007. Each column depicts data from a single control group. Each symbol is an individual animal. C: Thymus weights relative to brain weight (%) of control cynomolgus monkeys from 53 studies collected at a single CRO between 2002 and 2007. Each column depicts data from a single control group. Each symbol is an individual animal.

8.1.2. Normal Spleen Physiology The spleen plays a major role in humoral immunity through the generation of antibody responses, particularly in response to blood-borne pathogens. The spleen consists of two compartments: the red pulp and the white pulp. The red pulp, consisting of blood-filled sinusoids, is primarily responsible for the clearance of old or damaged red blood cells by splenic macrophages. The white pulp, consisting of T-lymphocytes, B-lymphocytes, and accessory cells, is responsible for the immunological function of the spleen. The white pulp consists of a periarteriolar lymphoid sheath (PALS) containing T-lymphocytes and sometimes B-lymphocyte follicles as well as a surrounding marginal zone (MZ) predominantly comprised of larger B-lymphocytes and antigen-presenting dendritic cells.

Endogenous glucocorticoids activate Type II adrenal steroid receptors to a greater extent in rat peripheral blood, thymus, and lymph node than in the spleen (Miller et al. 1990; Spencer et al. 1993). This is in contrast to exogenous glucocorticoids, which activate adrenal steroid Type II receptors equally in immune tissues, including the spleen (Miller et al. 1990, 1994). The bidirectional complexity of the sympathetic nervous system and the spleen is essential to innate and adaptive immune response (reviewed in Straub 2004). The spleen increases in weight with age, resulting in a relatively consistent ratio of organ weight to body weight over time (Cheung, Vovolka, and Terry 1981; Losco 1992). There is an increase in the number of reticular cells and macrophages in the

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splenic white pulp with age, and a corresponding decrease in the number and functional activity of lymphocytes (Cheung and Nadakavukaren 1983; Losco 1992). This decrease in splenic lymphocytes generally occurs in rats greater than approximately 20 months of age, and results in white pulp atrophy which is more prominent in the thymic-dependent T-lymphocyte areas (i.e., PALS) than in the B-lymphocyte areas (i.e., MZ). 8.1.3. Basal Lymphocyte Subpopulations Basal lymphocyte subsets in rat blood and lymphoid organs are summarized in Table 8. Lymphocyte subset proportions are generally similar between male and female rats, with the exception of NK cells (higher in male Long-Evans and mature male Fischer 344 rats compared to females; Page, Ben-Eliyahu, and Taylor 1995; Stefanski and Gruner 2006). Total lymphocyte counts and lymphocyte subsets are more variable in blood than in spleen (Nygaard and Lovik 2002). Subsets of lymphocytes, similar to other circulating leukocytes (see Clinical Pathology section above) undergo circadian cycling. The peak circulating cell counts (acrophase) of total and subclasses of lymphocytes (with the exception of NK cells), similar to all leukocytes, occur primarily between the 3rd and 7th hr of the light period (Depre´s-Brummer et al. 1997; McNulty et al 1990; Pelegri et al. 2003). In contrast, NK cells peak approximately 4 hr before the beginning of the light period (Pelegri et al. 2003) and reach their nadir approximately 9 hr after the beginning of the light period (McNulty et al. 1990). Lymphocyte surface markers exhibit circadian rhythms; expression of some markers peaks in the dark period, and others peak in the light period (Pelegri et al. 2003). The circadian rhythm of splenic lymphocytes is less pronounced than those of circulating lymphocytes. Splenic B-lymphocyte counts are highest at the beginning of the light period, and lowest a few hours after the beginning of the dark period (McNulty et al. 1990). The lack of circadian rhythms in thymic lymphocytes may be due to its predominance of immature cells (Alvarez and Sehgal 2005).

8.2. General Responses to Stress Stress-related effects on lymphocytes in circulation or in lymphoid organs are mostly attributable to cell trafficking or redistribution when the stressor is mild and/or transient. It is unlikely that these effects are the result of changes in cell survival because effects of mild acute stress on immune cell distribution are rapidly reversible upon cessation of the stress. Stress-related changes in lymphocytes are evaluated by measuring circulating leukocyte counts, and thymus and spleen weights and morphology. Changes in circulating lymphocyte counts generally occur sooner (e.g., within minutes to hours) than those of thymus or spleen. Circulating lymphocyte counts and thymic parameters are generally more sensitive than splenic parameters to most stressors (Pruett et al. 2007; Pruett et al. 2000). The duration, intensity, and type of stressor likely determine which parameters (blood, thymus, or both) are affected (Engler, Bailey,

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et al. 2004; Miller et al. 1994; Nygaard and Lovik 2002; Organ et al. 1989; Pruett et al. 1999; Pruett et al. 2000; Pruett 2003). 8.2.1. Thymus In the thymus, stress results in a decrease in the size of the cortex attributed to a loss of cortical lymphocytes, with the immature cortical lymphocytes being most affected (Zivkovic et al. 2005). Severe acute stress may result in profound and diffuse apoptosis characterized by the appearance of numerous ‘‘tingible-body’’ macrophages and cellular debris (i.e., a starry sky appearance). In contrast, low-grade chronic stress may produce obvious reduction in the size of the cortex but with limited to no evidence of apoptosis, presumably because cell loss occurred at the beginning of the stressor. Apoptosis of thymic lymphocytes begins at 2 hr after immobilization stress in rats, but thymus weights do not change up to 8 hr post-restraint (Hatanaka et al. 2001). Thymic lymphocyte counts and mass are decreased in rats after 21 days of daily forced swim stress. Thymic weights of mice are decreased by 12 and 15% at 12 and 24 hr, respectively, after a brief relocation of the cage rack (Drozdowicz et al. 1990). Effects on the thymus can be reversible. For example, a single dose of dexamethasone in mice results in thymic atrophy (indicated by decreased cell numbers and proliferation) that is most severe by day 3 posttreatment and is fully recovered by day 12. This transient response to corticosteroids has been documented by others (Cohen et al. 1992; Cohen 1992; Lundberg 1991). 8.2.2. Spleen In toxicity studies, stressors of sufficient magnitude to affect the spleen generally result in decreases in splenic weights accompanied by decreases in white pulp areas with or without obvious apoptosis of lymphocytes. Stress-related changes are generally less consistent and milder in the spleen compared to the thymus (Pruett et al. 2007). A single acute stress has been reported to cause transient effects on splenic weights in mice (Fukui et al. 1997). In rats exposed to 2 hr of stress for 7 consecutive days, white pulp volume is decreased due to decreased proliferation in T-lymphocyte areas. In addition, B-lymphocyte areas are decreased due to increased apoptosis (Kapitonova et al. 2009). As discussed previously, circulating B-lymphocytes can redistribute to lymphoid organs during stress and as a result may contribute to the variable morphology of the B-lymphocyte areas of the spleen under stress. Pharmacologic or toxicologic effects of a test article that increase spleen weights may mask stress-related splenic weight decreases (Avitsur, Stark, and Sheridan 2001; Avitsur et al. 2003). 8.2.3. Lymphocyte Subpopulations Subpopulations of lymphocytes in lymphoid organs and blood are altered by stress (Table 9). Stress results in redistribution of B-lymphocytes, causing decreased B-lymphocytes in bone marrow and increases in spleen (Dhabhar et al. 1995b). Stress results primarily in decreased survival of immature thymic cortical lymphocytes and function of mature T-

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TABLE 9.—Lymphocyte subset parameters in the blood, spleen, and thymus of the rat potentially affected by stress in routine toxicity studies. Rat blood

B-lymphocytes T-lymphocytes CD4þ CD8þ CD4:CD8 CD4þCD8þ NK cells

Rat spleen

Rat thymus

Percent

Stress effects

Percents

Stress effects

Percent

Stress effects

33–39% 49–55% 33–36% 17–28% 1.7:1–2.1:1 NR 0–10%

# # # # ! NR #"!

56–58% 36–40% 22–24% 14–16% NR NR 2–5%

"# ! ! ! NR NR #"!

Negligible 95–98% 5–7% 5–7% NR 85% Negligible

Negligible # " " NR # Negligible

NR ¼ not relevant.

lymphocytes, with a minor component of redistribution (Dominguez-Gerpe and Rey-Mendez 2001; Herold, McPherson, and Reichardt 2006; Irwin 1994; Padgett and Glaser 2003). Stress causes decreased circulating lymphocyte counts due primarily to decreased B-lymphocyte counts, with a lesser decrease in T-lymphocyte counts. In general, B-lymphocytes are more sensitive than T-lymphocytes to glucocorticoidmediated effects (Beer and Center 1980; Dhabhar et al. 1995b; Miller et al. 1994). Social stress also results in decreased B-lymphocytes, along with decreased CD4þ and CD8þ T-lymphocytes. NK cell numbers are variably affected (increased, decreased, or unchanged) depending on the stressor (Dhabhar et al. 1995b, 1996; Pruett 2003; Stefanski and Gruner 2006). Stress-related release of NK cells from bone marrow is mediated by peripheral b-adrenergic effects, while redistribution of circulating NK cells to lymphoid organs is mediated by glucocorticoids (Bauer 2005; Bosch et al. 2005; Dhabhar et al. 1996; Engler, Bailey, et al. 2004; Schedlowski et al. 1996). However, in one model of chronic continuous stress, circulating lymphocyte changes did not reflect lymphocyte changes seen in any of the other lymphoid organs or bone marrow (Organ et al. 1989). Homeostatic mechanisms (see Normal Thymus section above) may be responsible for the lack of correlation between thymic cellularity and other lymphoid organs and blood. 8.3. Catecholamines and Glucocorticoids 8.3.1. Catecholamines Catecholamines exert effects on lymphocytes and lymphoid organs primarily through sympathetic innervation of primary and secondary lymphoid organs (reviewed in D. L. Felten et al. 1985, 1987; D. L. Felten and Felten 1989; S. Y. Felten et al. 1992; Hori et al. 1995; Kohm and Sanders 2001; Nance and Sanders 2007; Williams et al. 1981). Catecholamines influence lymphocyte proliferation, cytokine secretion, antibody production, and cytolytic activity (Madden and Livant 1991) via b-adrenergic receptors on lymphoid cells (S. Y. Felten et al. 1992; Laukova et al. 2012; Madden 2003; Sanders and Kohm 2002). In the thymus, catecholamines inhibit T-lymphocyte development, acting via a- and b-adrenoreceptors (a-AR and b-AR)

on the surface of developing lymphoid and nonlymphoid cells (reviewed in Leposavic et al. 2006, 2008). Effects of catecholamines on T-lymphocyte development may be modulated by glucocorticoids (Pilipovic et al. 2010). Endogenous (splenic) catecholamines released due to stress or exogenous catecholamines can decrease splenic lymphocyte counts by redistribution of these cells to blood. (Ernstrom and Sandberg 1973; Ernstrom and Soder 1975; Iversen et al. 1994; Murray et al. 1993; Stevenson et al. 2001). In general, the contribution of this effect to altered lymphocyte counts during stress is modest relative to the parts played by lymphocyte depots in the thymus and lymph nodes. Catecholamines also play a complex neural regulatory role in lymphopoiesis as well as general hematopoiesis (see Clinical Pathology section above). Hematopoietic and nonhematopoietic cells in the bone marrow express a-AR and b-AR, and catecholamines influence myelopoiesis and lymphopoiesis in complex positive and negative regulatory patterns (extensively reviewed in Maestroni 2000). 8.3.2. Glucocorticoids Glucocorticoids primarily affect survival and trafficking/ redistribution of lymphocytes. In general, basal glucocorticoid concentrations promote lymphocyte survival, while increased glucocorticoids promote apoptosis (see Clinical Pathology section above). Immature lymphocytes in the rat are highly sensitive to glucocorticoid-induced apoptosis, whereas mature lymphocytes in circulation and secondary lymphoid organs are relatively resistant (Ahmed and Sriranganathan 1994; Berki et al. 2002; Cohen 1992; Xue et al. 1996). Although both mature and immature T-lymphocytes express glucocorticoid receptors, immature DP (CD4þ/CD8þ) lymphocytes in the thymus are more sensitive than the more mature single-positive CD4þ or CD8þ lymphocytes to glucocorticoid-dependent apoptosis. The reason for the lower sensitivity of single-positive thymocytes to glucocorticoid-induced apoptosis may be due to protective effects of CD28 signaling or expression of Bcl-2 and Notch (reviewed in Hernandez-Hoyos and Alberola-Ila 2003; Laky, Fleischacker, and Fowlkes 2006; van den Brandt, Wang, and Reichardt 2004; Yashiro-Ohtani, Ohtani, and Pear 2010). There are species differences in sensitivity of

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lymphocytes to glucocorticoids (Claman 1972). Sensitive species include the rat, rabbit, mouse, and hamster while resistant species include dog, ferret, Guinea pig, nonhuman primate, and human. Glucocorticoids can alter lymphocyte trafficking resulting in lymphocyte accumulation in secondary lymphoid organs, sometimes associated with decreased lymphocyte counts in blood. Glucocorticoids inhibit trafficking of bone marrow, lymph node, and splenic B-lymphocytes from lymphoid tissues into blood in rats (Beer and Center 1980; Cox and Ford 1982; Dhabhar et al. 1995a; Spry 1972). These effects of glucocorticoids are likely influenced by regulatory pathways mediated by the nervous system. Lesions of the hippocampus in rats causes typical glucocorticoid-related changes in circulating leukocyte counts (increased neutrophil counts, and decreased lymphocyte and eosinophil counts) as well as lymphoid organ size (decreased weights, Bratt et al. 2001). In addition, endogenous opioids may modulate stress-related lymphoid effects (Yin et al. 2000). Histologic changes in the thymus of rats after single or repeated doses of corticosterone that simulate stress include increased apoptosis of cortical lymphocytes, increased macrophages containing cell debris, and loss of cellularity and thinning of cortical areas (Kendall and al-Shawaf 1991; Sapolsky, Romero, and Munck 2000; Savino and Dardenne 2000; Zivkovic et al. 2005). These histologic changes are often associated with decreases in thymus weights and in numbers of immature DP (CD4þ/CD8þ) lymphocytes in the cortex (Ashwell, Lu, and Vacchio 2000; Pruett et al. 2007; Vacchio and Ashwell 2000). Similar changes are observed in the thymus of other species (Ahmed and Sriranganathan 1994; Gruver and Sempowski 2008; Sapolsky, Romero, and Munck 2000; Savino et al. 2007). Results of administration of corticosterone to rats at single doses simulating stress (1–5 mg/kg; intraperitoneal) include 20% decreases in spleen weight relative to body weight and histologic changes consisting of generalized lymphocyte depletion and decreased cellularity of B-lymphocyte areas (MZ and follicles of the PALS) by 24 hr after treatment. In mice, single intraperitoneal injections of dexamethasone do not induce apoptosis or alter numbers of T-lymphocytes in splenic white pulp at 48 hr (Ahmed and Sriranganathan 1994; Chen et al. 2004). At doses approximating the circulating glucocorticoid concentrations observed during stress, exogenous corticosterone causes decreased numbers of immature B-lymphocytes in the bone marrow, due in part to increased apoptosis and decreased proliferative capacity (Garvy, King et al. 1993; Garvy, Telford et al. 1993). Cotreatment with IL-7 isolated from bone marrow stromal cells partially protects immature B-lymphocytes from glucocorticoid effects (Valenzona et al. 1998). 8.4. Acute versus Chronic Effects of Stress on Immune Responses Stress may increase, decrease, or have no effect on immune responses depending of the duration and intensity of the

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stressor. In general, mild and/or acute stressors enhance immune responses while severe or long-term stressors can be immunosuppressive (Burns et al. 2002, 2003; Dhabhar 2000; Dragos and Tanasescu 2010; Fleshner et al. 1998; Glaser et al. 2000; Pressman et al. 2005; Silberman, Wald, and Genaro 2003; Tournier et al. 2001; Figure 4). Changes in the functional capacity of the immune system following acute and chronic stress have been discussed in a review article (Dhabhar 2009), and include decreased immune cell numbers and functions, increased immunosuppressive pathways, and increased pro-inflammatory cytokines. Effects of chronic stress on immune function have been well documented in humans (reviewed in Gouin, Hantsoo, and Kiecolt-Glaser 2008). These effects impact both the innate and adaptive immune system and include attenuated responses to vaccination, poorer wound healing, exaggerated release of inflammatory mediators, and premature aging of the immune system. Endogenous glucocorticoids are essential for antibody responses (Fleshner et al. 2001). As demonstrated in rodents, adrenalectomy or antiglucocorticoid receptor treatment decreases IgM and IgG responses. Partial recovery of IgM responses occurs with corticosterone replacement at basal concentrations, and complete recovery of antibody responses occurs with administration of additional corticosterone between 5 and 7 days postimmunization. In contrast, hydrocortisone at relatively high doses (100 mg/kg resulting in a peak serum concentration of 500 ng/ml) results in significant inhibition of the primary IgM response to an antigen (Dracott and Smith 1979a, 1979b).

9. CONCLUSIONS

AND

RECOMMENDATIONS

9.1. Minimizing Stress-related Effects on Toxicity Studies Numerous parameters evaluated during routine toxicity studies are sensitive to stress (Table 1). Some endpoints are fairly sensitive to stress (e.g., total body weight, circulating leukocyte counts, lymphoid and endocrine organ weights, and histology). In contrast, other changes (e.g., other routine pathology parameters such as clinical chemistry measurements and organ morphology as well as in-life endpoints) are less consistently affected or are not altered at all by stress. Accordingly, evaluation of data sets when considering the potential contribution of stress to study outcomes must rely on a weight-of-evidence approach. The impact of stress on toxicity studies is minimized by good study design. Major elements of suitable experimental design to limit the impact of stress include utilization of appropriate control groups, husbandry practices, habituation of individuals to known stresses (e.g., handling, housing conditions, experimental procedures) prior to initiating the study; standardization of testing methods both within and across studies; and removal of systematic bias. These practices also allow for more accurate assessment regarding whether, and to what extent, stress might have contributed to the biological outcomes of a given study.

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FIGURE 4.—Stress and the immune system. Diagram of the physiological and psychological adaptive responses to acute (minutes to hours) and chronic stress (months to years) in the context of the immune system. In general, the response of the immune system to acute stress is beneficial while the response to chronic stress is detrimental. Routine toxicity studies are often associated with varying sources of stress that can cause acute or chronic stress and as a result pathology findings may reflect a combination of acute and chronic responses. Reprinted with permission from F. S. Dhabhar and McEwen (2007).

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The most important study design feature is inclusion of appropriate control groups. Design of studies should consider incorporating a concurrent vehicle-dosed control group that undergoes the same procedures as test article– treated groups. In some instances, inclusion of an untreated (baseline) control cohort may be desirable to document the degree to which animal handling procedures and/or the treatment method itself will be perceived as a stress by test subjects. Having an appropriate study design does not control for compounds that have a significantly unpleasant taste or immediate side effect; these attributes may result in stress during dosing. Clinical signs such as salivation, struggling upon handling, and vocalization may indicate that the animals are experiencing stress. Proper husbandry practices can minimize deleterious stress to experimental animals. The most common practices include offering environmental enrichment, co-housing compatible individuals, and maintaining consistency in the types and conduct of necessary procedures. Training and habituation (i.e., pre-study acclimation) of experimental animals to potentially stressful procedures (e.g., daily restraint in nose-only inhalation cones, intermittent testing in water-filled mazes) is a useful means of dampening physiological responses to repeated stress prior to initiation of toxicity studies. Most consistent mild stressors result in habituation within several days to a week of their initial occurrence. Stress effects, particularly those on the thymus and reproductive system, can vary based on the sexual maturity of the subject. A surrogate measure of sexual maturity (e.g., total body weight and/or postnatal age, testis size, vaginal smear) should be included as part of criteria for stratifying animals into treatment groups so that bias in maturity-sensitive endpoints is minimized. Techniques involving manipulation of animals, especially when required for in-life data collection (e.g., fluid and tissue sampling, weight measurements, behavioral testing) must be made in a random or stratified (round-robin) order to prevent systematic bias from affecting results. Similar care must be taken to randomize the order in which animals are necropsied as well as the batches in which samples (e.g., blood, trimmed tissue specimens) are processed and/or analyzed to avoid the introduction of systematic bias into the results. 9.2. Parameters Routinely Measured Are Appropriate for Assessing the Role of Stress in Toxicity Studies In toxicologic research, it is difficult to isolate stressinduced changes from direct toxic changes. Toxic effects (e.g., clinical signs, end-organ toxicity) are often inherently stressful and can result in stress-related changes in the parameters discussed in this document as they are evaluated in routine toxicity studies. Therefore, a weight-of-evidence approach, based on standard parameters (e.g., in-life as well as clinical pathology and anatomic pathology endpoints) measured on routine toxicity studies, should be taken to determine whether the spectrum of findings supports an interpretation of

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primary (direct) toxicity or secondary (indirect) stress-related effects. Evaluation of stress-related hormones is not helpful in assessing the impact of stress on routine toxicity studies. Although stress hormones may be useful for studies designed to evaluate acute stress (minutes/hours), chronic stress is more typically observed during the conduct of toxicity studies. In acute stress, serum or plasma glucocorticoid concentrations generally are reflective of activation of the HPA axis and correlate fairly well with immediate increases in ACTH concentrations. In contrast, in chronic stress, ACTH and glucocorticoid concentrations can be low, normal, or increased, depending on the type, duration, and severity of the stress. Normal or low glucocorticoid concentrations are often observed in chronic disease states and other chronically stressful conditions (Mizoguchi et al. 2001, 2008; Silberman, Wald, and Genaro 2003; Turner-Cobb et al. 2010). The effects of chronic stress are well documented in human literature, but sparse in animal literature (TurnerCobb et al. 2010). In addition, eustress-related hormonal changes that occur with environmental enrichment (e.g., increases in glucocorticoids) cannot be differentiated from distress-related hormonal changes. For these reasons, the presence or lack of hormonal changes during routine toxicity studies does not add to the weight-of-evidence for determining whether or not effects were directly related to the test article or indirectly related to stress. Study design factors also contribute to the lack of utility for hormonal measurements on routine toxicity studies. Hormonal measurements of HPA axis activation are complicated by the inherent variability of hormonal concentrations among individuals, the impact of diurnal variations, pre-analytical influences of other study procedures, low numbers of animals per group in large animal (e.g., dog and nonhuman primate) studies, and blood volume limitations for rodents and nonhuman primates. In summary, for routine toxicity studies, the interpretation of all commonly collected data (e.g., clinical signs, body and organ weights, clinical pathology measurements, macroscopic and histologic pathology findings) is appropriate and sufficient for determining whether or not changes are directly related to test article exposure or are secondary consequences of stress. These data are assessed by toxicologic pathologists and toxicologists who understand the effects of stress on these endpoints. As physiological responses for toxicity and stress overlap to some degree, discrimination between toxicity and stress depends on a weight-of-evidence approach using the entire data set, rather than using individual endpoints. AUTHORS’ NOTE This review is a product of a Society of Toxicologic Pathology (STP) Working Group and has been reviewed and approved by the Scientific and Regulatory Policy Committee (SRPC) and Executive Committee (EC) of the Society. The article does not represent a formal best practice recommendation of the Society but provides expert guidance on key principles to consider in conducting regulated toxicity studies.

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ACKNOWLEDGMENTS The authors would like to thank Tim Vojt, The Ohio State University, for providing graphic design/medical illustration support; Stephen B. Pruett and Philip W. Harvey for providing external reviews; and Patrick Haley, Bernard Remandet, Katharine Sprugel, Michael Santostefano, Susan MacKenzie, Steven R. Frame, Scott Loveless, and the STP SRPC and EC for providing expert input.

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