Measurement of chromogranin A in porcine saliva: validation of a time-resolved immunofluorometric assay and evaluation of its application as a marker of acute stress

The objective of this study was to develop and validate a time-resolved immunofluorometric assay (TR-IFMA) for porcine salivary chromogranin A (CgA) measurements, using a species-specific antibody, and evaluate its behaviour in an acute stress model. Polyclonal antibodies were produced in rabbits immunized with a synthetic porcine fragment of CgA_359−379 and used to develop a sandwich TR-IFMA. This TR-IFMA was analytically validated and showed intra- and inter-assay coefficients of variation of 6.23% and 5.82%, respectively, an analytical limit of detection of 4.27 × 10⁻³ μg/ml and a limit of quantification of 24.5 × 10⁻³ μg/ml. The assay also demonstrated a high level of accuracy, as determined by linearity under dilution (r = 0.975) and recovery tests. When a model of experimental acute stress, in which animals were immobilized for 3 min with a nose snare (stressor stimulus), was applied, a significant increase (P < 0.05) in CgA levels in saliva was detected at 15 min post-stressor stimulus. These results indicate that the assay developed in this study could measure CgA in porcine saliva in a reliable way and that the concentrations of CgA in saliva samples of pigs increase after an acute stress situation.     

Changes in chromogranin A-immunoreactive guinea-pig pulmonary neuroendocrine cells after sensitization and challenge with ovalbumin

Immunohistochemical study with antisera to chromogranin A and neuron-specific enolase, a general marker for nerves and endocrine cells, was used to quantify changes in bronchial neuroendocrine cells in guineapigs sensitized and challenged with ovalbumin. Actively sensitized animals were killed 2, 6, 24, 48, 72, and more than 144 hours after being challenged by an aerosolized solution of ovalbumin. The number of chromogranin A-immunoreactive cells was significantly greater in sensitized but unchallenged animals and in sensitized animals killed 2 and 6 h after challenge when compared to controls; it decreased significantly in animals killed more than 24 h after challenge when compared to sensitized, unchallenged animals. The number of neuron-specific-enolase-immunoreactive cells did not change. We conclude that the peptide content of bronchial neuroendocrine cells increases during sensitization and in the early phase of a hypersensitivity reaction, and that the cells release their granule contents in the late phase of such a reaction. They may therefore play a role in immunoallergic events in the lung.     

Effects of pulsed extremely-low-frequency magnetic fields on skin wounds in the rat

Rats with skin-wounds surgically created on their backs were exposed immediately after surgery and every 12 h thereafter to pulsed, extremely-low-frequency magnetic fields. The shape of the pulse was a positive triangle (50 Hz, 8 mT peak). The rate of healing of skin wounds was evaluated macroscopically and by light and electron microscopy at 6, 12, 21, and 42 days after the operation. A significant increase in the rate of wound contraction was found in rats treated with magnetic fields. Forty-two days after surgery all treated animals show fully closed wounds, while control rats at the same time intervals still lacked a final 6% of the wound surface to be covered. Treated rats showed earlier cellular organization, collagen formation and maturation, and a very early appearance of newly formed vascular network.     

Intake of green tea inhibited increase of salivary chromogranin A after mental task stress loads

Background

Green tea has become renowned for its health benefits. In this study, we investigated the anti-stress effect of two kinds of green tea against a mental stress task load.

Methods

Warm water, ordinary green tea (Sagara), and shaded white tea, which contains more amino acid components than Sagara, were used as test samples in a randomized cross-over design study. Eighteen students (nine male and nine female) participated in three experimental trials on different days at intervals of seven days. Saliva was collected before beverage intake and after performing the mental stress load tasks. Concentration of chromogranin A (CgA) in the saliva was used as an index of autonomic nervous system activity.

Results

CgA level increased after the mental tasks, but intake of green tea inhibited this increase; the anti-stress effect was even greater after consumption of shaded white tea. Intake of shaded white tea also lowered Total Mood Disturbance (TMD) score on the Profile of Mood States (POMS); subjects in this condition tended to perform more calculations in the arithmetic task than those in the warm water treatment condition.

Conclusions

Salivary CgA concentration levels increased after mental stress load tasks, but ingestion of green tea inhibited this increase. This anti-stress effect was larger after the consumption of shaded white tea than after Sagara. Shaded white tea intake also lowered TMD score (POMS) and tended to improve performance on an arithmetic task compared to warm water, suggesting that shaded white tea might also improve mood during and after mental stress load.     

Effects of exposure to a circularly polarized 50-Hz magnetic field on plasma and pineal melatonin levels in rats

We sought to determine whether a 6-week exposure to a 50-Hz rotating magnetic field influences melatonin synthesis by 11–18 week-old Wistar-King male rats. Rats were exposed continuously to a rotating magnetic field at 1, 5, 50, or 250 μT (spatial vector rms) for 6 weeks, except for twice-weekly breaks of about 2 h for cleaning of cages and feeding. The rats were housed in exposure and sham-exposure facilities, which were located in the same room, under a 12:12 light-dark photoperiod (lights on at 06:00 h). The room was constantly illuminated by 4 small, dim red lights (< 0.07 lux in dark period). Levels of plasma and pineal gland melatonin were determined by radioimmunoassay. A significant decrease of melatonin was observed between the control group and groups exposed to a magnetic field at a flux density in excess of l μT during the night time, but no statistical differences were found among the exposed groups. These results indicate that subchronic exposure of albino rats to a 50-Hz rotating magnetic field influences melatonin production and secretion by the pineal gland. © 1993 Wiley-Liss, Inc.     

Distribution and coexistence of chromogranin A-, serotonin-and pancreastatin-like immunoreactivity in endocrine-like cells of the human anal canal

Summary The comparative distribution and coexistence of chromogranin A (CGA)-, serotonin (5-hydroxytryptamine; 5-HT)- and pancreastatin (PST)-like immunoreactivity in endocrine-like cells of the human anal canal was investigated by light-microscopic immunocytochemistry. The largest population of colorectal endocrine-like cells consisted of CGA-immunoreactive (ir) cells, followed by the 5-HT-ir and PST-ir cell population. In the anal transitional zone (ATZ), CGA-and 5-HT-immunoreactivity was equally distributed; ir-PST was confined to a smaller endocrine-like cell population. In the squamous zone and the perianal skin, Merkel cells in the basal layer of the epidermis and hair follicles exhibited ir-CGA and ir-PST but no ir-5-HT. Double immunofluorescence on identical sections revealed distinct coexistence patterns. In the colorectal zone, about 2/3 of the CGA-ir endocrine-like cells also stained for 5-HT, whereas in the ATZ epithelium, CGA- and 5-HT-immunoreactivity completely overlapped. No 5-HT-immunoreactivity could be detected in CGA-ir Merkel cells of the squamous zone of the anal canal and the perianal skin. PST-immunoreactivity was present in about 1/3 of the CGA-ir colorectal and anal transitional endocrine-like cells and in about 1/4 of the Merkel-cell population staining for CGA. These chemically heterogeneous phenotypes of the anal endocrine-like and Merkel cells may reflect a specific regulatory role of these cells in the various epithelial linings of the human anal canal and the perianal skin.     

Incidence and prognosis of patients with small intestinal neuroendocrine tumors in a population based nationwide study

Background & aimsSmall intestinal neuroendocrine tumours (SI-NETs) are the most frequent malignant tumours of the small intestine. Population based studies on SI-NETs are scarce. We aimed to examine the incidence, presentation of disease and prognosis of SI-NET and to determine patient prognosis in those undergoing emergency or elective surgery.MethodsThis was a retrospective population-based study. Information on all patients diagnosed with neuroendocrine tumours of the small intestine (excluding duodenum) from the beginning of the Icelandic Cancer Registry and the pathology departments in the country (1966–2017). Detailed phenotypic information was obtained from medical records on symptoms at diagnosis, treatment, recurrence and survival.ResultsA total of 113 patients with SI-NETs were identified, 3 patients were excluded due to lack of data and/or diagnostic error, leaving 110 patients for final analysis. The incidence of SI-NET was 0.78/100,000 and did not increase during the study period. A total of 42 % (n = 46) of patients were diagnosed incidentally. Long-term prognosis, after a landmark of 12 months, was better in patients who were diagnosed incidentally (HR 0.52; p = 0.03). Overall 89 % (n = 98) of cases underwent surgical resection of the primary tumor, 31 % (n = 30) patients acute or semi-acute surgery and 69 % (n = 68) elective surgery. Emergency surgery was associated with a 6-fold risk of death in the first 12 months after surgery (HR: 5.99; p = 0.01) and associated with more severe surgical complications. However, there was no difference in the long-term risk of death after the first 12 months (HR: 1.39; p = 0.27).ConclusionsThe incidence of SI-NETs has not changed significantly in the last decades. Incidentally diagnosed SI-NET was associated with a favorable long-term prognosis. Emergency surgery in patients with SI-NET was associated with a significantly worse short-term risk of mortality compared to those who underwent elective surgery.     

Carboxypeptidase E,an essential element of the regulated secretory pathway,is expressed and partially co-localized with chromogranin A in chicken thymus

Although the functions of hormones and neuropeptides in the thymus have been extensively studied, we still do not know whether these intra-thymic humoral elements are released in a stimulated manner via the regulated secretory pathway or in a constitutive manner. Carboxypeptidase E (CpE) and chromogranin A (CgA) are functional and structural hallmarks of the regulated secretory pathway in (neuro)endocrine cells. Whereas we have previously shown a CgA-positive neuroendocrine population in the chicken thymus, the current study assesses the expression of CpE in the thymus, both at the mRNA and the protein level. Our immunohistochemical studies provide evidence for the co-existence of CgA and CpE in identical neuroendocrine cells in the thymus. CpE and CgA dual-positive cells have primarily been found in the transition zone between the cortex and medulla of the thymus, an area known to contain numerous arterioles and to be innervated by the autonomic nervous system. Our findings suggest that the diffuse neuroendocrine system serves as a relay for nervous stimuli delivered by the sympathetic and/or parasympathetic nervous system. Thus, these newly defined neuroendocrine cells might play an important role in the immuno-neuro-endocrine cross-talk in the thymus, potentially enabling thymopoiesis to be fine-tuned via the regulated secretory pathway by a variety of physical and environmental factors.     

Cancer promotion in a mouse-skin model by a 60-Hz magnetic field: I. Experimental design and exposure system.

The rationale for selection of an animal model, the experimental design, and the design and evaluation of an exposure system used in studies of 60-Hz magnetic fields are described. The studies were conceived to assay development of cancer and immune responsiveness in mice exposed to magnetic fields. The exposure system utilized a quadrupole-coil configuration to minimize stray magnetic fields. Four square-wound coil provided a uniform field within a volume occupied by 16 animal cages. The magnetic field had a mean flux density of 2 mT that varied less than +/- 10% within the volume occupied by animals' cages. The flux density decreased to less than 0.1 microT at a distance of 2 m from the coils. In each exposure system 32 animals could be housed in plastic cages.     

Adenosine A2A and A2B receptor expression in neuroendocrine tumours: potential targets for therapy

The clinical management of neuroendocrine tumours is complex. Such tumours are highly vascular suggesting tumour-related angiogenesis. Adenosine, released during cellular stress, damage and hypoxia, is a major regulator of angiogenesis. Herein, we describe the expression and function of adenosine receptors (A(1), A(2A), A(2B) and A(3)) in neuroendocrine tumours. Expression of adenosine receptors was investigated in archival human neuroendocrine tumour sections and in two human tumour cell lines, BON-1 (pancreatic) and KRJ-I (intestinal). Their function, with respect to growth and chromogranin A secretion was carried out in vitro. Immunocytochemical data showed that A(2A) and A(2B) receptors were strongly expressed in 15/15 and 13/18 archival tumour sections. Staining for A(1) (4/18) and A(3) (6/18) receptors was either very weak or absent. In vitro data showed that adenosine stimulated a three- to fourfold increase in cAMP levels in BON-1 and KRJ-1 cells. The non-selective adenosine receptor agonist (adenosine-5'N-ethylcarboxamide, NECA) and the A(2A)R agonist (CGS21680) stimulated cell proliferation by up to 20-40% which was attenuated by A(2B) (PSB603 and MRS1754) and A(2A) (SCH442416) receptor selective antagonists but not by the A(1) receptor antagonist (PSB36). Adenosine and NECA stimulated a twofold increase in chromogranin A secretion in BON-1 cells. Our data suggest that neuroendocrine tumours predominantly express A(2A) and A(2B) adenosine receptors; their activation leads to increased proliferation and secretion of chromogranin A. Targeting adenosine signal pathways, specifically inhibition of A(2) receptors, may thus be a useful addition to the therapeutic management of neuroendocrine tumours.     

Effects of in vitro exposure to power frequency magnetic fields on UV-induced DNA damage of rat lymphocytes

The mechanisms of biological effects of 50/60 Hz (power frequency) magnetic fields (MF) are still poorly understood. There are a number of studies indicating that MF affect biochemical processes in which free radicals are involved, such as the biological objects' response to ultraviolet radiation (UVA). Therefore, the present study was aimed to assess the effect of 50 Hz MFs on the oxidative deterioration of DNA in rat lymphocytes irradiated in vitro by UVA. UVA radiation (150 J/m2) was applied for 5 min for all groups and 50 Hz MF (40 microT rms) exposure was applied for some of the groups for 5 or 60 min. The level of DNA damage was assessed using the alkaline comet assay, the fluorescence microscope, and image analysis. It has been found that the 1 h exposure to MF caused an evident increase in all parameters consistent with damaged DNA. This suggest that MF affects the radical pairs generated during the oxidative or enzymatic processes of DNA repair.     

Lack of a co-promotion effect of 60 Hz rotating magnetic fields on N-ethyl-N-nitrosourea induced neurogenic tumors in F344 rats

The present study was conducted to investigate the possible effect of 60 Hz magnetic fields as promoters of brain tumors initiated transplacentally by ethylnitrosourea (ENU) in F344 rats. One hundred twenty mated animals were divided into six different groups and exposed in utero on day 18 of gestation to a single intravenous dose of either Saline (vehicle control, Group I), or ENU 10 mg/kg (Groups II-VI). In the present study, a total of 480 offspring was used. The offspring in group II were given no further treatment while the offspring in Groups III-VI were exposed to four different intensities of magnetic fields. Animals received exposure to 60 Hz magnetic field at field strengths of 0 Tesla (sham control, T1, Group III), 5 muT (T2, Group IV), 83.3 muT (T3, Group V), or 500 muT (T4, Group VI), for 21 h/day from the age of 4 weeks to the age of 32 or 42 weeks. At histopathological examination, tumors of the nervous system were seen in all the ENU-treated groups. The tumor incidence of the ENU group at 32nd and 42nd week necropsy was higher than that of the vehicle control group. The incidence of glial tumors at 42nd week necropsy was higher than the 32nd week necropsy. However, there were no differences in the tumor incidence between the sham control (T1) and ENU + magnetic field exposure groups (T2-T4). In conclusion, there was no evidence that exposure of offspring to 60 Hz at magnetic field strengths up to 500 muT to the age of 32 or 42 weeks promoted ENU-initiated brain tumors in rats.     

Influence of weak static and 50 Hz magnetic fields on the redox activity of cytochrome-C oxidase

The effects of static and 50 Hz magnetic fields on cytochrome-C oxidase activity were investigated in vitro by strictly controlled, simultaneous polarographic measurements of the enzyme's high- and low-affinity redox reaction. Cytochrome-C oxidase was isolated from beef heart. Control experiments were carried out in the ambient geomagnetic and 50 Hz magnetic fields at respective flux densities of 45 and 1.8 μT. The experimentally applied fields, static and time-varying, were generated by Helmholtz coils at flux densities between 50 μT and 100 mT. Exposures were timed to act either on the combined enzyme-substrate interchange or directly on the enzyme's electron and proton translo-cations. Significant changes as high as 90% of the overall cytochrome-C oxidase activity resulted during exposure (1) to a static magnetic field at 300 μT or 10 mT in the high-affinity range, and (2) to a 50 Hz magnetic field at 10 or 50 mT in the low-affinity range. No changes were observed at other flux densities. After exposure to a change-inducing, static or time-varying field, normal activity returned. © 1993 Wiley-Liss. Inc.     

Modulation of cardiac rhythm in the humans exposed to extremely weak alternating magnetic fields

The effect of extremely weak alternating magnetic fields of various types with the amplitudes δ of 2 μT on the heart rate variability in humans was studied. Volunteers were placed in a large-volume coil system (2ω2ω2 m), which provided exposure of the whole body to extremely weak alternating magnetic fields uniform in their amplitude. It was shown for the first time that the exposure to such fields could both increase and decrease the magnitude of stress in humans. In particular, the field tuned to the nuclear spins of hydrogen atoms (amplitude, 1.6 μT and frequency, 76 Hz) decreased the Baevsky stress index, whereas the field tuned to the magnetic moments formed by the orbiting electrons in some atoms (amplitude, 0.192 μT and frequency, 3000 Hz) increased this index. These results provide a possible explanation for the mechanisms of adverse effects caused by extremely weak alternating magnetic fields of certain types of both technogenic and natural origins on the human cardiovascular system.     

Thermo-sensitive transient receptor potential vanilloid channel-1 regulates intracellular calcium and triggers chromogranin A secretion in pancreatic neuroendocrine BON-1 tumor cells

Transient receptor potential channels (TRPs) regulate tumor growth via calcium-dependent mechanisms. The (thermosensitive) capsaicin receptor TRPV1 is overexpressed in numerous highly aggressive cancers. TRPV1 has potent antiproliferative activity and is therefore potentially applicable in targeted therapy of malignancies. Recently, we characterized TRPM8 functions in pancreatic neuroendocrine tumors (NETs), however, the role of TRPV1 is unknown. Here, we studied the expression and the role of TRPV1 in regulating intracellular Ca²⁺ and chromogranin A (CgA) secretion in pancreatic NET BON-1 cell line and in primary NET cells (prNET). TRPV1 expression was detected by RT-PCR, Western blot and immunofluorescence. Intracellular free Ca²⁺ ([Ca²⁺]_i) was measured by fura-2; TRPV1 channel currents by the planar patch-clamp technique. Nonselective cation currents were analyzed by a color-coded plot method and CgA secretion by ELISA. Pancreatic BON-1 cells and NETs express TRPV1. Pharmacological blockade of TRPs by La³⁺ (100 μM) or by ruthenium-red (RuR) or by capsazepine (CPZ) (both at 10 μM) suppressed the capsaicin (CAP)- or heat-stimulated increase of [Ca²⁺]_i in NET cells. CAP (20 μM) also increased nonselective cation channel currents in BON-1 cells. Furthermore, CAP (10 μM) stimulated CgA secretion, which was inhibited by CPZ or by RuR (both 10 μM). La³⁺ potently reduced both stimulated and the basal CgA secretion. Our study shows for the first time that TRPV1 is expressed in pancreatic NETs. Activation of TRPV1 translates into changes of intracellular Ca²⁺, a known mechanism triggering the secretion of CgA. The clinical relevance of TRPV1 activation in NETs requires further investigations.     

50-Hz electromagnetic environment and the incidence of childhood tumors in Stockholm County

The magnetic fields from overhead power lines and other electromagnetic sources were determined at the birth and diagnosis dwellings of all tumor cases reported in the county of Stockholm during the years 1958-73 for individuals 0-18 years of age. The study was limited to 716 cases having a permanent address in the county both at time of birth and diagnosis. An equivalent number of controls was matched to the cases according to church district of birth, age, and sex. Outside each dwelling, the occurrence of visible electrical constructions (6-200-kV high-voltage wires, substations, transformers, electric railroads, and subways) within 150 m of the dwelling was noted. Also, the 50-Hz magnetic field was measured outside the main entrance of the dwelling. Visible 200-kv wires were noted at 45 of 2,098 dwellings and were found twice as frequently among cases as among controls (P less than .05). The magnetic field measured at the dwelling varied between 0.0004 to 1.9 microT (mean value 0.069 microT). The magnetic field was higher (0.22 microT) at dwellings with visible 200-kV wires than at those without such wires. Magnetic fields of 0.3 microT or more were measured at 48 dwellings, and were found twice as frequently among cases as among controls (P less than .05). The difference was most pronounced for dwellings of nervous system tumors and was less for leukemias.     

Effects of prenatal exposure to 50 Hz magnetic fields on development in mice: I. Implantation rate and fetal development

Pregnant CD1 mice were exposed or sham-exposed from day 0 to day 17 of gestation to a 50 Hz sinusoidal magnetic field at 20 mT (rms). Preimplantation and postimplantation survival were assessed and fetuses examined for the presence of gross external, internal, and skeletal abnormalities. There were no statistically significant field-dependent effects on preimplantation or postimplantation survival, sex ratio, or the incidence of fetuses with internal or skeletal abnormalities. Magnetic field exposure was, however, associated with longer and heavier fetuses at term, with fewer external abnormalities. The results lend no support to suggestions of increased rates of spontaneous abortion or congenital malformation following prenatal exposure to power frequency magnetic fields. © 1994 Wiley-Liss, Inc.     

基于氧化应激与细胞凋亡探究双酚A慢性暴露致小鼠肾毒性作用机制

双酚A (bisphenol A, BPA)被广泛应用于生产环氧树脂和聚碳酸酯塑料等制品,在强酸、强碱或高温条件下,BPA被释放出来,然后渗入环境中。在大多数生物液体中都检测到了不同浓度的BPA,BPA的存在已被证明与许多慢性疾病密切相关,包括慢性肾病(chronic kidney disease,CKD)。然而,关于BPA的有害作用及其对CKD的不良影响知之甚少。为了探讨BPA对动物肾毒性的作用机制,本研究通过向饮水中加入0.01、0.1和1 mg/L的BPA,暴露于雌性小鼠4周后,交配和怀孕的雌性小鼠持续接触BPA,直到断奶;F1代3周龄雄性仔鼠继续口服相同剂量的BPA,持续10周。结果表明,0.1mg/L和1mg/LBPA处理组小鼠的肾脏损伤严重,血清中肾脏功能指标尿素氮(urea nitrogen,UN)、肌酐(creatinine,CR)和尿酸(uric acid,UA)的含量均发生显著升高(P<0.05);肾脏组织形态结构被损害;肾脏抗氧化相关基因在mRNA和蛋白水平上的表达显著降低(P<0.05),包括谷胱甘肽硫转移酶(glutathione-S-transf...     

Studies on the possible biological effects of 50 Hz electric and/or magnetic fields: Evaluation of some glycolytic enzymes,glycolytic flux,energy and oxido-reductive potentials in human erythrocytes exposed in vitro to power frequency fields

An attempt has been made to understand whether 50 Hz electric and magnetic fields (EMFs) are involved in producing bioeffects by exposing human erythrocytes in vitro. The study evaluated some key glycolytic enzymes, glucose consumption, lactate production, energy charge, 2,3-diphosphoglycerate, and reduced glutathione levels. all of which are biochemical parameters significant to erythrocyte function. Cells exposed to individual or superimposed EMFs have not shown any significant difference compared with the controls. © 1993 Wiley-Liss. Inc.