The response of human PMN zeta potential to selected ligands of the cAMP signal pathway under low-dose β-radiation

The response of human PMN zeta potential to selected ligands of the cAMP signal pathway under low-dose β-radiation | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The response of human PMN zeta potential to selected ligands of the cAMP signal pathway under low-dose β-radiation Victor Zhirnov, Igor Iakovenko This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4557523/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The objective of this study was to examine the zeta potential (ZP) response of human neutrophils to selected ligands of the cAMP signalling pathway under low-dose β-radiation (LDBR). ZP was calculated from the electrophoretic mobility (EPM) of the cells. Cells were irradiated at a dose rate of 10 µGy/hour for one hour. Irradiation of cell suspensions was conducted by adding appropriate aliquots of 14 C-leucine to the incubation medium. EPM measurements were carried out in a radiation field. It was demonstrated that the LDBR and agonists of the AC system are capable of increasing the negative surface charge of human neutrophils. Conversely, the antagonist propranolol has been shown to reduce it. Furthermore, it has been demonstrated that radiation affects the response of the membrane ZP to some ligands of the AC pathway and vice versa. Therefore, LDBR can significantly alter the sensitivity of cells to drugs acting on the targets of these ligands. These results do not permit the conclusion that the effect of LDBR on ZP is mediated through the targets with which the studied ligands interact, since any of its components may undergo structural rearrangement, thereby altering the structural organisation of neutrophil membranes in the LDBR field. Low-dose β-radiation Human PMN Zeta potential db-cAMP Theophylline Forskolin Cholera toxin Propranolol Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Ionising radiation is a constant factor affecting biota on an ongoing basis [ 1 ]. In accordance with the principles of physical science, the mechanisms underlying the interaction of radiation with matter remain unaltered throughout the entire dose range [ 2 ]. The results of calculations and experiments indicate that in living organisms most of the damage caused by radiation is not directly related to the effect of radiation on organic molecules. Mainly, they are a consequence of the products of water radiolysis, namely reactive oxygen species, which interact with a larger number of atoms than the particles of primary radiation [ 3 ]. It is well established that an increase in the radiation load on the human organism affects the body's adaptive and homoeostatic mechanisms, which in turn determines the general functional state of cells [ 4 , 5 ]. Concurrently, the biological effects of radiation exhibit a pronounced discrepancy in both magnitude and quality, contingent upon the radiation field rate and the absorbed dose [6 Mothersill C, Rusin A, Seymour C. Towards a New Concept of Low Dose. Health Phys. 2019 Sep;117(3):330–336. doi: 10.1097/HP.0000000000001074 ]. The surface electrostatic charge of biomembranes plays a significant role in cellular signalling, influencing the activity of signalling proteins and the structuring of signalosomes [ 7 ]. Conversely, the negative surface charge of the cell membrane is influenced by the three-dimensional structure of proteins, as well as the different types of amino acids and lipids present on the cell membrane [ 8 ]. The surface membrane potential thus serves as an indicator of the structural and functional state of the cell during its interaction with endogenous and exogenous factors. Previous studies have demonstrated that exposure to low-dose β-radiation (LDBR) at microgray absorbed levels alters the functional response of leukocytes, specifically the chemokinesis of polymorphonuclear leukocytes (PMN) and the rosette formation ability of lymphocytes, in response to receptor ligands [ 9 ]. Furthermore, it has been demonstrated that LDBR elevate the absolute value of the zeta potential (ZP av ) of human blood cells due to alterations in the structural and functional state of the components of the plasma membrane [ 10 ]. The objective of the present study was to determine the influence of a low-dose rate β-radiation field on the zeta potential response of human blood PMN to ligands of the adenylate cyclase signal system. 2. Material and methods 2.1. Materials Isoprenaline, forskolin, propranolol, theophylline, and dibutyryl-cAMP (dbwere from Sigma Chemical Company, St Louis, MO, USA. Radioactive L- 14 С]-leucine (specific radioactivity is 318 mCi⋅mmol − 1 ) was from Amersham Pharmacia, Uppsala, Sweden. All other chemicals were reagent grade or highest purity available. Human blood was obtained fresh from Kiev regional center of blood. 2.2. Separation of Human PMN from Peripheral Blood Blood from healthy donors was drawn into tubes containing sodium citrate as anticoagulant. The blood was stored at 4°C and used during the week. The PMN were purified with a one-step method on a high-density (specific gravity, 1.095) Ficoll- Hypaque gradient, as described by Ferrante and Thong [ 11 ]. Briefly, heparinized blood obtained from the tail vein was gently layered onto the Ficoll-Hypaque mixture and centrifuged at 200xg for 50 min at room temperature. After centrifugation, the band of PMN, separated below the mononuclear cells and above the erythrocytes, was gently aspirated, and cells were washed with precooled Hanks balanced salt solution without calcium and magnesium. Residual erythrocytes were lysed in a buffer containing 0.15 M NH 4 C1, 10 mM KHCO 3 , and 0.1 mM disodium EDTA (pH 7.4) at 4°C for 3 min. Double volume of Hanks balanced salt solution with calcium and magnesium (HBSS) was added and the tubes were spin at 650xg for 10 minutes at 4°C to remove the lysed erythrocytes. The final PMN pellet was resuspended into desired volume of HBSS to achieve the final cell concentration of 1·10 6 cells/ml. The PMN concentration was determined with a standard haemocytometer chamber (Celeromics, Grenoble, France). The cell suspension contained at least 90% PMN, and the remaining cells were predominantly eosinophils. The viability of the purified PMN was 98% as determined by trypan blue dye exclusion test. 2.3. Electrophoretic Mobility of Cells The electrophoretic mobility of cells as indicator of their zeta potential was determined in the electrophoretic solution (КСl – 2.5; NaCl – 5.6; СаСl 2 – 2.0; glucose – 280.0; Tris-HСl – 10.0 mM, pH = 7.4) at room temperature using a cylindrical microelectrophoresis (12). Glucose was added to the buffer to keep the low ionic strength buffer isotonic with the cells. The investigation was carried out at a low ionic strength, as the influence of electrical field of the cell suspension will maximize any cellular changes that might be occurring (13). The zeta potential was calculated from the electrophoretic mobility value using Zeta for Windows. ( https://zeta-uploader.ru.malavida.com/windows/ ). 2.4. Cell Exposure to Drugs and Radionuclides Drug treatment was realized by cell incubation in the medium, which contained appropriate drug concentrations as pointed out in table 1 and figures. Drugs were added to the medium 10 min. before addition of radionuclides. The irradiation was performed by incubating the cells with 14 С-leucine (370 kBq∙l − 1 ) in the electrophoretic solution containing approximately 1⋅10 6 cells∙ml − 1 for one hour at 25 0 С. Aassuming that the radionuclides are uniformly distributed throughout the cell suspension, the dose rates were calculated as described previously [ 10 ]. 2.5. Data Analysis Statistical analysis of the results and the curve fitting was performed by means of the program of Statistica v6.0 for Windows. Significant statistical differences between two groups were evaluated using the unpaired Student t-test (p < 0.05). The data are given as means ± SEM (standard error of mean); n represents the number of the cell suspensions studied. 3. Results Previously, we have experimentally determined that nonradioactive leucine at a concentration in cell incubation medium equal to that of radioactive leucine (0.16 µM) used in the experiments did not influence the cell membrane ZP (data not shown). The data presented in Figur 1 show that in the dose range from 1 to 100 nGy, not exceeding the background value (~ 260 nGy), LDBR does not affect ZP. As the absorbed radiation dose increases, the curve passes through a maximum at 10 µGy. A decrease in ZP av at higher doses may indicate a tendency toward a reversal of the response, since at high doses there is a post-irradiation decrease in surface negative charge [ 14 , 15 ]. The dose of LDBR that produced the greatest ZP response was employed in subsequent experiments. Insert Fig. 1 here As demonstrated in Table, the β-radiation field (10 µGy·h − 1 ) resulted in an increase in the absolute value of zeta potential (ZP av ) of human PMN cells, with a range of 21 to 37% in comparison with the control. Insert Table here The full β-adrenergic receptor agonist isoprenaline also demonstrated a significant increase in ZP av relative to the control at all concentrations employed. The maximum effect is observed at the ligand concentration of 100 µM, with an increase of 44.3%. At other concentrations of isoprenaline, there is a dose-independent increase in ZP av by an average of 33%. The β-radiation field enhances the ZP av response to isoprenaline at a concentration of the latter of 100 µM compared to the control, but does not significantly change it in the range of 0.1–10 µM. The effect of the radiation field is the only one to manifest itself at a ligand concentration of 10 nM, despite the unidirectional influence of these factors on ZP value (Table, Fig. 2). Insert Fig. 2 here Forskolin, a direct activator of adenylate cyclase, was found to increase the ZP av of PMN by approximately 20% compared to control in a dose-independent manner at concentrations ranging from 100 nM to 100 µM. The β-radiation field, which increases ZP av by 27%, enhances the response to forskolin by approximately twofold (up to 40%), indicating an additive response nature (Table, Fig. 3). Furthermore, the inactive concentration of forskolin (10 nM) demonstrated intrinsic efficacy comparable to higher doses. This was evidenced by a greater response to this dose of forskolin in the radiation field than to the IR itself. Insert Fig. 3 here Theophylline, a competitive nonselective inhibitor of phosphodiesterase, which prevents cAMP degradation, changes ZP of human PMN similarly to isoprenaline. The β-radiation field does not change the ZP response to theophylline in concentration range studied (Table). The membrane-permeable cAMP analogue, db-cAMP, at concentrations of 1-100 µM increases ZP av of PMN about 30% relative to control, and the β-radiation field does not change this response. The concentrations of db-cAMP less than 1 µM does not cause the reliable changes in value of ZP but the radiation field potentiates the ZP response to the drug approximately 20% (Table, Fig. 4). Insert Fig. 4 here As illustrated in Table, cholera toxin, a constitutive activator of AC, at a concentration of 100 nM exhibits a similar effect to that of a β-radiation field alone, namely an increase in ZPav of PMN of approximately 20% (17.85 ± 0.56 mV against 14.93 ± 0.49 mV in control). The β-radiation field does not change the ZP response to cholera toxin (-17.97 ± 0.58 mV). Propranolol, an invers agonist of β-adrenoceptor, снижающий базальный уровень цАМФ в клетке, at a minimal concentration (10 nM) does not exert any influence. At 100 nM, however, it has been observed to decrease ZP by approximately 10% relative to the control (Table, Fig. 5). Insert Fig. 5 here The further increasing in drug concentration reduces ZP av by up to 27% dose-independently. The β-radiation field has been demonstrated to eliminate the effect of propranolol at 100 nM, and to decrease it at doses of 1 to 100 µM, regardless of the dose applied. 4. Discussion The findings of this study indicate that the ligands interacting with the various components of the adenylyl cyclase pathway alter the ZP of human PMN. Agonists of AC pathway elevate the ZP av of human PMN. Conversely, the antagonist propranolol has the effect of reducing the ZP av of human PMN. Furthermore, LDBR has been observed to alter ZP in a manner analogous to that observed with the agonists of the AC system. These results do not permit the conclusion that the effect of LDBR on ZP is mediated through the targets with which the studied ligands interact, since any of its components may undergo structural rearrangement, thereby altering the structural organisation of neutrophil membranes in the LDBR field. Furthermore, the reversibility of the effects of LDBR on the functional activity of cells [ 16 ] presents a significant challenge in assessing these effects on the whole organism when using external irradiation. In order to achieve this, it is necessary to record the cellular response of the organism in the field of the radiation source. An alternative approach is to administer the radionuclide to the organism at the requisite concentration. However, in this case, it is necessary to overcome the methodological difficulties of recording the effects of ionizing radiation associated with the preservation of the initial level of radionuclide in the measurement medium. Furthermore, cellular responses to the radiation under in vivo conditions may manifest at dose rates that differ from those observed under in vitro conditions. Nevertheless, the data obtained permit an integrated evaluation of the recorded change in ZP on the functional activity of cells in the LDBR field. As mentioned above, we demonstrated that LDBR-induced increases in ZP of lymphocytes are accompanied by a reduction in the rosette-forming ability of lymphocytes [ 9 ]. Forskolin has a comparable effect, while the PLA2 inhibitor quinacrine exerts an enhancing influence. The data presented here suggests that the LDBR field may be influencing the functioning of signalling pathways that regulate cell activity through the ZP [ 17 , 18 ]. So, with regard to function, increasing ZP av in cells is one of the defining characteristics of apoptosis in animal cells [ 19 ]. Consequently, it may be posited that an increase in ZP av in human PMN cells could be considered as a proapoptotic stimulus. However, agents that increase ZPav of human PMN and increase cAMP concentrations (isoprenaline, forskolin, db-cAMP, and cholera toxin) delay PMN apoptosis in the absence of pro-survival stimuli, but show the opposite effect in their presence [ 20 ]. This phenomenon can be attributed to the compartmentalisation of the signalling pathways of different signalosomes, which utilise the same secondary messenger, in this case cAMP [ 21 , 22 ]. Theophylline shows dual effects on apoptosis in spite of monodirectional influence on the ZP, depending on the concentration, increasing the cAMP pool of various signalosomes [ 23 ]. In contrast, a nonspecific antagonist of β 1 - и β 2− adrenergic receptors propranolol, reduces the negative charge on the membrane (Fig. 4), и induces apoptosis of neutrophils [ 24 , 25 ], and many other cells, apparently due to interaction with signalosomes with a unidirectional effect on apoptosis [ 26 ]. Consequently, the ZP of neutrophils is not a reliable indicator of their apoptosis. Consequently, the observed alteration in neutrophil ZP resulting from the ligands of the AC signaling pathway can be attributed to electrogenic structural rearrangements of the plasma membrane, which are a consequence of ligand-target interactions. The final result of these rearrangements, expressed in an increase or decrease in the surface charge of the neutrophil membrane, appears to reflect their general functional state, which in turn affects their biological behaviour. To illustrate, the surface charge of a cell can influence the adsorption of liposomes on the cell surface [ 27 ] and endocytosis [ 28 ]. With regard to neutrophils, it has been demonstrated that when granulocytes were labelled in vitro with radioactive diisopropylfluorophosphate and then returned to the circulation of the donor, approximately half of the labelled cells could not be found in the circulation at the conclusion of the infusion [ 30 , 31 ]. It can be posited that the remaining labelled cells (with low surface charge) exited the circulation in a random manner. The average proportion of labelled granulocytes that were infused and subsequently found in the circulating blood was 79% when epinephrine was administered in conjunction with the infusion of cells. This phenomenon can be attributed to an increase in the surface charge of cells, which in turn results in a reduction in intercellular adhesion [ 32 ]. Consequently, an increase in the fast population (with a high negative surface charge) is accompanied by an increase in PMN in the circulating blood. Regarding the mechanism of action of LDBR on ZP, its reversibility and lack of accumulation of secondary products of lipid peroxidation in cells have been previously demonstrated [ 9 , 16 , 33 ]. Consequently, the antioxidant activity of cells can inactivate excess reactive oxygen species induced by low-dose ionising radiation. Conversely, reactive oxygen species in a range of physiological concentrations regulate cellular functions through cellular signalling systems [ 34 ]. These processes are initiated by a reversible conformational rearrangement of membrane macromolecules. Furthermore, the cell responses to the imposed LDBR may also be mediated by the non-ionising radiation generated by β-radiation [ 35 ]. Conclusion In conclusion, the action of the imposed LDBR field on the surface potential of PMN is achieved through the structural and functional penetration of the plasma membrane. The radiation exerts an influence on the membrane ZP's response to certain ligands of the AC pathway. Consequently, the radiation can markedly alter the sensitivity of cells to drugs that target the sensitive components of this pathway with LDBR exposure. Conversely, some drugs, such as theophylline and propranolol, even at concentrations that do not elicit a cellular response when exposed to the target (≤ 50 µM), can neutralise the effect of LDBR on neutrophil ZP. It is also important to note that ZP responds only to electrogenic changes in the structure of the biological membrane. However, this does not exclude its influence on neutral conformational rearrangements of membrane components, which should be recorded by appropriate methods. Given that a LDBR can induce significant alterations to the structure and functional state of biomembranes, the utilisation of radioactive labels for the investigation of native functional activity of membrane-bound proteins is inherently problematic. Consequently, the validation of the derived results necessitates the application of non-radioisotope methodologies. The data obtained do not permit an assessment of their medical and biological significance, as the final effect of LDBR on ZP cells may be modified by a number of factors, including the influence of the cell microenvironment, the inclusion of adaptive mechanisms developed in the process of evolution, and others. The use of radionuclides enables the recording of spontaneously reversible changes in the structural and functional state of isolated cells that arise in the field of applied low-dose radiation. This is because post-radiation effects, traditionally studied by radiobiologists, are not recorded after the removal of irradiation if they have a non-damaging effect. Declarations The authors declare no conflict of interest, financial or otherwise in this work. The authors alone are responsible for the content and writing of the paper. The article was funded by National Academy of Sciences of Ukraine. Author Contribution V.Z. wrote the main manuscript text and prepared figures 1-5. I.I. performed the experimental part and prepared Table References Duarte GT, Volkova PY, Fiengo Perez F, Horemans N (2023) Chronic Ionizing Radiation of Plants: An Evolutionary Factor from Direct Damage to Non-Target Effects. Plants (Basel). 12, 1178. doi: 10.3390/plants12051178 . Matthews EP (2019) Radiation Physics, Biology, and Protection. Radiol Technol. 90, 471–485. PMID: 31088948. Baskar R, Dai J, Wenlong N, Yeo R, Yeoh KW (2014) Biological response of cancer cells to radiation treatment. 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ZP response (mV) of human PMN to ligands of adenylyl cyclase pathway under the β-radiation field (10 µGy h -1 ) Drugs Control 14 C Drug concentration, М 10 -8 10 -7 10 -6 10 -5 10 -4 Isoprenaline -13.53 ±0.34 - -18.12 ±0.73* -18.15 ±0.72* -18.09 ±0.79* -17.81 ±0.68* -19.52 ±0.77* Isoprenaline + 14 C -16.44 ±0.48* -16.27 ±0.55* & -17.29 ±0.57* -18.15 ±0.54* -18.66 ± 0.49* -21.44 ±0.61* & Forskolin -14.32 ±0.32 - -14.90 ±0.34 -17.64 ±0.50* -17.60 ±0.49* -17.12± 0.58* -17.29± 0.51* Forskolin + 14 C -18.24 ±0.51* -20.04 ±0.56٭ & -20.90 ±0.68* & -19.70 ±0.54* & -21.41 ±0.63* & -20.21 ±0.67* & Theophylline -14.79 ±0.31 - -18.71 ±0.48* -19.53 ±0.53* -19.18 ±0.55* -19.73 ±0.51* -19.52 ±0.57* Theophylline + 14 C -20.33 ±0.49* -19.01 ±0.51* -19.70 ±0.50* -19.01 ±0.44* -18.50 ±0.49* -18.64 ±0.58* Dibutiryl-cAMP -15.24 ±0.35 - -15.93± 0.55 -15.96 ±0.59 -20.25 ±0.56* -20.04 ±0.49* -20.19 ±0.41* Dibutiryl cAMP + 14 C -19.35 ±0.47* -19.67 ±0.49* & -19.13 ±0.37* & -19.18 ±0.39* -20.73 ±0.54* -19.53 ± 0.49* Propranolol -15.59 ±0.27 - -15.23 ±0.28 -13.87 ±0.41* -11.30 ±0.34* -11.34 ±0.35* -11.47 ±0.30* Propranolol + 14 C -21.77 ±0.51*. -21.57 ± 0.62* & -21.24 ±0.69* & -14.90 ±0.36 & -14.56 ±0.38 *& -14.21 ±0.34 *& The aliquot of 14 C solution was added in the cell suspension to obtain the final activity concentration 370 kBq∙l -1 . Drugs were added to the medium 10 min. before addition of radionuclides. The cells were incubated with radionuclides for 1 hour. Value of ZP was calculated from microelectrophoresis measurements of the PMN mobility in electrophoretic medium. * and & - significance difference from the control (background) or under the radiation field, consequently, p £ 0.05, n = 10. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4557523","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":317933302,"identity":"bc7d24b1-bd03-4c41-bff1-7b019a5bdcd3","order_by":0,"name":"Victor Zhirnov","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYDACZgbGx0CKh4GHsYFoLczGJGphYGCTBlM8xKo3OM78rLpwh50MA8/hxsc8DIflCWs5zGZ2e+aZZB4G3sZmY6AWQ4LOk2zmYbvN23aAh4GfsU1yBsNhwj4CaSmGamn/CdRiT1ALPzMPGzNYC29jG8MHhsOJRGhhM5YG+YWN52CzxAeD9GSCWtj4Dz/8DAwxe36e9IcfEiqsbQlqAQOQl9nALAOi1EO1jIJRMApGwSjACQCOKDCrKxilYAAAAABJRU5ErkJggg==","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Victor","middleName":"","lastName":"Zhirnov","suffix":""},{"id":317933303,"identity":"893af6e1-422b-4b23-8967-110f3a9bb557","order_by":1,"name":"Igor Iakovenko","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Igor","middleName":"","lastName":"Iakovenko","suffix":""}],"badges":[],"createdAt":"2024-06-10 10:51:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4557523/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4557523/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59201344,"identity":"61ec1a10-6be2-46bd-a76f-3e1e9a76fa39","added_by":"auto","created_at":"2024-06-27 15:11:51","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":100714,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4557523/v1/ef104a3c34ec12d08c9f92d9.jpg"},{"id":59200892,"identity":"82fa2eae-9143-4096-8c24-fb5215282857","added_by":"auto","created_at":"2024-06-27 15:03:52","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":170818,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4557523/v1/ee5ae1624639f6ffa757556d.jpg"},{"id":59200887,"identity":"7eba9b61-976e-46c1-ad91-cbfab1b65f45","added_by":"auto","created_at":"2024-06-27 15:03:51","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":156911,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4557523/v1/c6d108ce342fc2ea18bee622.jpg"},{"id":59201729,"identity":"947758ea-3d81-49be-866b-bb4633250d40","added_by":"auto","created_at":"2024-06-27 15:19:52","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":222779,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4557523/v1/e1eb845da801614036dba3d4.jpg"},{"id":59201345,"identity":"12ee0e79-c043-4c39-8a8c-0e043a542f1d","added_by":"auto","created_at":"2024-06-27 15:11:52","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":138079,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4557523/v1/7a7ab8c5f049b90f7471048d.jpg"},{"id":74143215,"identity":"ccc98428-fec7-4a88-b126-828e0fdfa36a","added_by":"auto","created_at":"2025-01-18 14:23:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1360593,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4557523/v1/8c57e12e-82c5-4747-953d-21446eef1d6f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eThe response of human PMN zeta potential to selected ligands of the cAMP signal pathway under low-dose β-radiation\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIonising radiation is a constant factor affecting biota on an ongoing basis [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. In accordance with the principles of physical science, the mechanisms underlying the interaction of radiation with matter remain unaltered throughout the entire dose range [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The results of calculations and experiments indicate that in living organisms most of the damage caused by radiation is not directly related to the effect of radiation on organic molecules. Mainly, they are a consequence of the products of water radiolysis, namely reactive oxygen species, which interact with a larger number of atoms than the particles of primary radiation [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. It is well established that an increase in the radiation load on the human organism affects the body's adaptive and homoeostatic mechanisms, which in turn determines the general functional state of cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Concurrently, the biological effects of radiation exhibit a pronounced discrepancy in both magnitude and quality, contingent upon the radiation field rate and the absorbed dose [6 Mothersill C, Rusin A, Seymour C. Towards a New Concept of Low Dose. Health Phys. 2019 Sep;117(3):330\u0026ndash;336. doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1097/HP.0000000000001074\u003c/span\u003e\u003cspan address=\"10.1097/HP.0000000000001074\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe surface electrostatic charge of biomembranes plays a significant role in cellular signalling, influencing the activity of signalling proteins and the structuring of signalosomes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Conversely, the negative surface charge of the cell membrane is influenced by the three-dimensional structure of proteins, as well as the different types of amino acids and lipids present on the cell membrane [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The surface membrane potential thus serves as an indicator of the structural and functional state of the cell during its interaction with endogenous and exogenous factors. Previous studies have demonstrated that exposure to low-dose β-radiation (LDBR) at microgray absorbed levels alters the functional response of leukocytes, specifically the chemokinesis of polymorphonuclear leukocytes (PMN) and the rosette formation ability of lymphocytes, in response to receptor ligands [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Furthermore, it has been demonstrated that LDBR elevate the absolute value of the zeta potential (ZP\u003csub\u003eav\u003c/sub\u003e) of human blood cells due to alterations in the structural and functional state of the components of the plasma membrane [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe objective of the present study was to determine the influence of a low-dose rate β-radiation field on the zeta potential response of human blood PMN to ligands of the adenylate cyclase signal system.\u003c/p\u003e"},{"header":"2. Material and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials\u003c/h2\u003e \u003cp\u003eIsoprenaline, forskolin, propranolol, theophylline, and dibutyryl-cAMP (dbwere from Sigma Chemical Company, St Louis, MO, USA. Radioactive L-\u003csup\u003e14\u003c/sup\u003eС]-leucine (specific radioactivity is 318 mCi\u0026sdot;mmol\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) was from Amersham Pharmacia, Uppsala, Sweden. All other chemicals were reagent grade or highest purity available. Human blood was obtained fresh from Kiev regional center of blood.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Separation of Human PMN from Peripheral Blood\u003c/h2\u003e \u003cp\u003eBlood from healthy donors was drawn into tubes containing sodium citrate as anticoagulant. The blood was stored at 4\u0026deg;C and used during the week. The PMN were purified with a one-step method on a high-density (specific gravity, 1.095) Ficoll- Hypaque gradient, as described by Ferrante and Thong [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Briefly, heparinized blood obtained from the tail vein was gently layered onto the Ficoll-Hypaque mixture and centrifuged at 200xg for 50 min at room temperature. After centrifugation, the band of PMN, separated below the mononuclear cells and above the erythrocytes, was gently aspirated, and cells were washed with precooled Hanks balanced salt solution without calcium and magnesium. Residual erythrocytes were lysed in a buffer containing 0.15 M NH\u003csub\u003e4\u003c/sub\u003eC1, 10 mM KHCO\u003csub\u003e3\u003c/sub\u003e, and 0.1 mM disodium EDTA (pH 7.4) at 4\u0026deg;C for 3 min. Double volume of Hanks balanced salt solution with calcium and magnesium (HBSS) was added and the tubes were spin at 650xg for 10 minutes at 4\u0026deg;C to remove the lysed erythrocytes. The final PMN pellet was resuspended into desired volume of HBSS to achieve the final cell concentration of 1\u0026middot;10\u003csup\u003e6\u003c/sup\u003e cells/ml. The PMN concentration was determined with a standard haemocytometer chamber (Celeromics, Grenoble, France). The cell suspension contained at least 90% PMN, and the remaining cells were predominantly eosinophils. The viability of the purified PMN was 98% as determined by trypan blue dye exclusion test.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Electrophoretic Mobility of Cells\u003c/h2\u003e \u003cp\u003eThe electrophoretic mobility of cells as indicator of their zeta potential was determined in the electrophoretic solution (КСl \u0026ndash; 2.5; NaCl \u0026ndash; 5.6; СаСl\u003csub\u003e2\u003c/sub\u003e\u0026ndash; 2.0; glucose \u0026ndash; 280.0; Tris-HСl \u0026ndash; 10.0 mM, pH\u0026thinsp;=\u0026thinsp;7.4) at room temperature using a cylindrical microelectrophoresis (12). Glucose was added to the buffer to keep the low ionic strength buffer isotonic with the cells. The investigation was carried out at a low ionic strength, as the influence of electrical field of the cell suspension will maximize any cellular changes that might be occurring (13). The zeta potential was calculated from the electrophoretic mobility value using Zeta for Windows. (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://zeta-uploader.ru.malavida.com/windows/\u003c/span\u003e\u003cspan address=\"https://zeta-uploader.ru.malavida.com/windows/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Cell Exposure to Drugs and Radionuclides\u003c/h2\u003e \u003cp\u003eDrug treatment was realized by cell incubation in the medium, which contained appropriate drug concentrations as pointed out in table 1 and figures. Drugs were added to the medium 10 min. before addition of radionuclides. The irradiation was performed by incubating the cells with \u003csup\u003e14\u003c/sup\u003eС-leucine (370 kBq∙l\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in the electrophoretic solution containing approximately 1\u0026sdot;10\u003csup\u003e6\u003c/sup\u003e cells∙ml\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e for one hour at 25 \u003csup\u003e0\u003c/sup\u003eС. Aassuming that the radionuclides are uniformly distributed throughout the cell suspension, the dose rates were calculated as described previously [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Data Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the results and the curve fitting was performed by means of the program of Statistica v6.0 for Windows. Significant statistical differences between two groups were evaluated using the unpaired Student t-test (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The data are given as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM (standard error of mean); n represents the number of the cell suspensions studied.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003ePreviously, we have experimentally determined that nonradioactive leucine at a concentration in cell incubation medium equal to that of radioactive leucine (0.16 \u0026micro;M) used in the experiments did not influence the cell membrane ZP (data not shown).\u003c/p\u003e \u003cp\u003eThe data presented in Figur 1 show that in the dose range from 1 to 100 nGy, not exceeding the background value (~\u0026thinsp;260 nGy), LDBR does not affect ZP. As the absorbed radiation dose increases, the curve passes through a maximum at 10 \u0026micro;Gy. A decrease in ZP\u003csub\u003eav\u003c/sub\u003e at higher doses may indicate a tendency toward a reversal of the response, since at high doses there is a post-irradiation decrease in surface negative charge [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. The dose of LDBR that produced the greatest ZP response was employed in subsequent experiments.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInsert Fig.\u0026nbsp;1 here\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs demonstrated in Table, the β-radiation field (10 \u0026micro;Gy\u0026middot;h\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) resulted in an increase in the absolute value of zeta potential (ZP\u003csub\u003eav\u003c/sub\u003e) of human PMN cells, with a range of 21 to 37% in comparison with the control.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInsert Table here\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe full β-adrenergic receptor agonist isoprenaline also demonstrated a significant increase in ZP\u003csub\u003eav\u003c/sub\u003e relative to the control at all concentrations employed. The maximum effect is observed at the ligand concentration of 100 \u0026micro;M, with an increase of 44.3%. At other concentrations of isoprenaline, there is a dose-independent increase in ZP\u003csub\u003eav\u003c/sub\u003e by an average of 33%. The β-radiation field enhances the ZP\u003csub\u003eav\u003c/sub\u003e response to isoprenaline at a concentration of the latter of 100 \u0026micro;M compared to the control, but does not significantly change it in the range of 0.1\u0026ndash;10 \u0026micro;M. The effect of the radiation field is the only one to manifest itself at a ligand concentration of 10 nM, despite the unidirectional influence of these factors on ZP value (Table, Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInsert Fig.\u0026nbsp;2 here\u003c/b\u003e \u003c/p\u003e \u003cp\u003eForskolin, a direct activator of adenylate cyclase, was found to increase the ZP\u003csub\u003eav\u003c/sub\u003e of PMN by approximately 20% compared to control in a dose-independent manner at concentrations ranging from 100 nM to 100 \u0026micro;M. The β-radiation field, which increases ZP\u003csub\u003eav\u003c/sub\u003e by 27%, enhances the response to forskolin by approximately twofold (up to 40%), indicating an additive response nature (Table, Fig.\u0026nbsp;3). Furthermore, the inactive concentration of forskolin (10 nM) demonstrated intrinsic efficacy comparable to higher doses. This was evidenced by a greater response to this dose of forskolin in the radiation field than to the IR itself.\u003c/p\u003e \u003cp\u003e \u003cb\u003eInsert Fig.\u0026nbsp;3 here\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTheophylline, a competitive nonselective inhibitor of phosphodiesterase, which prevents cAMP degradation, changes ZP of human PMN similarly to isoprenaline. The β-radiation field does not change the ZP response to theophylline in concentration range studied (Table).\u003c/p\u003e \u003cp\u003eThe membrane-permeable cAMP analogue, db-cAMP, at concentrations of 1-100 \u0026micro;M increases ZP\u003csub\u003eav\u003c/sub\u003e of PMN about 30% relative to control, and the β-radiation field does not change this response. The concentrations of db-cAMP less than 1 \u0026micro;M does not cause the reliable changes in value of ZP but the radiation field potentiates the ZP response to the drug approximately 20% (Table, Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInsert Fig.\u0026nbsp;4 here\u003c/b\u003e \u003c/p\u003e \u003cp\u003eAs illustrated in Table, cholera toxin, a constitutive activator of AC, at a concentration of 100 nM exhibits a similar effect to that of a β-radiation field alone, namely an increase in ZPav of PMN of approximately 20% (17.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.56 mV against 14.93\u0026thinsp;\u0026plusmn;\u0026thinsp;0.49 mV in control). The β-radiation field does not change the ZP response to cholera toxin (-17.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58 mV).\u003c/p\u003e \u003cp\u003ePropranolol, an invers agonist of β-adrenoceptor, снижающий базальный уровень цАМФ в клетке, at a minimal concentration (10 nM) does not exert any influence. At 100 nM, however, it has been observed to decrease ZP by approximately 10% relative to the control (Table, Fig.\u0026nbsp;5).\u003c/p\u003e \u003cp\u003e \u003cb\u003eInsert Fig.\u0026nbsp;5 here\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe further increasing in drug concentration reduces ZP\u003csub\u003eav\u003c/sub\u003e by up to 27% dose-independently. The β-radiation field has been demonstrated to eliminate the effect of propranolol at 100 nM, and to decrease it at doses of 1 to 100 \u0026micro;M, regardless of the dose applied.\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThe findings of this study indicate that the ligands interacting with the various components of the adenylyl cyclase pathway alter the ZP of human PMN. Agonists of AC pathway elevate the ZP\u003csub\u003eav\u003c/sub\u003e of human PMN. Conversely, the antagonist propranolol has the effect of reducing the ZP\u003csub\u003eav\u003c/sub\u003e of human PMN. Furthermore, LDBR has been observed to alter ZP in a manner analogous to that observed with the agonists of the AC system. These results do not permit the conclusion that the effect of LDBR on ZP is mediated through the targets with which the studied ligands interact, since any of its components may undergo structural rearrangement, thereby altering the structural organisation of neutrophil membranes in the LDBR field. Furthermore, the reversibility of the effects of LDBR on the functional activity of cells [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] presents a significant challenge in assessing these effects on the whole organism when using external irradiation. In order to achieve this, it is necessary to record the cellular response of the organism in the field of the radiation source. An alternative approach is to administer the radionuclide to the organism at the requisite concentration. However, in this case, it is necessary to overcome the methodological difficulties of recording the effects of ionizing radiation associated with the preservation of the initial level of radionuclide in the measurement medium. Furthermore, cellular responses to the radiation under \u003cem\u003ein vivo\u003c/em\u003e conditions may manifest at dose rates that differ from those observed under \u003cem\u003ein vitro\u003c/em\u003e conditions. Nevertheless, the data obtained permit an integrated evaluation of the recorded change in ZP on the functional activity of cells in the LDBR field. As mentioned above, we demonstrated that LDBR-induced increases in ZP of lymphocytes are accompanied by a reduction in the rosette-forming ability of lymphocytes [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Forskolin has a comparable effect, while the PLA2 inhibitor quinacrine exerts an enhancing influence. The data presented here suggests that the LDBR field may be influencing the functioning of signalling pathways that regulate cell activity through the ZP [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. So, with regard to function, increasing ZP\u003csub\u003eav\u003c/sub\u003e in cells is one of the defining characteristics of apoptosis in animal cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Consequently, it may be posited that an increase in ZP\u003csub\u003eav\u003c/sub\u003e in human PMN cells could be considered as a proapoptotic stimulus. However, agents that increase ZPav of human PMN and increase cAMP concentrations (isoprenaline, forskolin, db-cAMP, and cholera toxin) delay PMN apoptosis in the absence of pro-survival stimuli, but show the opposite effect in their presence [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. This phenomenon can be attributed to the compartmentalisation of the signalling pathways of different signalosomes, which utilise the same secondary messenger, in this case cAMP [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Theophylline shows dual effects on apoptosis in spite of monodirectional influence on the ZP, depending on the concentration, increasing the cAMP pool of various signalosomes [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In contrast, a nonspecific antagonist of β\u003csub\u003e1\u003c/sub\u003e- и β\u003csub\u003e2\u0026minus;\u003c/sub\u003eadrenergic receptors propranolol, reduces the negative charge on the membrane (Fig.\u0026nbsp;4), и induces apoptosis of neutrophils [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and many other cells, apparently due to interaction with signalosomes with a unidirectional effect on apoptosis [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Consequently, the ZP of neutrophils is not a reliable indicator of their apoptosis.\u003c/p\u003e \u003cp\u003eConsequently, the observed alteration in neutrophil ZP resulting from the ligands of the AC signaling pathway can be attributed to electrogenic structural rearrangements of the plasma membrane, which are a consequence of ligand-target interactions. The final result of these rearrangements, expressed in an increase or decrease in the surface charge of the neutrophil membrane, appears to reflect their general functional state, which in turn affects their biological behaviour. To illustrate, the surface charge of a cell can influence the adsorption of liposomes on the cell surface [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] and endocytosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. With regard to neutrophils, it has been demonstrated that when granulocytes were labelled \u003cem\u003ein vitro\u003c/em\u003e with radioactive diisopropylfluorophosphate and then returned to the circulation of the donor, approximately half of the labelled cells could not be found in the circulation at the conclusion of the infusion [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. It can be posited that the remaining labelled cells (with low surface charge) exited the circulation in a random manner. The average proportion of labelled granulocytes that were infused and subsequently found in the circulating blood was 79% when epinephrine was administered in conjunction with the infusion of cells. This phenomenon can be attributed to an increase in the surface charge of cells, which in turn results in a reduction in intercellular adhesion [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Consequently, an increase in the fast population (with a high negative surface charge) is accompanied by an increase in PMN in the circulating blood.\u003c/p\u003e \u003cp\u003eRegarding the mechanism of action of LDBR on ZP, its reversibility and lack of accumulation of secondary products of lipid peroxidation in cells have been previously demonstrated [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Consequently, the antioxidant activity of cells can inactivate excess reactive oxygen species induced by low-dose ionising radiation. Conversely, reactive oxygen species in a range of physiological concentrations regulate cellular functions through cellular signalling systems [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. These processes are initiated by a reversible conformational rearrangement of membrane macromolecules. Furthermore, the cell responses to the imposed LDBR may also be mediated by the non-ionising radiation generated by β-radiation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eConclusion\u003c/b\u003e \u003c/p\u003e \u003cp\u003eIn conclusion, the action of the imposed LDBR field on the surface potential of PMN is achieved through the structural and functional penetration of the plasma membrane. The radiation exerts an influence on the membrane ZP's response to certain ligands of the AC pathway. Consequently, the radiation can markedly alter the sensitivity of cells to drugs that target the sensitive components of this pathway with LDBR exposure. Conversely, some drugs, such as theophylline and propranolol, even at concentrations that do not elicit a cellular response when exposed to the target (\u0026le;\u0026thinsp;50 \u0026micro;M), can neutralise the effect of LDBR on neutrophil ZP. It is also important to note that ZP responds only to electrogenic changes in the structure of the biological membrane. However, this does not exclude its influence on neutral conformational rearrangements of membrane components, which should be recorded by appropriate methods. Given that a LDBR can induce significant alterations to the structure and functional state of biomembranes, the utilisation of radioactive labels for the investigation of native functional activity of membrane-bound proteins is inherently problematic. Consequently, the validation of the derived results necessitates the application of non-radioisotope methodologies. The data obtained do not permit an assessment of their medical and biological significance, as the final effect of LDBR on ZP cells may be modified by a number of factors, including the influence of the cell microenvironment, the inclusion of adaptive mechanisms developed in the process of evolution, and others. The use of radionuclides enables the recording of spontaneously reversible changes in the structural and functional state of isolated cells that arise in the field of applied low-dose radiation. This is because post-radiation effects, traditionally studied by radiobiologists, are not recorded after the removal of irradiation if they have a non-damaging effect.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eThe authors declare no conflict of interest, financial or otherwise in this work. 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Braz J Phys. 40(1):38\u0026ndash;46. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003edoi.org/10.1590/S0103-97332010000100007\u003c/span\u003e\u003cspan address=\"10.1590/S0103-97332010000100007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Table","content":"\u003cp\u003e\u003cstrong\u003eTable.\u0026nbsp;\u003c/strong\u003eZP response (mV) of human PMN to ligands of adenylyl cyclase pathway under the \u0026beta;-radiation field (10 \u0026micro;Gy h\u003csup\u003e-1\u003c/sup\u003e)\u003c/p\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eDrugs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003eControl\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e\u003csup\u003e14\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"5\"\u003e\n \u003cp\u003eDrug concentration, М\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003csup\u003e-8\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003csup\u003e-7\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003csup\u003e-6\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003csup\u003e-5\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e10\u003csup\u003e-4\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIsoprenaline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e-13.53\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-18.12\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.73*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-18.15\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.72*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-18.09\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.79*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-17.81\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.68*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.52\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.77*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eIsoprenaline\u0026nbsp;+\u0026nbsp;\u003csup\u003e14\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-16.44\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.48*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-16.27\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.55*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-17.29\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.57*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-18.15\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.54*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-18.66\u003c/p\u003e\n \u003cp\u003e\u0026plusmn; 0.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-21.44\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.61*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eForskolin\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e-14.32\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-14.90\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-17.64\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.50*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-17.60\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-17.12\u0026plusmn;\u003c/p\u003e\n \u003cp\u003e0.58*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-17.29\u0026plusmn;\u003c/p\u003e\n \u003cp\u003e0.51*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eForskolin\u0026nbsp;+\u0026nbsp;\u003csup\u003e14\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-18.24\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.51*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-20.04\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.56٭\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-20.90\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.68*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.70\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.54*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-21.41\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.63*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-20.21\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.67*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTheophylline\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e-14.79\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-18.71\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.48*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.53\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.53*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.18\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.55*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.73\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.51*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.52\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.57*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eTheophylline\u0026nbsp;+\u0026nbsp;\u003csup\u003e14\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-20.33\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.01\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.51*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.70\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.50*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.01\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.44*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-18.50\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-18.64\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.58*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDibutiryl-cAMP\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e-15.24\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.35\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-15.93\u0026plusmn;\u003c/p\u003e\n \u003cp\u003e0.55\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-15.96\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.59\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-20.25\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.56*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-20.04\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-20.19\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.41*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eDibutiryl cAMP\u0026nbsp;+\u0026nbsp;\u003csup\u003e14\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.35\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.47*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.67\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.49*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.13\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.37*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.18\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.39*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-20.73\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.54*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-19.53\u0026nbsp;\u0026plusmn;\u003c/p\u003e\n \u003cp\u003e0.49*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePropranolol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\"\u003e\n \u003cp\u003e-15.59\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-15.23\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-13.87\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.41*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-11.30\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.34*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-11.34\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.35*\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-11.47\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.30*\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003ePropranolol\u0026nbsp;+\u0026nbsp;\u003csup\u003e14\u003c/sup\u003eC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-21.77\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.51*.\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-21.57\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;\u0026nbsp;0.62*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-21.24\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.69*\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-14.90\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.36\u003csup\u003e\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-14.56\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.38\u003csup\u003e*\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd\u003e\n \u003cp\u003e-14.21\u003c/p\u003e\n \u003cp\u003e\u0026plusmn;0.34\u003csup\u003e*\u0026amp;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eThe aliquot of \u003csup\u003e14\u003c/sup\u003eC solution was added in the cell suspension to obtain the final activity concentration 370 kBq∙l\u003csup\u003e-1\u003c/sup\u003e. Drugs were added to the medium 10 min. before addition of radionuclides. The cells were incubated with radionuclides for 1 hour. Value of ZP was calculated from microelectrophoresis measurements of the PMN mobility in electrophoretic medium.\u0026nbsp;* and \u003csup\u003e\u0026amp;\u003c/sup\u003e - significance difference from the control (background) or under the radiation field, consequently, p \u0026pound; 0.05, n = 10.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Low-dose β-radiation, Human PMN, Zeta potential, db-cAMP, Theophylline, Forskolin, Cholera toxin, Propranolol","lastPublishedDoi":"10.21203/rs.3.rs-4557523/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4557523/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe objective of this study was to examine the zeta potential (ZP) response of human neutrophils to selected ligands of the cAMP signalling pathway under low-dose β-radiation (LDBR). ZP was calculated from the electrophoretic mobility (EPM) of the cells. Cells were irradiated at a dose rate of 10 \u0026micro;Gy/hour for one hour. Irradiation of cell suspensions was conducted by adding appropriate aliquots of \u003csup\u003e14\u003c/sup\u003eC-leucine to the incubation medium. EPM measurements were carried out in a radiation field. It was demonstrated that the LDBR and agonists of the AC system are capable of increasing the negative surface charge of human neutrophils. Conversely, the antagonist propranolol has been shown to reduce it. Furthermore, it has been demonstrated that radiation affects the response of the membrane ZP to some ligands of the AC pathway and vice versa. Therefore, LDBR can significantly alter the sensitivity of cells to drugs acting on the targets of these ligands. These results do not permit the conclusion that the effect of LDBR on ZP is mediated through the targets with which the studied ligands interact, since any of its components may undergo structural rearrangement, thereby altering the structural organisation of neutrophil membranes in the LDBR field.\u003c/p\u003e","manuscriptTitle":"The response of human PMN zeta potential to selected ligands of the cAMP signal pathway under low-dose β-radiation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-27 15:03:47","doi":"10.21203/rs.3.rs-4557523/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9846e707-5b26-4739-bd87-10756f14fe24","owner":[],"postedDate":"June 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-01-18T14:23:27+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-27 15:03:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4557523","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4557523","identity":"rs-4557523","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}