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Chapter 39:  Systems Physiology of Aging and Selected Disorders of Homeostasis

George A. Kuchel

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Learning objectives.

• AN INTEGRATIVE APPROACH TO THE PHYSIOLOGY OF AGING
• HOMEOSTASIS IN A HISTORICAL CONTEXT
• HOMEOSTATIC REGULATION IN OLD AGE
• SPECIFIC HOMEOSTATIC CHALLENGES
• ACKNOWLEDGMENT
• FURTHER READING
• Full Chapter
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Describe general features of physiological aging shared across tissues and organ systems including loss of complexity, increased heterogeneity, loss of resilience, homeostenosis, diminished physiological reserves, diminished end-organ responsiveness, loss of negative feedback, and allostatic load.

Understand the concepts of homeostasis and homeostenosis.

Describe the impact of aging on homeostatic mechanisms which help maintain a normal physiologic body temperature and fluid/salt balance in the face of lowered or increased ambient temperature, as well depletion or overload of fluids and sodium.

Describe the clinical features—including epidemiology, symptoms, signs, results of diagnostic tests and treatment—of hypothermia, hyperthermia, and water and sodium excess or depletion.

Describe the impact of aging on the ability to maintain a normal blood pressure in the face of orthostasis, meal ingestion, hypovolemia, and volume overload.

In addition to tissue- and organ-specific changes, shared features of physiological aging include loss of complexity, increased heterogeneity, loss of resilience, homeostenosis, diminished physiological reserves, diminished end-organ responsiveness, loss of negative feedback, and allostatic load.

Homeostasis reflects the aggregate effect of varied mechanisms that maintain normal physiologic constancy in the face of different extrinsic challenges. Aging is associated with impaired homeostasis, or homeostenosis, in the form of diminished capacity to respond to varied challenges.

Aging is associated with a failure of several different homeostatic mechanisms that enhance the risk of hypothermia in the face of decreased ambient temperature.

Aging is associated with a failure of homeostatic mechanisms that enhance the risk of hyperthermia and heatstroke in the face of increased ambient temperature.

The clinical presentation of hypothermia and hyperthermia may be subtle in older adults, requiring a high index of suspicion and careful supportive management in order to avoid the high rate of mortality associated with these conditions in late life.

Aging is also associated with homeostatic deficits when confronted with the assumption of the upright posture, eating, hypovolemia or a fluid challenge, increased or decreased sodium level, increased or decreased glucose level, bladder filling or bladder outlet obstruction, major burns or trauma, bed rest, or exercise.

“Besides more or less obvious physical changes in old age, physiological investigation may reveal increasing limitation of the effectiveness of homeostatic devices which keep the bodily conditions stable.”

Walter Bradford Cannon (1871–1945)

All organ systems undergo physiological changes with aging. However, the rate and nature of such changes varies both among organ systems and across individuals. This book includes individual chapters that address physiological changes associated with aging within the context of specific organ systems or tissues (see Part V: Organ Systems and Diseases).

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Homeostatic medicine: a strategy for exploring health and disease

• Review article
• Open access
• Published: 26 September 2022
• Volume 1 , article number  16 , ( 2022 )

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• Songlin Wang 1 , 2 , 3 &
• Lizheng Qin 1 , 2 , 4  

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Homeostasis is a process of dynamic balance regulated by organisms, through which they maintain an internal stability and adapt to the external environment for survival. In this paper, we propose the concept of utilizing homeostatic medicine (HM) as a strategy to explore health and disease. HM is a science that studies the maintenance of the body’s homeostasis. It is also a discipline that investigates the role of homeostasis in building health, studies the change of homeostasis in disease progression, and explores ways to restore homeostasis for the prevention, diagnosis and treatment of disease at all levels of biological organization. A new dimension in the medical system with a promising future HM focuses on how homeostasis functions in the regulation of health and disease and provides strategic directions in disease prevention and control. Nitric oxide (NO) plays an important role in the control of homeostasis in multiple systems. Nitrate is an important substance that regulates NO homeostasis through the nitrate-nitrite-NO pathway. Sialin interacts with nitrate and participates in the regulation of NO production and cell biological functions for body homeostasis. The interactions between nitrate and NO or sialin is an important mechanism by which homeostasis is regulated.

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1 Introduction

Medicine is a systematic study of human life. The definition of life varies greatly, depending on one’s perspective, however, life is generally defined as an open system (Abel 2011 ) characterized by energy metabolism, stimulus-response compatibility, and self-reproduction. Basically, all living things are made up of matter, thus, any movement of living things should obey the general laws of physics as described in the second law of thermodynamics, that is, all systems have a spontaneous tendency towards disorder and a high-entropy state. For the system to maintain order and a low entropy state, the living body needs to constantly ingest substances with a high entropy state in the environment and convert them into a low-entropy state by consuming energy and excreting metabolic wastes (Chirumbolo and Vella 2021 ). Therefore, the activities of the living body involve a series of physical and chemical reactions, and these reactions must take place under the corresponding reaction conditions. To ensure the orderliness and efficiency of these reactions, an organism should maintain a relatively stable internal state, which is referred to as homeostasis. Organisms are in a constantly changing environment, but regardless of how the environment changes, they must maintain their internal stability for normal physiological functions to occur; such a process is called homeostasis regulation (Billman 2020 ).

Simply put, homeostasis is a self-regulating process through which organisms remain stable and constantly adapt to the changing external environment, thus leading to better survival. Health and disease are opposites that are interrelated with each other, and homeostasis regulation is the key factor in their mutual transformation. There are two aspects to health. One is that each system of the body maintains a normal physiological equilibrium while the other is that organisms are able to adapt to environmental changes, external stimuli, and pathogenic factors. Any disruption to the regular equilibrium regulation leads to disease, followed by a series of metabolic, functional, and structural changes that manifest as abnormal symptoms, signs, and behaviors (Lopez-Otin and Kroemer 2021 ). In other words, homeostasis is an intersection of health and disease; thus, good maintenance of homeostasis is a prerequisite for health, whereas disruption of homeostasis will inevitably lead to disease.

Therefore, we propose the concept of homeostatic medicine, as a science of the homeostasis of molecules, cells, organs, and the whole body. Homeostatic medicine is a comprehensive discipline with the basic idea of maintaining human health and preventing and diagnosing diseases by maintaining the equilibrium of the internal environment required by the human system for survival ( Fig.  1 ) . Homeostatic medicine focuses on the strategy of homeostasis regulation at the molecular, cellular, organ, systems, and external environmental levels. The ultimate goal of homeostatic medicine is to study the role of homeostasis in health and disease and to regulate homeostasis for the purpose of maintaining health and treating diseases. Different from traditional “symptomatic treatment,” homeostatic medicine focuses more on the destruction of homeostasis as the cause of the disease, which is “etiological treatment”. The idea of homeostatic medicine calls for further research on the relationship between health and disease and provides new ideas and strategies for health maintenance and disease prevention and treatment.

The conception of homeostatic medicine. Homeostatic medicine is a science of the homeostatic of molecules, cells, organs, and the whole body. Homeostasis is closely related to health and disease. The main idea of homeostatic medicine is to maintain human health and to prevent or treat diseases by modulating the homeostasis balance. Created with BioRender.com

Homeostatology is a subject that focuses on the concept of homeostatic medicine but is not limited to biology or medicine. The research subjects of homeostatology are physics, psychology, and even sociology, which is a new scientific research idea and methodology for predicting and controlling various systems by studying self-stability phenomena. Homeostatology generalizes various self-stable phenomena in the energy transfer, information exchange and matter exchange and proposes the basic structure of system auto-stability. Briefly, antagonistic information exchange exists among the elements in the system, and the loop formed by this information exchange constitutes the fundamentals of equilibrium. Through homeostatology, people can better understand the principles of the world, and rational use of homeostatological methods is conducive to the research and progress of various disciplines.

2 The history of the concept of homeostasis

The concept of homeostasis has experienced a dialectic period. The original theory of body equilibrium can be traced back to 460 BC when Hippocrates first proposed the Four Humors theory to describe the balanced state of the human body. He proposed that the human body consisted of four major fluids or humors, that is, blood, phlegm, yellow bile, and black bile. These four major humors must be maintained in equilibrium in order to promote good health (Santacroce et al. 2021 ). Compared to modern medicine, the Four Humors theory has many limitations, however, this theory remained for nearly two thousand years, influencing both Western and Eastern medicine. Hippocrates further suggested that the body can heal itself, and it is the doctor’s responsibility to clear obstacles to return the patient’s body to its natural state (Billman 2020 ). This view has been maintained until today, and for this theory, Hippocrates is regarded as “the father of Western medicine”.

With the constant exploration of body functions, the mechanism of homeostatic regulation has been further explained. French physiologist Claude Bernard (1813–1878) proposed the theory of “homeostasis of internal environment” (Adolph 1961 ). The theory holds that living systems have internal stability, which can buffer and protect the body from the constantly changing external environment. He stated that the human body was a collection of body fluids and cells in the internal environment and he believed that the stability and independence of the internal environment are necessary prerequisites for the survival of the body. However, Bernard’s viewpoint is flawed because he believed that the internal environment of the body was unchangeable and independent of the external environment, which was later proved to be inaccurate (Gross 2009 ).

Based on Claude Bernard’s theory of “homeostasis of internal environment,” Walter B. Cannon (1871–1945), an American medical scientist, improved this theory by introducing the concept of dynamic balance. Dynamic balance is a self-regulation process indicating that organisms can maintain stability while adapting to the changing environment (Cooper 2008 ). In Cannon’s book, The Wisdom of the Body , he explained that homeostasis requires two components at the same time: internal stability within a certain range and the ability to remain stable by regulating variables (Billman 2020 ). Canon’s theory of homeostasis has been recognized in modern medicine and has elaborated a good theoretical foundation for the study of homeostasis.

Cybernetics was first proposed by Norbert Wiener (1894–1964) in 1948. He defined cybernetics as a science that focuses on general laws of control and communication in machines, life and society. Its core idea is to study how dynamic systems maintain equilibrium under the changing environment (Wiener 1948 ). It has been widely applied to a variety of complex control systems with multiple factors, such as the economic regulation system, the resource allocation system, the ecological and environmental system, and the computer logic system. The feedback control theory in cybernetics refers to a regulation process in which the difference between the result and the standard is identified through information feedback and corrective measures are taken to stabilize the system at the target state, which corresponds to the negative feedback regulation mechanism in homeostasis regulation. Moreover, the adaptive theory refers to the automatic adjustment of its own structure or behavior parameters according to the changes of external conditions before the environmental conditions have affected the control object, so as to maintain the original function of the system, which corresponds to the feed-forward adjustment mechanism in homeostasis regulation (Benjamin et al. 2020 ).

Similar to the concept of homeostasis in Western medicine, traditional Chinese medicine pursues the theory of Yin and Yang. Yin and Yang in Equilibrium and The Pursuit of Balance were first proposed in Huangdi’s Class ic on Medicine (Huang Di Nei Jing). Yin and Yang in Equilibrium implies opposing and restricting forces, which eventually harmonize to form a relative dynamic balance. The Pursuit of Balance emphasized the importance of maintaining a balance. Therefore, it is more important to restore balance than to remove the causes of a disease. Furthermore, excessive treatment should be avoided so as not to break the balance between Yin and Yang (Maiese 2006 ). Clearly, though there is great difference in Eastern and Western medicine, the understanding of the role of homeostasis in health and disease is quite close.

At the beginning of the life circle, the regulation of homeostasis plays an important role in organisms. It would not be exaggerating to say that homeostasis regulation distinguishes living things from non-living things. It provides a basis for life to maintain a state of low entropy, and is an important mechanism of evolution in organisms. The prevailing view is that life began in the ocean. The ancient ocean was a potassium-rich environment, which can be viewed as a precursor to potassium-rich intracellular fluids. As the environment evolved, the ocean changed into a sodium-rich environment, which is viewed as a precursor to extracellular fluid. The existence of cell membranes and sodium/potassium pumps provides the structural basis for the homeostasis regulation of potassium ions and the membrane potential in cells and makes it possible to conduct electrical signals in nerves and muscles (Sieck 2017 ). As the structure of living organisms became more complex, single-celled organisms began to metabolize and cooperate to better maintain the low entropy state of the system. In prokaryotes, biofilm systems and a sense of community emerged, while in eukaryotes, primitive intercellular signaling systems emerged, and this primitive homeostasis regulatory mechanism underlies morphogenesis, dynamic homeostasis, regeneration, and reproduction (Torday 2013 ). Thus, homeostasis regulation is involved in each key node of biological evolution and is an important factor in promoting the evolution of organisms.

3 Homeostasis regulation

The most important mechanism in homeostasis regulation is the feedback system, which has four main components: (1) the variable to be controlled, (2) a sensor that monitors the variable, and (3) a comparator or central processing unit where the information provided by the sensor is fed back into the system. The information is compared with the specified value, and (4) effectors are used to regulate the desired control variables (Billman 2020 ). These parts form a closed loop that feeds the signal back. In a negative feedback process, the activity of the effector is opposite to the changes in the variables to buffer them. In a positive feedback process, the activity of the effector is identical to its changes to achieve rapid changes in the state of the body by amplifying a control signal (Goldstein and Kopin 2017 ). Nevertheless, it is noteworthy that the four components only represent the vitals of the feedback system; the feedback system in biology is much more complicated, involving the superposition and nesting of multiple feedback pathways.

Taking blood pressure as an example, most of the time, our blood pressure is maintained within a relatively stable range through feedback regulation. The receptors for blood pressure in the body are baroreceptors on the aortic arch and carotid sinuses which respond to changes in arterial pressure. The solitary tract in the medulla oblongata of the brain processes signals from pressure receptors and acts on the effectors located in the blood vessels and heart by regulating the neural activity of the sympathetic and parasympathetic nerves (Dworkin and Dworkin 1995 ). When the blood pressure is elevated, the baroreceptors are activated, and the regulation of the solitary tract nucleus decreases sympathetic activity and increases the vascular diameter. In addition, increased parasympathetic activity lowers the heart rate and stroke output, thus lowering the blood pressure. The opposite occurs when the blood pressure is below a set point. Through this negative feedback regulation, fluctuations in blood pressure can be effectively buffered so that the body’s blood pressure can remain relatively stable all day, despite changes in the environment or behavior (Humphrey and Schwartz 2021 ).

Feed forward regulation, another important mechanism of homeostasis regulation, refers to the assessment and adjustment of impending changes before they actually occur (Carpenter 2004 ). Several levels of regulation are involved in homeostasis. The first level is the effector responsible for receiving higher-level regulatory signals and variables. Feedback regulation, also known as autonomic regulation, is the second level of this system, which processes signals detected by receptors and initiates adjustments to the first level. The third level is located in the central nervous system, which processes information transmitted from the second level. It can integrate the changing information of the environment to coordinate the physiological behavior of various feedback systems (Goodman 1980 ). Under certain conditions, this regulation can be unconscious or regulated by subjective consciousness. Taking blood pressure regulation as an example, when faced with danger or challenge, the cardiac output rate and blood pressure can be raised through the central nervous system to respond to impending environmental changes, the process of which is not regulated by consciousness. However, when faced with cold weather, clothes are actively added to maintain body temperature. In this case, the regulation process is completed through the intervention of consciousness and experience (Billman 2020 ).

Homeostasis is a key regulatory mechanism in health and disease. Normal homeostasis is fundamental for maintaining health and ensuring various physiological functions. In contrast, disease progression is typically accompanied by an imbalance in homeostasis. These changes have adverse effects on the body and eventually cause functional disorders and organic lesions. As mentioned above, each physiological indicator is maintained within a prescribed range through homeostasis regulation. However, this range is not fixed, and the body can adjust the default physiological indicators to adapt to different needs under certain circumstances (Kotas and Medzhitov 2015 ). For example, the default setting point for body temperature is approximately 37 °C. In conditions of inflammation or infection, the setting point of the temperature can increase to 40 °C to increase the basal metabolic rate and defend against infection, a process commonly known as “fever” (Morrison 2016 ). When the external stimuli are removed, the setting point of the body temperature gradually returns to normal. In this case, fever helps the body to cope with extreme challenges. However, prolonged high fever can cause structural and functional destruction of tissues and organs and, in severe cases, multiple organ failure and even death. In short, when the adjustment of the setting point deviates from the acceptable range, it causes irreversible effects on the homeostasis of the body, leading to a pathological state.

Homeostatic medicine is the science of the homeostasis of molecules, cells, organs, and the whole body. The basic idea is to maintain human health, to prevent or treat diseases by maintaining homeostasis. Homeostatic medicine systematically studies the mechanism of homeostasis regulation and summarizes a series of strategies to guide clinical treatment. Homeostatic medicine integrates the concept of homeostasis in both Eastern and Western medicine and summarizes the role of homeostasis regulation in health and disease. The destruction of homeostasis is the essence of disease. Therefore, homeostatic medicine mainly focuses on restoring homeostasis to eliminate the cause of disease. The goal of homeostatic medicine is to study changes in homeostasis in disease and to incorporate existing medical methods to treat or alleviate the disease ( Fig. 1 ) . Homeostatic medicine comprises three key steps. The first is to understand the mechanism of homeostasis regulation and its role in maintaining health. The next is to analyze the causes and intervention factors of homeostasis imbalance in the process of disease. Finally, it consolidates the information gained in the first two steps and restores the homeostasis of the body through reasonable interventions. Given the crucial role of homeostasis in health and disease, homeostatic medicine has a wide range of applications in coping with various diseases.

4 Fifty percent concept in homeostasis

The reserve function of organisms is an important mechanism for homeostasis regulation, which is reflected in all levels of the body. Approximately 50% of the physiological potential of the body is not utilized, which is known as the reserve function of the body. Only in response to abnormal conditions or external challenges can the physiological potential of surplus reserves be mobilized, which makes it possible for the organism to deal with overload work, thus facilitating the rapid recovery of homeostasis (Atamna et al. 2018 ). The reserve function of an organism is closely related to its health and disease states. For example, aging causes a decline in the reserve capacity of the body’s tissues and organs, including the functional decline of the immune, muscular, and nervous systems. This reduction in reserve function leads to the decreased ability to respond to external stimuli, increased susceptibility to infection, and a longer recovery time from diseases (Iliodromiti et al. 2016 ).

The metabolic efficiency of an organism depends on the number and activity of enzymes, and excessive enzymes are the basis of the metabolic reserve. For example, with an excessive quantity of glycolytic enzymes, the body can respond to the energy demand of overloaded physiological activities and increase these to the maximum capacity when necessary to maintain homeostasis (Eanes et al. 2006 ). In nerve cells, neuronal activity depends on the maintenance of the membrane potential by sodium/potassium ATP pumps. The sodium/potassium ATP pump shows a large reserve capacity to ensure neuronal homeostasis under highly intensive neural activity (Howarth et al. 2012 ). In addition, the amount of mitochondrial DNA (mtDNA) in a cell is much higher than the number of mitochondria. An average of 2000–10,000 copies of mtDNA is estimated to be contained in a nucleated cell with 1000 mitochondria. This redundant mtDNA has a reserve capacity that can reduce the impact of mutations on energy metabolism through the excess mtDNA reserves (Miller et al. 2003 ). The regulation of reserve function in homeostasis can also be reflected at the cellular level. In a healthy state, most cells of the human body are in a static state of division. In response to adverse stimuli, these static cells can quickly enter the cell cycle to repair damaged tissues and functions. For example, in the process of liver regeneration after toxic injury or hepatectomy, epithelial and parenchymal cells can respond quickly, rapidly divide, and differentiate to restore the quality and function of the liver (Campana et al. 2021 ). At the organ level, in the case of the kidney, the concept of the renal functional reserve has been proposed for decades, which refers to the difference between the glomerular filtration rate at rest and at maximum capacity. In general, utilization of the kidney accounts for only approximately 50% of the total potential. The renal filtration rate is considerably increased in pregnancy, isolated kidney, or hypertensive diabetic nephropathy, and the presence of the renal reserve allows the serum creatinine and glomerular filtration rate to remain normal in the presence of renal damage until more than 50% of renal units are lost (Palsson and Waikar 2018 ).

The effect of the reserve function on homeostasis is one of the focuses of homeostatic medicine. Making full use of the 50% reserve potential to maintain a steady state is an important scientific issue. Sometimes, not only is it difficult to completely cure a disease or restore the body to a healthy level, but also excessive treatment can cause serious side effects. Benefiting from the reserve function, healthy homeostasis can theoretically be maintained by restoring 50% of the function of the body or by maintaining half of the organ’s health. For example, in kidney transplant donors, a single kidney is sufficient to meet normal functional demands through compensation (Steiger 2011 ). Therefore, it may be more reasonable to aim for the health of one kidney than the more difficult goal of two healthy kidneys. The previous concept states that health occurs only if all organs are intact. In contrast, homeostatic medicine adheres to the 50% treatment concept, the goal of which is to restore at least 50% of physiological function and to maintain homeostasis by making the most use of reserve function.

5 Nitrogen oxygen and Sialin-two important regulators in homeostasis

Nitric oxide (NO) is perceived as a harmful atmospheric pollutant. However, Furchgott et al. proposed that NO is a signaling molecule in the cardiovascular system, which won the Nobel Prize in 1998. Since then, it has been noted that NO is an important signaling molecule in mammals and plays a variety of important physiological functions in various systems of the body, including the nervous, digestive, urinary, reproductive, immune, and cardiovascular (Moncada et al. 1991 ). As an important molecule in homeostasis regulation, NO plays a key role in maintaining homeostasis in the body. For example, as signaling molecules, NO can regulate the dynamic balance of the endocrine system by regulating the neuroendocrine and autonomic nervous systems. In the case of dehydration and bleeding stress, NO plays a protective role and restores the autonomic neuro-humoral balance (Krukoff 1999 ). However, in some cases, NO plays a dual role. For example, NO can inhibit tissue inflammation by inhibiting leukocyte recruitment (Kubes et al. 1991 ). However, excessive NO production will cause oxidative stress in cells and further promote inflammation (Krol and Kepinska 2020 ). Therefore, maintenance of NO homeostasis in vivo contributes to the regulation of systemic homeostasis. NO is synthesized internally by nitric oxide synthase (NOS). Moreover, when endogenous NO synthesis is impaired, such as in endothelial dysfunction, exogenous nitrate intake can effectively maintain NO homeostasis (Kapil et al. 2020a ) ( Fig.  2 ) .

The relationship between nitrate, NO homeostasis. and system homeostasis. NO is an important molecule to maintain body homeostasis. Through the synthesis of endogenous NO by nitric oxide and enzymes and the intake of exogenous nitrate, the level of NO in the body can be maintained in a relatively stable range. The homeostasis of NO is of great significance to the homeostasis of the body. Insufficient NO will cause endothelial dysfunction and metabolic disorder, while excessive NO will induce oxidative stress and promote inflammation. Created with BioRender.com

Nitrate is widely distributed in nature, particularly in green vegetables. After a nitrate-rich meal, nitrate is rapidly absorbed into the blood in the upper digestive tract. Approximately 25% of the nitrate is absorbed by the salivary glands and secreted into the oral cavity by the saliva. This process is called intestinal salivary circulation, which is conducive to maintaining nitrate levels in vivo. Nitrate in the saliva is reduced to nitrite by nitrate-reducing bacteria inhabiting the oral cavity. These nitrites are then swallowed and reabsorbed into the blood, where they can be reduced to NO by various enzymes in the blood and tissues. This process is known as the nitrate-nitrite-NO pathway. Through the nitrate-nitrite-NO pathway, nitrate performs a variety of NO-like physiological functions (Ma et al. 2018 ), including protecting the digestive system by increasing blood flow and regulating intestinal flora (Hu et al. 2020a ; Wang et al. 2020a ), alleviating obesity by adjusting fat metabolism (Ma et al. 2020a ) and assisting tumor therapy by increasing sensitivity to cisplatin chemotherapy (Feng et al. 2021a ). Moreover, nitrate can significantly upregulate the expression of a variety of cellular signaling pathways, including the MAPK signaling pathway (Feng et al. 2021b ), the PI3K-Akt signaling pathway, the mTOR and WNT signaling pathway (Jiang et al. 2014 ), glutathione metabolism, and the cell cycle (Jia et al. 2011 ), which play important roles in cell regeneration, cell metabolism, and disease progression ( Fig.  3 ) .

Signaling pathways and functions of nitrate on the regulation of homeostasis. Through the nitrate-nitrite-NO pathway, nitrate plays an important role in regulating homeostasis of cells from multiple perspectives, including regulating metabolism, promoting cell regeneration, and inhibiting disease progression

Sialin is a nitrate transporter in mammalian cell membranes that plays a key role in the beneficial effects of nitrate and homeostasis maintenance of NO (Qin et al. 2012 ). There is an interaction between nitrate and sialin, which is usually accompanied by elevated sialin expression in important organs. The high expression of sialin promotes a range of cellular biological functions. Nitrates improve mitochondrial function and reduce aging of mesenchymal stem cells by upregulating sialin expression. Oral administration of inorganic compounds can directly regulate the M1/M2 ratio of macrophages, thus preventing non-alcoholic fatty liver disease. In addition, sialin is highly expressed in human salivary glands, and salivary gland function is an important factor in the physiological function of nitrate (Qu et al. 2016 ). Interestingly, nitrate can regulate salivary gland function. In rats with xerostomia caused by ovariectomy, inorganic nitrate upregulated the expression of aquaporin-5 in the salivary glands, thus increasing salivary secretion and effectively reducing the fibrosis area and acinous atrophy of salivary gland tissues (Xu et al. 2018 ). In a salivary gland radiation injury model of miniature pigs, nitrate increased the expression of sialin in acinar cells, which further promoted the entry of nitrate into cells. This nitrate-sialin loop activates the EGFR-Akt-MAPK signaling pathway, thereby promoting the proliferation of acinar and ductal cells and reducing apoptosis (Feng et al. 2021b ). The nitrate-sialin loop preserves radiation-lost salivary gland cells by regulating autophagy within salivary gland cells. It follows that the mutual effects of sialin and nitrate are beneficial for maintaining NO homeostasis and further improving systemic homeostasis ( Fig.  4 ) . Therefore, the nitrate-sialin loop and the regulatory effect of nitrate on NO homeostasis may be a promising research direction, and the mechanism by which nitrate regulates systemic homeostasis deserves further study.

The mechanism of sialin on the regulation of homeostasis. Sialin is a membrane transporter of nitrate and plays an important role in homeostasis regulation. There is a positive feedback loop between nitrate and sialin, which is beneficial for nitrate to enter cells. Sialin can also regulate the function of mitochondria to improve the antioxidant stress ability of mesenchymal stem cells. Moreover, sialin is involved in the regulation of inflammatory phenotypes of macrophages to prevent non-alcoholic fatty liver disease. Created with BioRender.com

6 Homeostasis and disease

6.1 homeostasis in tumors.

The occurrence and development of tumors are closely related to dyshomeostasis. Proto-oncogenes are a group of genes that play important roles in cell proliferation and differentiation. Normally, proto-oncogenes are strictly regulated by another group of genes called tumor suppressor genes, and the dynamic balance between them is conducive to maintaining the number of cells and normal physiological function. However, the balance between proto-oncogenes and tumor suppressor genes can be disrupted by gene mutations under the action of stimulating factors, and the overexpression of proto-oncogenes may lead to abnormal cell proliferation and tumor formation (Pitot 1993 ). At present, the mainstream treatments for cancer are surgery, radiotherapy, chemotherapy, and immunotherapy, which aim to reduce tumor volume, block its invasiveness, and induce tumor cell death. Homeostasis regulation is an important factor in tumor occurrence and development. Therefore, homeostatic medicine aims to combine the concept of homeostasis regulation with tumor treatment. It can improve the resistance of normal tissues and organs to tumors by restoring their original homeostasis or inhibiting the growth of tumors by destroying the homeostasis of tumor tissue to improve the therapeutic effect on tumors and obtain twice the result with half the effort. In addition, it is controversial whether all tumors in the body must be eliminated for the sole purpose of treatment. Considering the negative effects of total tumor removal, one of the therapeutic goals should be to control tumor growth and maintain homeostasis in the body.

Inorganic nitrate can improve the sensitivity of oral squamous cell carcinoma to chemotherapy by decreasing the expression of REDD1 (regulated in development and DNA damage responses 1), which may be associated with improved NO homeostasis in hypoxic tumor tissues (Feng et al. 2021a ). Inorganic nitrates also showed considerable protective effects against the side effects of tumor radiotherapy. Inorganic nitrate effectively relieved the decline of salivary gland function after radiation injury in rats (Feng et al. 2021b ; Li et al. 2021a ), and it relieved colitis in rats after systemic radiation by regulating the homeostasis of intestinal flora (Wang et al. 2020b ). These results suggest that inorganic nitrates may improve the postoperative quality of life of patients with tumors after radiotherapy. The anti-radiation effects of nitrate are not limited to this. Even at low radiation intensities, such as after cone-beam computed tomography (CBCT) examination, nitrate shows an inhibitory effect on radiation-induced oxidative stress (Yang et al. 2022 ).

6.2 Homeostasis in cardiovascular disease

Homeostasis plays a key role in the health and disease of the cardiovascular system. Mechanobiological stability can be maintained in healthy blood vessels through multiple levels of negative feedback. However, the progression of cardiovascular disease is usually associated with overregulation of the mechanical biological balance or unstable positive feedback (Humphrey and Schwartz 2021 ). Generally, the cardiovascular system can adapt to changes in the internal and external environments of the body through functional and structural regulation. For example, a continuous increase in cardiac output causes dilation of the central artery, which reduces resistance to blood flow and thus reduces the heart’s workload. These regulations are beneficial for maintaining homeostasis (Humphrey 2008 ). However, long-term constriction or dilation of blood vessels causes excessive dilation and thinning of blood vessels, even aneurysm or arterial rupture. To enhance vascular stress intensity, blood vessels begin to develop fibrosis. Eventually, excessive fibrosis of the blood vessels leads to vascular sclerosis and hemadostenosis. These changes cause atherosclerosis and hypertension, which further increase stress on the cardiovascular system (Schwartz et al. 2018 ). This abnormal positive feedback is one of the pathogenic mechanisms underlying cardiovascular disease.

At present, the treatment of cardiovascular disease mainly focuses on alleviating the symptoms of patients. These include reducing the blood volume by using diuretics, limiting vasoconstriction by inhibiting the renin-angiotensin pathway and calcium channels, or lowering cholesterol synthesis to alleviate atherosclerosis by using statins (Takebe et al. 2018 ). Although these treatments have been widely used in clinical practice and have achieved a certain efficacy, many drugs may cause unexpected side effects that are not conducive to their long-term application.

Ideally, homeostatic treatment should comprehensively consider the mechanisms of vascular biomechanics, cell signal transduction, immunobiology, and other aspects of homeostasis regulation to avoid the disruption of homeostasis. By means of identifying and blocking therapeutic targets of disease processes, homeostatic medicine improves the state of a disease by restoring the dynamic balance of the cardiovascular system. Inorganic nitrate can maintain nitric oxide homeostasis through the nitrate-nitrite-nitric oxide pathway and has shown beneficial effects in a variety of cardiovascular diseases (Kapil et al. 2020b ). A significant antihypertensive effect was still observed after 1 year of use of inorganic nitrate, proving that inorganic nitrate does not have the same drug resistance as organic nitrate (Munzel et al. 2005 ). Moreover, no adverse effects such as syncope or postural hypotension were observed during nitrate use. In addition, compared to healthy volunteers, more significant hypotensive effects of inorganic nitrate were observed in hypertensive patients with impaired endothelial function, suggesting that inorganic nitrate may contribute to ameliorating endothelial homeostasis disorders (Kapil et al. 2015 ).

6.3 Homeostasis in metabolism diseases

Energy balance is mainly regulated by the central nervous system, including the neuroendocrine center located in the hypothalamus and the solitary nucleus located in the upper brainstem (Matafome and Seica 2017 ). Obesity is a metabolic disorder characterized by the excessive accumulation of body fat and is associated with an increased prevalence of various diseases. Metabolic homeostatic disorders are the most important causes of obesity (Lustig et al. 2022 ). Normally, the foraging behavior of the human body is dynamically balanced, and the desire to consume food increases to meet physiological needs when energy stores decrease. However, obese patients often exhibit a state of hedonism. To promote sensory pleasure, they consume food beyond energy balance, even when energy reserves are sufficient (Rossi and Stuber 2018 ). In addition, overeating alters the sensitivity of the dietary reward center to dopamine, further exacerbating the disruption of energy metabolism homeostasis and promoting the progression of obesity (Kessler et al. 2016 ).

Homeostatic medicine improves metabolic diseases by understanding the mechanism of homeostasis imbalance in metabolic diseases to eliminate the imbalance between energy uptake and metabolism. Inorganic nitrates ameliorate metabolic diseases, and their beneficial effects may be related to the promotion of the nitrate-nitrite-NO balance, thus maintaining NO homeostasis and regulating microbial homeostasis. In eNOS-deficient mice with impaired NO synthesis, inorganic nitrates decreased body fat and improved glucose homeostasis (Carlstrom et al. 2010 ). Liver senescence and metabolic dysfunction are also important causes of metabolic disease. Daily intake of nitrate can effectively restore the liver metabolic capacity of d-galactose-induced aging mice and naturally aging mice by preventing the aging of liver cells and the degeneration of glucose and lipid metabolism (Wang et al. 2018 ). Moreover, by activating the nitrate-nitrite-NO pathway, inorganic nitrates can promote the conversion of white adipose tissue to brown adipose tissue (Roberts et al. 2015 ). Inorganic nitrates can also reduce high-fat diet-induced obesity in mice and ameliorate glucose and lipid metabolism disorders by activating the NO pathway and regulating gut microbiota (Ma et al. 2020b ).

6.4 Homeostasis in immune and infectious diseases

The immune system, which can maintain homeostasis by immune surveillance, sensing metabolic changes, and controlling inflammation caused by external stimuli, is the most important line of defense of the body against harmful external stimuli and antigens (Weisberg et al. 2021 ). The body immunity maintains a dynamic balance under the interaction of various cellular and humoral immunities. Disorder in immune homeostasis is closely related to disease, and the development of a disease is the result of immune regulation failure (Kennedy 2010 ).

Take COVID-19 circulating worldwide as an example, which also disrupts the homeostasis of the immune system. COVID-19 is an infectious disease caused by SARS-COV-2 and characterized by acute respiratory failure. After infection, cells infected with SARS-COV-2 are recognized by macrophages and dendritic cells. Macrophages produce a large number of proinflammatory cytokines and chemokines, leading to the recruitment of inflammatory cells such as neutrophils to the site of the infection. The over release of pro-inflammatory cytokines may lead to cytokine storms, which can cause tissue damage, organ failure, and death. Severe symptoms are most common in elderly patients and are often accompanied by complications, while the symptoms in young people are often mild. This is because the homeostasis regulation of the immune system changes with the aging of the body. Compared to young patients, elderly patients have a lower response and clearance ability to viral infections, while the inflammatory response to infections is more active. Immune features are the main reason for the high severity and mortality of COVID-19 in elderly patients (Fulop et al. 2017 ). Homeostasis also plays an indispensable role in autoimmune diseases. Immune cells regulate and restrict each other to maintain the homeostasis of the immune system. For example, regulatory T cells can limit the activity of effector T cells, and reduced function of regulatory T cells can lead to autoimmune responses of effector T cells to tissues, such as rheumatoid arthritis (Kotschenreuther et al. 2022 ).

Dietary nitrate pretreatment can deliver NO to the vascular system and reduce the inflammatory response of leukocytes to acute chemokines through the nitrate-nitrite-NO pathway (Jadert et al. 2012 ). In mice susceptible to atherosclerosis, inorganic nitrate intake rescued NO homeostasis, thus inhibiting neutrophil activation and increasing IL-10 levels. Nitrate reduced the number of macrophages in atherosclerotic plaques and inhibited their inflammatory activity by reversing NO deficiency caused by endothelial dysfunction (Khambata et al. 2017 ). In liver oxidative stress injury induced by ischemia-reperfusion, inorganic nitrate intake increased NO levels in the plasma and liver and relieved liver oxidative stress by upregulating nuclear factor erythroid 2-related factor 2 (NRF2)-related molecules and increasing antioxidant enzyme activity (Li et al. 2021b ). In addition, the homeostasis of microorganisms in the human body has important regulatory roles in infectious diseases as well as in inflammatory processes. Inorganic nitrates can regulate the homeostasis of intestinal bacteria, alleviate dextran DSS-induced enteritis, improve colon length, and maintain the body weight of mice (Hu et al. 2020 ).

In summary, homeostasis regulation plays an important role in the health maintenance and disease prevention in different systems. Any disruption of homeostasis causes body dysfunction and ultimately leads to disease. Therefore, restoring the body’s homeostasis is key to reversing disease states. Homeostatic medicine integrates the mechanisms of homeostasis regulation at the molecular, cellular, organ, and systemic levels. By understanding the regulatory mechanism of homeostasis in health and disease states, the imbalance in homeostasis can be reversed through reasonable intervention. In this way, homeostatic medicine contributes to the maintenance of health and the treatment of disease. Homeostatic medicine, which has a very broad development prospect, is a new medical system that is expected to provide a new strategy for medical research and disease treatment. Regulation of NO homeostasis is closely related to the homeostasis of multiple systems in vivo. Inorganic nitrate, which can provide a variety of beneficial physiological functions through the nitrate-nitrite-NO pathway, is an important substance that regulates NO homeostasis. As a key protein for nitrate to enter cells, sialin interacts with nitrate and participates in the regulation of NO production and body homeostasis. Sialin can independently mediate a range of cellular functions. Therefore, the interaction between NO, nitrate, and sialin is an important mechanism for homeostasis regulation, which is of great concern in the study of homeostatic medicine.

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Why Homeostasis Is Important to Everyday Activities

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In this case study, a college student named "Blake" winds up in the emergency room after he experiences a panic attack brought on by drinking a mixture of beverages containing caffeine and alcohol. His panic attack results in a severe episode of hyperventilation. The alcohol he has consumed has the added effect of making the situation worse by impairing Blake's perception and judgment. Through this case study, students learn about acid/base chemistry as they explore hyperventilation, the Bohr effect, the Haldane effect, and how alcohol and stimulants such as caffeine can affect the acid-base balance in the body. This case was originally designed for a flipped classroom, and the associated videos, including one developed by the author, contain foundational information to lead students through basic chemistry and help them connect daily activities to homeostasis and the Bohr effect. Originally written for a general biology course in which general chemistry concepts are discussed, the case could easily be modified for use in an anatomy and physiology course.

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• Describe the difference between an acid and a base.
• Apply pH to blood chemistry.
• Analyze and predict changes in oxygen binding affinity as it relates to the Bohr effect.
• Compare and contrast the Bohr and Haldane effects.
• Apply respiratory and metabolic acidosis to acid-base chemistry.

homeostasis; pH; acids; bases; Bohr effect; Haldane effect; caffeine; panic attack;, stimulants; energy drinks; alcohol

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• Acids, Bases and pH This video explains pH as the power of hydrogen and how increases in the hydronium ion (or hydrogen ion) concentration can lower the pH and create acids. The video also explains how the reverse is true. In addition, an analysis of a strong acid and strong base is included. Running time: 8:53 min. Produced by Bozeman Science, 2013.
• Acid-Base Chemistry, pH, and the Human Body Created by the author specifically for this case, this brief video applies the definitions of acids, bases, and pH to a scenario in which blood chemistry is altered in our body. Running time: 4:07 min. Created by Brian J. Dingmann for the National Center for Case Study Teaching in Science, 2017.
• Bohr Effect vs. Haldane Effect This video takes a close look at how some friendly competition for hemoglobin allows the body to more efficiently move oxygen and carbon dioxide around. Running time: 13:52 min. Produced by the Khan Academy, 2012.

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The Concept Map - Intracerebral Hemorrhagic Stroke lg ...

Patient care with stroke assesment lg ..., mitral stenosis: symptoms, signs, and homeostatic disturbance lg ..., concept mapping for deteriorating patient and risk factors of haemorrhagic stroke lg ..., health and homeostasis lg ..., the case study of greta balodis: pathophysiology and signs of stroke lg ....

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Glycyrrhizic acid induced acquired apparent mineralocorticoid excess syndrome with a hyperadrenergic state: a case report

• John Szendrey 1 ,
• Anthony Poindexter 2 &
• Gregory Braden 2  

Journal of Medical Case Reports volume  18 , Article number:  358 ( 2024 ) Cite this article

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Syndrome of apparent mineralocorticoid excess (AME) is characterized by excessive MR stimulation despite low levels of aldosterone. 11Beta-hydroxysteroid dehydrogenase-2 (11βDSH-2) inactivates cortisol to cortisone, preventing cortisol-induced MR activation. Genetic defects in 11βDSH-2 cause AME through accumulation of cortisol in the distal nephron, leading to MR activation induced hypertension, hypokalemia and metabolic alkalosis. Acquired AME can occur due to the ingestion of glycyrrhizic acid, found in licorice root, which inhibits 11βDSH-2 and has additional effects on cortisol homeostasis through inhibition of 11βDSH-1.

We present a case of acquired AME with a hyperadrenergic symptoms induced by ingestion of Advanced Liver Support, a nutritional supplement produced by Advanced BioNutritionals (R) , in a 65-year-old Caucasian female who presented with accelerated hypertension, hypokalemia, metabolic alkalosis and adrenergic symptoms. Cessation of the licorice-containing supplement resulted in complete resolution of the patient's hypertension, symptoms and abnormal lab values. To our knowledge this is the first reported case of AME from this supplement, and the first to describe accompanying hyperadrenergic symptoms.

Conclusions

Glycyrrhizic acid is increasingly being found in unregulated nutritional supplements and has the potential to induce a reversable syndrome of AME. Acquired AME should be suspected in individuals who present with hypertension along with hypokalemia, metabolic alkalosis and low plasma renin and serum aldosterone levels.

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Cases of hypertension with hypokalemia and metabolic alkalosis are typical for syndromes of excessive mineralocorticoid receptor stimulation, the most classic of which is primary aldosteronism. The syndrome of apparent mineralocorticoid excess (AME), or pseudo-hyperaldosteronism, is characterized by hypertension with hypokalemia with low plasma renin and serum aldosterone levels. This rare inherited syndrome has been reported in individuals found to have mutations in 11β-hydroxysteroid dehydrogenase type 2 (11βDSH-2). 11βDSH-2 is expressed in many tissues which express mineralocorticoid receptors (MR), including the distal nephron, salivary glands, skin, and select regions of the brain such as the paraventricular nucleus [ 1 , 2 , 3 , 4 ]. Cortisol has equal binding affinity to MR to that of aldosterone [ 5 ], but within the distal nephron it is rapidly converted by 11βDSH-2 to the inactive metabolite cortisone, preventing cortisol mediated MR activation. Loss-of-function of 11βDSH-2 increases local cortisol levels, increasing MR activation. In the distal nephron, MR activation increases expression of the epithelial sodium channel (ENaC), leading to sodium and water retention.

Licorice consumption is a well described secondary cause of hypertension by inducing acquired AME [ 6 , 7 ]. Licorice root contains high concentrations of glycyrrhizic acid, which inhibits 11βDSH-2 causing acquired AME syndrome. Glycryrrhizic acid is also an antagonist of the other 11β hydroxysteroid dehydrogenase, 11βDSH-1, expressed within the liver, pancreas, adipose tissue and throughout the brain where it acts as a bidirectional catalyst for the interconversion of cortisone and cortisol. Glycyrrhizic acid is found in a variety of candies, chewing tobacco, traditional medicines, and increasingly in nutritional supplements. We report a case of acquired AME due to ingestion glycyrrhizic acid containing Advanced Bionutritionals (R) Advanced Liver Support nutritional supplement. Our patient’s presentation is unique as they also displayed hyperadrenergic symptoms, which have not been reported to our knowledge in other AME cases.

A 65-year-old Caucasian female with a past medical history of asthma presented to the emergency department after noting a systolic blood pressure of 220 mmHg at home. Earlier in the day she had been diagnosed with hypertension by her primary care physician and prescribed Olmesartan 20 mg daily. In the emergency room she complained of a mild headache and chest tightness. She also noted symptoms of anxiety, tremor, diaphoresis, and agitation which had been present for the past few months. Her medications included olmesartan 20 mg daily, budesonide-formoterol inhaler and fluticasone nasal spray needed, and an over-the-counter nutritional supplement.

She had a blood pressure of 197/89 mmHg, a heart rate of 104 beats/min, and a respiratory rate of 13 breaths/min. She appeared in moderate distress. She had a benign cardiac and respiratory exam and had no focal neurologic deficits. An EKG showed normal sinus rhythm without evidence of ischemia. Serial high-sensitivity troponins were completed which were in normal reference range. Her complete blood count and serial high sensitivity troponins were in the normal reference range. Her serum sodium was 141 mmol/L, potassium 3.1 mmol/L, chloride 101 mmol/L, bicarbonate 30 mmol/L, blood urea nitrogen of 13 mmol/L, and creatinine of 0.7 mg/dL with an estimated glomerular filtration rate of 96 mL/min/1.73 m 2 . Bloodwork completed 4 months prior showed sodium 133 mmol/L, a potassium level of 4.6 mmol/L and bicarbonate level of 27 mmol/L. The patient was diagnosed with hypertensive urgency and 5 mg of amlodipine daily was initiated.

Two days later the patient attended outpatient nephrology follow up and was found to be persistently hypertensive with a blood pressure of 188/86 mmHg despite a regimen of olmesartan 20 mg daily and amlodipine 5 mg daily. She again noted mild headache and persistent adrenergic symptoms of tremors, anxiety and agitation. She was diagnosed with accelerated hypertension. On investigation for secondary causes of hypertension found she was euthyroid with a TSH of 2.24 µIU/mL (reference range 0.4–4.2 µIU/mL) and free T4 of 1.18 ng/dL. Her serum fasting free metanephrines and normetanephrines were normal at 22.9 pg/mL (reference range 0–88 pg/mL) and 51.5 pg/mL (reference range 0–285.2 pg/mL). Her plasma renin activity was reduced at 0.437 ng/mL/hr (reference range 0.167–5.38 ng/mL/hr) and her serum aldosterone level was less than 1 ng/dL (reference range 4–10 ng/dL). olmesartan had been discontinued prior to drawing renin and aldosteorne levels.

The patient's presentation of hypertension with hypokalemia and metabolic alkalosis, and low plasma renin and serum aldosterone levels suggested the diagnosis of AME or Liddle syndrome. Liddle syndrome was excluded given her prior normal baseline metabolic panels. The patient was noted to be taking 4 tablets daily of Advanced Liver Support produced by Advanced Bionutritionals (R) (Nacros, Georgia) for the prior 4 months, which contains amongst its ingredients 250 mg of Licorice root extract per tablet for a total daily dose of 1,000 mg. The patient was diagnosed with hypertension secondary to glycryrrhizic acid induced acquired AME. Her supplement was stopped, and she had resolution of her hypertension, adrenergic symptoms, hypokalemia and her metabolic alkalosis on follow-up 2 weeks later, and her blood pressure medications were discontinued without further hypertension. She remained without hypertension of metabolic abnormalities on 3 month and 1 year follow-up. Confirmatory testing for AME via a 24-h urinary cortisol/cortisone ratio was not completed given the patients return to normotension along with her resolution of hypokalemia and metabolic alkalosis.

We present a case of acquired AME secondary to ingestion of Advanced BioNutritionals (R) Advanced Liver Support supplement. To our knowledge, this is the first reported case of acquired AME from this licorice root containing nutritional supplement. Our patient was consuming 1,000 mg daily of licorice root extract containing at least 20% glycryrrhizic acid, the pharmacologically active component of licorice root. Cases of licorice induced AME are rare in the United States, as the FDA regulates the maximum glycyrrhizin content which can be added to food products. Furthermore, many nutritional supplements which contain licorice are deglycyrrhizinated, free of the active ingredient which causes AME. However as in this case, there are still a variety of glycyrrhizin containing nutritional supplements available on the market (Table  1 ). Acquired AME has been observed with doses of glycryrrhizic acid greater than 217 mg/day [ 6 ], although there is significant variability among individuals in their sensitivity to licorice mediated AME. One proposed explanation for differences in susceptibility to glycyrrhizic acid is genetic variations in the epithelial sodium channel (ENaC) [ 7 ]. ENaC is responsible for MR-regulated sodium retention within the distal nephron, and individuals who have more prolific ENaC activity or expression may be more suspectable to glycyrrhizic acid induced hypertension. As the patient's hypertension and metabolic abnormalities had rapidly resolved, confirmatory testing for AME via 24 h urinary cortisol/cortisone ratio testing was not performed.

Glycryrrhizic acid induces AME through inhibition of 11βDSH-2, a mechanism which parallels the effects of loss-of-function mutations seen in inherited AME. Glycryrrhizic acid has additional effects on cortisol metabolism, as it is also an antagonist of the other hydroxysteroid dehydrogenase isoenzyme, 11βDSH-1. 11βDSH-1 is a NADPH/NADP + mediated bidirectional catalyst of cortisol and cortisone conversion [ 8 ]. The direction of 11βDSH activity is regulated through the ratio of NADPH/NADP + , with an increase in NADPH increasing reduction of cortisone to cortisol. Its kinetics from tissue samples typically favor reduction of cortisone to cortisol, resulting in a net increase in local cortisol levels. 1βDSH-1 is expressed in the liver, adipose tissue, muscle, pancreas, and throughout the brain [ 2 , 9 ] and is responsible for approximately 30% of daily cortisol production through the salvage of cortisone within the splanchnic circulation [ 10 ].

We believe our patient had hyperadrenergic symptoms due to glycryrrhizic acid mediated changes in cortisol expression and signaling within the CNS. To our knowledge, this is the first case reported of licorice induced AME to also note hyperadrenergic symptoms. Our patient complained of significant agitation, tremors, and anxiety which resolved along with her AME after cessation of her nutritional supplement. We suspect her symptoms could be secondary changes in local cortisol levels within the CNS, altering local glucocorticoid receptor and MR signaling. 11βDSH-2 is expressed in the paraventricular nucleus (PVN) of rats, and inhibition of 11βDSH-2 with glycryrrhizic acid has been shown to increase sympathetic outflow from the PVN through glucocorticoid dependent activation of MR [ 4 ]. While it remains unclear whether humans express 11βDSH-2 within our PVN, this mechanism would explain our patient’s increased adrenergic symptoms. A second explanation for our patient’s adrenergic symptoms could be through inhibition of 11βDSH-1. As 11βDSH-1 is a bidirectional catalyst, its inhibition could also result in increased local cortisol levels based upon the local metabolome and balance of cofactors NADPH/NADP + . Further investigation of the endocrine and paracrine effects of 11βDSH-1 and 11βDSH-2 within the CNS is required and could lead to a better understanding of the body's regulation and role of steroids in the brain.

Glycyrrhizic acid ingestion can induce an acquired AME syndrome, which in this case presented in tandem with previously undescribed hyperadrenergic symptoms. AME and other excess mineralocorticoid activity syndromes should be suspected when hypertension is present with metabolic alkalosis and hypokalemia.

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Abbreviations

• Apparent mineralocorticoid excess

11β Hydroxysteroid dehydrogenase type 1

11β Hydroxysteroid dehydrogenase type 2

Epithelial sodium channel

Hypothalamic pituitary adrenal gland axis

Mineralocorticoid receptor

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Szendrey, J., Poindexter, A. & Braden, G. Glycyrrhizic acid induced acquired apparent mineralocorticoid excess syndrome with a hyperadrenergic state: a case report. J Med Case Reports 18 , 358 (2024). https://doi.org/10.1186/s13256-024-04674-1

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Male autism spectrum disorder is linked to brain aromatase disruption by prenatal BPA in multimodal investigations and 10HDA ameliorates the related mouse phenotype

• Christos Symeonides   ORCID: orcid.org/0009-0009-9415-4097 1 , 2 , 3   na1 ,
• Kristina Vacy   ORCID: orcid.org/0009-0000-5330-5260 4 , 5   na1 ,
• Sarah Thomson   ORCID: orcid.org/0000-0002-5120-3997 4 ,
• Sam Tanner   ORCID: orcid.org/0009-0003-9363-0756 4 ,
• Hui Kheng Chua   ORCID: orcid.org/0000-0002-6047-4027 4 , 6 ,
• Shilpi Dixit   ORCID: orcid.org/0000-0003-4837-0548 4 ,
• Toby Mansell   ORCID: orcid.org/0000-0002-1282-6331 2 , 7 ,
• Martin O’Hely   ORCID: orcid.org/0000-0002-0212-1207 2 , 8 ,
• Boris Novakovic   ORCID: orcid.org/0000-0002-5623-9008 2 , 8 ,
• Julie B. Herbstman 9 , 10 ,
• Shuang Wang   ORCID: orcid.org/0000-0002-1693-6888 9 , 11 ,
• Jia Guo   ORCID: orcid.org/0000-0002-9774-9856 9 , 11 ,
• Jessalynn Chia 4 ,
• Nhi Thao Tran   ORCID: orcid.org/0000-0002-0396-9760 4   nAff28 ,
• Sang Eun Hwang   ORCID: orcid.org/0009-0009-7271-7493 4 ,
• Kara Britt   ORCID: orcid.org/0000-0001-6069-7856 12 , 13 , 14 ,
• Feng Chen 4 ,
• Tae Hwan Kim   ORCID: orcid.org/0009-0000-6163-3483 4 ,
• Christopher A. Reid 4 ,
• Anthony El-Bitar 4 ,
• Gabriel B. Bernasochi   ORCID: orcid.org/0000-0002-3966-2074 4 , 15 ,
• Lea M. Durham Delbridge   ORCID: orcid.org/0000-0003-1859-0152 15 ,
• Vincent R. Harley   ORCID: orcid.org/0000-0002-2405-1262 12 , 16 ,
• Yann W. Yap 6 , 16 ,
• Deborah Dewey   ORCID: orcid.org/0000-0002-1323-5832 17 ,
• Chloe J. Love   ORCID: orcid.org/0000-0002-2024-4083 8 , 18 ,
• David Burgner   ORCID: orcid.org/0000-0002-8304-4302 2 , 7 , 19 , 20 ,
• Mimi L. K. Tang 2 , 15 ,
• Peter D. Sly   ORCID: orcid.org/0000-0001-6305-2201 8 , 21 , 22 ,
• Richard Saffery   ORCID: orcid.org/0000-0002-9510-4181 2 ,
• Jochen F. Mueller   ORCID: orcid.org/0000-0002-0000-1973 23 ,
• Nicole Rinehart   ORCID: orcid.org/0000-0001-6109-3958 24 ,
• Bruce Tonge   ORCID: orcid.org/0000-0002-4236-9688 25 ,
• Peter Vuillermin   ORCID: orcid.org/0000-0002-6580-0346 2 , 8 , 18 ,
• the BIS Investigator Group ,
• Anne-Louise Ponsonby   ORCID: orcid.org/0000-0002-6581-3657 2 , 3 , 4   na2 &
• Wah Chin Boon 4 , 26   na2  

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• Autism spectrum disorders
• Epigenetics and behaviour

Male sex, early life chemical exposure and the brain aromatase enzyme have been implicated in autism spectrum disorder (ASD). In the Barwon Infant Study birth cohort ( n  = 1074), higher prenatal maternal bisphenol A (BPA) levels are associated with higher ASD symptoms at age 2 and diagnosis at age 9 only in males with low aromatase genetic pathway activity scores. Higher prenatal BPA levels are predictive of higher cord blood methylation across the CYP19A1 brain promoter I.f region ( P  = 0.009) and aromatase gene methylation mediates ( P  = 0.01) the link between higher prenatal BPA and brain-derived neurotrophic factor methylation, with independent cohort replication. BPA suppressed aromatase expression in vitro and in vivo. Male mice exposed to mid-gestation BPA or with aromatase knockout have ASD-like behaviors with structural and functional brain changes. 10-hydroxy-2-decenoic acid (10HDA), an estrogenic fatty acid alleviated these features and reversed detrimental neurodevelopmental gene expression. Here we demonstrate that prenatal BPA exposure is associated with impaired brain aromatase function and ASD-related behaviors and brain abnormalities in males that may be reversible through postnatal 10HDA intervention.

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Introduction.

Autism spectrum disorder (ASD or autism) is a clinically diagnosed neurodevelopmental condition in which an individual has impaired social communication and interaction, as well as restricted, repetitive behavior patterns 1 . The estimated prevalence of ASD is approximately 1–2% in Western countries 2 , with evidence that the incidence of ASD is increasing over time 3 . While increased incidence is partly attributable to greater awareness of ASD 4 , other factors including early life environment, genes and their interplay are important 5 . Strikingly, up to 80% of individuals diagnosed with ASD are male, suggesting sex-specific neurodevelopment underlies this condition 5 .

Brain aromatase, encoded by CYP19A1 and regulated via brain promoter I.f 6 , 7 , 8 converts neural androgens to neural estrogens 9 . During fetal development, aromatase expression within the brain is high in males 10 in the amygdala 11 , 12 . Notably, androgen disruption is implicated in the extreme male brain theory for ASD 13 , and postmortem analysis of male ASD adults show markedly reduced aromatase activity compared to age-matched controls. Furthermore, CYP19A1 aromatase expression was reduced by 38% in the postmortem male ASD prefrontal cortex 14 , as well as by 52% in neuronal cell lines derived from males with ASD 15 . Environmental factors, including exposure to endocrine-disrupting chemicals such as bisphenols, can disrupt brain aromatase function 16 , 17 , 18 .

Early life exposure to endocrine-disrupting chemicals, including bisphenols, has separately been proposed to contribute to the temporal increase in ASD prevalence 19 . Exposure to these manufactured chemicals is now widespread through their presence in plastics and epoxy linings in food and drink containers and other packaging products 20 . Although bisphenol A (BPA) has since been replaced by other bisphenols such as bisphenol S in BPA-free plastics, all bisphenols are endocrine-disrupting chemicals that can alter steroid signaling and metabolism 21 . Elevated maternal prenatal BPA levels are associated with child neurobehavioral issues 20 including ASD-related symptoms 22 , 23 , with many of these studies reporting sex-specific effects 20 , 22 , 23 , 24 . Furthermore, studies in rodents have found that prenatal BPA exposure is associated with gene dysregulation in the male hippocampus accompanied by neuronal and cognitive abnormalities in male but not female animals 20 , 23 , 24 . One potential explanation is that epigenetic programming by bisphenols increases aromatase gene methylation, leading to its reduced cellular expression 16 and a deficiency in aromatase-dependent estrogen signaling. If such is the case, it is possible that estrogen supplementation, such as with 10-hydroxy-2-decenoic acid (10HDA), a major lipid component of the royal jelly of honeybees, may be relevant as a nutritional intervention for ASD. Indeed, 10HDA is known to influence homeostasis through its intracellular effects on estrogen responsive elements that regulate downstream gene expression 25 , 26 , as well as its capacity to influence neurogenesis in vitro 27 .

Here, we have investigated whether higher prenatal BPA exposure leads to an elevated risk of ASD in males and explore aromatase as a potential underlying mechanism. We demonstrate in a preclinical (mouse) model that postnatal administration of 10HDA, an estrogenic fatty acid, can ameliorate ASD-like phenotypes in young mice prenatally exposed to BPA.

Human studies

We examined the interplay between prenatal BPA, aromatase function and sex in relation to human ASD symptoms and diagnosis in the Barwon Infant Study (BIS) birth cohort 28 . By the BIS cohort health review at 7-11 years (mean = 9.05, SD = 0.74; hereafter referred to as occurring at 9 years), 43 children had a pediatrician- or psychiatrist- confirmed diagnosis of ASD against the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-5) criteria, as of the 30th of June 2023. ASD diagnosis was over-represented in boys with a 2.1:1 ratio at 9 years (29 boys and 14 girls; Supplementary Table  1 ). In BIS, the DSM-5 oriented autism spectrum problems (ASP) scale of the Child Behavior Checklist (CBCL) at age 2 years 29 predicted diagnosed autism strongly at age 4 and moderately at age 9 in receiver operating characteristic (ROC) curve analyses; area under the curve (AuC) of 0.92 (95% CI 0.82, 1.00) 30 and 0.70 (95% CI 0.60, 0.80), respectively. The median CBCL ASP score in ASD cases and non-cases at 9 years was 51 (IQR = 50, 58) and 50 (IQR = 50, 51), respectively. Only ASD cases with a pediatrician-confirmed diagnosis of ASD against the DSM-5, as verified by the 30th of June 2023, were included in this report. We thus examined both outcomes (ASP scale and ASD diagnosis) as indicators of ASD over the life course from ages 2 to 9 years (Supplementary Table  1 ). Quality control information for the measurement of BPA is presented in Supplementary Table  2 .

BPA effects on ASD symptoms at age 2 years are most evident in boys genetically predisposed to low aromatase enzyme activity

Of the 676 infants with CBCL data in the cohort sample, 249 (36.8%) had an ASP score above the median based on CBCL normative data (Supplementary Table  1 ). From a whole genome SNP array (Supplementary Methods), a CYP19A1 genetic score for aromatase enzyme activity was developed based on five single nucleotide polymorphisms (SNPs; rs12148604, rs4441215, rs11632903, rs752760, rs2445768) associated with lower estrogen levels 31 . Among 595 children with prenatal BPA and CBCL data, those in the top quartile of the genetic predisposition score, that is, children with three or more variants associated with lower levels of estrogens were classified as ‘low aromatase activity’ with the remaining classified as ‘high aromatase activity’ (Fig.  1 ). Regression analyses stratified by this genetic score and child’s sex were performed and an association between high prenatal BPA exposure (top quartile (>2.18 μg/L) and greater ASP scores was only seen in males with low aromatase activity, with a matched OR of 3.56 (95% CI 1.13, 11.22); P  = 0.03 (Supplementary Table  4 ). These findings were minimally altered following adjustment for additional potential confounders. Among males with low aromatase activity, the fraction with higher than median ASP scores attributable to high BPA exposure (the population attributable fraction) was 11.9% (95% CI 4.3%, 19.0%). These results indicate a link between low aromatase function and elevated ASP scores. A sensitivity analysis using an independent weighted CYP19A1 genetic score confirmed these findings. For the additional score, the Genotype-Tissue Expression (GTEx) portal was first used to identify the top five expression quantitative trait loci (eQTLs; rs7169770, rs1065778, rs28757202, rs12917091, rs3784307) for CYP19A1 in any tissue type that showed a consistent effect direction in brain tissue. A functional genetic score was then computed for each BIS participant by summing the number of aromatase-promoting alleles they carry across the five eQTLs, weighted by their normalized effect size (NES) in amygdala tissue. This score captures genetic contribution to cross-tissue aromatase activity with a weighting towards the amygdala, a focus in our animal studies. The score was then reversed so that higher values indicate lower aromatase activity and children in the top quartile were classified as ‘low aromatase activity’ with the remaining classified as ‘high aromatase activity’. Again, a positive association between prenatal BPA exposure and ASP scores was only seen in males with low aromatase activity, with a matched OR of 3.74 (95% CI 1.12, 12.50); P  = 0.03. Additional adjustment for individual potential confounders provided matched ORs between 3.13 to 3.85 (Supplementary Table  5 ).

Conditional logistic regression models were run where participants were matched on ancestry and time of day of urine collection and, for ASD diagnosis at 9 years, each case within these matched groups was individually matched to eight controls based on nearest date of and age at year 9 interview. BPA was classified in quartiles with the top quartile above 2.18 μg/L as high BPA exposure vs the other three quartiles. ‘Low aromatase enzyme activity’ means being in the top quartile and ‘high aromatase enzyme activity’ means being in the lower three quartiles of an unweighted sum of the following genotypes associated with lower estrogen levels 31 (participant given 1 if genotype is present, 0 if not): CC of rs12148604, GG of rs4441215, CC of rs11632903, CC of rs752760, AA of rs2445768. ‘Greater ASD symptoms’ represents a T-score above 50 (that is, above median based on normative data) on the DSM-5-oriented autism spectrum problems scale of the Child Behavior Checklist for Ages 1.5-5 (CBCL). Data are OR ± 95% CI. Source data are provided as a Source Data file. * Since there were only two ASD cases at age 9 in the girls with low aromatase enzyme activity group, the regression model was not run.

BPA effects on ASD diagnosis at 9 years are most evident in boys genetically predisposed to low aromatase enzyme activity

In subgroup analyses where we stratified by child’s sex and unweighted CYP19A1 genetic score, the results were consistent with those found at 2 years. A positive association between high prenatal BPA exposure and ASD diagnosis was only seen in males with low aromatase activity, with a matched OR of 6.24 (95% CI 1.02, 38.26); P  = 0.05 (Supplementary Table  4 ). In this subgroup, the fraction of ASD cases attributable to high BPA exposure (the population attributable fraction) was 12.6% (95% CI 5.8%, 19.0%). In a sensitivity analysis where the weighted CYP19A1 genetic score was used, a similar effect size was observed in this subgroup; matched OR = 6.06 (95% CI 0.93, 39.43), P  = 0.06 (Supplementary Table  4 ).

Higher prenatal BPA exposure predicts higher methylation of the CYP19A1 brain promoter PI.f in human cord blood

We investigated the link between BPA and aromatase further by evaluating epigenetic regulation of the aromatase gene at birth in the same BIS cohort. CYP19A1 (in humans; Cyp19a1 in the mouse) has eleven tissue-specific untranslated first exons under the regulation of tissue-specific promoters. The brain-specific promoters are PI.f 6 , 7 , 8 and PII 17 . For a window positioned directly over the primary brain promoter PI.f, higher BPA was positively associated with average methylation, mean increase = 0.05% (95% CI 0.01%, 0.09%); P  = 0.009 (Fig.  2 ). Higher BPA levels predicted methylation across both PI.f and PII as a composite, mean increase per log 2 increase = 0.06% (95% CI 0.01%, 0.10%); P  = 0.009. Methylation of a control window, comprising the remaining upstream region of the CYP19A1 promoter and excluding both PI.f and PII brain promoters, did not associate with BPA, P  = 0.12. These findings persisted after adjustment for the CYP19A1 genetic score for aromatase enzyme activity. Thus, higher prenatal BPA exposure was associated with increased methylation of brain-specific promoters in CYP19A1 . Sex-specific differences were not observed. While these effects were identified in cord blood, methylation of CYP19A1 shows striking concordance between blood and brain tissue (Spearman’s rank correlation across the whole gene: ρ = 0.74 (95% CI 0.59, 0.84); over promoter PI.f window: ρ = 0.94 (95% CI 0.54, 0.99) 32 . Thus, prenatal BPA exposure significantly associates with disruption of the CYP19A1 brain promoter and hence likely the level of its protein product, aromatase.

Visualized using the coMET R package. A Association of individual CpGs along the region of interest with BPA exposure, overlaid with three methylation windows: a 2 CpG window positioned directly on promoter PII, and 7 and 15 CpG windows overlapping PI.f. The red shading reflects each CpG’s level of methylation (beta value). B The CYP19A1 gene, running right to left along chromosome 15, and the positions of both brain promoters. Orange boxes indicate exons. C A correlation matrix for all CpGs in this region. Highlighted in tan are the two CpGs located within the PII promoter sequence and the single CpG located within PI.f. For the 7 CpG window over promoter PI.f, higher BPA associated positively with methylation, mean increase = 0.05% (95% CI 0.01%, 0.09%); P  = 0.009, after adjustment for relevant covariates including cell composition. The BPA-associated higher methylation of the brain promoter PI.f region remained evident when the window was expanded to 15 CpGs (mean increase = 0.06%, 95% CI [0.01%, 0.11%], P  = 0.04). For PII, the BPA-associated mean methylation increase was 0.07%, 95% CI [-0.02%, 0.16%], P  = 0.11). BPA also associated positively with methylation across both PI.f and PII as a composite, mean increase = 0.06% (95% CI 0.01%, 0.10%); P  = 0.009. For the remainder of CYP19A1 , excluding both PI.f and PII brain promoters, there was no significant association, P  = 0.12. Higher CYP19A1 brain promoter methylation leads to reduced transcription 17 . All statistical tests are two sided. Source data are provided as a Source Data file.

Replication of the association between higher BPA levels and hypermethylation of the CYP19A1 brain promoter

Previously, the Columbia Centre for Children’s Health Study-Mothers and Newborns (CCCEH-MN) cohort (Supplementary Table  3 ) found BPA increased methylation of the BDNF CREB-binding region of promoter IV both in rodent blood and brain tissue at P28 and in infant cord blood in the CCCEH-MN cohort 33 . In rodents, BDNF hypermethylation occurred concomitantly with reduced BDNF expression in the brain 33 . Re-examining the CCCEH-MN cohort, BPA level was also associated with hypermethylation of the aromatase brain promoter P1.f (adjusted mean increase 0.0040, P  = 0.0089), replicating the BIS cohort finding.

Molecular mediation of higher BPA levels and hypermethylation of BDNF through higher methylation of CYP19A1

In BIS, we aimed to reproduce these BDNF findings and extend them to investigate aromatase methylation as a potential mediator of the BPA- BDNF relationship. A link between aromatase and methylation of the BDNF CREB-binding region is plausible given that estrogen (produced by aromatase) is known to elevate brain expression of CREB 34 , 35 . In BIS, male infants exposed to BPA (categorized as greater than 4 µg/L vs. rest, following the CCCEH-MN study) had greater methylation of the BDNF CREB-binding site (adjusted mean increase = 0.0027, P  = 0.02). This was also evident overall (adjusted mean increase = 0.0023, P  = 0.006), but not for females alone (adjusted mean increase = 0.0019, P  = 0.13). We then assessed whether methylation of aromatase promoter P1.f mediates this association. In both cohorts, aromatase methylation was positively associated with BDNF CREB-binding-site methylation in males (BIS, adjusted mean increase = 0.07, P  = 0.0008; CCCEH-MN, adjusted mean increase = 0.91, P  = 0.0016). In the two overall cohorts, there was evidence that the effect of increased BPA on BDNF hypermethylation was mediated partly through higher aromatase methylation (BIS, indirect effect, P  = 0.012; CCCEH-MN, indirect effect, P  = 0.012).

Prenatal programming laboratory studies—BPA effects on cellular aromatase expression in vitro, neuronal development as well as behavioral phenotype in mice

Bpa reduces aromatase expression in human neuroblastoma sh-sy5y cell cultures.

To validate the findings of our human observational studies on BPA and aromatase expression, we began by studying the effects of BPA exposure on aromatase expression in the human neuroblastoma cell line SH-SY5Y (Fig.  3A ). Indeed, the aromatase protein levels more than halved in the presence of BPA 50 μg/L ( P  = 0.01; Fig.  3B ) by Western Blot analysis.

A Western Blot (1 representative blot) demonstrates that increasing BPA concentrations reduced immunoblotted aromatase protein signals (green fluorescence, 55 kDa) in lysates from human-derived neuroblastoma SH-SY5Y cells. Each sample was normalized to its internal house keeping protein β-Actin (red fluorescence, 42 kDa). B Aromatase immunoblotted signals in SH-SY5Y cells treated with vehicle or BPA ( n  = 3 independent experiments/group). Five-day BPA treatment of SH-SY5Y cells leads to a significant reduction in aromatase following 50 mg/L(MD = 89, t(6) = 4.0, P  = 0.01) and 100 mg/L (MD = 85, t(6) = 3.9, P  = 0.01) BPA treatment, compared to vehicle. C BPA treatment (50 µg/kg/day) of Cyp19 -EGFP mice at E10.5-E14.5 results in fewer EGFP+ neurons in the medial amygdala (MD = -5334, t(4) = 5.9, P  = 0.004) compared to vehicle mice, n  = 3 mice per treatment. Independent t -tests were used and where there were more than two experimental groups ( B ), P -values were corrected for multiple comparisons using Holm-Sidak. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file. Note: UT = untreated.

The effects of prenatal BPA exposure on aromatase-expressing neurons within the amygdala of male mice

There is a prominent expression of aromatase within cells of the male medial amygdala (MeA) 11 . To visualize aromatase-expressing cells, we studied genetically modified, Cyp19 -EGFP transgenic mice harboring a single copy of a bacterial artificial chromosome (BAC) encoding the coding sequence for enhanced green fluorescent protein (EGFP) inserted upstream of the ATG start codon for aromatase ( Cyp19a1 ) 11 (see Methods). As shown, EGFP (EGFP+) expression in male mice was detected as early as embryonic day (E) 11.5 (Supplementary Fig.  1 ), indicating that aromatase gene expression is detectable in early CNS development.

To study the effects of prenatal BPA exposure on brain development, pregnant dams were subject to BPA at a dose of 50 μg/kg/day via subcutaneous injection, or a vehicle injection during a mid-gestation window of E10.5 to E14.5, which coincides with amygdala development. This dose matches current USA recommendations 36 , 37 as well as the Tolerable Daily Intake (TDI) set by the European Food Safety Authority (EFSA) at the time that the mothers in our human cohort were pregnant 28 , 38 . In these experiments, we observed that prenatal BPA exposure led to a 37% reduction ( P  = 0.004) in EGFP+ neurons in the MeA of male EGFP+ mice compared to control mice (Fig.  3C ). These results are consistent with our findings in SH-SY5Y cells that indicate that BPA exposure leads to a marked reduction in the cellular expression of aromatase.

Prenatal BPA exposure at mid-gestation influences social approach behavior in male mice

Next, we evaluated post-weaning social approach behavior (postnatal (P) days P21-P24) using a modified three-chamber social interaction test 39 (Fig.  4C ). As shown, male mice with prenatal exposure to BPA were found to spend less time investigating sex- and age-matched stranger mice, when compared with vehicle-treated males (with a mean time ± SEM of 101.2 sec ± 11.47 vs. 177.3 s ± 26.97, P  = 0.0004; Fig.  4A ). Such differences were not observed for female mice prenatally exposed to BPA (Fig.  4A ). As a control for these studies, we found that the presence of the EGFP BAC transgene is not relevant to behavioral effects in the test (Supplementary Fig.  2 ), and the proportions of EGFP transgenic mice were not significantly different across BPA-exposed and vehicle-exposed cohorts.

Sociability is the higher proportion of time spent in the stranger interaction zone compared to the empty interaction zone. In the three chamber social interaction test ( A ) BPA-exposed mice ( n  = 30, MD = 75 s, t(96) = 3.7  P  = 0.0004) spent less time investigating the stranger mouse as compared with male control ( n  = 21) mice. B Male ArKO ( n  = 8, MD = 43 s, t(30) = 2.3, P  = 0.03) mice also spent less time with the stranger compared to male WT littermates ( n  = 9). C A schematic of the 3-Chamber Sociability Trial. Created with BioRender.com. D Male BPA-exposed mice ( n  = 12, MD = 8.2, U = 11, P  = 0.048.) spent more time grooming compared to control ( n  = 5) mice. There were no differences between female BPA-exposed ( n  = 9) and female control ( n  = 6) mice. Independent t -tests were used P -values were corrected for multiple comparisons using Holm-Sidak. For ( C ), a Mann–Whitney U test was used. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file. Note: Veh = vehicle.

To determine if the effects of prenatal BPA exposure were developmentally restricted, we delivered subcutaneous injections (50 µg/kg/day) of BPA to pregnant dams at early (E0.5–E9.5), mid (E10.5–E14.5), and late (E15.5–E20.5) stages of gestation. From these experiments, we found that while male pups exposed to BPA in mid-gestation developed a social approach deficit, such behavioral impairments were not observed for early or late gestation BPA exposure (Supplementary Fig.  3 ). In addition, we performed experiments in which BPA was available to dams by voluntary, oral administration (50 µg/kg/day) during mid-gestation. As shown, a social approach deficit was again observed in male mice (Supplementary Fig.  4 ), consistent with results from prenatal (mid-gestation) BPA exposure by subcutaneous injections. Thus, we find that prenatal BPA exposure at mid-gestation (E10.5-E14.5) in mice leads to reduced social approach behavior in male, but not female offspring. Notably, the amygdala of embryonic mice undergoes significant development during mid-gestation 40 .

Aromatase knockout (ArKO) male mice have reduced social behavior

Having demonstrated that prenatal BPA exposure reduces aromatase expression in SH-SY5Y cells and affects the postnatal behavior of mice, we next asked if the aromatase gene ( Cyp19a1 ) is central to these phenotypes. To address this, we performed social approach behavioral testing (Supplementary Fig.  5 ) on aromatase knockout (ArKO) mice 41 which have undetectable aromatase expression. The social preference towards the stranger interaction zone compared to the empty zone was only evident for the wildtype ( P  = 0.003 Fig.  4B ) but not the ArKO ( P  = 0.45 Fig.  4B ). This male-specific social interaction deficit is similar to the BPA exposed pups. Further, postnatal estrogen replacement could reverse the ArKO reduction in sociability seen in males ( P  = 0.03 Supplementary Fig.  5 ) resulting in a similar stranger-to-empty preference in the E2-treated ArKO as observed for wildtype. The female ArKO pups did not have a sociability deficit (Supplementary Fig.  5 ).

Further, we did not observe any behavioral differences between ArKO vs WT (or BPA exposed vs unexposed) mice of both sexes in Y-maze test. All groups were able to distinguish the novel arm from the familiar arm. All groups spent significantly more time in the novel arm compared to the familiar arm (Supplementary Fig.  6 ), excluding major short-term memory, motor and sensory intergroup difference contributions.

Prenatal exposure to BPA affects repetitive behavior in male mice

Using the water squirt test, we have previously reported that male ArKO, but not female ArKO mice displayed excessive grooming, a form of repetitive behavior, compared to WT mice 42 . Thus, we conducted the water squirt test on BPA-exposed mice to find that male but not female mice exhibited excessive grooming behavior ( P = 0.048; Fig. 4D). Thus, male prenatal BPA-exposed mice and ArKO mice, but not females, exhibited such repetitive behaviors compared to control mice.

The development of the MeA is altered in male ArKO mice as well as in prenatal BPA-exposed male mice

The development and function of the amygdala are highly relevant to human brain development and ASD 43 , 44 . Notably, the medial amygdala (MeA) is central to emotional processing 45 , and this tissue is a significant source of aromatase-expressing neurons. Given that aromatase function in the amygdala is significant for human cognition 46 and behavior 12 , 47 , and that aromatase is highly expressed in the mammalian MeA, as particularly observed in male mice 11 , we investigated changes to the structure and function of this brain region. We performed stereology analyses on cresyl violet (Nissl)-stained sections of male MeA, we observed a 13.5% reduction in neuron (defined by morphology, size, and presence of nucleolus) number. Compared to the vehicle-exposed males, BPA-exposed males had significantly reduced total neuron number (mean count of 91,017 ± SEM of 2728 neurons vs 78,750 ± SEM of 3322 neurons, P  = 0.046; Supplementary Fig.  7 ).

We further examined the characteristics of cells within this amygdala structure in detail using Golgi staining (Fig.  5A, B ). We found that the apical and basal dendrites in the MeA were significantly shorter in male BPA-exposed mice vs. vehicle-treated mice (apical: 29.6% reduction, P  < 0.0001; basal, P  < 0.0001). This phenotype was also observed for male ArKO vs. WT mouse brains (apical 35.0% reduction, P  < 0.0001; basal 31.9% reduction, P  < 0.0001; Fig.  5A ). Dendritic spine densities of apical and basal dendrites of male ArKO mice, as well as male mice exposed to BPA, were also significantly reduced (KO vs WT apical, P  = 0.01; KO vs WT basal, P  < 0.0001; BPA treated vs vehicle treated apical P  < 0.0001; BPA treated vs vehicle treated basal, P  = 0.004; Fig.  5B ). The dendritic lengths (Fig.  5A ) and spine densities (Fig.  5B ) for apical and basal neurites within the MeA of female ArKO mice or BPA-exposed mice were not significantly different compared to control. Thus, in the context of aromatase suppression by prenatal BPA-exposure, or in ArKO mice lacking aromatase, we find that the apical and basal dendrite features within the MeA are affected in a sexually dimorphic manner.

A Golgi staining showed shorter apical and basal dendrites in male BPA-exposed (apical: n  = 27β = −136 μ m, 95% CI [−189, −83], P  = 6.0 × 10 −7 ; basal: n  = 27 neurons β =-106, 95% CI [−147, −64], P  = 5.1 × 10 −7 ) and ArKO mice (apical: n  = 27 β = −194, 95% CI [−258, −130], P  = 2.9 × 10 −9 ; basal: n  = 27, β = −121, 95% CI [−143, −100], P  = 1.2 × 10 −29 ) compared to male vehicle (apical n  = 27, basal n  = 27) or WT (apical n  = 27, basal n  = 29). Female BPA-exposed mice had longer basal dendrites vs. vehicle ( n  = 26 neurons/group, β = 133, 95% CI [76, 191], P  = 5.5 × 10 −6 ), while female ArKO mice had shorter basal dendrites vs. WT ( n  = 22 neurons/group, β = −45, 95% CI [−91, −0.2], P  = 0.049). Significant sex-by-BPA-treatment interaction effects were observed for apical ( P  = 0.0002) and basal ( P  = 3.0 × 10 −11 ) dendritic lengths, and a sex-by-genotype interaction for basal length ( P  = 0.003) but not apical length ( P  = 0.19). B Golgi staining showed male BPA-exposed (apical: n  = 39, β = −7.0, 95% CI [−10.1, −4.0], P  = 5.5 × 10 −6 ; basal: n  = 90, β = −3.4, 95% CI [−5.7, −1.1], P  = 0.004) and ArKO (apical: n  = 51, β = −6.8, 95% CI [−12.2, −1.3], P  = 0.01; basal: n  = 97, β = −3.8, 95% CI [−5.4, −2.2], P  = 5.2 × 10 −6 ) mice had lower spine densities on apical and basal dendrites vs. vehicle (apical n  = 46, basal n  = 106) or WT (apical n  = 53, basal n  = 109) mice. Female mice exhibited no spine density differences for BPA exposure (apical n  = 74, basal n  = 106) vs. vehicle (apical n  = 61, basal n  = 103) and ArKO (apical n  = 94, basal n  = 86) vs. WT (apical n  = 88, basal n  = 83). There was a significant sex-by-BPA-treatment interaction for apical spine density ( P  = 0.0005) but not basal ( P  = 0.99), and no significant sex-by-genotype interactions (apical: P  = 0.08; basal: P  = 0.19). For golgi staining experiments, 3 mice/group with 6–9 neuron measures/mouse. Spine count datapoints represent the number of spines on a single 10μm concentric circle. C c-Fos fluorescent immunostaining in adult male PD-MeA revealed fewer c-Fos+ve cells in BPA-exposed ( n  = 3) vs. vehicle mice ( n  = 3; mean difference MD = 3687, t (4) = 16.12, P  < 0.0001) and ArKO ( n  = 4) vs. WT mice ( n  = 4; MD = −10237; t (4) = 6.48, P  = 0.0002). Early postnatal estradiol restored ArKO c-Fos to WT levels ( n  = 4; MD = −3112; t(4) = 1.97, P  = 0.08). D Microelectrode array electrophysiology showed a lower rate of change in EPSP over 1-4 volts in male BPA-exposed mice ( n  = 5 mice, n  = 11 slices) vs. vehicle ( n  = 7 mice, n = 12 slices; P  = 0.02). Generalized estimating equations were used clustering by mouse ( A , B ) or voltage input ( D ) and assuming an exchangeable correlation structure. For ( C ), independent t -tests were used and where there were more than two experimental groups (ArKO analysis), P -values were corrected for multiple comparisons using Holm–Sidak. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file.

Prenatal BPA exposure or loss of aromatase in ArKO male mice leads to amygdala hypoactivation and alters behavioral response to a novel social stimulus

The amygdala, a social processing brain region, is hyporesponsive in ASD (see review ref. 48 ). A post-mortem stereology study reported that adolescents and adults diagnosed with ASD feature an ~15% decrease in the numbers of neurons within the amygdala 43 . Also, functional MRI studies report amygdala hypoactivation in participants with ASD compared to controls 49 . Given that the amygdala is a significant source of aromatase-expressing neurons, we next conducted a series of studies to explore how aromatase deficiency influences the male mouse amygdala, using a combination of c-Fos immunohistochemistry, Golgi staining of brain sections, as well as electrophysiological analyses.

To investigate amygdala activation responses after interacting with a stranger mouse, we performed c-Fos immunohistochemistry (a marker for neuronal activation 50 ; Supplementary Fig.  8 ). As shown, prenatal BPA-exposed mice featured 58% fewer c-Fos positive neurons than in the amygdala of vehicle-exposed mice brains ( P  < 0.0001; Fig.  5C ). Similarly, we found that the MeA of ArKO mice had a marked deficit of 67% c-Fos-positive neurons when compared with WT ( P  = 0.0002) mice, which was ameliorated by early postnatal estradiol replacement (Fig.  5C ). Therefore, prenatal BPA exposure or loss of aromatase expression in ArKO mice leads to amygdala hypoactivation.

Next, we measured the synaptic excitability (I/O curve) of the MeA using multiple electrode analysis, with excitatory postsynaptic potential (EPSP) output indicative of electrical firing by local neurons. As shown, compared to corresponding controls, we find that MeA excitability (I/O curve) is significantly reduced in male mice prenatally exposed to BPA as well as in male ArKO mice (Figs.  5 D and 9D ). As shown, at 4-volt input, BPA treatment resulted in a 22.8% lower ( P  = 0.02) excitatory EPSP output than the vehicle treatment, while a 21% reduction ( P  = 0.03) in signal was observed for male ArKO mice compared to male WT mice. Thus, prenatal BPA exposure leads to functional hypoactivation of the amygdala of male mice, and this pattern is also evident in male ArKO mice.

Prenatal BPA exposure or loss of aromatase in ArKO male mice leads to abnormalities in neuronal cortical layer V as well as brain function

It has been reported that individuals with ASD show distinct anatomical changes within the somatosensory cortex, including in neurons of cortical layer V 51 . We previously reported that layer V within the somatosensory cortex is disrupted in ArKO mice 52 . Thus, we performed Golgi staining to study the apical and basal dendrites of neurons within layer V of the somatosensory cortex following prenatal BPA exposure, as well as in ArKO mice. As shown, we found that apical and basal dendrite lengths of layer V cortical neurons were significantly decreased in male mice prenatally exposed to BPA, compared with vehicle-treated mice (apical P  = 0.04; basal P  < 0.0001, Fig.  6A ). Such reductions in dendrites were also reported in male ArKO vs. WT mice (apical P  < 0.0001; basal P  = 0.02; Fig.  6A ). Furthermore, we found that dendritic spine densities on apical dendrites were also reduced (BPA-exposed mice vs. vehicle, P  = 0.04; ArKO vs. WT mice, P  = 0.01 (Fig.  6B ).

A Golgi staining showed shorter apical and basal dendrites in male BPA-exposed (apical: n  = 36, β =-350μm, 95% CI [−679, −20], P  = 0.04; basal: n  = 36, β = −217, 95% CI [−315, −119], P  = 1.4 × 10 −5 ) and ArKO mice (apical: n  = 35, β = −541.9, 95% CI [−666, −417], P = 1.3 × 10 −17 ; basal: n  = 35, β = −163, 95% CI [−308, −17], P  = 0.02) compared to male vehicle (apical n  = 35, basal n  = 36) or WT(apical n  = 36, basal n  = 36). B Golgi staining showed male BPA-exposed (apical: n  = 186, β = −4.7, 95% CI [−9.2, −0.2], P  = 0.04; basal: n  = 40 β = −6.7, 95% CI [−16, 2.8], P  = 0.17) and ArKO (apical: n  = 148 β = −4.4, 95% CI [−7.7, −1.0], P  = 0.01; basal: n  = 51 β = −5.2, 95% CI [−14.4, 4.1], P  = 0.27) mice had lower spine densities on apical but not on basal dendrites vs. vehicle (apical n  = 189, basal n  = 56) or WT mice (apical n  = 185, basal n  = 55). For golgi staining experiments, 3 mice/group with 9–12 neuron measures/mouse. Spine count datapoints represents the number of spines on a single 10 μm concentric circle. C Representative photomicrographs of golgi stained cortical neurons, scale bar is 100μm. D Electrocorticograms (ECoG) revealed an increased in the average spectral power at 8 Hz in BPA-exposed ( n  = 4; * a 8 Hz MD = −0.5; t(325) = 3.4  P  = 0.01) mice and (E) 4–6 Hz in ArKO mice ( n  = 4; * b 4 Hz MD = −0.2; t(120) = 4.3, P  = 0.0006; * c 5 Hz MD = −0.2, t (120) = 6.1, P  < 0.0001; * d 6 Hz MD = −0.2, t (120) = 5.2  P  < 0.0001) vs. vehicle ( n  = 7) or WT ( n  = 4)mice. Generalized estimating equations were used clustering by mouse (Panels A, B) and assuming an exchangeable correlation structure. For ( D ) and(E) Independent t test were used used, P -values were corrected for multiple comparisons using Holm-Sidak. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file.

To explore the effects of reduced aromatase on cortical activity, we performed electrocorticography (ECoG) recordings from mice in both experimental models (Fig.  6C ). As shown, spectral analysis revealed an increased power in the range of 4–6 Hz for ArKO vs. WT mice (4 Hz  P  = 0.0006, 5 Hz  P  < 0.0001, 6 Hz  P  < 0.0001; Fig.  6D ) and at 8 Hz for BPA-exposed vs. vehicle mice ( P  = 0.01; Fig.  6C ). These data indicate that BPA-exposure or loss of aromatase in ArKO mice affects cortical activity, a result which is reminiscent of cortical dysfunction evidenced by EEG recordings on human participants diagnosed with ASD 53 .

Molecular docking simulations indicate 10HDA is acting as a ligand at the same site as BPA on Estrogen Receptors α and β

It has been reported that BPA interferes with estrogen signaling through its competitive interaction and binding with estrogen receptors α (ERα) and β (ERβ) 54 . To explore this in the context of our findings, we used high-resolution in silico 3D molecular docking simulations to model the binding affinity of the natural ligand 17β-estradiol, the putative ligand BPA, as well as a putative therapeutic ligand of interest 10HDA with ERα (Protein Data Bank (PDB) ID: 5KRI) and ERβ (PDB ID: 1YYE). As shown, our spatial analysis indicated that all three ligands have robust binding affinity (Fig.  7 ; Supplementary Movie  1 ). However, while docking alignment revealed that the predicted fit for 10HDA is strikingly similar to that of 17β-estradiol 25 , 55 , BPA showed a greater mismatch (Fig.  7D ), consistent with previous reports that BPA is 1000-fold less estrogenic than the native ligand 56 . Thus, at least for ERα and Erβ, we find that 10HDA may be effective as a competitive ligand that could counteract the effects of BPA on estrogen signaling within cells.

In silico molecular docking analysis of estrogen receptor β (ERβ, Protein Data Bank (PDB) ID: 1YYE; encoded by the ES gene) using the DockThor platform, showing binding predictions for ( A ) the native ligand 17β-estradiol (E2), ( B ) bisphenol A (BPA), ( C ) Trans-10-hydroxy-2-decenoic acid (10HDA), and ( D ) E2 and BPA (left) and E2 and 10HDA (right) superimposed for spatial alignment comparison. While the molecular affinities of BPA and 10HDA for Erβ were comparable (−9.2 vs. −7.9, respectively), 10HDA aligns better with the binding conformation of the endogenous ligand E2, which activates the receptor. BPA is previously reported as sub-optimally estrogenic 106 — >1000-fold less compared to natural estradiol 54 , 56 —whereas 10HDA has an estrogenic role in nature 25 , 55 . Thus, 10HDA may compensate for E2 deficiency caused by a reduction in aromatase enzyme, and in competition with binding by BPA. Please see Supplementary Movie 1 for a video of the above molecular docking of ERβ with E2 superimposed with BPA and Supplementary Movie 2 for the above molecular docking of ERβ with E2 superimposed with 10HDA.

In vitro effects of BPA and 10HDA in primary fetal cortical cell cultures from male brains

Examining male fetal primary cortical culture, BPA alone shortened neurite lengths (Fig.  8A , BPA; quantified in Fig.  8B-C ) and decreased the spine density. BPA treatment reduced both neurite length ( P  = 0.0004) and spine densities ( P  < 0.0001; Fig.  8A , BPA; quantified in Fig.  8B–C ). Co-administration with 10HDA ameliorated these adverse effects of BPA (Fig.  8A , 10HDA + BPA; quantified in Fig.  8B, C ).

A Representative photomicrographs of primary cultures of embryonic (ED15.5) mouse cortical neurons, red staining is βIII tubulin and green is aromatase. Scale bar is 100μm. B Compared to the BPA group, the vehicle group (β = 79.9, 95% CI [36, 124], P  = 0.0004) and BPA + 10HDA group (β = 174, 95% CI [102, 247], P  = 2.4 × 10 −7 ) have significantly longer neurites. The BPA + 10HDA group (β = 94, 95% CI [8, 180], P  = 0.03) has longer neurites compared to the vehicle group, and there is no difference between the vehicle and 10HDA groups. C Compared to the BPA group, the vehicle group(β = 16, 95% CI [11, 21], P  = 1.7 × 10 −8 ) and BPA + 10HDA group (β = 30, 95% CI [21, 40], P  = 8.4 × 10 −10 ) have significantly higher spine densities. The BPA + 10HDA group (β = 14, 95% CI [4, 24], P  = 0.006) has a higher spine density compared to the vehicle group, and there is no difference between the vehicle and 10HDA groups. n  = 10 neurons/group. Primary cortical cell culture was obtained from 12 male mouse embryoes. Spine count datapoints represent the number of spines on a single 10μm concentric circle. Generalized estimating equations were used clustering by mouse and assuming an exchangeable correlation structure. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file.

In vivo effects of 10HDA on BPA mouse model

Guided by our findings in cultured neurons, we next investigated the effects of postnatal 10HDA administration on mice prenatally exposed to BPA at mid-gestation, as follows. After weaning, pups (six litters, 3 weeks of age) were administered daily injections of 10HDA (0 and 500 μg/kg/day; dissolved in saline, i.p.) for 3 weeks, following which pups were assessed for behavioral phenotypes. Strikingly, 10HDA treatment significantly improved social interaction (Fig.  9A ). To determine whether the effect of 10HDA administration is permanent, all treatments were withdrawn for 3 months, and mouse behaviors were subsequently re-tested. Withdrawal of 10HDA treatment in BPA-exposed male mice resulted in a deficit in social interaction (Fig.  9B ), and this deficit was once again ameliorated by a subsequent 10HDA treatment (Fig.  9C ) at 5 months of age, in adulthood. Taken together, these data demonstrate that continuous, postnatal 10HDA administration is effective for ameliorating social interaction deficits in male mice following prenatal BPA exposure.

A 10HDA treatments increased social approach in males ( n  = 10/group, MD = 41.14, U  = 11, P  = 0.03,) but not females ( n  = 8/group), compared to saline controls. After 3 months of treatment withdrawal ( B ), male mice (saline n  = 8, 10HDA n  = 7) no longer spent more time interacting with strangers, compared to vehicle treatment. C When male mice ( n  = 8/group) were subsequently treated with a second round of 10HDA, social approach behavior was once again significantly elevated (MD = 39.2, U  = 5, P  = 0.003), indicative of a rescue of this behavioral effect. D Compared to the WT Saline ( n  = 10) group (β = 57.1, 95% CI [47.4, 66.8]), EPSP increases at a 21% lower rate with increasing input in the ArKO Saline ( n  = 14) group (β = 45.1 μV, 95% CI [40.1, 50.1], P  = 0.03). No differences in slope were detected when comparing the WT Saline group with each of the other two treatment (WT n  = 18, KO n  = 12) groups. Mann–Whitney U tests were used and for ( D ), Generalized estimating equations were used clustering by voltage input ( D ) and assuming an exchangeable correlation structure. All statistical tests were two-sided. Plots show mean ± SEM. Source data are in a Source Data file. Note: Sal = saline, w/d = withdrawal.

Next, we wanted to determine if hypoactivity arising from the absence of aromatase in the amygdala may be influenced by 10HDA. To address this question, we studied ArKO mice using multiple-electrode analyses, following 3 weeks of treatment with 10HDA (500 μg/kg/day, i.p.). As shown in Fig, 9D , the electrical activity of the male ArKO amygdala treated with 10HDA was similar to male WT activity levels, whereas saline-treated male ArKO amygdala showed significantly lower activity ( P  = 0.03) when stimulated by an input/output paradigm, suggesting that 10HDA treatment was effective to compensate the absence of aromatase. Therefore, we interpret these results to suggest that 10HDA restores signaling deficits arising from aromatase deficiency. Given that prenatal BPA exposure suppresses aromatase, 10HDA supplementation may be relevant to aromatase-dependent signaling in that context as well.

Transcriptomic studies of the fetal brain cortex and cortical cell cultures

MiSeq Next-Gen Sequencing was performed on the transcriptome libraries generated from the brain cortex of the E16.5 fetuses after maternal mid-gestation BPA or vehicle exposure. The action of 10HDA was analyzed by RNAseq of transcriptome libraries from total RNA extracted from primary mouse fetal cortical cultures treated with vehicle or 10HDA. Firstly, pathway analysis of the RNAseq data was performed for Gene Ontology (GO) categories using the clusterProfileR R package. No individual pathways in the BPA analysis survived correction for multiple comparisons using an agnostic (non-candidate) approach. Further candidate investigation using the binomial test showed a significant inverse effect of BPA and 10HDA on pathways previously linked to autism 57 , with 10HDA treatment counteracting the effects of BPA on these pathways (Supplementary Fig.  9 ). Based on our Golgi staining experimental findings relating to altered dendrite morphology, we further assessed the category “dendrite extension” as a candidate pathway. Genes in this pathway were downregulated by BPA (Supplementary Figs.  10 A, 9A) and upregulated by 10HDA (Supplementary Figs.  9B , 10B ). More broadly, Fisher’s exact test showed a significant BPA-associated down-regulation ( P  = 0.01), and 10HDA-associated up-regulation ( P  = 0.0001), of pathways with the terms “axon” and “dendrite”. Notably, the majority (82%; 9 of the top 11 available) mid gestational biological processes whose activity is overrepresented in induced pluripotent stem cells of autism cases vs. controls 57 were impacted by BPA, and in the opposite direction to 10HDA ( P  = 0.03; Supplementary Fig.  9 ).

Next, we performed pathway enrichment analysis, also using a candidate pathway approach, of the RNAseq data using Ingenuity (Fig.  10 ). Strikingly, the effects of BPA and 10HDA on gene expression were diametrically opposed across many functional domains (Fig.  10 ). For example, the canonical pathways “Synaptogenesis Signaling pathway” and “CREB signaling” were downregulated by BPA but upregulated by 10HDA. Similarly, key brain functions, e.g., growth of neurites and neural development were down regulated by BPA and reciprocally upregulated by 10HDA (Fig.  10 ). Taken together, prenatal BPA exposure is detrimental to gene expression through a mechanism that may be ameliorated by postnatal 10HDA administration. The full list of differentially expressed genes can be found in Supplementary Dataset  1 .

The Canonical pathways and Disease and Function—Brain pathway databases were selected in Ingenuity. Several key signaling pathways and brain functions were downregulated ( Z -score less than zero) by BPA and also upregulated ( Z -score greater than zero) by 10HDA. 10HDA downregulated four brain disorder related pathways—hyperactive behavior, seizures, seizure disorder and behavioral deficit. Colored boxes indicate significant ( P  < 0.05, Fisher’s Exact Test with Benjamani-hochberg) changes in z -score. Boxes shaded in gray indicate non-significant gene expression changes ( P  = 0.05 or greater). Source data are provided as a Source Data file.

Here, we report that prenatal BPA exposure leads to ASD endophenotypes in males, and that this involves the actions of the aromatase gene, as well as its functions in brain cells. Our multimodal approach, incorporating both human observational studies and preclinical studies with two mouse models, offer significant insight into how prenatal programming by BPA disrupts aromatase signaling to cause anatomical, neurological, as well as behavioral changes reminiscent of ASD in males.

In the Barwon Infant Study (BIS) human birth cohort the adverse effect of high prenatal BPA exposure on ASD symptoms (ASP score) at age 2, and clinical ASD diagnosis at age 9, was particularly evident among males with a low aromatase enzyme activity genetic score. Further, studying cord blood gene methylation as an outcome we have demonstrated that BPA exposure specifically methylated the offspring CYP19A1 brain promoter in the BIS cohort, replicated in the CCCEH-MN cohort. Previously, in a meta-analysis of two epigenome-wide association studies (EWAS), two CpGs in the region around the brain promoters PI.f and PII were significantly associated ( P  < 0.05) with ASD in both EWAS 58 . Past work 16 , and our findings of BPA reduced aromatase expression in a neuronal cell culture and reduced aromatase-eGFP expression in mouse brain, are consistent with the brain-specific suppression of aromatase expression. We also demonstrated that BPA exposure led to a reduction in steady-state levels of aromatase in a neuronal cell line. Further, we replicated past work that higher prenatal BPA levels are associated with BDNF hypermethylation 33 , previously demonstrated to be associated with lower BDNF expression in males 33 .

We find that prenatal BPA exposure at mid-gestation in mice induces ASD-like behaviors in male but not female offspring, concomitant with cellular, anatomical, functional, and behavioral changes (Supplementary Fig.  11 and Supplementary Fig.  12 ). We found that these features were also observed in male ArKO mice, and this is important because aromatase expression is disrupted in BPA-treated male mice. In the Y-maze test, both the ArKO and BPA offspring did not show any differences with the respective control indicating that there are no major memory, sensory or motor issues in these animals. Given the distinct parallels between the effects of BPA that suppresses aromatase, as well as ArKO mice that lack aromatase, we surmise from our studies that BPA disrupts aromatase function to influence the male mouse brain which manifests as: (i) reduced excitatory postsynaptic potentials in the amygdala, (ii) reduced neuron numbers as well as dendritic lengths and spine densities for neurons within the MeA, (iii) altered cortical activity as recorded by ECoG concomitant with decreased dendritic length and spine density in layer IV/V somatosensory cortical neurons, as well as (iv) enhanced repetitive behaviors and reduced social approach to a stranger. These results in mice are consistent with studies with human participants that report abnormal neuronal structure in these comparative regions within the brains of individuals with ASD 51 . Furthermore, we investigated our gene expression dataset and found that the majority of the top biological processes over-represented in cells derived from ASD cases compared to non-cases in a human pluripotent stem cell analysis with a focus on mid gestational brain development 57 were impacted in opposite directions by BPA compared to 10HDA in our gene expression studies on the male mouse brain (Supplementary Fig.  9 ). Of note, the sexually dimorphic effect we report is consistent with work of others demonstrating that prenatal BPA exposure of rodents led to dysregulation of ASD-related genes with neuronal abnormalities, and learning and memory problems only in males 59 .

In our investigations of BPA, we recognized that 10HDA may be a suitable compound as a ligand in the context of brain ER signaling 27 because of its positive effect on gene expression through stimulation of estrogen responsive DNA elements 25 and its role in neurogenesis 27 —characteristics that altogether may compensate for a relative lack of aromatase-generated neural estrogens. Administration of 10HDA alongside BPA protected neuronal cells in culture from the adverse sequelae observed for BPA alone at the same dose. Three weeks of daily postnatal 10HDA treatment significantly enhanced the sociability of the male BPA-exposed mice and dendrite morphology in primary cell culture. The adverse decrease in dendrite lengths and spine densities of the BPA-exposed mice was also corrected by 10HDA administration (Fig.  8 ). Furthermore, postnatal 10HDA treatment restored amygdala electrical activity in the ArKO mice, indicating that 10HDA likely acts downstream of, rather than directly upon, the aromatase enzyme, given that ArKO mice lack functional aromatase. Transcriptomic analyses revealed that 10HDA upregulated, whereas BPA downregulated, gene expression for fetal programming such as for synaptogenesis and growth of neurites. Some of these pathways could be activated by factors downstream of aromatase, such as 17β-estradiol (Supplementary Fig.  13 ). In this study, the ArKO model was useful because it provided an estrogen deficient comparison 41 . We were able to demonstrate that early postnatal E2 administration restored both MeA neural activation and social preference behavior in the ArKO males.

The molecular docking simulations indicate that ERα and ERβ both comprise docking sites for 10HDA and BPA, however, 10HDA is strongly estrogenic 25 , 55 while BPA is greater than 1000-fold less potent than natural estrogen 54 . Such differences in binding are likely relevant to the diverse transcriptomic effects observed in the cells we analyzed by RNAseq.

Strengths of this study include the multimodal approach to test the hypothesis of the interplay of BPA, male sex, and aromatase suppression. In our human epidemiological studies, extensive information was available to allow confounding to be accounted for using matched analyses for the BPA-ASD cohort finding, and findings persisted after adjustment for further individual confounders. Using a modern causal inference technique, molecular mediation 60 we demonstrate in both birth cohorts that aromatase gene promoter I.f methylation underlies the known effect of higher prenatal BPA on BDNF hypermethylation. Other key features that support an underlying causal relationship include: the consistency of the findings across studies in this program (Supplementary Fig.  11 ); and the consistency with which our experimental laboratory work maps to prior studies of people with ASD (summarized in Supplementary Fig.  12 ) in relation to neuronal and structural abnormality in the amygdala 43 and abnormality in amygdala connectivity 44 , and resting-state cortical EEG 53 . Our findings are also consistent with past work indicating reduced prefrontal aromatase levels in individuals with ASD at postmortem 14 , 15 . The finding that BPA-associated gene methylation patterns in the BIS cohort were not sex-specific but that BPA-associated ASD symptoms and clinical diagnosis were more evident in males with a low genetic aromatase score would be consistent with the male vulnerability to BPA reflecting not differential epigenetic programming, but a greater vulnerability to reduced aromatase function in the developing male brain. This is reinforced by the ArKO model which resulted in an ASD-like phenotype in males not females. We have provided experimental evidence not only on the adverse neurodevelopment effects of BPA, but also experimental evidence of the alleviation of the behavioral, neurophysiological, and neuroanatomical defects following postnatal treatment with 10HDA. A human randomized controlled prevention trial that achieved bisphenol A elimination during pregnancy, with a resultant reduction in ASD among male offspring, would be a useful next step to provide further causal evidence of BPA risks but the feasibility and ethics of such an undertaking would be considerable. We demonstrate that postnatal administration of 10HDA may be a potential therapeutic agent that counteracts the detrimental impacts on distinct gene expression signatures directly impacted by prenatal BPA exposure. Furthermore, 10HDA may ameliorate deficits in ArKO mice which further suggests its utility as a replacement therapy for aromatase deficiency.

Two limitations of our human study were that BPA exposure was measured in only one maternal urine sample at 36 weeks, and that the assay may have low sensitivity 61 . We partially redressed the latter by focusing on categorical BPA values, as recommended 61 , and undertook a matched ASD analysis where determinants for BPA variation, such as the urine collection time of day, were matched to reduce misclassification. Also, functional gene expression studies were unavailable for human samples in our study, but whilst the misclassification introduced by a reliance on a SNP based score would likely lead to an underestimation, an effect was still found among males with a low genetic aromatase score. It would be useful in further studies to consider altered aromatase function with a combined epigenetic-genetic score to reflect environment-by-epigenetic and genetic determinants of low aromatase function. Direct brain EWAS measures were not available, but for the key brain promoter PI.f region of CYP19A1 , the brain-blood correlation is very high: Spearman’s rho= 0.94, 95% CI [0.80, 0.98] 32 . Although ASD symptoms (ASP score) at 2 years were based on parent report, we have previously reported that a higher ASP score was predictive of later ASD diagnosis by age 4 30 . ASD diagnosis at age 9 was verified to meet DSM-5 criteria by pediatrician audit of medical records, thereby reducing diagnostic misclassification.

In our preclinical studies, we performed the 10HDA studies and some of the BPA mechanistic studies only on male animals because our extensive laboratory and human studies, with more than 25 analyses, demonstrated that BPA exposure had significantly more adverse effects in males than females. In addition, we performed the RNASeq on the cortex of fetal mice exposed to BPA in vivo, whereas 10HDA was performed in vitro on cortical primary cell culture. This would likely increase the variability between the BPA RNASeq and the 10HDA RNASeq, yet many of the same pathways were impacted but in opposing directions. Furthermore, the changes we observed in our RNAseq data could be due to changes in the cell type or cell state. This could be clarified by future single cell RNAseq experiments now that this specific issue has been identified.

The BPA exposure of mouse dams under our experimental conditions (50 µg/kg bodyweight) matches the current Oral Reference Dose set by the United States Environmental Protection Agencyc, the current safe level set by the U.S. Food and Drug Administration (FDA) 37 , as well as the Tolerable Daily Intake set by the European Food Safety Authority 38 at the time that the mothers in our human cohort were pregnant 28 . The EFSA set a new temporary TDI of 4 µg/kg bodyweight in 2015 62 and, in December 2021, recommended further reducing this by five orders of magnitude to 0.04 ng/kg 63 ; although this was subsequently revised to 0.2 ng/kg in EFSA's scientific opinion published in 2023 64 . Therefore, the timing of this new evidence is particularly pertinent and provides direct human data to support the reduced TDI.

Consistent with typical human exposure in other settings 20 , BPA exposure in our birth cohort was substantially lower than the above, and yet we see adverse effects. Assuming fractional excretion of 1 65 and average daily urine output of 1.6 L 65 , the median urinary bisphenol concentration of 0.68 µg/L—for which we see increased odds of ASD diagnosis—equates to a total daily intake of just 13 ng/kg, given a mean maternal bodyweight of 80.1 kg at time of urine collection. Notably, while we find an adverse association at 13 ng/kg, we do not have sufficient participants with lower exposure to evaluate a safe lower limit of exposure below this. Our findings in cell culture, with concentration 5 µg/L, parallels these human findings in terms of dose response. Although there are limitations in translating concentrations across body compartments without a stronger understanding of pharmacokinetics of BPA, 5 µg/L corresponds to the 90th and 95th percentile of BPA in urine in our human cohort, and allowing for a standard factor of 10 for variability in human sensitivity used when setting TDIs 38 indicates relevance down to at least 0.5 µg/L, below the median urine concentration. Our findings in laboratory animal studies, with exposure of 50 µg/kg bodyweight, are a little higher, as they were designed to correspond to the then current recommendations 36 , 37 , 38 , but implications nevertheless have relevance within the range of exposure in our cohort. Allowing for standard factors of 10 for interspecies variability 38 and variability in human sensitivity 38 , our animal study findings support a TDI at 500 ng/kg or below, which corresponds to the upper 0.5% of our human cohort. The findings of the human study, also allowing for a factor of 10 for variability in human sensitivity 38 , therefore, support a TDI at or below 1.3 ng/kg.

Despite bans on its use in all infant products by the European Union in 2011 and the U.S. FDA in 2012, BPA remains widespread in the environment 66 . The main source of human exposure to BPA is dietary contamination 68 . Bisphenols are used in the production of common food contact materials, and migrate from those materials during use 69 , including polycarbonate food and beverage containers and the epoxy linings of metal food cans, jar lids, and residential drinking water storage tanks and supply systems 64 . Additional sources of exposure include BPA-based dental composites and sealant epoxies, as well as thermal receipts 64 . BPA levels in pregnant women have previously been reported to be higher for young mothers, smokers, lower education, and lower income 70 . A substantial proportion of ASD cases might be prevented at the population level if these findings were causal and prenatal maternal BPA exposure were reduced. Here, exposures in the top quartile of BPA (>2.18 ug/L) correspond to a population attributable fraction (PAF) for males with low aromatase of 12.6% (95% CI 5.8%, 19.0%) although this estimate is imprecise as it is based on low case numbers. The only other available study with data on BPA exposure (>50 ug/L) and ASD provides an estimate in all children of 10.4% 67 . These studies have misclassification issues (e.g., a single urine measure for BPA and, in the Stein et al. study, an ASD diagnosis derived from health care sources 67 ) but these misclassifications are likely non-differential and thus would bias findings towards the null. Additionally, we need to consider that the above findings of RfD/TDI and PAF are based on BPA alone. Factoring in that most exposures occur as part of a chemical mixture adds additional concern 71 . For example prenatal valproic acid exposed mice (an established ASD mouse model) also have a lower brain aromatase expression 72 .

In summary, this multimodal program of work has shown an adverse effect of higher maternal prenatal BPA on the risk of male offspring ASD by a molecular pathway of reduced aromatase function, which plays a key role in sex-specific early brain development. Overall, these findings add to the growing evidence base of adverse neurodevelopmental effects from bisphenol and other manufactured chemical exposure during pregnancy. The case is compelling and supports broader evidence on the need to further reduce BPA exposure, especially in pregnancy. We also envision that our findings will contribute to new interventions for the prevention and/or amelioration of ASD targeting this specific pathophysiological pathway and we have identified one possible neuroprotective agent—10HDA—that has strong laboratory support. This agent now warrants further study, including human safety and efficacy evaluation.

The human Barwon Infant Study cohort study was approved by the Barwon Health Human Research Ethics Committee, and families provided written informed consent. Parents or guardians provided written informed consent at prenatal recruitment and again when the child was 2 years of age. The human Columbia Center for Children’s Environmental Health Mothers and Newborn cohort study was approved by the Institutional Review Boards of Columbia University and the Centers for Disease Control and Prevention, and all participants in the study provided informed consent. All procedures involving mice were approved by the Florey Institute of Neuroscience and Mental Health animal ethics committee and conformed to the Australian National Health and Medical Research Council code of practice for the care and use of animals for scientific purposes and All experiments were designed to minimize the number of animals used, as well as pain and discomfort. This work adheres to the ARRIVE essential 10 guidelines.

The Barwon Infant Study birth cohort

Participants.

From June 2010 to June 2013, a birth cohort of 1074 mother–infant pairs (10 sets of twins) were recruited using an unselected antenatal sampling frame in the Barwon region of Victoria, Australia 28 . Eligibility criteria, population characteristics, and measurement details have been provided previously 28 ; 847 children had prenatal bisphenol A measures available (Supplementary Table  1 ).

Bisphenol A measurement

We used a direct injection liquid chromatography tandem mass spectrometry (LC-MS/MS) method, as previously described in detail 73 . In summary, a 50 µL aliquot of urine was diluted in milli-Q water and combined with isotopically-labeled standards and b-glucuronidase (from E. Coli -K12). Samples were incubated for 90 min at 37 °C to allow for enzymatic hydrolysis of bisphenol conjugates before quenching the reaction with 0.5% formic acid. Samples were centrifuged before analysis, which was performed using a Sciex 6500 + QTRAP in negative electrospray ionization mode. The BPA distribution and quality control attributes for the application of this method to the Barwon Infant Study (BIS) cohort are shown in Supplementary Table  2 .

Child neurodevelopment

Between the ages of seven and ten, a health screen phone call was conducted to gather information on autism spectrum disorder (ASD) diagnoses and symptomology. Out of the 868 individuals who responded to the health screen, 80 had an ASD diagnosis reported by their parents/guardians or were identified as potentially having ASD. The parent-reported diagnoses were confirmed by pediatric audit of the medical documentation to verify an ASD diagnosis as per DSM-5 guidelines. Participants that had a parent-reported diagnosis and then a verified pediatrician diagnosis by 30 June 2023 and whose diagnoses occurred before the date of their 9-year health screen were included as ASD cases in this study’s analyses ( n  = 43). Participants were excluded if (i) their parent/guardian responded with ‘Yes’ or ‘Under Investigation’ to the question of an ASD diagnosis on the year-9 health screen but their diagnosis was not verified by 30 June 2023 ( n  = 26), or (ii) they had a verified diagnosis of ASD by 30 June 2023 but their date of diagnosis did not precede the date of their year-9 health screen ( n  = 15). The DSM-5-oriented autism spectrum problems (ASP) scale of the Child Behavior Checklist for Ages 1.5-5 (CBCL) administered at 2-3 years was also used as an indicator of autism spectrum disorder.

Whole genome SNP arrays

Blood from the umbilical cord was gathered at birth and then transferred into serum coagulation tubes (BD Vacutainer). Following this, the serum was separated using centrifugation as described elsewhere 74 . Genomic DNA was extracted from whole cord blood using the QIAamp DNA QIAcube HT Kit (QIAGEN, Hilden, Germany), following manufacturer’s instructions. Genotypes were measured by Erasmus MC University Medical Center using the Infinium Global Screening Array-24 v1.0 BeadChip (Illumina, San Diego, CA, USA). The Sanger Imputation Service (Wellcome Sanger Institute, Hinxton, UK) was used for imputing SNPs not captured in the initial genotyping using the EAGLE2 + PBWT phasing and imputation pipeline with the Haplotype Reference Consortium reference panel 75 . Detailed methods are provided elsewhere 76 .

Genome-wide DNA methylation arrays and analysis methods can be found in the Supplementary Methods.

Center for Children’s Environmental Health (CCCEH) epigenetic investigations

The study participants consisted of mothers and their children who were part of the prospective cohort at the Columbia Center for Children’s Environmental Health Mothers and Newborn (CCCEH-MN) in New York City (NYC). They were enrolled between the years 1998 and 2003, during which they were pregnant. The age range for these women was between 18 and 35, and they had no prior history of diabetes, hypertension, or HIV. Furthermore, they had not used tobacco or illicit drugs and had initiated prenatal care by the 20th week of their pregnancy. Every participant gave informed consent, and the research received approval from the Institutional Review Boards at Columbia University as well as the Centers for Disease Control and Prevention (CDC) 33 .

Epigenetic methods have been previously described 77 . Briefly, DNA methylation was measured in 432 cord blood samples from the CCCEH-MN cohort using the 450 K array (485,577 CpG sites) and in 264 MN cord blood samples using the EPIC array (866,895 CpG sites) (Illumina, Inc., San Diego, CA, USA).

BPA measures in the CCCEH were based on spot urine samples collected from the mother during pregnancy (range, 24–40 weeks of gestation; mean, 34.0 weeks) 33 , 78 .

Other statistical analysis

Maternal urinary BPA concentrations were corrected for specific gravity to control for differences in urine dilution. Given a high proportion of the sample (46%) had BPA concentrations that were not detected or below the limit of detection (LOD), a dichotomous BPA exposure variable was formed using the 75th percentile as the cut-point. Dichotomizing the measurements in this way is also likely to give similar results regardless of whether indirect or direct analytical methods were used 79 . This is desirable since indirect methods might be flawed and underestimate human exposure to BPA 61 .

To evaluate whether autism spectrum problems at 2 years could be used as a proxy for later ASD diagnosis, receiver operating characteristic (ROC) curve analyses were used. CBCL ASP at age 2 years predicted diagnosed autism strongly at age 4 and moderately at age 9 with an area under the curve of 0.92 (95% CI 0.82, 1.00) and 0.70 (95% CI 0.60, 0.80), respectively.

According to the normative data of the CBCL, T-scores greater than 50 are above the median. Due to a skewed distribution, ASP measurements were dichotomized using this cut point, which has respective positive and negative likelihood ratios of 2.68 and 0.00 in the prediction of verified ASD diagnosis at 4 years and 1.99 and 0.49 in the prediction of verified ASD diagnosis at 9 years.

A CYP19A1 genetic score for aromatase enzyme activity was developed based on five genotypes of single nucleotide polymorphisms (CC of rs12148604, GG of rs4441215, CC of rs11632903, CC of rs752760, AA of rs2445768) that are associated with sex hormone levels 31 . Participants were classified as ‘low activity’ if they were in the top quartile, that is, they had three or more genotypes associated with lower levels of estrogen and as ‘high activity’ otherwise. Conditional logistic regression model analyses investigating the association between prenatal BPA levels and (i) early childhood ASP scores and (ii) verified ASD diagnosis at 9 years were conducted in the full sample, repeated after stratification by child’s sex (assigned at birth based on visible external anatomy), and repeated again after further stratifying by the CYP19A1 genetic score. Matching variables included child’s sex (in the full sample analysis only), ancestry (all four grandparents are Caucasian vs not) and time of day of maternal urine collection (after 2 pm vs before). Within these matched groups, we additionally matched age-9 ASD cases and non-cases based on the date of the health screen and child’s age at the health screen using the following procedure. Each case was matched to a single non-case based on nearest date of and age at health screen. Once all cases had one matched non-case, a second matched non-case was allocated to each case, and so on until all cases had 8 matched non-cases (8 was the most possible in the boys with high aromatase activity sub-sample and so this number was used across all sub-samples). The order by which cases were matched was randomly determined at the start of each cycle.

The guidelines for credible subgroup investigations were followed 80 . Only two categorical subgroup analyses were conducted, and these were informed a priori by previous literature and by initial mouse study findings. The adverse BPA effects in males with low aromatase enzyme activity (as inferred from the CYP19A1 genetic score) were expected to be of higher magnitude, based on the prior probabilities from the laboratory work. A systematic approach was used to evaluate non-causal explanations and build evidence for causal inference, considering pertinent issues such as laboratory artefacts that are common in biomarker and molecular studies 81 .

A second CYP19A1 genetic score was developed for use in sensitivity analyses. The Genotype-Tissue Expression (GTEx) portal was used to identify the top five expression quantitative trait loci (eQTLs) for aromatase in any tissue type that showed a consistent effect direction in brain tissue. A functional genetic score was then computed for each BIS participant by summing the number of aromatase-promoting alleles they carry across the five eQTLs (AA of rs7169770, CC of rs1065778, AA of rs28757202, CC of rs12917091, AA of rs3784307), weighted by their normalized effect size (NES) in amygdala tissue. The score was then reversed so that higher values indicate lower aromatase activity. The score thus captures genetic contribution to reduced cross-tissue aromatase activity with a weighting towards the amygdala, a focus in our animal studies. The variable was dichotomized using the 75th percentile as the cut-point and the above stratified analyses were repeated with this new weighted score replacing the original, unweighted score.

For the human epigenetic investigations, we used multiple linear regression and mediation 60 approaches. As in past work 33 , BPA was classified as greater than 4 μg/L vs less than 1 μg/L in the CCCEH-MN cohort. A comparable classification was used for the BIS cohort, with greater than 4 μg/L vs the rest. In both cohorts the regression and mediation analyses were also adjusted for sex, gestational age, self-reported ethnicity, and cord blood cell proportions. In the BIS cohort, ethnicity was defined as all four grandparents are Caucasian vs not (see Table  S3 ). For the CCCEH-MN cohort, ethnicity was defined as Dominican vs African American 33 . We used statistical software packages R v3.6.3 82 and Stata 15.1 83 .

LABORATORY STUDIES

Shsy-5y cell culture study, bpa treatment on aromatase expression in cell culture.

Human neuroblastoma SHSY-5Y cells were chosen because they were known to express aromatase and SH-SY5Y have been used in ASD research 84 . SHSY-5Y cells (CRL-2266, American Type Culture Collection, Virginia, USA) were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (10313-021, Gibco-life technologies, New York (NY), USA) supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) (12003C-500 mL, SAFC Biosciences, Kansas, USA), 1% penicillin streptomycin (pen/strep) (15140-122, Gibco-life technologies, NY, USA) and 1% L-Glutamine (Q) (25030-081, Gibco-life technologies, NY, USA) at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 . SHSY-5Y cells were grown in 175 cm 2 cell culture flasks (T-175) (353112, BD Falcon, Pennsylvania, USA). Cells were passaged when the seeding density of the T-175 flasks was reached (roughly 80-90% confluence). Cells were passaged by aspirating media from flasks and flasks were then washed once with 10 mL of DPBS (14190-136 Gibo-life technologies, NY, USA) to remove the FBS (inhibits the actions of trypsin). Next, cells were incubated with trypsin (2 mL/T-175 flask) at 37 °C for 5 min to detach cells from the flask wall. To prevent further action of trypsin, media (8 mL/T-175 flask) was added, and contents were pipetted up and down to disperse cell clumps. The cell suspension was then transferred to a 15 mL centrifuge tube (430791, Corning CentriStar, Massachusetts, USA) and centrifuge (CT15RT, Techcomp, Shanghai, China) for 5 min at 1000 RPM at room temperature (RT). The media was then aspirated from these tubes and the cell pellet resuspended in 1 mL of media. Cell viability counts were performed using a hemocytometer (Hausser Scientific, Pennsylvania, USA) to determine the number of live versus dead cells in solution. Two μL of cell suspension was diluted with media (98 μL) and then trypan blue (100 μL) (T8154, Sigma-Aldrich Co., St. Louis, MO, USA) (which labeled dead cells) in a sterile microcentrifuge tube (MCT-175-C-S, Axygen, California, USA). Ten μL of this solution was loaded into the hemocytometer and imaged using a light microscope (DMIL LED, Leica, Germany). Dead cells appeared blue under the microscope because these cells take up the dye whereas live cells were clear (i.e., unstained). Cells were counted in the outer four squares located in each chamber (two chambers, eight squares), with their dimensions known. The average of the eight counts was multiplied by the dilution factor and by 104, yielding the concentration of cells/mL solution. Average cell counts were plotted against treatment groups using GraphPad Prism 9.0 (GraphPad Software, Inc., San Diego, CA).

Bisphenol A (BPA) (239658-50 G, Sigma-Aldrich Co., St. Louis, MO, USA) was used for cell treatment. Prior to treatment, stock solutions of each drug were prepared as stated below. BPA was dissolved in pure ethanol (EA043-2.5 L, Chem supply, South Australia, Australia) and the final concentration of the stock solutions was 0.0435 g/mL. Cells in T-175 flasks were randomly assigned to receive treatment with BPA at a dosage of 100 μg/L, 50 μg/L, 25 μg/L or 0 μg/L (vehicle). There was also a no treatment (no vehicle added) flask.

Cell treatment, protein assay, SDS-Page, and western blotting methods can be found in the Supplementary Methods.

Animal studies

Two colonies of mice, maintained at the Florey Institute, were used in this study. The Aromatase knockout (ArKO) mouse model and the Aromatase-enhanced fluorescent green protein (Cyp19-EGFP) transgenic mouse model. Animals were monitored daily except for weekends. If animals showed general clinical signs, an animal technician or a vet was consulted for advice and euthanasia performed as required.

Mice were maintained under specific pathogen-free (SPF) conditions on a 12 h day/night cycle, with ad libitum water and soybean-free food (catalog number SF06-053, Glen Forrest Stockfeeders, Glen Forrest, Western Australia, Australia). Facial tissues were provided for nesting material, and no other environmental enrichment was provided. The room temperature ranged from 18 °C-23 °C and the humidity ranged from 45%-55%.

The sex of mice was determined by SRY genotyping if fetal, otherwise sex was determined by examining the anogenital region around PND9 and again at weaning. Sex was confirmed by inspecting the gonads during dissection. Cyp19 -EGFP mice were toe and tail clipped for identification and genotyping at PND9, ArKO mice were ear notched and tailed clipped at two weeks of age. The oligonucleotide sequences (custom oligos, Geneworks, Australia) for ArKO, GFP and SRY genotyping can be found in Supplementary Data File  2 .

Aromatase knockout (ArKO) mouse model

The ArKO mouse is a transgenic model having a disruption of the Cyp19a1 gene. Exon IX of the Cyp19a1 gene was replaced with a neomycin-resistant cassette 41 . Homozygous Knockout (KO) and wild-type (WT) offspring were bred by mating heterozygous (het) ArKO parents and then PCR genotyped. ArKO mice were backcrossed onto a C57BL/6 J background strain, >10 generations (obtained from Animal Resources Centre, Western Australia) and the colony maintained at the Florey institute.

Aromatase-enhanced fluorescent green protein ( Cyp19 -EGFP) transgenic mouse model

The Cyp19-EGFP mouse model (backcrossed onto the FVBN background strain >10 generations, obtained from Animal Resources Centre, Western Australia) is a transgenic model having a bacterial artificial chromosome containing the full length of the Cyp19a1 gene with an Enhanced Green Fluorescent Protein (EGFP) gene inserted upstream of the ATG start codon 11 . Thus, EGFP expression is an endogenous marker for Cyp19a1 expression. This allows for the visualization and subsequent localization of EGFP as the marker for aromatase without the use of potentially nonspecific aromatase antibodies 11 . We have previously characterized this transgenic model and its brain expression of EGFP 11 . Based on our characterization studies, this transgenic model does not have phenotypes that are significantly different to wildtype mice.

Early postnatal 17β-estrodiol treatment

Mice were allocated into three groups: (1) WT mice receiving a sham implantation; (2) ArKO mice receiving a sham implantation; and (3) ArKO mice undergoing implantation with a 17β-estradiol pellet (sourced from Innovative Research America). This estradiol pellet was designed to release 0.2 mg of 17β-estradiol steadily over a period of 6 weeks. A corresponding sham pellet, identical in size but devoid of E2, was implanted in the control groups.

The implantation procedure was carried out on postnatal day 5. For anesthesia, mice were exposed to 2% isoflurane (IsoFlo, Abbott Laboratories, VIC, Australia) within an induction chamber. The efficacy of anesthesia was confirmed by the lack of response to foot-pinch stimuli. During the surgical procedure, mice were maintained on a heated pad to regulate body temperature. A small, 5 mm incision was made in the dorsal region for the subcutaneous insertion of the pellet, preceded by an injection of Bupivacane in the same area. Following the implantation, the incision was carefully sutured. Post-surgery, mice were placed in a thermal cage (Therma-cage, Manchester, UK) for recovery and monitoring until they regained consciousness and could be returned to their respective litters. Any mice exhibiting complications such as opened stitches were excluded from the study.

BPA injection administration treatment

Plugged FVBN dams were randomly assigned, blocking by weight gain at E9.5 and litter/cage where applicable, to receive daily scruff subcutaneous injections (24 G x 1”, Terumo, Somerset, New Jersey, USA) of BPA (239658-50 G, Sigma-Aldrich Co., St Louis, MO, USA) in ethanol and peanut oil (Coles, Victoria, Australia), either between E0.5-E9.5, E10.5-E14.5 or E15.5-birth at a dosage of 50 μg/kg (deemed as the safe consumption level by the Food and Drug Administration, FDA) 37 or 0 μg/kg (vehicle) of maternal body weight. The injection volume was 1.68 μL/g bodyweight. Mice were weighed directly before each injection. BPA and vehicle exposed litters did not differ in litter size (Supplementary Fig.  14 ).

10HDA injection administration treatment

Cyp19 -EGFP or ArKO mice were randomly assigned by blocking on sex and litter to receive daily intraperitoneal injections (31 G x 1”, Terumo, Somerset, New Jersey, USA) of 500 μg/kg 10HDA (Matreya, USA) in saline or vehicle saline for 21 consecutive days. The injection volume was 2.1 μL/g bodyweight. Mice were weighed directly before each injection.

BPA oral administration treatment

Plugged FVBN dams were exposed to jelly at E9.5. The jelly contained 7.5% Cottee’s Raspberry Cordial (Coles, Victoria, Australia) and 1% bacteriological agar (Oxoid, Australia) in milli-Q water. The pH was increased to between 6.5-7.5 with a pallet of NaOH to allow the jelly to set. Dams were then randomly assigned, blocking by weight gain at E9.5 and litter/cage where applicable, to receive a daily dose of jelly, which contained either ethanol or BPA dissolved in ethanol, at a dosage of 50 μg/kg or 0 μg/kg (vehicle) of maternal body weight. Dams received doses between E10.5-E14.5, and only dams that were observed to have consumed all the jelly each day were included in the study.

Behavioral paradigms

Three-chamber social interaction test.

The three-chamber social interaction test is extensively used to investigate juvenile and adult social interaction deficits, including in sociability 85 , 86 . BPA exposed pups were habituated in the experimental room on P21, directly after weaning. Following a two-to-three-day habituation, testing was conducted from P24 to P27, as only a maximum of ten mice could be tested during the light phase per day. ArKO mice treated with estrogen or sham pallet began habituation at PND28-29, with testing at PND31-33. Both male and female mice were tested. Testing was performed in a dedicated room for mouse behavior studies; no other animals were present in the room at the time of acclimatization and testing. The temperature of the room was maintained at approximately 21 °C.

The test apparatus, a three-chambered clear plexiglass, measuring 42 cm x 39 cm x 11 cm, had two partitions creating a left, right (blue zones), and center chamber (green zone) in which mice could freely roam via two 4 cm x 5 cm openings in the partitions (Supplementary Fig.  15 ). The two side chambers contained two empty wire cages. A 1 cm wide zone in front of each wire cage was defined as the interaction zone (yellow zones). The chamber was set on a black table for white mice, and on a white covering for black mice to aide tracking.

Each test consisted of two consecutive 10-min trials, a habituation trial (T1) and a sociability trial (T2). T1 allowed the test mouse to habituate, and any bias for either empty interaction zone was noted. For T2, a C57BL/6 J novel stranger mouse matched with the test mouse for age and gender was introduced into the cage on the opposite side to which the test mouse demonstrated an interaction zone bias. Thus, any evidence of sociability is bolstered as interaction zone bias would have to be overcome.

For each trial, the test mouse began in the center chamber, and its activity, both body center point, and nose point was tracked and quantified by TopScan Lite (Clever Sys Inc., Reston VA, USA). In this study, the key measure extracted was the average duration of the nose point in each interaction zone.

Social approach and sociability were analyzed. We define social approach as the time the test mouse’s nose point was tracked in the stranger cage interaction zone. Sociability is the higher proportion of time the test mouse to spends with the nose point in the stranger cage interaction zone compared to the empty cage interaction zone.

Details on the Y-maze and grooming methods can be found in the Supplementary Methods.

Golgi staining

Mice had not undergone any behavioral testing. For Golgi staining and analysis, Wild Type (WT) and Knockout (ArKO) and Cyp19 -EGFP littermate males (aged P65-P70); one mouse from n  = 3 litters for each genotype) were deeply anesthetized with isopentane rapidly decapitated and fresh whole brain tissues were collected. Brains were first washed with milli-Q water to remove excess blood and then directly placed in the solution obtained from the FD Rapid GolgiStain TM Kit (FD Neuro-Technologies, Inc., MD, USA). Brains were stored at room temperature in the dark and the solutions were replaced after 24 hours, and the tissues were kept in the solution for two weeks. After two weeks, tissues were transferred into solution C for a minimum of 48 hours at room temperature. For sectioning, brains were frozen rapidly by dipping into isopentane pre-cooled with dry ice, and 100 µm thick coronal sections were cut at -22 °C and mounted on 1% gelatin-coated slides. The sections were then air dried in the dark at room temperature. When sections were completely dry, slides were further processed and rinsed with distilled water and placed in the solution provided in the kit for 10 min and washed again with distilled water followed by dehydration for 5 min each in 50%, 75%, 95%, and 100 % ethanol. Sections were further processed in xylene and mounted with Permount.

Neuron Tracing

Neuron tracing was conducted on the amygdala and somatosensory cortex of BPA-exposed mice (exposed ED10.5-14.4) and untreated ArKO mice. Neuron tracing in the amygdala was conducted in both male and female mice, and in the somatosensory cortex, only in male mice. Stained slides were coded to ensure that morphological analysis was conducted by an observer who was blind to the animals’ treatment. Morphological analysis followed a previously described protocol 87 with the following modifications: layer V pyramidal cells of the somatosensory cortex, which were fully impregnated and free of neighboring cells or cellular debris, were randomly selected for analysis (Supplementary Fig.  16 ). Golgi-stained coronal sections containing medial amygdala and somatosensory cortical area were visualized under Olympus BX51 microscope. Neuronal tracing was carried out with the help of Neurolucida and Neuroexplorer software (MicroBright Field Inc., Williston, USA). Up to three pyramidal cells in the MeA and four pyramidal cells in the somatosensory cortex per section over 3 sections (9 (MeA) and 12 (cortex) cells per animal respectively) were sampled 88 , 89 . For Sholl analysis 90 , concentric circles were placed at 10 µm intervals starting from the center of the cell body and the parameters i) total dendritic length (sums of the length of individual branches) of apical and basal dendrites of pyramidal cells and ii) number of spines (protrusions in direct contact with the primary dendrite) and their density (number of spines per 10 µm) were recorded.

Neuron selection criteria: Neurons were selected based on the following criteria. They had to be fully stained, and the cell body had to be in the middle third of the section thickness. The dendrites of the neuron had to be unobscured by the other nearby neuron. Also, neurons had to possess tapering of the majority of the dendrites towards their ends. Representative images of neurons from vehicle and BPA-exposed adult mice can be found in Supplementary Fig.  17 .

Visualizing c-Fos activation to conspecific exposure (amygdala)

Stranger exposure paradigm procedure.

Cyp19-EGFP mice of both sexes as well as male ArKO mice, together with male WT littermates were utilized in this study. Mice had not undergone any other behavior testing. All test mice were acclimatized to the testing room in individual cages for three nights prior to testing. All mice were age P24 on the day of testing, which was performed between 10 am-2 pm. Testing was performed in a dedicated room for mouse behavior studies and no other animals were present in the room at the time of acclimatization and testing. The temperature of the room was maintained at approximately 21 °C.

On the day of testing, each mouse cage containing the isolated test mouse was placed on a stage (a trolley). The lid containing food and water was removed and immediately following, a sex-/age-matched C57Bl/6 J stranger mouse or a novel object (new 1 mL syringe) was placed into the cage and a clean, empty lid was placed on the top. New gloves were used to handle each syringe to avoid transferring another mouse’s olfactory signature to it. The 10 min trial began as soon as the cage lid was shut. After 10 min had elapsed, the stranger or the novel object was removed, the test mouse with home cage was returned to its original location with the original cage lid with food and water for 2 hours prior to perfusion. Once it was established that there was a difference in c-fos expression between stranger exposure and novel object exposure in the medial amygdala, BPA and vehicle exposed (ED10.5-14.5) Cyp19-EGFP mice as well as estrogen and sham pallet treated ArKO and WT mice were exposed to an age and sex matched stranger as described above. C-fos expression was quantified in male mice only.

Histology and stereological analysis methods can be found in the Supplementary Methods.

Neuron count brain collection, staining, brain region delineation and stereology can be found in the Supplementary Methods.

Electrophysiological studies

Microelectrode array electrophysiology.

Male mice aged 8 weeks weighing between 15 and 20 g were used for this study. They had not undergone any behavioral testing prior to electrophysiology. We studied synaptic activity parameters such as the Input/Output (I/O) curve. Stimulation of the glutamatergic synapses terminate in the basolateral amygdala (BLA) and the basomedial amygdala (BMA), which were integrated with multiple inputs that compute to produce an output (field excitatory postsynaptic potential, fEPSP). I/O curve serves as an index of synaptic excitability of large neuronal populations. Mice were anesthetized with isoflurane (IsoFlo TM , Abbott Laboratories, Victoria, Australia) and decapitated. The whole brains were quickly removed and placed in ice-cold, oxygenated (95% O 2 , 5% CO 2 ) cutting solution (composition in mmol/L: 206 sucrose, 3 KCl, 0.5 CaCl 2 , 6 MgCl 2 -H 2 O, 1.25 NaH 2 PO 4 , 25 NaHCO 3, and 10.6 D-glucose). Coronal brain amygdala slices (300 µm) were prepared with a VT 1200 S tissue slicer (Leica) and quickly transferred to 34  o C carbogen bubbled artificial CSF (aCSF) (composition in mmol/L: 126 NaCl, 2.5 KCl, 2.4 CaCl 2 , 1.36 MgCl 2 -H 2 O, 1.25 NaH 2 PO 4 , 25 NaHCO 3, and 10 D-glucose) for 30 min. After further recovery of 1 h equilibrium in oxygenated aCSF at room temperature, the slices were transferred to a submission recording chamber, an MEA chip with 60 electrodes spaced 200 μm apart (60 MEA 200/30 iR-Ti: MCS GnbH, Reutlingen, Germany). The slice was immobilized with a harp grid (ALA Scientific Instruments, New York, USA) and was continuously perfused with carbogenated aCSF (3 mL/min at 32 °C). fEPSPs produced in BLA and BMA were by stimulation of a randomly chosen electrode surrounding the target area with a biphasic voltage waveform (100 μs) at intermediated voltage intensity. The electrode could only be chosen if it produced a fair number of fEPSPs in the surrounding recording electrodes. The width of the EPSP wave ranged from 20 to 30 ms was selected. We chose slices where BLA and BMA were greatly represented according to Allen Mouse Brain Atlas 91 . Care was taken to choose the stimulating electrode in the same region from one slice to the other. The peak-to-peak amplitude of fEPSP in BLA and BMA was recorded by a program of LTP-Director and analyzed using LTP-Analyzer (MCS GnbH, Reutlingen, Germany).

Electrocorticogram (ECoG)

Electrocorticogram recordings.

Male mice aged 8 weeks were used for this study. Mice had not undergone any behavioral testing prior to ECoG recording. For ECoG, surgeries were performed as previously described 92 . Mice were anesthetized with 1–3% isoflurane and two epidural silver ‘ball’ electrodes implanted on each hemisphere of the skull. Electrodes were placed 3 mm lateral of the midline and 0.5 mm, caudal from bregma. A ground electrode was placed 2.5 mm rostral from bregma and 0.5 mm lateral from the midline. Mice were allowed to recover for at least 48 hours after surgery. ECoGs were continuously recorded in freely moving mice for a 4–6-hour period during daylight hours following a standard 30-min habituation period. Signals were band-pass filtered at 0.1 to 40 Hz and sampled at 1 kHz using the Pinnacle EEG/EMG tethered recording system (Pinnacle Technology Inc, KS). Power spectrums were calculated using Hann window with a resolution of 1 Hz using Sirenia Pro analysis software (Pinnacle Technology Inc) on stable 30-min periods of ECoG recordings.

Primary Cortical Cultures

Neuroprotective effect of 10hda against injury induced by bpa on embryonic mouse cortical neurons.

Primary cortical neurons were obtained from male Cyp19 -EGFP mouse embryos at gestational day 15.5. Embryos were genotyped for SRY to determine sex, and only male embryos were used. Cells were seeded in 24-well plates containing 12 mm glass coverslips, coated with 100 µg/mL poly D-lysine to a density of ~0.45 x 10 6 cells/well and incubated in a humidified CO 2 incubator (5% CO 2 , 37 °C). Cells were pre-treated with vehicle (DMSO), 1 mM 10HDA (Matreya, PA,USA), 25 nM BPA and 1 mM 10HDA with 25 nM BPA. For each group, 10 neurons were measured, and the experiments were duplicated. Each replicate was from a separate culture.

Cells were fixed in 4% paraformaldehyde and stained with mouse anti-βIII tubulin monoclonal primary antibody (1:1000; cat #ab41489, Abcam, United Kingdom) and goat anti-mouse secondary antibody, Alexa Fluor 488, (1:2000; cat#A11017; Invitrogen, USA) to label neuronal cells. Aromatase was stained using Rabbit anti-aromatse Antibody (1:2000 cat# A7981; Sigma Aldrich, St. Louis, MO, USA) and donkey anti-rabbit Alexa594 (1:2000; cat# A-21207; Invitrogen, USA). Cell nucleus was stained with Hoechst 33258 solution (Sigma 94403 (2 µg/mL)). Images were captured using an Olympus IX51 microscope (X40 objective). Neurites were quantified using Neurolucida and Neuroexplorer software (MicroBright Field Inc, Williston, USA) as described in the neuron tracing section.

RNA extraction

Total RNA was extracted using PARIS kit (cat#: AM1921, Invitrogen™PARIS™ Kit) according to the protocol supplied by the manufacturer. cDNA libraries were generated using the SureSelect.

Strand-Specific RNA Library Prep for Illumina Multiplexed Sequencing kit (Agilent Technologies, CA, USA), according to manufacturer’s instructions.

In vivo effects of BPA on Fetal brain cortical RNA seq

Pregnant Cyp19-EGFP dams were injected subcutaneously with BPA or vehicle ED10.5-14.5 as described in previous section, and culled on ED15.5 by isoflurane overdosed. Fetuses were harvested and placed in chilled PBS. Embryo brain cortical tissue was dissected from fetuses, snap frozen in liquid nitrogen and stored in −80°C until RNA extraction. The sex of fetuses was determined by visual assessment of the gonads and Sry (a male-specific gene) genotyping. Each RNA seq run, 6 cDNA libraries (derived from total RNA samples with 3 biological samples per group), were analyzed by MidSeq Nano run, 50 bp, Single end read on the Illumina platform. Because of undetectable levels of Cyp19a1 RNA in the fetal brain, Cyp19a1 RNA levels were not included. This is consistent in that Aromatase+ cells represent <0.05% of neurons in the adult mouse brain 93 . Subsequent in vivo transcriptomic analyses were completed in males only. Read quality was then assessed with FastQC. The sequence reads were then aligned against the Mus musculus genome (Build version GRCm38). The Tophat aligner (v2.0.14) was used to map reads to the genomic sequences. Sequencing data were then summarized into reads per transcript using Feature counts 94 . The transcripts were assembled with the StringTie tool v1.2.4 using the reads alignment with Mus_musculus.GRCm38 and reference annotation based assembly option (RABT) using the Gencode gene models for the mouse GRCm38/mm10 genome build. Normalisation and statistical analysis on the count data were executed using EdgeR (version edgeR_3.14.2 in R studio, R version 3.14.2). The data were scaled using trimmed mean of M-values (TMM) 95 and differentially expressed genes between all treatment group (Benjamini–Hochberg false discovery rate >0.1). Differentially expressed genes (DEGs) were identified by comparing mice exposed to 50 μg/kg/day BPA with those exposed to the vehicle.

In vitro effects of 10HDA in primary cell culture RNASeq

Primary brain cortical neurons were obtained from C57BL/6 mouse embryos at GD 15.5. Neuronal cell cultures were treated with vehicle (DMSO) or 1 mM 10HDA (Matreya, PA,USA) as described above. The libraries were sequenced with 50 bp single end reads using an Illumina Hiseq and read quality assessed using FastQC. Untrimmed reads were aligned to mouse mm10 genome using Subjunc aligner (version 1.4.4) within the Subread package 96 . Sequencing data were then summarized into reads per transcript using Feature counts 94 and the Gencode gene models for the mouse GRCm38/mm10 genome build (August 2014 freeze) 97 . Normalisation and statistical analysis on the count data were executed using EdgeR (version edgeR_3.4.2 in R studio, R version 3.0.2) 98 after removing features with less than 10 counts per million for at least 3 of the samples. The data were scaled using trimmed mean of M-values (TMM) 95 and differentially expressed genes between all treatment group (Benjamini–Hochberg false discovery rate >0.1). Annotation was added using the ensemble mouse gene annotation added using bioMart package 99 . Differentially expressed genes were identified by comparing cells exposed to 10HDA with those exposed to the vehicle.

Pathway analysis

The BPA and 10HDA differential expression data for enriched pathways were analyzed using Ingenuity (QIAGEN) and tested against the Canonical Pathway Library, Brain Diseases and Functions Library and the Brain Disorders pathway libraries. We included the top 8 Canonical pathways. Then we included only pathways which were p  < 0.05 in both the BPA and the 10HDA data for the Brain Diseases and Functions pathway libraries, and included all P -values for the Brain Disorders pathway library ( p  > 0.05 are in gray).

An additional analysis of the gene expression data was performed using the c lusterProfileR R package 100 , which provides a range of statistical tests to detect pathways from a query gene set. The test used here was Gene Set Enrichment Analysis (GSEA) 101 , and the genes were tested against the Gene Ontology pathway database (specifically, GO: Biological Process) 102 .

Computational Molecular Docking

The DockThor molecular docking platform 103 was used to assess binding affinities between estrogen receptor beta (encoded by ESR2 gene) and the ligands 17-beta estradiol (E2; the native ligand), BPA, and 10HDA.DockThor takes as input 3D molecular structures for a putative receptor-ligand pair and employs a genetic-algorithm-based optimization strategy to identify optimal binding position within a specified search region. The crystal structures of estrogen receptor alpha (Erα) and beta (Erβ) were sourced from the Protein Data Bank (PDB) with respective PDB IDs: Erα - 5KRI and Erβ - 1YYE. For each ligand, the search grid was restricted to the known estrogen receptor beta ligand-binding domain, centered at x  = 30, y  = 35, z  = 40, with total grid size of x  = 25, y  = 28 and z  = 22. Default settings were used for the optimization procedure.

Statistical analysis

Researchers were blind to treatment during the conduct of the experiment and the outcome assessment but not during statistical analysis.

Mean, standard deviation (SD), and standard error of the mean (SEM) were calculated with GraphPad Prism version 9.4 (GraphPad Software).

Data were tested for equal variances and normality using the Shapiro Wilk test. As electrophysiology, Golgi staining and primary cell culture experiments utilized several data points per animal, observations were not independent, and this non-independence was accounted for in our analyses. We used generalized estimating equations (GEEs) in R version 4.1.2. in a marginal modeling approach that estimates population-averaged effects while treating the covariance structure as a nuisance. We specified the covariance structure as exchangeable (that is, assumed equal correlation between pairs of measurements on the same animal). Given the small number of clusters (i.e. animals), bootstrapped standard errors were estimated using 200 repeats to maintain a conservative type 1 error rate 104 . An interaction term was added to the amygdala Golgi staining study to assess a sex * genotype or sex * BPA exposure interaction.

Where data were normally distributed, parametric tests were conducted. For more than two groups, a one-way ANOVA was conducted with Holm-Sidak post hoc FDR correction, with alpha set to 0.05. Otherwise, unpaired two-tailed Student’s t -tests were used to compare two variables. In cases where normality was not assumed, a Mann-Whitney (comparing two groups) or Kruskal-Wallis with Dunn’s post hoc (comparing three or more groups) was used. In the case of the three-chamber data, a two-way mixed ANOVA was used to assess group x cage side interaction (stranger cage interaction zone vs empty cage interaction zone) with post hoc testing adjusted by the Holm-Sidak method.

Comparisons made are indicated on the Figure legends, and p -values < 0.05 were considered significant. All tests were two-sided (two-tailed) where applicable.

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The BIS data including all data used in this paper are available under restricted access for participant privacy. Access can be obtained by request through the BIS Steering Committee by contacting Anne-Louise Ponsonby, The Florey institute of Neuroscience and Mental Health, [email protected]. Requests to access cohort data will be responded to within two weeks. Requests are then considered on scientific and ethical grounds and, if approved, provided under collaborative research agreements. Deidentified cohort data can be provided in Stata or CSV format. Additional project information, including cohort data description and access procedure, is available at the project’s website https://www.barwoninfantstudy.org.au . Source data underlying Figs.  1 – 6 , 8 – 10 and Supplementary Figs.  2 , 3 – 7 , 14 have been provided as a Source Data file with this paper. The RNAseq data discussed in this publication have been deposited in NCBI’s Gene Expression Omnibus 105 and are accessible through GEO Series accession numbers; fetal brain expression with and without prenatal BPA exposure, GSE266401 and primary cortical culture treated with and without 10HDA, GSE266400   Source data are provided with this paper.

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Acknowledgements

The authors thank the BIS participants for their generous contribution to this project. The authors also thank current and past cohort staff. The establishment work and infrastructure for the BIS was provided by the Murdoch Children’s Research Institute, Deakin University, and Barwon Health, supported by the Victorian Government’s Operational Infrastructure Program. We thank all the children and families participating in the study, and the BIS fieldwork team. We acknowledge Barwon Health, Murdoch Children’s Research Institute, and Deakin University for their support in the development of this research. We thank Dr Shanie Landen for statistical advice, and Alex Eisner for independent statistical review of the analyses in the manuscript. We thank Soumini Vijayay and Kristie Thompson for human BPA lab measurement and Dr Steve Cheung for assistance preparing the primary cortical culture. We thank Chitra Chandran, Georgia Cotter, Stephanie Glynn, Oliver Wood and Janxian Ng for manuscript preparation. Manuscript editor Julian Heng (Remotely Consulting, Australia) provided professional editing of this article. This multimodal project was supported by funding from the Minderoo Foundation. Funding was also provided by the National Health and Medical Research Council of Australia (NHMRC), the NHMRC-EU partnership grant for the ENDpoiNT consortium, the Australian Research Council, the Jack Brockhoff Foundation, the Shane O’Brien Memorial Asthma Foundation, the Our Women’s Our Children’s Fund Raising Committee Barwon Health, The Shepherd Foundation, the Rotary Club of Geelong, the Ilhan Food Allergy Foundation, GMHBA Limited, Vanguard Investments Australia Ltd, and the Percy Baxter Charitable Trust, Perpetual Trustees, Fred P Archer Fellowship; the Scobie Trust; Philip Bushell Foundation; Pierce Armstrong Foundation; The Canadian Institutes of Health Research; BioAutism, William and Vera Ellen Houston Memorial Trust Fund, Homer Hack Research Small Grants Scheme and the Medical Research Commercialisation Fund. This work was also supported by Ms. Loh Kia Hui. This project received funding from a NHMRC-EU partner grant with the European Union’s Horizon 2020 Research and Innovation Programme, under Grant Agreement number: 825759 (ENDpoiNTs project). This work was also supported by NHMRC Investigator Fellowships (GTN1175744 to D.B., APP1197234 to A.-L.P., and GRT1193840 to P.S.). The study sponsors were not involved in the collection, analysis, and interpretation of data; writing of the report; or the decision to submit the report for publication.

Author information

Nhi Thao Tran

Present address: The Ritchie Centre, Department of Obstetrics and Gynaecology, School of Clinical Sciences, Monash University, Clayton, Australia

These authors contributed equally: Christos Symeonides, Kristina Vacy.

These authors jointly supervised this work: Anne-Louise Ponsonby, Wah Chin Boon.

Authors and Affiliations

Minderoo Foundation, Perth, Australia

• Christos Symeonides

Murdoch Children’s Research Institute, Parkville, Australia

Christos Symeonides, Toby Mansell, Martin O’Hely, Boris Novakovic, David Burgner, Mimi L. K. Tang, Richard Saffery, Peter Vuillermin, Fiona Collier, Anne-Louise Ponsonby, Sarath Ranganathan, Lawrence Gray & Anne-Louise Ponsonby

Centre for Community Child Health, Royal Children’s Hospital, Parkville, Australia

Christos Symeonides, Sarath Ranganathan & Anne-Louise Ponsonby

The Florey Institute of Neuroscience and Mental Health, Parkville, Australia

Kristina Vacy, Sarah Thomson, Sam Tanner, Hui Kheng Chua, Shilpi Dixit, Jessalynn Chia, Nhi Thao Tran, Sang Eun Hwang, Feng Chen, Tae Hwan Kim, Christopher A. Reid, Anthony El-Bitar, Gabriel B. Bernasochi, Anne-Louise Ponsonby, Anne-Louise Ponsonby & Wah Chin Boon

School of Population and Global Health, The University of Melbourne, Parkville, Australia

Kristina Vacy

The Hudson Institute of Medical Research, Clayton, Australia

Hui Kheng Chua & Yann W. Yap

Department of Pediatrics, The University of Melbourne, Parkville, Australia

Toby Mansell & David Burgner

School of Medicine, Deakin University, Geelong, Australia

Martin O’Hely, Boris Novakovic, Chloe J. Love, Peter D. Sly, Peter Vuillermin, Fiona Collier & Lawrence Gray

Columbia Center for Children’s Environmental Health, Columbia University, New York, NY, USA

Julie B. Herbstman, Shuang Wang & Jia Guo

Department of Environmental Health Sciences, Columbia University, New York, NY, USA

Julie B. Herbstman

Department of Biostatistics, Columbia University, New York, NY, USA

Shuang Wang & Jia Guo

Department of Anatomy and Developmental Biology, Monash University, Clayton, Australia

Kara Britt & Vincent R. Harley

Breast Cancer Risk and Prevention Laboratory, Peter MacCallum Cancer Centre, Melbourne, Australia

Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Australia

Faculty Medicine, Dentistry & Health Sciences, University of Melbourne, Parkville, Australia

Gabriel B. Bernasochi, Lea M. Durham Delbridge, Mimi L. K. Tang, Leonard C. Harrison & Sarath Ranganathan

Sex Development Laboratory, Hudson Institute of Medical Research, Clayton, Australia

Vincent R. Harley & Yann W. Yap

Departments of Paediatrics and Community Health Sciences, The University of Calgary, Calgary, Canada

Deborah Dewey

Barwon Health, Geelong, Australia

Chloe J. Love, Peter Vuillermin, Fiona Collier & Lawrence Gray

Department of General Medicine, Royal Children’s Hospital, Parkville, Australia

David Burgner

Department of Pediatrics, Monash University, Clayton, Australia

Child Health Research Centre, The University of Queensland, Brisbane, Australia

Peter D. Sly

WHO Collaborating Centre for Children’s Health and Environment, Brisbane, Australia

Queensland Alliance for Environmental Health Sciences, The University of Queensland, Brisbane, Australia

Jochen F. Mueller

Monash Krongold Clinic, Faculty of Education, Monash University, Clayton, Australia

Nicole Rinehart

Centre for Developmental Psychiatry and Psychology, Monash University, Clayton, Australia

Bruce Tonge

School of BioSciences, Faculty of Science, The University of Melbourne, Parkville, Australia

Wah Chin Boon

Walter and Eliza Hall Institute, Parkville, Australia

Leonard C. Harrison

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the BIS Investigator Group

• , Toby Mansell
• , Martin O’Hely
• , David Burgner
• , Mimi L. K. Tang
• , Peter D. Sly
• , Richard Saffery
• , Jochen F. Mueller
• , Peter Vuillermin
• , Fiona Collier
• , Anne-Louise Ponsonby
• , Leonard C. Harrison
• , Sarath Ranganathan
•  & Lawrence Gray

Contributions

Conceptualization—laboratory experiments: W.C.B., N.R., B.T., L.M.D.D. Laboratory experiments and analysis: W.C.B., K.V., S.D., H.K.C., J.C., F.C., CR, T.K., G.B.B., A.E.-B., S.E.H., N.T.T., K.B. Supervision of lab data collection: W.C.B., K.V., S.D., C.R. Laboratory statistical analysis: W.C.B., K.V., F.C., C.R., S.Th., V.H., Y.W.Y. Design and conduct of the Barwon Infant Study: C.S., A.-L.P., P.V., D.B., P.S., C.L., M.L.K.T., BIS Investigator Group. Design, conduct and analysis of the CCCEH-MN study: J.B.H., S.W., J.G. Design and conduct of BPA study measures in BIS: J.M., C.S., A.-.L.P. Design, conduct, and analysis of gene methylation studies: S.Ta., B.N., T.M., R.S., D.D., A.-L.P. Human studies statistical analysis: C.S., S.Th., A.-.L.P., S.Ta., K.V., M.O.H. Writing—reports and original draft: C.S., K.V., S.Th., S.Ta., A.-.L.P., W.C.B. Writing—editing: all authors. Results interpretation: all authors. Kara Britt did the laboratory experiment—estrogen pellet implantation.

Corresponding author

Correspondence to Wah Chin Boon .

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Competing interests.

W.C.B. is a co-inventor on ‘Methods of treating neurodevelopmental diseases and disorders’, USA Patent No. US9925163B2, Australian Patent No. 2015271652. This has been licensed to Meizon Innovation Holdings. A.-L.P. is a scientific advisor and W.C.B. is a board member of the Meizon Innovation Holdings. The remaining authors declare no competing interests.

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Symeonides, C., Vacy, K., Thomson, S. et al. Male autism spectrum disorder is linked to brain aromatase disruption by prenatal BPA in multimodal investigations and 10HDA ameliorates the related mouse phenotype. Nat Commun 15 , 6367 (2024). https://doi.org/10.1038/s41467-024-48897-8

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DOI : https://doi.org/10.1038/s41467-024-48897-8

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• v.32(2); 2021 Jun

Malignant Hyperthermia Syndrome: A Clinical Case Report

Isabel sánchez-molina acosta.

1 Hospital Comarcal de Laredo, Laredo, Cantabria, Spain

Guillermo Velasco de Cos

2 Hospital Universitario Marqués de Valdecilla, Santander, Cantabria, Spain

Matilde Toval Fernández

Malignant hyperthermia is a pharmacogenetic disorder. It manifests as a hypercatabolic skeletal muscle syndrome linked to inhaled volatile anesthetics or depolarizing muscle relaxants. Its clinical signs and symptoms are tachycardia, hyperthermia, hypercapnia, acidosis, muscle rigidity, rhabdomyolysis, hyperkalemia, arrhythmia and renal failure. Mortality without specific treatment is 80% and decreases to 5% with the use of dantrolene sodium.

This article presents the case of a 39-year-old patient admitted to the Intensive Care Unit for malignant hyperthermia after surgery for septoplasty plus turbinoplasty.

INTRODUCTION

Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder of the skeletal musculature, characterized by an anesthesia-related hypermetabolic state (1, 2).

The pathophysiological mechanism is associated with mutation of the RYR1, CACNS1S and STAC3 genes (3, 4), responsible for controlling intracellular calcium homeostasis.

In susceptible individuals, the triggering stimulus causes hyperactivation of the receptors, resulting in uncontrolled release of Ca ++ from the endoplasmic reticulum (ER) of muscle cells, leading to increased intracytoplasmic Ca ++ , responsible for enzymatic activation leading to decreased ATP and O 2 consumption and increased anaerobic metabolism, resulting in increased heat and lactic acidosis (5).

Clinical signs and symptoms during the crisis is characterized by tachycardia, hypercapnia, arrhythmia, muscular contracture, cyanosis, metabolic and respiratory acidosis, lactic acidosis, hyperthermia, coagulopathy and rhabdomyolysis (3,6,7).

Mortality without treatment amounts to 80%, decreasing to 5% with supportive measures and effective treatment, which consists of the suspension of halogenated agents, hyperventilation with 100% O 2 and the administration of dantrolene sodium (DS), a muscle relaxant that inhibits the release of Ca ++ from the ER by acting on RYR1 (2,8,9).

Diagnosis is purely clinical, while post-event confirmation is made by the halothane-caffeine contracture test (CHCT) or genetic study of the mutations of the genes involved (3,10).

CLINICAL CASE

A 39-year-old male patient, with no personal history of interest, was admitted for scheduled surgery for septoplasty plus turbinoplasty. Anesthetic induction was performed with midazolam, propofol and remifentanil. Neuromuscular relaxation was performed with succinylcholine (100 mg) and rocuronium (50 mg), and hypnosis with desflurane, due to difficult manual ventilation.

The surgery was uneventful and the patient was afebrile. At the end of the surgery, a rapidly progressive rise of EtCO 2 (CO 2 at the end of expiration) was observed, reaching values of up to 130 mmHg, tachycardia and axillary hyperthermia 39.5 º C. When malignant hyperthermia was suspected, desflurane was stopped, physical maneuvers were performed to cool the patient and specific treatment was started with dantrolene sodium, with an initial dose of 250 mg i.v. (2.5 mg/kg), plus continuous perfusion with propofol and cisatracurium. A bladder catheter was placed, diuretics were prescribed and temperature was monitored.

Initial laboratory tests showed mixed acidosis, hyperkalemia, hypocalcemia and renal failure, so calcium bicarbonate, dextrose 5% and insulin were administered, improving EtCO 2 and temperature.

Once the patient was stabilized, it was decided to transfer him to the Intensive Care Unit (ICU).

On arrival at the ICU the patient was under the effects of anesthesia, presenting isochoric and normoreactive pupils, muscle hypertrophy, normothermic, tachycardic, good bilateral ventilation, bladder catheterization with myoglobinuria and no edema. He was maintained on mechanical ventilation.

A new analytical control was performed, highlighting: severe hypoglycemia 0.83 mmol/L, hypocalcemia, normalization of hyperkalemia, mild renal failure creatinine 140 μmol /L and persistence of acidosis. After 6 hours, a progressive increase in transaminases, lactate dehydrogenase (LDH) and creatinine kinase (CK) was detected, with a maximum value at 48 hours after the onset of the crisis of 112 860 U/L ( Table 1 ).

Timeline of laboratory tests

pCO 2 : partial pressure of carbon dioxide (mmHg); pO 2 : partial pressure of oxygen (mmHg); SaO2: oxygen saturation; CK: creatinine kinase; LDH: lactate dehydrogenase; ALT: alanine aminotransferase; AST: aspartate aminotransferase.

Serum therapy was increased with resolution of ionic alterations and renal function, but hepatic alterations and muscle destruction persisted for days. A new dose of dantrolene was not required.

The patient had a subsequent good evolution, allowing withdrawal of sedation and extubation at 24 hours. He was discharged from the ICU 48 hours after the crisis, with adequate blood glucose levels.

A genetic study was requested from the reference laboratory, where the c.6856C>G p.(Leu-2286Val) mutation in the RYR1 gene was detected. Massive sequencing was used for analysis, using Agilent’s CCP17 Sure Select panel. The analysis was performed on the Illumina NextSeq sequencer. This mutation is described in the clinical database of the American College of medical genetics and genomics as a probably pathogenic variant associated with malignant hyperthermia.

MH is a pharmacogenetic alteration that manifests as a hypermetabolic response after exposure to inhaled anesthetics (isoflurane, halothane, sevoflurane, desflurane and enflurane), and muscle relaxants such as succinylcholine (1), although it can also be produced by heat, infections, emotional stress, statin therapy and strenuous exercise (3). This reaction occurs in individuals with a certain genetic predisposition. Since susceptible patients do not present phenotypic alterations before anesthesia, it is impossible to diagnose them before exposure or before specific tests are performed.

Anesthetics: nitrous oxide, local anesthetics, propofol, etomidate, thiopental, ketamine, opioids, benzodiazepines, non-depolarizing muscle relaxants are considered safe in MH susceptible patients (2,7,9).

The incidence of MH is 1/50 000 to 1/250 000 in adults and 1/15 000 in children. The actual prevalence is difficult to define because there are patients with no or mild clinical reactions. In addition, the penetrance of the inherited trait is variable and incomplete (1,2).

MH has an autosomal dominant pattern of inheritance. Most of the cases described are due to mutations in three genes: RYR1 (ryanodine receptor type 1), CACNS1S (dihydropyridine receptor), and STAC3. It is estimated that 70% of cases are caused by mutations in the RYR1 gene (1,11,12,13). As discussed above, our patient was heterozygous for the RYR1 gene mutation.

Ryanodine receptors are large (560 kDa) ion channels involved in intracellular calcium release, especially in the sarcoplasmic reticulum. There are three isoforms that are variably distributed in tissues, with the RYR1 isoform predominating in skeletal muscle. In the case of our patient, the mutation described above is associated with a missense-type change that predicts the substitution of an amino acid leucine for valine at position 2286 of the protein.

When the receptor is mutated, it releases excess calcium once activated by anesthetic agents. This results in sustained muscle contraction, altered calcium homeostasis and a hypermetabolic state, especially anaerobic, with lactate production, increased temperature and CO 2 and oxygen consumption. This leads to rhabdomyolysis, hyperkalemia, hypocalcemia, myoglobinuria, elevated CK and hypernatremia (9).

Ionic disturbances are due to loss of function of the cell membrane, on the one hand there is a release of enzymes and electrolytes, especially potassium into the intercellular space. On the other hand, this release is compensated by a flow of water into the cellular interior which causes a state of hypovolaemia in patients, resulting in a haemoconcentration of various analytes such as sodium.

MH may appear early with succinylcholine or late with inhaled anesthetics. In the case described, it was attributed to the mixture of succinylcholine and desflurane. One of the earliest clinical manifestations of MH that should alert the anesthesiologist is increased EtCO 2 . Hypercapnia is the most specific symptom, being found in 90% of cases (14).

Other associated signs may include cyanosis, metabolic and respiratory acidosis, lactic acidosis and increased CK. Peak CK values are reached hours after the onset of the crisis. In this case, the patient reached values of 112 860 U/L 48 hours after surgery, with a subsequent decrease.

The diagnosis should be confirmed using The Clinical Grading Scale (CSG) for MH developed by Larach (9). A score above 50 classifies the episode as almost certainly malignant hyperthermia, as was the case presented.

Following the European Malignant Hyperthermia Group Guidelines (EMHG) (15), once the condition is diagnosed, treatment should be initiated as soon as possible, progressively decreasing the anesthetic agent. The drug used for MH is dantrolene sodium. Dantrolene is a muscle relaxant that acts at the level of the RYR1 receptor, decreasing intracellular calcium availability and slowing massive skeletal muscle contraction. Initially 2.5 mg/kg should be administered as an intravenous bolus, and this dose should be repeated every 3-5 min. until the signs are controlled, maintaining thereafter the administration of 1 mg/kg every 6 hours to prevent recurrence of crises. In the case presented, only one dose was needed, without presenting subsequent crises. Simultaneously, treatment of hyperthermia, hyperkalemia, acidosis, renal failure and arrhythmias should be started (2,15). Once the crisis has been controlled, the patient should be monitored and transferred to the ICU for at least 24 hours, due to the risk of relapse.

Confirmation of MH is performed by HCT and is indicated when a patient has had a previous suspicious reaction or in patients with a family history (10).

The genetic susceptibility described MH justifies the performance of the genetic study to search for the presence of mutations and subsequent genetic counseling in families with a history (13).

LEARNING POINTS

The laboratory has a key role in early diagnosis for the administration of effective treatment: dantrolene sodium.

The most common clinical manifestations are nonspecific and mild, but associated with exposure to triggering agents, will be sufficient for initial suspicion of MH.

Triggering agents are inhaled anesthetics and depolarizing muscle relaxants.

Confirmation of susceptibility will depend on the result of the halothane-caffeine contracture test (HCTC), indicated three months after the crisis.

The genetic study of the disease aims at a presymptomatic diagnosis, without the need for biopsy, and at assessing new mutations.

ORIGINAL RESEARCH article

Longitudinal transcriptomic analysis reveals persistent enrichment of iron homeostasis and erythrocyte function pathways in severe covid-19 ards.

• 1 Department of Internal Medicine, Division of Pulmonary, Critical Care, and Sleep Medicine, The Ohio State University, Columbus, OH, United States
• 2 Clinical Trials Management Office, College of Medicine, The Ohio State University, Columbus, OH, United States
• 3 The Center for RNA Biology, College of Medicine, The Ohio State University, Columbus, OH, United States

Introduction: The acute respiratory distress syndrome (ARDS) is a common complication of severe COVID-19 and contributes to patient morbidity and mortality. ARDS is a heterogeneous syndrome caused by various insults, and results in acute hypoxemic respiratory failure. Patients with ARDS from COVID-19 may represent a subgroup of ARDS patients with distinct molecular profiles that drive disease outcomes. Here, we hypothesized that longitudinal transcriptomic analysis may identify distinct dynamic pathobiological pathways during COVID-19 ARDS.

Methods: We identified a patient cohort from an existing ICU biorepository and established three groups for comparison: 1) patients with COVID-19 ARDS that survived hospitalization (COVID survivors, n = 4), 2) patients with COVID-19 ARDS that did not survive hospitalization (COVID non-survivors, n = 5), and 3) patients with ARDS from other causes as a control group (ARDS controls, n = 4). RNA was isolated from peripheral blood mononuclear cells (PBMCs) at 4 time points (Days 1, 3, 7, and 10 following ICU admission) and analyzed by bulk RNA sequencing.

Results: We first compared transcriptomes between groups at individual timepoints and observed significant heterogeneity in differentially expressed genes (DEGs). Next, we utilized the likelihood ratio test to identify genes that exhibit different patterns of change over time between the 3 groups and identified 341 DEGs across time, including hemoglobin subunit alpha 2 ( HBA1, HBA2 ), hemoglobin subunit beta ( HBB ), von Willebrand factor C and EGF domains ( VWCE ), and carbonic anhydrase 1 ( CA1 ), which all demonstrated persistent upregulation in the COVID non-survivors compared to COVID survivors. Of the 341 DEGs, 314 demonstrated a similar pattern of persistent increased gene expression in COVID non-survivors compared to survivors, associated with canonical pathways of iron homeostasis signaling, erythrocyte interaction with oxygen and carbon dioxide, erythropoietin signaling, heme biosynthesis, metabolism of porphyrins, and iron uptake and transport.

Discussion: These findings describe significant differences in gene regulation during patient ICU course between survivors and non-survivors of COVID-19 ARDS. We identified multiple pathways that suggest heme and red blood cell metabolism contribute to disease outcomes. This approach is generalizable to larger cohorts and supports an approach of longitudinal sampling in ARDS molecular profiling studies, which may identify novel targetable pathways of injury and resolution.

Introduction

From emergence to January 2024, coronavirus disease 2019 (COVID-19) has led to over 7 million deaths worldwide ( 1 ). Acute respiratory distress syndrome (ARDS) is one complication of severe COVID-19 and contribute to COVID-19 related death ( 2 , 3 ). Among hospitalized patients with COVID-19, nearly one-third develop ARDS ( 4 ). Autopsy studies of COVID-19 reveal evidence of ARDS in most decedents ( 5 , 6 ). Epidemiologic data from 2020 suggests at least a five-fold increase in ARDS-related deaths during the height of the COVID-19 pandemic ( 7 ). ARDS is a form of acute, non-cardiogenic, hypoxemic respiratory failure, characterized by bilateral lung infiltrates ( 8 ) that accounts for 10% of ICU admissions ( 9 ) with mortality rates ranging from around 30-50% ( 9 ). ARDS is notoriously heterogeneous, affecting varied patient populations with lung injury from various causes. One current focus of ARDS research is the identification of distinct patient subgroups that display varied outcomes and responses to targeted therapies ( 10 – 13 ). While clinical trials have demonstrated the efficacy of corticosteroids as treatment in COVID-19 ARDS ( 14 , 15 ), studies evaluating immune modulating therapies in diverse ARDS populations have yielded mixed results ( 16 , 17 ). Despite decades of clinical trials in ARDS, supportive care remains the primary treatment approach in diverse ARDS populations ( 18 ), and effective, targeted therapeutics for ARDS are lacking.

A better understanding of ARDS pathobiology may inform the limitations of current treatments, and the COVID-19 global pandemic has renewed the importance and urgency of new approaches to ARDS research. Recent studies have employed molecular profiling tools, such as RNA-sequencing, single-cell RNA-seq, and proteomics, with varied study design to better understand gene signatures that correlate with disease severity among COVID-19 ARDS patients. In this respect, multiple studies have highlighted the importance of type I interferon signaling and acute pro-inflammatory mediators in peripheral blood mononuclear cells (PBMCs) collected from patients early in disease course that correlate with COVID-19 disease severity ( 19 – 21 ), suggesting that PBMC analysis provides valuable insight into the dysregulated biology of COVID-19. As COVID-19 is dynamic with an evolving disease course over hours and days, other groups have examined transcriptomic data from patients at multiple time-points with varied sampling schema, including defined clinical stages (treatment, convalescence, and rehabilitation) ( 20 ), early and late ICU time points ( 22 ), and sampling from admission through two months of follow-up ( 23 ). Optimal strategies for patient sampling during the course of COVID-19 ARDS illness and recovery remain unclear. Our approach of longitudinal patient sampling provides opportunities to identify mechanisms that may contribute to disease progression or resolution.

To better understand how dynamic gene expression correlates with clinical outcomes among a group of patients with COVID-19 ARDS, we performed RNA-sequencing of patient PBMCs collected at 4 fixed time intervals across ICU admission. We hypothesized that longitudinal analysis may identify distinct transcriptomic changes compared to analysis at single time points to better characterize dynamic processes of injury and repair in COVID-19 ARDS. Here, we established three groups for comparison, including patients with COVID-19 ARDS that survived hospitalization, patients with fatal COVID-19 ARDS, and ARDS patients without COVID-19 as a control comparison group. We analyzed differential gene expression at each individual time point and performed longitudinal analysis to identify unique patterns of dynamic gene expression throughout acute illness.

Study design and identification of patient cohort

Patients with ARDS with available longitudinal peripheral blood samples were identified from the Ohio State University Intensive Care Unit Registry (BuckICU), a pre-existing, IRB-approved (IRB #2020H0175) biorepository that enrolls patients within 48 hours of admission to the intensive care units at the Ohio State University Wexner Medical Center and the Arthur G. James Cancer Hospital and Richard J. Solove Research Institute with acute respiratory failure and/or suspicion of sepsis. For inclusion in the BuckICU biorepository, acute respiratory failure is defined by an increase in supplemental oxygen requirement to maintain oxygen saturation (SpO2) greater than 92% or the need for adjunctive respiratory support, including high flow nasal cannula, non-invasive positive pressure ventilation or mechanical ventilation. To screen patients for enrollment, BuckICU defines suspicion of sepsis as meeting SIRS criteria ((any two or more of White Blood Cell count > 12 or < 4 x 10 9 /L, heart rate > 90 beats per minute, respiratory rate > 20 breaths per minute, or temperature > 38°C or < 36°C) and clinical suspicion of infection (collection of any clinical culture specimen OR initiation of antibiotics)) ( 24 ). Following completion of the study protocol, patient cases are adjudicated by two pulmonary and critical care physicians. While SIRS criteria were used for screening purposes, Sepsis-3 guidelines were used to define sepsis during case adjudication. Sepsis-3 defines sepsis as organ dysfunction caused by dysregulated host response to infection represented by an increase in Sequential Organ Failure Assessment (SOFA) score of at least 2 points due to infection ( 25 , 26 ). For this study, we identified patients between May 2020 and June 2021 who required mechanical ventilation at ICU admission with ARDS as defined by the Berlin definition ( 8 ), with at least 3 available longitudinal blood samples (days 1, 3, and 7). As this study aimed to identify differences in gene expression with correlation to COVID-19 ARDS mortality, we selected 9 patients with ARDS and positive SARS-CoV-2 upper respiratory tract testing by PCR and an additional 4 patients with ARDS and negative SARS-CoV-2 testing to characterize differences in gene expression that may be specific to COVID-19 status. As shown in Figure 1 , we defined 3 groups: 1) patients with COVID-19 ARDS who survived hospitalization (COVID survivors, n = 4); 2) patients with COVID-19 ARDS who did not survive hospitalization (COVID non-survivors, n = 5); and 3) patients with ARDS from other causes (ARDS controls, n = 4).

Figure 1 Experimental design. Patients were identified from the Ohio State University Intensive Care Unit Registry (BuckICU). Subjects with available longitudinal biosamples with COVID-19 ARDS or non-COVID-19 ARDS controls were selected for inclusion in the study.

RNA extraction, RNA-seq library construction and sequencing

For gene expression profiling, we used longitudinal, banked, peripheral blood mononuclear cells (PBMCs), isolated by Ficoll (Sigma, Cytiva 17-1440-03) density gradient centrifugation of whole blood, lysed in Trizol (Invitrogen, 15596026), and stored at -80°C. We extracted RNA using the Direct-zol RNA Miniprep Plus kit (Zymo Research, R2071) followed by RNA cleanup with the Monarch RNA Cleanup Kit (New England BioLabs, #T2040, 50 μg) with additional in tube DNaseI treatment, per the manufacturer’s protocols. Total RNA was quantified using the Invitrogen Qubit RNA HS Assay kit (Invitrogen, Carlsbad, CA) and RNA quality was assessed by RNA integrity scoring using Agilent 2100 Bioanalyzer and/or 2200 TapeStation (Agilent, Santa Clara CA). RNA seq libraries were prepared using kits from New England Biolabs (Ipswich, MA) with 100 ng total RNA by targeted depletion of rRNA (NEB E#E6310x). Fragmentation and amplification were done using NEBNext Ultra II Directional (stranded) RNA Library Prep Kit (NEB#E7760L) and NEBNext (E64490S/L). Samples were sequenced to a depth of 40 million paired-end 150 bp clusters on the Illumina NovaSeq platform (Illumina, Inc., San Diego, CA) through the Ohio State University Genomics Shared Resource.

Sequence alignment, gene count generation, and differential expression analysis

RNA sequencing sample reads underwent pre-alignment quality control with fastqc followed by alignment to the human genome GRCh38.96 (GCF_000001405.26) using HiSAT2 (v2.1.0) to obtain feature counts for each sample. Read alignment was performed through the Ohio Supercomputer Center ( 27 ). These counts were used for Differential Expression Analysis using the DESeq2 package in R (version 4.3.2). Lowly expressed genes were removed, and counts underwent normalization prior to differential expression analysis. Data visualizations were performed in R. We used the likelihood ratio test (LRT) to compare groups with or without time as an interaction term. Genes were determined to be significantly expressed based on adjusted p < 0.05 (false discovery rate). A variance stabilizing transformation (VST) was performed for normalization to create heat maps or cluster diagrams.

Ingenuity pathway analysis

We utilized Ingenuity Pathway Analysis (IPA v23.0, Qiagen) software to analyze the resultant gene expression data to identify canonical pathways common to the DEGs in each comparison. DESeq2 results were uploaded to IPA along with their corresponding adjusted p values, as the LRT does not provide fold change results. Pathways were filtered for significance at a -log10 p value of 1.3, which corresponds to an FDR of 0.05.

Subject cohort characteristics

Subject characteristics are shown in Table 1 . Per group definitions, all subjects in the COVID non-survivors group did not survive hospitalization, while all subjects in the COVID survivors group survived critical illness. All patients met ARDS criteria during ICU admission, required mechanical ventilation with low tidal volume ventilation, and received standard ICU supportive care. At Day 1, P/F ratios were similar across groups. Subjects with COVID-19 showed decreased P/F at Day 3, compared to Day 1, and COVID-19 survivors showed improvements in P/F later in ICU admission, compared to COVID-19 non-survivors. ARDS controls and COVID-19 non-survivors had significantly higher SOFA scores at Day 1 with higher SOFA scores throughout ICU admission compared to COVID-19 survivors. Further, vasopressor requirements were higher in the ARDS controls and COVID-19 non-survivors compared to COVID-19 survivors. All ARDS controls had sepsis from a bacterial infection as a risk factor leading to ARDS. These patients received treatment per current sepsis care guidelines ( 28 , 29 ), including source control, early antibiotics, and volume resuscitation. All COVID-19 subjects tested positive for SARS-CoV-2 by upper respiratory tract nucleic acid amplification test, and all subjects were unvaccinated against SARS-CoV-2. Some COVID-19 patients had positive bacterial cultures during the study period, but COVID-19 was determined to be the primary risk factor for ARDS. There were differences in steroid exposure among groups, as all patients in the COVID-19 survivors group received dexamethasone, while 4 of 5 COVID-19 non-survivors and no ARDS Controls received dexamethasone.

Table 1 Values shown as median (range) or percentage of subjects.

Analysis of differential gene expression at single time points highlights heterogeneity of host responses during ARDS

Prior to analyzing dynamic gene expression patterns across ICU admission, we first performed differential expression analysis at each time point (days 1, 3, 7, and 10), comparing the three groups (COVID survivors, COVID non-survivors, and ARDS controls). Prior studies had analyzed transcriptomes of biospecimens from early time points of severe COVID-19 and identified genes associated with interferon signaling, T cell activation, and acute inflammation that correlated with worse outcomes. Comparing our 3 groups at each collection day, we identified 150 differentially expressed genes (DEGs) at Day 1, 803 DEGs at Day 3, 514 DEGs at Day 7, and 172 DEGs at Day 10. We next identified common differences in gene expression across time points by comparing the lists of DEGs and observed that the majority of DEGs identified at each timepoint were unique ( Figure 2A ). For example, differences in gene expression profiles peaked at Day 3. However, of the 803 DEGs identified at Day 3, only 82 of these transcripts were determined to be significantly different at the other time points. We observed greatest overlap between Days 1 and 3 (43 common DEGs) and Days 7 and 10 (62 common DEGs).

Figure 2 Differential expression analysis on individual days. (A) Venn diagram showing overlap of significant differentially expressed genes for each day. (B) Heatmap of the 150 significant DEGs (FDR < 0.05) identified at Day 1 with column clustering by gene expression pattern. (C) Ingenuity pathway analysis (IPA) of canonical pathways identified by DEGs at Day 1. The length of the bar indicates statistical significance of each pathway using -log10 BH multiple correction p-value.

As prior studies have characterized differential expression at early time points in COVID-19, we further examined the 150 DEGs identified at Day 1. The top 20 DEGs identified at Day 1 between the three groups are shown in Table 2 . Clustering of DEGs revealed correlation with our pre-defined comparison groups apart from one non-survivor, who displayed a pattern of expression similar to COVID survivors ( Figure 2B ). Considering all COVID-19 patients, we observed increases in interferon-stimulated genes, including interferon alpha-inducible protein 6 (IFI6), interferon alpha-inducible protein 27, mitochondrial (IFI27) ( Table 2 ). Ingenuity pathway analysis (IPA) of DEGs revealed canonical pathways primarily related to interferon signaling ( Figure 2C ).

Table 2 Top 20 most significant DEGs identified at Day 1.

Longitudinal analysis of gene expression reveals additional host response mechanisms

As we observed significant heterogeneity of differential gene expression at each time point, we next compared dynamic gene expression across time. As opposed to identifying differences at one timepoint, this approach allows for identification of transcripts that show differential patterns of expression across time (i.e. Which genes change over time? Are those patterns of change different between groups)? Here we identified 341 genes with significant differential expression across timepoints (p < 0.05). The top 20 significant DEGs identified by our longitudinal analysis are shown in Table 3 . Notably, this approach only determines significance and does not specify fold change. After filtering for significant DEGs, we next performed additional visualizations to determine directionality and patterns of change. First, clustering analysis identified transcripts that demonstrate similar patterns of temporal change. Of the 341 DEGs, 314 showed similar temporal patterns ( Figure 3 ). The top 20 genes demonstrating the most significant differences include hemoglobin subunit alpha 2 (HBA1, HBA2), hemoglobin subunit beta (HBB), von Willebrand factor C and EGF domains (VWCE), and carbonic anhydrase 1 (CA1) ( Table 3 ). Among COVID survivors, the 314 genes showed downregulation of expression over time with a nadir by Day 7, compared to the COVID non-survivors and ARDS controls. The ARDS control patients demonstrated a pattern of increase in temporal gene expression, and the COVID non-survivors demonstrated delayed increases in these significantly changed genes over time ( Figure 3 ).

Table 3 Top 20 most significant DEGs identified by longitudinal analysis.

Figure 3 Clustering diagram demonstrates differential dynamic gene expression across time. Using the likelihood ratio test, we compared differences in dynamic gene expression across 3 patient groups, COVID-19 ARDS survivors, COVID-19 ARDS non-survivors, and ARDS controls and 4 time points with Day 1 as reference. DEGs were clustered by similar patterns of gene expression. 314 of the 341 DEGs identified by the longitudinal analysis showed a similar pattern of dynamic change. Significance determined by the adjusted p-value from DESeq2 analysis.

To better understand dynamic gene expression at the level of individual genes, we used box plots to visualize gene expression of the top 20 genes identified by the LRT. As expected, these genes demonstrate a similar pattern of change that was observed in the clustering analysis ( Figure 3 ). Specifically, we observe the most variability in gene expression at day 1 for the COVID survivors, compared to the other two comparison groups. Further, differences in gene expression between groups increases across time ( Figure 4 ). Together, these findings suggest that differences in host response across disease course may better inform underlying biological processes contributing to outcomes than differential gene expression at ICU admission.

Figure 4 The top 20 differentially expressed genes identified by longitudinal analysis. Following identification of significantly differentially expressed genes by longitudinal analysis, we plotted the 20 genes with most significant differences to observe individual patterns of change. Here, we plotted normalized gene expression by variance stabilizing transformation (y-axis) against time (days 1, 3, 7, and 10 on the x-axis). Colored boxes represent the 3 comparison groups.

Perturbations in hematopoiesis and erythrocyte function pathways correlated with COVID-19 fatality

To identify common biological pathways among the significant DEGs observed in our LRT comparisons, we utilized Ingenuity Pathway Analysis (IPA) to elucidate the pathways engaged among COVID survivors, non-survivors, and ARDS controls. As we compared 3 groups, this analysis does not specify fold-change differences or directionality and describes only significance and pathway enrichment, determined by the number of DEGs involved in each pathway. Among the most represented pathways, we identified iron homeostasis signaling, erythrocyte interaction with oxygen and carbon dioxide, transcriptional activity of SMAD2/SMAD3:SMAD4 heterotrimer, deubiquitination, erythropoietin signaling, heme biosynthesis, metabolism of porphyrins, and iron uptake and transport among the top 12 IPA canonical pathways ( Figure 5 ). These results suggest that oxygen carrying capacity and metabolism of heme may be important modulators of disease course in COVID-19 ARDS.

Figure 5 Ingenuity pathway analysis. A bubble chart shows the top pathway categories of the IPA canonical pathways of significant genes (adjusted p < 0.05) detected across all days simultaneously. The size and color intensity of bubbles indicates the number of genes overlapping each pathway.

Here we performed longitudinal RNA sequencing analysis of PBMCs from patients with ARDS at a single center during the COVID-19 pandemic (May 2020 – June 2021) and compared temporal gene expression changes between COVID-19 ARDS survivors and non-survivors, as well as non-COVID ARDS patients, across 10 days of ICU admission. Longitudinal analysis revealed 341 transcripts with significantly different patterns of dynamic gene expression over time with most significant differences in pathways of iron homeostasis, heme biosynthesis, and erythrocyte function, that remained upregulated throughout ICU course in fatal COVID-19 ARDS compared to survivors of COVID-19 ARDS. Enriched gene signatures of hemoglobin metabolism have previously been described in blood leukocytes from septic patients ( 30 ). In vitro studies have shown induction of hemoglobin genes during cellular stress in murine macrophages ( 31 ) and human PBMCs ( 32 ). The role of heme in macrophages is complex ( 32 – 34 ) and additional data are needed to support strategies targeting heme biosynthesis in ARDS patients. Notably, our study employing longitudinal sampling and analysis identified distinct pathway regulation throughout disease course that was not identified at single time points. This study highlights the dynamic nature of COVID-19 ARDS and represents a novel approach towards better understanding of COVID-19 ARDS pathobiology.

The COVID-19 pandemic increased the global incidence of severe viral pneumonia, acute respiratory failure, and ARDS ( 7 ). Clinicians and researchers alike observed unique features of COVID-19 ARDS and raised the question: Is COVID-19 ARDS somehow different than “regular” ARDS ( 35 , 36 )? As a syndrome, ARDS is known for its heterogeneity and efforts to identify subtypes and subgroups of ARDS patients predate the pandemic ( 10 , 13 ). Our study identified a cohort of ARDS patients with COVID-19 and varied clinical outcomes and compared to a group of patients with ARDS and bacterial sepsis as the primary ARDS risk factor. Our differential expression analysis on Day 1 of study enrollment was characterized by significant activation of immune system pathways and interferon signaling consistent with prior reports in studies of severe COVID-19 ( 37 – 40 ). Dysregulated interferon signaling has been implicated in COVID-19 as an important driver of pathology ( 21 ). Compared to healthy controls, interferon responses are elevated in COVID-19 ( 41 ). However, when compared to other viral infections or among COVID-19 patients with varied clinical course, interferon responses are more variable ( 42 ). Subjects in our comparison group of non-COVID-19 ARDS had sepsis from bacterial sources as their primary risk factor for ARDS. Host responses to bacterial versus viral infection yield different transcriptomic signatures ( 43 – 45 ), which may account for the significant differences in interferon responses in our analysis. While multiple interferon-stimulated genes showed significant upregulation in our analysis, one specific gene, interferon-inducible protein 27 (IFI27), showed significant upregulation at study enrollment in COVID-19 ARDS compared to non-COVID-19 ARDS. This recapitulates findings of other study that suggest robust and specific upregulation of IFI27 in COVID-19. A re-analysis of nine independent cohort studies that analyzed peripheral blood gene expression across multiple infections, including COVID-19, influenza, and bacterial pneumonia, described a COVID-19-specific gene signature comprised of 149 genes, among which IFI27 was the sole gene directly associated with the interferon response ( 46 ). A single-center cohort study comparing peripheral blood transcriptomes at one time point showed IFI27 was highly upregulated in COVID-19, even when compared to influenza and seasonal coronaviridae ( 42 ). Despite reports of varied interferon responses, robust induction of IFI27 may represent a peripheral blood gene signature unique to COVID-19 infection. Notably, our study, like the above referenced COVID-19 transcriptomics studies, focused on mixed populations of peripheral blood cells, which may reflect transcriptomic signatures driven by a single cell type or changes in relative cell populations. Further, PBMCs represent transcriptional signatures in peripheral blood, which may differ from the lung transcriptome ( 47 ).

Despite multiple prior reports of transcriptomics in COVID-19, our study is unique in our sampling and analysis strategy. In opposition to single time-point studies or limited longitudinal sampling, we analyzed peripheral blood gene expression throughout acute illness at multiple short intervals. While our study is limited by the small sample size and single-center design, it provides proof of concept that longitudinal molecular profiling can be valuable for identifying dynamic molecular mechanisms that function during ARDS disease course. In ARDS studies, analysis at individual timepoints allows for identification of relative differences in gene expression between groups. However, this design is vulnerable to baseline patient heterogeneity and lead-time bias. While longitudinal sampling is subject to the same obstacles, our approach allows each patient to function as their own baseline control and focuses on dynamic gene expression during acute illness. We identified gene signatures related to iron homeostasis, erythropoietin signaling, heme biosynthesis, and iron uptake that we grouped into processes contributing to erythropoiesis, as well as upregulation of erythrocyte function in fatal COVID-19 compared to COVID-19 survivors. Among ARDS controls, we observed early upregulation of these genes compared to the other groups with decreased rate of change between days 7 and 10. Among the COVID-19 non-survivors, gene expression related to erythropoiesis and erythrocyte function showed late increased compared to COVID-19 survivors, which showed relatively low expression throughout ICU course. Other groups have employed sampling at multiple time points with various strategies and have also identified gene expression correlating with pathways of hemopoiesis, reactive oxygen species, and erythrocyte functioning ( 20 , 22 , 23 ). Zheng et al. examined longitudinal transcriptomes but defined three ad hoc clinical stages (treatment, convalescence, and rehabilitation) as opposed to consistent time intervals; they identified early downregulation of genes related to humoral immunity and type I interferon response and upregulated gene expression related to hemopoiesis, regulation of inflammatory response, mRNA splicing via spliceosome, and epithelial cell proliferation during COVID-19 recovery ( 20 ). Another study applied longitudinal multi-omics with 2 – 7 times points from 0 – 55 days after admission and demonstrated increased protein catabolism, erythrocyte differentiation, ferroptosis, and organelle disassembly in clusters primarily corresponding to COVID non-survivors compared to COVID survivors ( 23 ). As our study and others have identified common pathways of iron homeostasis and erythrocyte function, it is intriguing to hypothesize that interventions targeting effective erythropoiesis, iron handling, and erythrocyte function represent a novel therapeutic strategy in COVID-19 ARDS. Indeed, studies have demonstrated perturbations in iron handling and ferroptosis (iron-dependent cell death) due to COVID-19 in humans ( 48 ), animal models ( 49 ), and human cells ( 50 ), suggesting that COVID-19 may uniquely impact iron homeostasis and downstream pathways during disease course. Further, observational human studies, including ours, may be capturing a compensatory mechanism of increased erythropoiesis and erythrocyte function in severe COVID-19. Steroid treatment with dexamethasone is a common and accepted treatment in severe COVID-19 ( 14 , 15 ) and may impact iron metabolism pathways ( 51 ). In our cohort, steroid exposure was high in the COVID-19 groups, as 8 of 9 subjects received dexamethasone. However, ARDS controls did not receive steroid treatment. All patients in our cohort met sepsis criteria, recognizing the frequent overlap between critical illness syndromes, such as ARDS and sepsis. Among septic patients, heme metabolism and iron homeostasis genes are enriched in white blood cells (neutrophils and PBMCs) compared to controls ( 30 ), and transcriptomic differences in heme biosynthesis genes correlated with sepsis subgroups or endotypes ( 52 ). In non-erythroid cells, hemoglobin scavenges free radicals and functions in nitric oxide metabolism ( 53 ). Cellular damage through reactive oxygen species is a known pathologic mechanism in both sepsis and ARDS. Our study and others ( 23 , 52 ) demonstrate increased heme-related transcript expression correlated with worse outcomes in certain critically ill populations. We postulate that upregulation of these transcripts in non-erythroid cells represents a compensatory mechanism due to ongoing oxidative stress. As iron and heme metabolism are foundational biologic processes, it is unclear if these pathways represent viable interventional targets during COVID-19 ARDS.

Our study supports a novel patient sampling strategy and demonstrates a unique analysis of dynamic gene expression. We focused on patients with severe COVID-19 ARDS as a subgroup of ARDS patient to gain insights into disease pathology. Our findings suggest an approach for future studies, generalizable to larger COVID-19 and ARDS cohorts. Our study is limited by several factors. First, all patients were enrolled from a single center for analysis, which may not be representative of the general population. As we analyzed multiple samples from each subject, the sample size of the cohort was small. While we attempted to select age- and sex-matched subjects, the sample size does not allow for proper control of these potentially confounding variables. Despite the small cohort size, we were able to identify several biologically relative pathways, reflected in correlation with prior studies, and provide new insights via our longitudinal approach. We defined outcomes by mortality in this study. While our COVID-19 ARDS non-survivors demonstrate impaired oxygenation throughout ICU stay, consistent with worsened ARDS, this group also had baseline higher SOFA scores that increased over time and increased vasopressor requirements, which may confound our findings. Further, this study used ARDS as a primary inclusion criterion, but severe COVID-19 is a systemic disease. All patients in our cohort met criteria for sepsis, and we did not attempt to differentiate COVID-19 ARDS, sepsis, or both as independent groups. Our transcriptomic findings in peripheral blood cells may be related to systemic responses instead of lung-specific pathology related to COVID-19 or ARDS. Disease classification in critical illness is currently limited by often overlapping syndrome-based paradigms ( 54 , 55 ). However, transcriptomic studies have the potential to identify dysregulated host response pathways during critical illness that inform clinical disease course beyond established definitions of ARDS or sepsis ( 56 ).

Patients with ARDS from COVID-19 may represent a subgroup of ARDS patients with distinct molecular pathophysiology that drive disease outcomes. Our study identified differences in dynamic expression of genes related to iron homeostasis and erythrocyte function that correlate to survival in COVID-19 ARDS. Our findings are supported by prior studies of molecular profiling in COVID-19 and suggest that iron handling and ferroptosis may be putative mechanisms of ongoing lung injury during SARS-CoV-2 infection. Additional research is needed to examine the therapeutic potential of these pathways. Finally, our study suggests that short interval longitudinal sampling during acute illness may uncover novel mechanisms of injury, repair, and resolution during COVID-19 ARDS. This approach should be considered further for future ARDS molecular profiling study design.

Data availability statement

The data presented in the study are deposited in the Gene Expression Omnibus (https://www.ncbi.nlm.nih.gov/geo/), accession number GSE273149 .

Ethics statement

The studies involving humans were approved by The Ohio State University Biomedical Sciences Institutional Review Board. The studies were conducted in accordance with the local legislation and institutional requirements. The human samples used in this study were acquired from an existing biorepository that collects and stores human biosamples and data from critically-ill patients at Ohio State University. The biorepository project utilizes broad consent to collect and store samples for use in secondary analyses. Written informed consent for participation was not required from the participants or the participants’ legal guardians/next of kin in accordance with the national legislation and institutional requirements.

Author contributions

ME: Formal Analysis, Validation, Visualization, Writing – original draft, Writing – review & editing, Data curation, Investigation. FJ: Data curation, Investigation, Writing – review & editing, Methodology. DF: Investigation, Methodology, Writing – review & editing, Supervision. LL: Investigation, Writing – review & editing, Data curation. SC: Data curation, Investigation, Writing – review & editing. KH: Data curation, Investigation, Writing – review & editing. SK: Data curation, Investigation, Writing – review & editing. GS: Data curation, Investigation, Writing – review & editing. SP: Data curation, Investigation, Writing – review & editing, Conceptualization, Methodology. JH: Methodology, Writing – review & editing, Funding acquisition, Project administration, Resources, Supervision. RM: Writing – review & editing, Funding acquisition, Methodology, Project administration, Resources, Supervision. JE: Methodology, Writing – review & editing, Data curation, Investigation, Writing – original draft. JB: Methodology, Writing – original draft, Writing – review & editing, Conceptualization, Formal Analysis, Funding acquisition, Project administration, Visualization.

The authors declare financial support was received for the research, authorship, and/or publication of this article. This project was supported, in part, by the National Center for Advancing Translational Sciences (NCATS) of the National Institutes of Health (NIH) under Grant Numbers UM1TR004548 and KL2TR002734. This project was also supported by NIH R01HL142767 awarded to J.A.E., R01HL141195 awarded to J.C.H., P01HL114453, R01HL097376, R01HL081784, and R01HL096376 awarded to R.K.M., K08HL169725 awarded to J.S.B., and the Ohio State University Office of Research 2020 COVID-19 Seed Funding Program and the Department of Internal Medicine 2021 Junior Investigator Award, both awarded to J.S.B.

Acknowledgments

We thank the Ohio State University Clinical Trials Management Office and Critical Care Clinical Trials group, specifically Brent Oleksak, Preston So, and Madison So, for collection and processing of biospecimens and clinical data. We thank all clinical staff that helped in sample collection. We thank Pearlly Yan and Estela Puchulu-Campanella from the Ohio State Shared Genomics Resource for sample processing and sequencing services. We are forever grateful to the patients and their family members who participated in this research that would not have been possible without their support.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

Author disclaimer

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Keywords: COVID - 19, ARDS (acute respiratory disease syndrome), RNA seq analysis, longitudinal analysis, SARS-CoV-2

Citation: Eltobgy M, Johns F, Farkas D, Leuenberger L, Cohen SP, Ho K, Karow S, Swoope G, Pannu S, Horowitz JC, Mallampalli RK, Englert JA and Bednash JS (2024) Longitudinal transcriptomic analysis reveals persistent enrichment of iron homeostasis and erythrocyte function pathways in severe COVID-19 ARDS. Front. Immunol. 15:1397629. doi: 10.3389/fimmu.2024.1397629

Received: 07 March 2024; Accepted: 17 July 2024; Published: 05 August 2024.

Reviewed by:

Copyright © 2024 Eltobgy, Johns, Farkas, Leuenberger, Cohen, Ho, Karow, Swoope, Pannu, Horowitz, Mallampalli, Englert and Bednash. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY) . The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

*Correspondence: Joseph S. Bednash, [email protected]

Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.

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Mechanistic insights into the biological effects and antioxidant activity of walnut ( juglans regia l.) ellagitannins: a systematic review.

1. Introduction

2. methodology, 2.1. eligibility criteria, 2.2. information sources, 2.3. search strategy, 2.4. selection process, 2.5. data items, 3. results and discussion, 3.1. phytochemical composition, 3.1.1. walnut sample preparation and ets and ea extraction, 3.1.2. separation and characterization of walnut ets and ea, 3.1.3. walnut ets and ea derivatives, 3.2. metabolism of walnut ets and ea, 3.2.1. urolithin biosynthesis from ets and ea metabolism, 3.2.2. influence of individual metabotype and gm on urolithin biosynthesis, 3.2.3. bioavailability of urolithins and their metabolites after walnut intake, 3.2.4. pharmacokinetics of uro-a and uro-b after walnut intake, 3.2.5. metabolic compounds derived from walnuts, 3.3. antioxidant activity of ets and their metabolites, 3.4. anti-inflammatory activity of ets and their metabolites, 3.4.1. preclinical studies, 3.4.2. clinical studies, 3.5. cardiometabolic activity of ets and their metabolites, 3.5.1. weight, waist circumference, visceral adiposity, 3.5.2. gut health, 3.6. neuroprotection activity of ets and their metabolites, 3.6.1. preclinical studies, 3.6.2. clinical studies, 3.7. antitumoral potential of ets and their metabolites, 3.7.1. preclinical studies, 3.7.2. clinical studies, 3.8. other potential therapeutic effects of ets and their metabolites, 3.8.1. hepatoprotective effects, 3.8.2. bone health, 3.8.3. anti-aging effects, preclinical studies, clinical studies, 3.8.4. antimicrobial activity, 4. strength, limitations, and future prospects, 5. conclusions, author contributions, informed consent statement, data availability statement, conflicts of interest, abbreviations.

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Mateș, L.; Banc, R.; Zaharie, F.A.; Rusu, M.E.; Popa, D.-S. Mechanistic Insights into the Biological Effects and Antioxidant Activity of Walnut ( Juglans regia L.) Ellagitannins: A Systematic Review. Antioxidants 2024 , 13 , 974. https://doi.org/10.3390/antiox13080974

Mateș L, Banc R, Zaharie FA, Rusu ME, Popa D-S. Mechanistic Insights into the Biological Effects and Antioxidant Activity of Walnut ( Juglans regia L.) Ellagitannins: A Systematic Review. Antioxidants . 2024; 13(8):974. https://doi.org/10.3390/antiox13080974

Mateș, Letiția, Roxana Banc, Flaviu Andrei Zaharie, Marius Emil Rusu, and Daniela-Saveta Popa. 2024. "Mechanistic Insights into the Biological Effects and Antioxidant Activity of Walnut ( Juglans regia L.) Ellagitannins: A Systematic Review" Antioxidants 13, no. 8: 974. https://doi.org/10.3390/antiox13080974

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