Hypothesis-driven weight of evidence evaluation indicates styrene lacks endocrine disruption potential.

Styrene is among the U.S. EPA's List 2 chemicals for Tier 1 endocrine screening subject to the agency's two-tiered Endocrine Disruptor Screening Program (EDSP). Both U.S. EPA and OECD guidelines require a Weight of Evidence (WoE) to evaluate a chemical's potential for disrupting the endocrine system. Styrene was evaluated for its potential to disrupt estrogen, androgen, thyroid, and steroidogenic (EATS) pathways using a rigorous WoE methodology that included problem formulation, systematic literature search and selection, data quality evaluation, relevance weighting of endpoint data, and application of specific interpretive criteria. Sufficient data were available to assess the endocrine disruptive potential of styrene based on endpoints that would respond to EATS modes of action in some Tier 1-type and many Tier 2-type reproductive, developmental, and repeat dose toxicity studies. Responses to styrene were inconsistent with patterns of responses expected for chemicals and hormones known to operate via EATS MoAs, and thus, styrene cannot be deemed an endocrine disruptor, a potential endocrine disruptor, or to exhibit endocrine disruptive properties. Because Tier 1 EDSP screening results would trigger Tier 2 studies, like those evaluated here, subjecting styrene to further endocrine screening would produce no additional useful information and would be unjustified from animal welfare perspectives.

Endocrine characteristics and risk factors of type 2 diabetes complicated with gastrointestinal autonomic neuropathy: A single-center retrospective study.

To analyze endocrine characteristics and risk factors of type 2 diabetes mellitus (T2DM) gastrointestinal autonomic neuropathy. A total of 202 patients with T2DM with obesity who were hospitalized at our institute between January 2019 and June 2021 were selected. Based on adrenocorticotropic hormone (ACTH) levels, 102 patients were placed in the ACTH abnormal group and 100 patients were placed in the non-ACTH abnormal group. Ninety-five healthy adults without diabetes, hyperlipidemia, osteoporosis, or endocrine system abnormalities who were examined at our hospital during the same period were selected as the control group. Fasting plasma glucose (FPG), fasting insulin (FINS), triglycerides (TG), total cholesterol (TC), homeostasis model assessment of insulin resistance (HOMA-IR), ACTH level, body mass index (BMI), and bone mineral density (BMD) were measured to evaluate endocrine characteristics and risk factors. BMI, FPG, FINS, HOMA-IR, TG, TC, and ACTH levels in the abnormal ACTH group were significantly higher than those in the other 2 groups, while BMD was significantly lower than that in the other 2 groups (all P < .05). BMI, FPG, FINS, HOMA-IR, TG, TC, and ACTH in the non-ACTH abnormal group were significantly higher than those in the control group, whereas BMD was significantly lower than that in the control group (all P < .05). The plasma ACTH level in patients with abnormal ACTH levels was significantly positively correlated with BMI, FPG, FINS, HOMA-IR, TG, and TC and negatively correlated with BMD (all P < .05). Multivariate regression analysis showed that BMI, advanced age, FINS, TG, and FPG were risk factors for ACTH abnormalities in patients with diabetes (odds ratio > 1, all P < .05). BMI, advanced age, FINS, TG and FPG are the risk factors of abnormal ACTH in T2DM patients with gastrointestinal autonomic neuropathy.

Weight of evidence for cross-species conservation of androgen receptor-based biological activity.

The U.S. Environmental Protection Agency's Endocrine Disruptor Screening Program (EDSP) is tasked with assessing chemicals for their potential to perturb endocrine pathways, including those controlled by androgen receptor (AR). To address challenges associated with traditional testing strategies, EDSP is considering in vitro high-throughput screening assays to screen and prioritize chemicals more efficiently. The ability of these assays to accurately reflect chemical interactions in nonmammalian species remains uncertain. Therefore, a goal of the EDSP is to evaluate how broadly results can be extrapolated across taxa. To assess the cross-species conservation of AR-modulated pathways, computational analyses and systematic literature review approaches were used to conduct a comprehensive analysis of existing in silico, in vitro, and in vivo data. First, molecular target conservation was assessed across 585 diverse species based on the structural similarity of ARs. These results indicate that ARs are conserved across vertebrates and are predicted to share similarly susceptibility to chemicals that interact with the human AR. Systematic analysis of over 5000 published manuscripts was used to compile in vitro and in vivo cross-species toxicity data. Assessment of in vitro data indicates conservation of responses occurs across vertebrate ARs, with potential differences in sensitivity. Similarly, in vivo data indicate strong conservation of the AR signaling pathways across vertebrate species, although sensitivity may vary. Overall, this study demonstrates a framework for utilizing bioinformatics and existing data to build weight of evidence for cross-species extrapolation and provides a technical basis for extrapolating hAR-based data to prioritize hazard in nonmammalian vertebrate species.

Investigating endocrine-disrupting properties of chemicals in fish and amphibians: Opportunities to apply the 3Rs.

Many regulations are beginning to explicitly require investigation of a chemical's endocrine-disrupting properties as a part of the safety assessment process for substances already on or about to be placed on the market. Different jurisdictions are applying distinct approaches. However, all share a common theme requiring testing for endocrine activity and adverse effects, typically involving in vitro and in vivo assays on selected endocrine pathways. For ecotoxicological evaluation, in vivo assays can be performed across various animal species, including mammals, amphibians, and fish. Results indicating activity (i.e., that a test substance may interact with the endocrine system) from in vivo screens usually trigger further higher-tier in vivo assays. Higher-tier assays provide data on adverse effects on relevant endpoints over more extensive parts of the organism's life cycle. Both in vivo screening and higher-tier assays are animal- and resource-intensive and can be technically challenging to conduct. Testing large numbers of chemicals will inevitably result in the use of large numbers of animals, contradicting stipulations set out within many regulatory frameworks that animal studies be conducted as a last resort. Improved strategies are urgently required. In February 2020, the UK's National Centre for the 3Rs and the Health and Environmental Sciences Institute hosted a workshop ("Investigating Endocrine Disrupting Properties in Fish and Amphibians: Opportunities to Apply the 3Rs"). Over 50 delegates attended from North America and Europe, across academia, laboratories, and consultancies, regulatory agencies, and industry. Challenges and opportunities in applying refinement and reduction approaches within the current animal test guidelines were discussed, and utilization of replacement and/or new approach methodologies, including in silico, in vitro, and embryo models, was explored. Efforts and activities needed to enable application of 3Rs approaches in practice were also identified. This article provides an overview of the workshop discussions and sets priority areas for follow-up. Integr Environ Assess Manag 2022;18:442-458. © 2021 The Authors. Integrated Environmental Assessment and Management published by Wiley Periodicals LLC on behalf of Society of Environmental Toxicology & Chemistry (SETAC).

A Multiplexed Microfluidic Platform toward Interrogating Endocrine Function: Simultaneous Sensing of Extracellular Ca2+ and Hormone.

Extracellular Ca2+ ([Ca2+]ex) is an important regulator of various physiological and pathological functions, including intercellular communication for synchronized cellular activities (e.g., coordinated hormone secretion from endocrine tissues). Yet it is rarely possible to concurrently quantify the dynamic changes of [Ca2+]ex and related bioactive molecules with high accuracy and temporal resolution. This work aims to develop a multiplexed microfluidic platform to enable monitoring oscillatory [Ca2+]ex and hormone(s) in a biomimetic environment. To this end, a low-affinity fluorescent indicator, Rhod-5N, is identified as a suitable sensor for a range of [Ca2+]ex based on its demonstrated high sensitivity and selectivity to Ca2+ in biomedical samples, including human serum and cell culture medium. A microfluidic chip is devised to allow for the immobilization of microscale subjects (analogous to biological tissues), precise control of the perfusion gradient at sites of interest, and integration of modalities for fluorescence measurement and enzyme-linked immunosorbent assay. As this analytical system is demonstrated to be viable to quantify the dynamic changes of Ca2+ (0.2-2 mM) and insulin (15-150 mU L-1) concurrently, with high temporal resolution, it has the potential to provide key insights into the essential roles of [Ca2+]ex in the secretory function of endocrine tissues and to identify novel therapeutic targets for human diseases.

Endocrine profile of the VCD-induced perimenopausal model rat.

During the transition to menopause, women experience a variety of physical and psychological symptoms that are directly or indirectly linked to changes in hormone secretion. Establishing animal models with intact ovaries is essential for understanding these interactions and finding new therapeutic targets. In this study, we assessed the endocrine profile, as well as the estrous cycle, in the 4-vinylcyclohexene diepoxide (VCD)-induced follicular depletion rat model in 10-day intervals over 1 month to accurately establish the best period for studies of the transition period. Twenty-eight-day-old female rats were injected daily with VCD or oil s.c. for 15 days and euthanized in the diestrus phase approximately 70, 80, 90 and 100 days after the onset of treatment. The percentage of rats showing irregular cycles and the plasma level of FSH increased only in the 100-day VCD group. Plasma anti-Müllerian hormone (AMH) and progesterone were lower in all VCD groups compared to control groups, while estradiol remained unchanged or higher. As in control groups, dihydrotestosterone (DHT) progressively decreased in the 70-90-day VCD groups; however, it was followed by a sharp increase only in the 100-day VCD group. No changes were found in plasma corticosterone, prolactin, thyroid hormones or luteinizing hormone. Based on the estrous cycle and endocrine profile, we conclude that 1) the time window from 70 to 100 days is suitable to study a perimenopause-like state in this model, and 2) regular cycles with low progesterone and AMH and normal FSH can be used as markers of the early/mid-transition period, whereas irregular cycles associated with higher FSH and DHT can be used as markers of the late transition period to estropause.

Principal component and correlation analysis of biochemical and endocrine markers in a healthy pediatric population (CALIPER).

BACKGROUND: Reference intervals (i.e. normative ranges) established from a healthy reference population are essential to accurately interpret disease biomarkers. Biomarker concentration may partially depend on associations with other biomarkers due to various physiological and pathophysiological processes. In this study, a robust correlation analysis was performed to identify physiological biomarker associations in the healthy pediatric CALIPER cohort. METHODS: Population reference values for 35 biochemical and 20 fertility/endocrine markers were analyzed for correlations in all subjects, male adolescents, female adolescents, and young children. Associations between biomarkers were assessed by Spearman's rank correlation and a multivariate analysis technique, principal component analysis (PCA). RESULTS: Of 197, 90, 59, and 32 significant correlations between biochemical markers in all subjects, male adolescents, female adolescents, and children, respectively, 23, 19, 16, and 9 were moderately strong (r > 0.5 or r < -0.5). Of 98, 24, 33, and 16 significant correlations between fertility/endocrine markers in all subjects, male adolescents, female adolescents, and children, respectively, 17, 8, 11, and 5 were moderately strong. Results were agreeable between Spearman's rank method and PCA. In some cases, biomarker correlations differed between sexes. CONCLUSIONS: Using PCA, this study provides for the first time an extensive analysis of circulating biomarker associations in a healthy pediatric cohort. These data can inform future studies of potential confounding factors or particular variables that should be considered in test result interpretation for specific diseases.

Overcoming endocrine resistance in metastatic hormone receptor-positive breast cancer.

Endocrine therapy has historically formed the basis of treatment of metastatic hormone receptor-positive breast cancer. The development of endocrine resistance has led to the development of newer endocrine drug combinations. Use of the CDK4/6 inhibitors has significantly improved progression-free survival in this group of patients. There are multiple studies of the use of P13K inhibitors and mTOR inhibitors for use as subsequent lines of therapy, particularly for endocrine resistance. The optimal sequencing of therapy should be based on medical comorbidities, prior adjuvant therapies, quality of life, side-effect profile, and disease-free interval.

Global DNA methylation changes spanning puberty are near predicted estrogen-responsive genes and enriched for genes involved in endocrine and immune processes.

Background: The changes that occur during puberty have been implicated in susceptibility to a wide range of diseases later in life, many of which are characterized by sex-specific differences in prevalence. Both genetic and environmental factors have been associated with the onset or delay of puberty, and recent evidence has suggested a role for epigenetic changes in the initiation of puberty as well. Objective: To identify global DNA methylation changes that arise across the window of puberty in girls and boys. Methods: Genome-wide DNA methylation levels were measured using the Infinium 450K array. We focused our studies on peripheral blood mononuclear cells (PBMCs) from 30 girls and 25 boys pre- and post-puberty (8 and 14 years, respectively), in whom puberty status was confirmed by Tanner staging. Results: Our study revealed 347 differentially methylated probes (DMPs) in females and 50 DMPs in males between the ages of 8 and 14 years (FDR 5%). The female DMPs were in or near 312 unique genes, which were over-represented for having high affinity estrogen response elements (permutation P < 2.0 × 10-6), suggesting that some of the effects of estrogen signaling in puberty are modified through epigenetic mechanisms. Ingenuity Pathway Analysis (IPA) of the 312 genes near female puberty DMPs revealed significant networks enriched for immune and inflammatory responses as well as reproductive hormone signaling. Finally, analysis of gene expression in the female PBMCs collected at 14 years revealed modules of correlated transcripts that were enriched for immune and reproductive system functions, and include genes that are responsive to estrogen and androgen receptor signaling. The male DMPs were in or near 48 unique genes, which were enriched for adrenaline and noradrenaline biosynthesis (Enrichr P = 0.021), with no significant networks identified. Additionally, no modules were identified using post-puberty gene expression levels in males. Conclusion: Epigenetic changes spanning the window of puberty in females may be responsive to or modify hormonal changes that occur during this time and potentially contribute to sex-specific differences in immune-mediated and endocrine diseases later in life.

Bone marrow adipose tissue is an endocrine organ that contributes to increased circulating adiponectin during caloric restriction.

The adipocyte-derived hormone adiponectin promotes metabolic and cardiovascular health. Circulating adiponectin increases in lean states such as caloric restriction (CR), but the reasons for this paradox remain unclear. Unlike white adipose tissue (WAT), bone marrow adipose tissue (MAT) increases during CR, and both MAT and serum adiponectin increase in many other clinical conditions. Thus, we investigated whether MAT contributes to circulating adiponectin. We find that adiponectin secretion is greater from MAT than WAT. Notably, specific inhibition of MAT formation in mice results in decreased circulating adiponectin during CR despite unaltered adiponectin expression in WAT. Inhibiting MAT formation also alters skeletal muscle adaptation to CR, suggesting that MAT exerts systemic effects. Finally, we reveal that both MAT and serum adiponectin increase during cancer therapy in humans. These observations identify MAT as an endocrine organ that contributes significantly to increased serum adiponectin during CR and perhaps in other adverse states.

The history of the discovery of vitamin D and its daughter steroid hormone.

It is largely through historical accident in the interval of 1920-1940 that vitamin D(3) became classified as a vitamin rather than as a steroid hormone. The formal definition of a vitamin is that it is a trace dietary constituent required to produce the normal function of a physiological process or processes. The emphasis here is on trace and the fact that the vitamin must be supplied regularly in the diet; this implies that the body is unable to metabolically synthesize the vitamin in question. However, the ultraviolet exposure of 7-dehydrocholesterol present in the skin results in the photochemical production of vitamin D(3). Thus, vitamin D(3) becomes a true vitamin only when the animal or human does not have regular access to sunlight or ultraviolet light. Under normal physiological circumstances, all mammals, including humans, can generate, via ultraviolet exposure of 7-dehydrocholesterol present in the skin, adequate quantities of vitamin D(3) to meet their nutritionally defined requirements. There is a vibrant historical record beginning in 1650 and culminating in 1963 concerned with the determination of the chemical structures of vitamin D(3) and vitamin D(2). A surprising aspect concerning vitamin D(3) is that it is itself biologically inert. There are no known essential biological actions or contributions that rely specifically on the molecule vitamin D(3). While chemists had certainly appreciated the strong structural similarity between the vitamins D and other steroids, this correlation was never widely acknowledged in the biological, clinical, or nutritional sciences until 1965-1970. The biological role of vitamin D(3) is to serve as a substrate for the liver 25-hydroxylase which produces 25-hydroxyvitamin D(3) [25(OH)D(3)]. 25(OH)D(3) in turn serves as the substrate for the kidney proximal tubule 25(OH)D(3)-1α-hydroxylase enzyme which produces the steroid hormone 1α,25(OH)(2)-vitamin D(3) [1α,25(OH)(2)D(3)].

An approach to detecting estrogenic endocrine disruption via choriogenin expression in an estuarine model fish species.

A large body of work has established a link between endocrine disrupting compounds (EDCs) and a number of abnormalities in fishes. However, most EDC studies use several standard laboratory denizens to assess impacts, so assumptions about sensitivity are primarily based on these few species. Additionally, existing methods rely on obtaining sufficient plasma to measure EDC biomarkers. Our objectives were (a) to establish a new model species for estuarine fishes, (b) to evaluate endocrine impacts with a highly sensitive and specific biomarker, and (c) to develop a method for the analysis of this biomarker in small fish that do not possess sufficient blood plasma for protein measurement. As such, we created a polyclonal antibody (Ab) to the estrogen-responsive proteins chorion (Ch) and choriogenin (Chg) in Menidia beryllina, found throughout coastal North America and already utilized in EPA Whole Effluent Testing. We then validated the Ab by using it to measure the response to aqueous ethinylestradiol (EE2) through the development an ELISA using Menidia whole body homogenate (WBH). Sensitivity of the Ab to Menidia WBH is greater than that of the commercially available option. ELISA sensitivity, with a detection limit of 5 ng/ml and a working range of 22.6-1370.9 ng/ml, is comparable to ELISAs developed to measure plasma Chg. To our knowledge this is the first ELISA method developed for the detection of Chg using WBH. Including additional model species and methods allowing the evaluation of alternative sample matrices will contribute to an enhanced understanding of inter-species differences in EDC response.

Expression of ezrin in human embryonic, fetal, and normal adult tissues.

Ezrin, which cross-links the cytoskeleton and plasma membrane, was involved in a wide variety of cellular processes. Here, to investigate the distribution of ezrin, tissue microarray technology was employed to perform immunohistochemical experiments on human embryos, fetuses at 4 to 22 weeks' gestation, and adult tissue specimens. Results showed that ezrin was widely expressed in the gastrointestinal tract throughout the human developmental stages studied. At 6 to 8 weeks' gestation, ezrin was found in epithelial cells, and this staining pattern was particularly pronounced in the brush border of mature absorptive cells lining the villus in later developmental stages and adult tissues. Throughout neural development, ezrin was only expressed in the neural tube at 4 weeks' gestation. Ezrin was also detected in the cortex and medulla of the adrenal gland at 8 to 12 weeks' gestation, whereas its immunoreactivity was increased from the zona glomerulosa through the zona reticularis and was essentially undetectable in the adrenal medulla of adult tissues. Significant expression of ezrin was seen throughout development in the kidney, spleen, lymph nodes, and cells of stratified squamous epithelia. However, ezrin was undetectable in lung, liver, heart, and blood vessels. These results demonstrated that the expression pattern of ezrin was highly time specific and tissue specific.

Phenolics: occurrence and immunochemical detection in environment and food.

Phenolic compounds may be of natural or anthropogenic origin and be present in the environment as well as in food. They comprise a large and diverse group of compounds that may be either beneficial or harmful for consumers. In this review first a non-exhausting overview of interesting phenolics is given, in particular with regards to their presence in environment and food. For some of these compounds, beneficial, toxicological and/or optionally endocrine disrupting activities will be presented. Further, immunochemical detection and/or isolation methods developed will be discussed, including advantages and disadvantages thereof in comparison with conventional analytical methods such as HPLC, GC, MS. A short overview of new sensor-like methods will also be included for present and future application.

Fate of nonylphenol and 17beta-estradiol contained in composted sewage sludge after land application.

Many environmental problems caused by endocrine disrupters (EDs) have been reported. Because little is known about the fate of EDs accumulated in sewage sludge, we carried out a study to clarify the fate of EDs in composted sludge after its application to soil. Nonylphenol (NP) and 17beta-estradiol (E2) were measured for leachate and soil. High concentrations of NP and E2 were detected in the leachate at the early stage, but they decreased rapidly. Also, the high contents of NP and E2 in soil decreased significantly within 300 days. Because the decrease of NP and E2 in the soil was much larger than that of NP and E2 in the leachate, there must have been a physicochemical or biological decomposition mechanism in the soil layer. We also tried to clarify the transfer of NPs to plants from compost. In the experimental conditions of this study, the transfer of NPs to plants from compost was not observed.

Expression profiling of peroxisome proliferator-activated receptor-delta (PPAR-delta) in mouse tissues using tissue microarray.

Peroxisome proliferator-activated receptor-delta (PPAR-delta) is known as a transcription factor involved in the regulation of fatty acid oxidation and mitochondrial biogenesis in several tissues, such as skeletal muscle, liver and adipose tissues. In this study, to elucidate systemic physiological functions of PPAR-delta, we examined the tissue distribution and localization of PPAR-delta in adult mouse tissues using tissue microarray (TMA)-based immunohistochemistry. PPAR-delta positive signals were observed on variety of tissues/cells in multiple systems including cardiovascular, urinary, respiratory, digestive, endocrine, nervous, hematopoietic, immune, musculoskeletal, sensory and reproductive organ systems. In these organs, PPAR-delta immunoreactivity was generally localized on the nucleus, although cytoplasmic localization was observed on several cell types including neurons in the nervous system and cells of the islet of Langerhans. These expression profiling data implicate various physiological roles of PPAR-delta in multiple organ systems. TMA-based immunohistochemistry enables to profile comprehensive protein localization and distribution in a high-throughput manner.

Pleomorphic extra-renal manifestation of the glomerular podocyte marker podocalyxin in tissues of normal beagle dogs.

Podocalyxin (PC) was initially identified as a major sialoprotein on the apical surface of glomerular podocytes to perform the filtration barrier function. Later, it was reported to be expressed in endothelial cells, megakaryotes/platelets, and hemangioblasts, the common progenitor cells of the hematopoietic and endothelial cells. Recently, increasing numbers of reports have indicated that PC is not merely a molecule restricted at renal glomerulus, angiogenic or hematopoietic system. To further elucidate the expression pattern and address the possible physiological role of PC in adult mammals, we conducted an extensive study by immunohistochemistry and immunofluorescence staining on various tissues of healthy adult beagle dogs. By combinatory usage of two different anti-podocalyxin antibodies recognizing distinct epitopes in PC, we have demonstrated that (1) PC is expressed in renal tubules, mesothelium, myocardium, striated muscles in tongue, esophagus and extraocular region, myoepithelial cells in esophagus and salivary glands, neurons, and ependyma, etc.; (2) there are at least three forms of PC proteins, depending upon the accessibility of two different PC antibodies, expressed in different organs/systems; and (3) a particular form of PC is distributed in a vesicle-like compartment in certain organs/systems, such as the central nervous system.

Small molecule signaling in Caenorhabditis elegans.

Whereas the C. elegans genome was sequenced many years ago, the role of small molecule signals in its biology is still poorly understood. A recent publication reports the identification of two steroidal signaling molecules that regulate C. elegans reproductive development and dauer diapause via the nuclear receptor DAF-12. The two compounds, named dafachronic acids, represent the first endogenous ligands identified for any of the 284 nuclear receptors in C. elegans.

Molecular mechanisms of intercellular communication in the hormonal and neural systems.

This paper reviews our studies that have addressed the molecular mechanisms underlying the biosynthesis and reception of extracellular signaling molecules and integrative mechanisms of extracellular-intracellular signaling transmission in biological systems. We introduced recombinant DNA technology into the neuroendocrine system and established the concept that a single peptide precursor encompasses multiple biologically active peptides and brings about coordinate functions in various biological systems. We then developed a novel functional cloning of membrane receptors and ion channels by combining an oocyte expression system with electrophysiology. We molecularly elucidated not only various peptide receptors, including the first demonstration of the molecular entity of a G protein-coupled peptide receptor (GPCR), substance K receptor, and also diverse members of both G protein-coupled metabotropic type and NMDA type of neurotransmitter glutamate receptors. We demonstrated many novel synaptic mechanisms involving distinct types of glutamate receptors in brain function and dysfunction. These include the mechanisms underlying segregation of light-dark signals in visual transmission, discrimination and memory formation in olfactory transmission, and motor co-ordination in the cerebellum, basal ganglia and the retinal network.

Rethinking the opiate system? Morphine and morphine-6-glucuronide as new endocrine and neuroendocrine mediators.

Since the 80s, intrigued by presence of morphine precursors in some mammalian cells, different laboratories were able to characterize morphine and morphine precursors in animal tissues. Endogenous morphine studies continued during 90s and this alkaloid was successfully characterized from more organs and fluids of vertebrates, including brain, adrenal gland, heart, cerebrospinal fluid and urine. Then, in the last three years a high rate of publications dealing with this topic emerged, leading to a better understanding of the endogenous morphine system. In this regard, this article comment all the new data recently collected on this rising subject and replace the morphine and its derivative, morphine-6-glucuronide, in the mammalian physiology.