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SINGLE TREATMENT (HORMONAL IMPRINTING) OF NEWBORN RATS WITH SEROTONIN INCREASES THE SEROTONIN CONTENT OF CELLS IN ADULTS
Cell Biology International 2002, Vol. 26, No. 8, 663–668 doi:10.1006/cbir.2002.0916, available online at http://www.idealibrary.com on
SINGLE TREATMENT (HORMONAL IMPRINTING) OF NEWBORN RATS WITH SEROTONIN INCREASES THE SEROTONIN CONTENT OF CELLS IN ADULTS GYO } RGY CSABA1, PE u TER KOVA u CS1 and E u VA PA u LLINGER2 1
Department of Genetics, Cell and Immunobiology, Semmelweis University and 2Molecular Immunological Research Group, Hungarian Academy of Sciences, Budapest, Hungary Received 10 October 2001, accepted 5 November 2001
Hormonal imprinting takes place perinatally at the first encounter between the hormone and its target receptor, causing the finishment of the maturation of receptor–signal transduction system. In the presence of an excess of the target hormone or related molecules faulty imprinting develops with life-long consequences. In earlier experiments single neonatal treatment with minute dose of IL-6 caused also prolonged stimulation of IL-6 production. In the present experiment newborn female and male rats were treated with 20 g serotonin (hormonal imprinting) and were studied for serotonin content of different cell types in adult age. Serotonin content was measured by flow cytometry and its localization was determined by confocal microscopy. Serotonin content was detected in white blood cells (lymphocytes, monocytes and granulocytes); in lymphocytes, monocytes (macrophages), granulocytes and mast cells of peritoneal fluid and thymic lymphocytes. Serotonin was present in all cell types of control animals studied. Serotonin content extremely elevated in the white blood cells and also increased in the peritoneal cells of neonatally treated female animals. There was no elevation in thymic lymphocytes. The mean values of male animals remained at the control level. The experiments call attention to the life-long effect of the perinatal hormonal imprinting manifested presently in the elevation of serotonin content and point to the gender differences of serotonin imprinting. Considering the role of serotonin in mood and psychiatric diseases, the observations could have 2002 Elsevier Science Ltd. All rights reserved. some clinical importance. K: hormonal imprinting; neonatal treatment; serotonin; digoxin; white blood cells; mast cells; thymus.
INTRODUCTION Hormonal imprinting takes place in the perinatal critical period, when the developing receptor meets the target hormone (Csaba, 1980). As a consequence of imprinting the receptor–signal transduction system finishes its maturation establishing the binding capacity and response, characteristic to the adult cell (Csaba, 1994). However, in the critical period the excess of the target hormone or an amount of molecules similar to it (members of the same hormone family, synthetic hormone analogues, drugs or environmental pollutants with similar structure) result in faulty 1065–6995/02/$-see front matter
imprinting causing life-long alteration of hormone binding and response (Csaba, 1991, 2000). Receptorial hormonal imprinting was demonstrated at a unicellular level and in mammals alike. In addition, in unicellular Tetrahymena hormonal imprinting provoked the overproduction of the imprinter hormone in many generations after imprinting (Csaba and Kova´cs, 1999, 2000). This gave the idea to study the effect of perinatal hormonal imprinting on the hormone production of adult’s cells in mammals which had been proven in case of the cytokine, IL-6 (Csaba and Kova´cs, 2001). In the present experiments a tissue hormone (and neurotransmitter) serotonin and a steroid 2002 Elsevier Science Ltd. All rights reserved.
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Cell Biology International, Vol. 26, No. 8, 2002
like cardioactive glycoside, digoxin was chosen for proving the prolonged hormone production stimulatory effect of imprinting.
MATERIALS AND METHODS Newborn male and female animals of our closed (Charles River originated) Wistar breed, serotonin (Sigma, U.S.A.) or digoxin (Sigma) was given in the dose of 20 g or 0.5 g/ animal, respectively, subcutaneously under the skin of the neck. When the animals were 2 months old, isotonic Na citrate was injected (during ether anaesthesia) into the peritoneal cavity which was regained after 30 s. After that, blood was obtained by cardiac puncture into isotonic Na citrate solution. The thymus was removed, minced in normal saline and the cells were filtered on gauze. Before labelling, erythrolysis was done (by using Becton Dickinson FACS Lysing Solution) in the case of blood and peritoneal cells. The lysed red blood cell remnants were removed by washings in PBS and the cells were fixed in 4% paraformaldehyde solution. After that, the cells were permeabilized with 0.1% saponin. The serotonin or digoxin content of permeabilized cells were detected with antibody to serotonin (produced in rabbit, Sigma, U.S.A.) or antibody to digoxin (produced in mouse, Sigma, U.S.A.) used as primary antibodies, and FITC-labelled anti-rabbit FITC- IgG (Sigma) or anti-mouse FITC-IgG (Human, Go¨do¨llo¨), as secondary antibodies, respectively. For controlling the specificity, autofluorescence of the cells and aspecificity of the secondary antibodies were detected. The measurement was done in a FACSCalibur flow cytometer (Becton Dickinson, San Jose, U.S.A.), using 10,000 cells for each measurement. For the measurement and analysis CellQuest 3.1 program was used. During the evaluation, cell populations had been separated on the basis of size and granulation and defined by ‘gating’. In the identical cell populations the serotonin content inside the cells had been compared. The numerical comparison of detected values was done by the comparison of percentual changes of geometric mean channel values to the control groups. Each experiment was repeated at least three times. After the flow cytometric analysis the cells were subjected to confocal microscopic analysis in a BioRad MRC 1024 confocal laser scanning microscope, equipped with krypton-argon mixed gaslaser as a light source, at an excitation wavelength
Fig. 1. Serotonin content in the white blood cells.
of 480 nm line. All experiments were repeated twice.
RESULTS Serotonin treatment In the blood of females lymphocytes, monocytes and granulocytes were studied. In each neonatally treated group the serotonin content was significantly elevated (Fig. 1). In the granulocyte group of controls there were two subpopulations, considering the cell’s serotonin content, which was higher in one of the groups, however in the treated animals both group had higher than control serotonin level. In the blood of males there was no significant difference between the control and treated animals, considering the lymphocytes, monocytes and granulocytes (Fig. 1). The values strongly deviated, however always above the control level Confocal microscopy supported the data of flow cytometry, demonstrating stronger serotonin fluorescence in the cells of females (Fig. 2(C) and (D)). Serotonin fluorescence was located periferically in the controls, accumulating in one pole of the cell. In the neonatally treated animals the fluorescence was diffuse, filling the whole cytoplasm. In the peritoneal fluid of females, lymphocytes, monocytes–granulocytes–macrophages and mast cells were separately identified. In each group of cells elevation of serotonin content was observed (Fig. 3). The values strongly deviated, so the differences were not mathematically significant however, all values of treated animals were above the control level. In males there was not signicant difference between control and treated. Confocal microscopy supported the quantitative data, demonstrating more intense fluorescence in the cells of the neonatally treated female animals. In the controls only the mast cells showed intense fluorescence, while in other cells this was weak and patchy. In the neonatally treated animals peritoneal mast cells were
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Fig. 2. Confocal microscopic pictures on the localization of serotonin in the cells of the thymus, blood and peritoneal fluid. There is no difference in the fluorescence intensity and localization of control (A) and treated (B) thymic cells, however, in the blood the control cell’s (C) fluorescence is paler and periferically localized, related to the treated ones (D). In the peritoneal fluid of controls (E,F) only the mast cells are strongly fluorescent, while in the neonatally treated animals (G,H) also the lymphocytes, granulocytes and monocytes.
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Fig. 3. Serotonin content in the cells of the peritoneal fluid.
Fig. 4. Serotonin content in the cells of the thymus.
strongly fluorescent as well as many of the lymphatic cells and granulocytes (Fig. 2(E),(F) and (G),(H)). In the thymus there was no quantitative difference between the cells of treated and control male as well as female animals (Fig. 4), and this was justified also with confocal microscopy (Fig. 2(A),(B)). However serotonin was present in the thymocytes, demonstrated by the strong fluorescence of controls and treated alike. Digoxin treatment There was not immunologically demonstrable digoxin present in the cells studied, at all.
DISCUSSION The serotonin (5HT) content of mast cells and basophilic granulocytes is well known (Selye, 1965; Yong, 1997). There are also data on the serotonin content of lymphocytes and monocytes (macrophages) (Teniukov and Gordon, 1984; Fuchs et al., 1988). Our observations support these earlier data demonstrating the presence of 5HT in these cell types using both confocal microscopic and flow cytometric methods. Considering these observations our model seemed to be suitable to study the effect of hormonal imprinting on hormone (serotonin) content.
Hormonal imprinting is a biological phenomenon which takes place in the perinatal critical period, when a hormone meets the target receptor at first and as a consequence of this, the receptor– signal transduction system finishes its maturation reaching the binding characteristics typical for adults (Csaba, 1980, 1991, 1994, 2000). In the presence of excess amount of the target hormone or molecules similar, however not identical with the target hormone (members of the same hormone family, synthetic hormone-like molecules, drugs, environmental pollutants) can disturb the normal receptor system maturation causing faulty imprinting. The effect of (faulty) imprinting is life-long and it is manifested in morphological, biochemical, genetical and behavioral alterations alike (Bern et al., 1973, 1984; Iguchi, 1992; Tchernitchin and Tchernitchin, 1992; Gray-Nelson et al., 1994). Hormonal imprinting can be observed from a very low level of phylogeny (unicellular animals) to mammals, having evolutionary importance (Csaba, 2000). However, the mechanism of imprinting is not known. Blalock’s theory on the genetic parallellism of hormone and receptor (Blalock and Smith, 1984) gave the idea to study the effect of hormonal imprinting on hormone production. In the unicellular Tetrahymena which is able to produce hormones characteristic to mammals (LeRoith et al., 1983), not only the receptors are influenced by hormonal imprinting, but also the hormone (insulin, pituitary hormones, IL-6) production is stimulated up to many generations after treatment (Csaba and Kova´cs, 1999, 2000, 2001). This was the reason why we studied in an earlier experiment the effect of neonatal treatment with a minute dose (10 ng) of the cytokine, IL-6 to the IL-6 production of cells in adult rats. Though only confocal microscopic evaluation was used, mast cells, which in resting stage do not contain IL-6, massively produced it after neonatal IL-6 treatment and also the increase of IL-6 amount was estimated in adult’s lymphatic cells. In the present experiments, a single dose of serotonin was the imprinter and peritoneal, blood and thymic cells were studied in adult age. An extreme elevation in the serotonin content of blood lymphocytes, monocytes and granulocytes and an expressed elevation in the same cells of the peritoneal fluid was observed, however only in female animals. In addition there was also an increase in the serotonin content of the peritoneal mast cells. At the same time there was no change in thymic lymphocytes and also the mean of the serotonin content of each cell type was unaltered in males.
Cell Biology International, Vol. 26, No. 8, 2002
This shows that females were sensitive to imprinting in contrast to males which did not react to it. The gender difference in case of serotonin is well known. It was demonstrated in human investigations that in mood and anxiety disorders—in which 5-HT has an important role—there is a female preponderance (Liechti et al., 2001). Platelet 5-HT concentration is significantly higher in male than female in healthy persons and shizophrenic patients (Carlsson et al., 1985). These male–female differences are valid also in the case of rats (Ferrari et al., 1999; Muck et al., 1999; Haider and Haleem, 2000). This can explain, why was difference also in female and male serotonin imprinting. In peritoneal lymphocytes as well as in monocytes–macrophages– granulocytes there were also a very expressed elevation with values always above the control level, however this was not statistically significant, because of the high standard deviation. Similarly, the unaltered mean value in case of males also hid a very expressed deviation from the mean, which also has to be explained. In men, a serotonin transporter gene polymorphism was demonstrated (Heils et al., 1997), which can affect not only the brain, but other cell’s serotonin content (Greenberg et al., 1999). If we suppose that this polymorphism is present in rats, too, the high standard deviation in the effect of imprinting can be explained. There is no question that the single neonatal treatment (hormonal imprinting) caused a life-long effect on the serotonin content of the cells studied. However, the problem is that on the basis of the experiments we can not decide, whether hormone production was influenced for life by the serotonin imprinting, or the function of the serotonin transporter (Heils et al., 1997). Namely, it is known that serotonin can be taken up and secreted by mast cells (Kova´cs and Csaba, 1977; Purcell and Hanahoe, 1990) and other serotonin containing cells (Kanner and Bendahan, 1985; Jackson et al., 1988). We also can not explain, why peritoneal and blood cells (of females) reacted to imprinting, while thymic lymphocytes do not react to it. However, cells of similar origin can react differently to stimuli, depending on the milieu in which they are living. In addition, in case of lymphocytes there are many types with different immunological functions and their progenitors could have been differently influenced neonatally. For resolving these problems further experiments are needed. Digoxin is a cardioctive plant glycoside, with a steroid-like structure, however it is also present endogeneously (Castaneda-Hernandez, 1989; Ghione et al., 1993a,b). Nevertheless, it is not
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known what are the cells in the mammalian organism which can produce digoxin, as its presence was only demonstrated in the blood heart, adrenal cortex, amniotic fluid and human milk and others were not investigated before. Imprinting with digoxin can cause life-long alterations of steroid receptor binding and sexual behavior (Csaba and Inczefi-Gonda, 1998; Karabe´lyos and Csaba, 1998), since it can disturb the normal development of steroid receptors (similar to other molecules having steroid-like character, as e.g. benzpyrene) given in the critical period of receptor development. However, there were no data on the presence or absence of digoxin in white blood cells or mast cells and the absence of it in these cells was supported by our present results. The negativity of the experiments with digoxin (and positivity in case of serotonin) demonstrates that perinatal hormonal imprinting is not able to open closed genes (for producing materials not characteristic to the cell; digoxin at present case) and it works only, if the gene is routinely functioning in the given cell. Considering that serotonin has an important role in different brain processes the troubles of which are manifested in certain psychiatric diseases, our observations could have some clinical importance.
ACKNOWLEDGEMENTS This work was supported by the National Research Fund (OTKA-T-029002), Hungary. The authors thank Ms Katy Kallay and Ms Angela Koza´k for the expert technical assistance. REFERENCES B HA, G RA, K S, 1973. Long term effects of prenatal hormone administration. Science 181: 189–190. B HA, J LA, M T, Y PN, 1975. Exposure of neonatal mice to steroids: long term effects on the mammary gland and other reproductive structures. J Ster Biochem 6: 673–676. B JE, S EM, 1984. Hydropathic anticomplementarity of amino acids based on the genetic code. Biochem Biophys Res Com 121: 203–207. C M, S K, E E, C A, 1985. Rat brain serotonin: biochemical and functional evidence for a sex difference. J Neur Transm 63: 297–313. C-H G, 1989. Evidence for the existence of the same endogeneous digitalis-like factor in some mammalian species. Comp Biochem Physiol 94C: 49–53. C G, 1980. Phylogeny and ontogeny of hormone receptors: the selection theory of receptor formation and hormonal imprinting. Biol Rev 55: 47–63.
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C G, 1994. Phylogeny and ontogeny of chemical signaling: Origin and development of hormone receptors. Int Rev of Cytol 155: 1–48. C G, 1991. Interaction between the genetic programme and environmental influences in the perinatal critical period. Zool Sci 8: 813–825. C G, 2000. Hormonal imprinting: its role during the evolution and development of hormones and receptors. Cell Biol Int 24: 407–414. C G, I-G A, 1998. Fetal digoxin treatment enhances the binding capacity of thymic glucocorticoid receptors in adult female rats. Gen Pharm 30: 647–649. C G, K´ P, 1999. Localization of -endorphin in Tetrahymena by confocal microscopy. Induction of the prolonged production of the hormone by hormonal imprinting. Cell Biology Int 23: 695–702. C G, K´ P, 2000. Insulin uptake, localization and production in previously insulin treated and untreated Tetrahymena. Data on the mechanism of hormonal imprinting. Cell Biochem Funct 18: 161–167. C G, K´ P, 2001. Effect of neonatal interleukin-6 (IL-6) treatment (hormonal imprinting) on the IL-6 content and localization of the peritoneal, blood and thymic cells of adult rats. A confocal microscopic analysis. Cell Biol Int. 25: 1179–1182. F PF, L S, T H, G P, W CA, H RW, 1999. The influence of gender and age on neonatal rat hypothalamic 5HT1A and 5HT2A receptors. Cell Mol Neurobiol 19: 775–784. F BA, C KS, M AE, 1988. Norepinephrine and serotonin content of the murine spleen: its relationship to lymphocyte beta-adrenergic receptor density and the humoral immune response in vivo and in vitro. Cell Immunol 117: 339–351. G S, B S, B S, M U, B J, 1993a. Evidence of marked digoxin-like immunoreactivity in the human adrenal cortex: results of an immunohistochemical study. Eur J Histochem 37: 273–276. G S, B S, D S, P A, P L, M U, 1993b. Endogeneous digitalis-like activity in the newborn. J Cardiovasc Pharm 22: 25–28. G-N K, S Y, E B, S T, ML JA, 1994. Exposure to diethylstilbestrol during a critical developmental period of the mouse reproductive tract leads to persistent induction of two estrogen-regulated genes. Cell Growth Diff 5: 115–119. G BD, T TJ, H SJ, L Q, B D, M DL, 1999. Genetic variation in the serotonin
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promoter region affects serotonin uptake in human blood platelets. Amer J Med Gen 88: 83–87. H S, H DJ, 2000. Decreases of brain serotonin following a food restriction schedule of 4 weeks in female and male rats. Med Sci Mon 6: 1061–1067. H A, M R, L KP, 1997. The human serotonin transporter gene polymorphism—basic research and clinical implications. J Neur Transm 104: 1005–1014. I T, 1992. Cellular effect of early exposure to sex hormones and antihormones. Int Rev Cytol 139: 1–57. J JC, W RF, B WH, R TL, 1988. Specific uptake of serotonin by murine macrophages. Life Sci 42: 1641–1650. K BI, B A, 1985. Transport of 5hydroxytryptamine in membrane vesicles from rat basophilic leukemia cells. Biochim Biophys Acta 816: 403– 410. K´ C, C G, 1998. Effect of fetal digoxin exposure (imprinting) on the sexual behavior of adult rats. Gen Pharm 31: 367–369. K´ P, C G, 1977. Biogenic amine and amine precursor uptake by mast cells. Acta Morph Hung 25: 19–23. LR D, S J, B M, F LA, K BT, R J, 1983. Are messenger molecules in microbes the ancestors of the vertebrate hormones and tissue factors? Fed Proc 42: 2602–2607. L ME, G A, V FX, 2001. Gender differences in the subjective effects of MDMA. Psychopharm 154: 161–168. M-S D, P N, J M, 1999. Sex differences, season of birth and platelet 5-HT levels in schizophrenic patients. J Neur Transm 106: 337–347. P WM, H TH, 1990. Differential release of histamine and 5-hydroxytryptamine from rat mast cells: the contribution of amine uptake to the apparent pattern of secretion. Agents Act 30: 38–40. S H, 1965. The Mast Cells. Washington, Butterworths. T A, T N, 1992. Imprinting of paths of heterodifferentiation by prenatal or neonatal exposure to hormones, pharmaceuticals, pollutants and other agents and conditions. Med Sci Res 20: 391–397. T VV, G DS, 1984. Localization of catecholamines, histamine and acetylcholinesterase in human peripheral blood structures. Arkh Anat Gist Embriol 87: 78–81. Y LC, 1997. The mast cell: origin, morphology, distribution, and function. Exp Tox Path 49: 409–424.
SINGLE TREATMENT (HORMONAL IMPRINTING) OF NEWBORN RATS WITH SEROTONIN INCREASES THE SEROTONIN CONTENT OF CELLS IN ADULTS GYO } RGY CSABA1, PE u TER KOVA u CS1 and E u VA PA u LLINGER2 1
Department of Genetics, Cell and Immunobiology, Semmelweis University and 2Molecular Immunological Research Group, Hungarian Academy of Sciences, Budapest, Hungary Received 10 October 2001, accepted 5 November 2001
Hormonal imprinting takes place perinatally at the first encounter between the hormone and its target receptor, causing the finishment of the maturation of receptor–signal transduction system. In the presence of an excess of the target hormone or related molecules faulty imprinting develops with life-long consequences. In earlier experiments single neonatal treatment with minute dose of IL-6 caused also prolonged stimulation of IL-6 production. In the present experiment newborn female and male rats were treated with 20 g serotonin (hormonal imprinting) and were studied for serotonin content of different cell types in adult age. Serotonin content was measured by flow cytometry and its localization was determined by confocal microscopy. Serotonin content was detected in white blood cells (lymphocytes, monocytes and granulocytes); in lymphocytes, monocytes (macrophages), granulocytes and mast cells of peritoneal fluid and thymic lymphocytes. Serotonin was present in all cell types of control animals studied. Serotonin content extremely elevated in the white blood cells and also increased in the peritoneal cells of neonatally treated female animals. There was no elevation in thymic lymphocytes. The mean values of male animals remained at the control level. The experiments call attention to the life-long effect of the perinatal hormonal imprinting manifested presently in the elevation of serotonin content and point to the gender differences of serotonin imprinting. Considering the role of serotonin in mood and psychiatric diseases, the observations could have 2002 Elsevier Science Ltd. All rights reserved. some clinical importance. K: hormonal imprinting; neonatal treatment; serotonin; digoxin; white blood cells; mast cells; thymus.
INTRODUCTION Hormonal imprinting takes place in the perinatal critical period, when the developing receptor meets the target hormone (Csaba, 1980). As a consequence of imprinting the receptor–signal transduction system finishes its maturation establishing the binding capacity and response, characteristic to the adult cell (Csaba, 1994). However, in the critical period the excess of the target hormone or an amount of molecules similar to it (members of the same hormone family, synthetic hormone analogues, drugs or environmental pollutants with similar structure) result in faulty 1065–6995/02/$-see front matter
imprinting causing life-long alteration of hormone binding and response (Csaba, 1991, 2000). Receptorial hormonal imprinting was demonstrated at a unicellular level and in mammals alike. In addition, in unicellular Tetrahymena hormonal imprinting provoked the overproduction of the imprinter hormone in many generations after imprinting (Csaba and Kova´cs, 1999, 2000). This gave the idea to study the effect of perinatal hormonal imprinting on the hormone production of adult’s cells in mammals which had been proven in case of the cytokine, IL-6 (Csaba and Kova´cs, 2001). In the present experiments a tissue hormone (and neurotransmitter) serotonin and a steroid 2002 Elsevier Science Ltd. All rights reserved.
664
Cell Biology International, Vol. 26, No. 8, 2002
like cardioactive glycoside, digoxin was chosen for proving the prolonged hormone production stimulatory effect of imprinting.
MATERIALS AND METHODS Newborn male and female animals of our closed (Charles River originated) Wistar breed, serotonin (Sigma, U.S.A.) or digoxin (Sigma) was given in the dose of 20 g or 0.5 g/ animal, respectively, subcutaneously under the skin of the neck. When the animals were 2 months old, isotonic Na citrate was injected (during ether anaesthesia) into the peritoneal cavity which was regained after 30 s. After that, blood was obtained by cardiac puncture into isotonic Na citrate solution. The thymus was removed, minced in normal saline and the cells were filtered on gauze. Before labelling, erythrolysis was done (by using Becton Dickinson FACS Lysing Solution) in the case of blood and peritoneal cells. The lysed red blood cell remnants were removed by washings in PBS and the cells were fixed in 4% paraformaldehyde solution. After that, the cells were permeabilized with 0.1% saponin. The serotonin or digoxin content of permeabilized cells were detected with antibody to serotonin (produced in rabbit, Sigma, U.S.A.) or antibody to digoxin (produced in mouse, Sigma, U.S.A.) used as primary antibodies, and FITC-labelled anti-rabbit FITC- IgG (Sigma) or anti-mouse FITC-IgG (Human, Go¨do¨llo¨), as secondary antibodies, respectively. For controlling the specificity, autofluorescence of the cells and aspecificity of the secondary antibodies were detected. The measurement was done in a FACSCalibur flow cytometer (Becton Dickinson, San Jose, U.S.A.), using 10,000 cells for each measurement. For the measurement and analysis CellQuest 3.1 program was used. During the evaluation, cell populations had been separated on the basis of size and granulation and defined by ‘gating’. In the identical cell populations the serotonin content inside the cells had been compared. The numerical comparison of detected values was done by the comparison of percentual changes of geometric mean channel values to the control groups. Each experiment was repeated at least three times. After the flow cytometric analysis the cells were subjected to confocal microscopic analysis in a BioRad MRC 1024 confocal laser scanning microscope, equipped with krypton-argon mixed gaslaser as a light source, at an excitation wavelength
Fig. 1. Serotonin content in the white blood cells.
of 480 nm line. All experiments were repeated twice.
RESULTS Serotonin treatment In the blood of females lymphocytes, monocytes and granulocytes were studied. In each neonatally treated group the serotonin content was significantly elevated (Fig. 1). In the granulocyte group of controls there were two subpopulations, considering the cell’s serotonin content, which was higher in one of the groups, however in the treated animals both group had higher than control serotonin level. In the blood of males there was no significant difference between the control and treated animals, considering the lymphocytes, monocytes and granulocytes (Fig. 1). The values strongly deviated, however always above the control level Confocal microscopy supported the data of flow cytometry, demonstrating stronger serotonin fluorescence in the cells of females (Fig. 2(C) and (D)). Serotonin fluorescence was located periferically in the controls, accumulating in one pole of the cell. In the neonatally treated animals the fluorescence was diffuse, filling the whole cytoplasm. In the peritoneal fluid of females, lymphocytes, monocytes–granulocytes–macrophages and mast cells were separately identified. In each group of cells elevation of serotonin content was observed (Fig. 3). The values strongly deviated, so the differences were not mathematically significant however, all values of treated animals were above the control level. In males there was not signicant difference between control and treated. Confocal microscopy supported the quantitative data, demonstrating more intense fluorescence in the cells of the neonatally treated female animals. In the controls only the mast cells showed intense fluorescence, while in other cells this was weak and patchy. In the neonatally treated animals peritoneal mast cells were
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Fig. 2. Confocal microscopic pictures on the localization of serotonin in the cells of the thymus, blood and peritoneal fluid. There is no difference in the fluorescence intensity and localization of control (A) and treated (B) thymic cells, however, in the blood the control cell’s (C) fluorescence is paler and periferically localized, related to the treated ones (D). In the peritoneal fluid of controls (E,F) only the mast cells are strongly fluorescent, while in the neonatally treated animals (G,H) also the lymphocytes, granulocytes and monocytes.
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Fig. 3. Serotonin content in the cells of the peritoneal fluid.
Fig. 4. Serotonin content in the cells of the thymus.
strongly fluorescent as well as many of the lymphatic cells and granulocytes (Fig. 2(E),(F) and (G),(H)). In the thymus there was no quantitative difference between the cells of treated and control male as well as female animals (Fig. 4), and this was justified also with confocal microscopy (Fig. 2(A),(B)). However serotonin was present in the thymocytes, demonstrated by the strong fluorescence of controls and treated alike. Digoxin treatment There was not immunologically demonstrable digoxin present in the cells studied, at all.
DISCUSSION The serotonin (5HT) content of mast cells and basophilic granulocytes is well known (Selye, 1965; Yong, 1997). There are also data on the serotonin content of lymphocytes and monocytes (macrophages) (Teniukov and Gordon, 1984; Fuchs et al., 1988). Our observations support these earlier data demonstrating the presence of 5HT in these cell types using both confocal microscopic and flow cytometric methods. Considering these observations our model seemed to be suitable to study the effect of hormonal imprinting on hormone (serotonin) content.
Hormonal imprinting is a biological phenomenon which takes place in the perinatal critical period, when a hormone meets the target receptor at first and as a consequence of this, the receptor– signal transduction system finishes its maturation reaching the binding characteristics typical for adults (Csaba, 1980, 1991, 1994, 2000). In the presence of excess amount of the target hormone or molecules similar, however not identical with the target hormone (members of the same hormone family, synthetic hormone-like molecules, drugs, environmental pollutants) can disturb the normal receptor system maturation causing faulty imprinting. The effect of (faulty) imprinting is life-long and it is manifested in morphological, biochemical, genetical and behavioral alterations alike (Bern et al., 1973, 1984; Iguchi, 1992; Tchernitchin and Tchernitchin, 1992; Gray-Nelson et al., 1994). Hormonal imprinting can be observed from a very low level of phylogeny (unicellular animals) to mammals, having evolutionary importance (Csaba, 2000). However, the mechanism of imprinting is not known. Blalock’s theory on the genetic parallellism of hormone and receptor (Blalock and Smith, 1984) gave the idea to study the effect of hormonal imprinting on hormone production. In the unicellular Tetrahymena which is able to produce hormones characteristic to mammals (LeRoith et al., 1983), not only the receptors are influenced by hormonal imprinting, but also the hormone (insulin, pituitary hormones, IL-6) production is stimulated up to many generations after treatment (Csaba and Kova´cs, 1999, 2000, 2001). This was the reason why we studied in an earlier experiment the effect of neonatal treatment with a minute dose (10 ng) of the cytokine, IL-6 to the IL-6 production of cells in adult rats. Though only confocal microscopic evaluation was used, mast cells, which in resting stage do not contain IL-6, massively produced it after neonatal IL-6 treatment and also the increase of IL-6 amount was estimated in adult’s lymphatic cells. In the present experiments, a single dose of serotonin was the imprinter and peritoneal, blood and thymic cells were studied in adult age. An extreme elevation in the serotonin content of blood lymphocytes, monocytes and granulocytes and an expressed elevation in the same cells of the peritoneal fluid was observed, however only in female animals. In addition there was also an increase in the serotonin content of the peritoneal mast cells. At the same time there was no change in thymic lymphocytes and also the mean of the serotonin content of each cell type was unaltered in males.
Cell Biology International, Vol. 26, No. 8, 2002
This shows that females were sensitive to imprinting in contrast to males which did not react to it. The gender difference in case of serotonin is well known. It was demonstrated in human investigations that in mood and anxiety disorders—in which 5-HT has an important role—there is a female preponderance (Liechti et al., 2001). Platelet 5-HT concentration is significantly higher in male than female in healthy persons and shizophrenic patients (Carlsson et al., 1985). These male–female differences are valid also in the case of rats (Ferrari et al., 1999; Muck et al., 1999; Haider and Haleem, 2000). This can explain, why was difference also in female and male serotonin imprinting. In peritoneal lymphocytes as well as in monocytes–macrophages– granulocytes there were also a very expressed elevation with values always above the control level, however this was not statistically significant, because of the high standard deviation. Similarly, the unaltered mean value in case of males also hid a very expressed deviation from the mean, which also has to be explained. In men, a serotonin transporter gene polymorphism was demonstrated (Heils et al., 1997), which can affect not only the brain, but other cell’s serotonin content (Greenberg et al., 1999). If we suppose that this polymorphism is present in rats, too, the high standard deviation in the effect of imprinting can be explained. There is no question that the single neonatal treatment (hormonal imprinting) caused a life-long effect on the serotonin content of the cells studied. However, the problem is that on the basis of the experiments we can not decide, whether hormone production was influenced for life by the serotonin imprinting, or the function of the serotonin transporter (Heils et al., 1997). Namely, it is known that serotonin can be taken up and secreted by mast cells (Kova´cs and Csaba, 1977; Purcell and Hanahoe, 1990) and other serotonin containing cells (Kanner and Bendahan, 1985; Jackson et al., 1988). We also can not explain, why peritoneal and blood cells (of females) reacted to imprinting, while thymic lymphocytes do not react to it. However, cells of similar origin can react differently to stimuli, depending on the milieu in which they are living. In addition, in case of lymphocytes there are many types with different immunological functions and their progenitors could have been differently influenced neonatally. For resolving these problems further experiments are needed. Digoxin is a cardioctive plant glycoside, with a steroid-like structure, however it is also present endogeneously (Castaneda-Hernandez, 1989; Ghione et al., 1993a,b). Nevertheless, it is not
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known what are the cells in the mammalian organism which can produce digoxin, as its presence was only demonstrated in the blood heart, adrenal cortex, amniotic fluid and human milk and others were not investigated before. Imprinting with digoxin can cause life-long alterations of steroid receptor binding and sexual behavior (Csaba and Inczefi-Gonda, 1998; Karabe´lyos and Csaba, 1998), since it can disturb the normal development of steroid receptors (similar to other molecules having steroid-like character, as e.g. benzpyrene) given in the critical period of receptor development. However, there were no data on the presence or absence of digoxin in white blood cells or mast cells and the absence of it in these cells was supported by our present results. The negativity of the experiments with digoxin (and positivity in case of serotonin) demonstrates that perinatal hormonal imprinting is not able to open closed genes (for producing materials not characteristic to the cell; digoxin at present case) and it works only, if the gene is routinely functioning in the given cell. Considering that serotonin has an important role in different brain processes the troubles of which are manifested in certain psychiatric diseases, our observations could have some clinical importance.
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