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Increased apoptosis of adult rat lymphocytes after single neonatal vitamin a treatment (hormonal imprinting). A flow cytometric analysis
Life Scimm, Vol. 61, No. 23, pa. PL 33%342,1S97 03pylQ$I 0 1997 mcvicr scieaec Inc. Printed in the USA. Au rights ln32Lm5/97 $17.00 + .a0
PI1 soo24.3205(97)oo94o-5
PlL4RM4COLOGYLET AccelenafedCommunicatian INCREASED APOPTOSIS OF ADULT RAT LYMPHOCYTES AFTER SINGLE NEONATAL VITAMIN A TREATMENT (HORMONAL IMPRINTING). A FLOW CYTOMETRIC ANALYSIS. Annamtia Gail’,
J. Bocsi*, A. Falus’, B. Szende’ and G. Csaba”
‘Department of Genetics, Cell and Immunobialogy and *lst Department of Pathology and Experimental Cancer Research, Semmelweis University of Medicine, H-l 445 Budapest, POB 370, Hungary (Submitted May 29, 1997,accepted June 20, 1997; receivedin final form September11,1!297)
Abstract. Newborn rats were treated with a single dose of vitamin A (retinol) and apoptosis of peripheral lymphocytes was studied by flow cytometry in adult age. Vitamin A treatment (hormonal imprinting) caused a moderate, however significant elevation in the number of apoptotic lymphocytes after three months. Dexamethasone or Concanavalin-A alone did not influence apoptosis significantly. However, in the neonatally retinol treated rats dexamathasone significantly elevated the quantity of apoptotic lymphocytes related to the control or Concanavalin-A treated control cells. The results call attention to the prolonged effect of hormonal imprinting in a new index and to the possible dangerous effects in human, neonatally treated with vitamin A. o 1997Elsevier Science Inc.
Key Words: apoptasis, programmed cell death, hormonal imprinting, retinoids, vitaminA Introduction Apoptosis participates in many developmental processes during the intrauterine life as well, as in adults. Morphogenetic hormones as regulators of the development can influence apoptosis as this is done by dexamethasone, a synthetic glucocorticoid (l-4). However, nonhormone molecules having hormone-like effects and intracellular receptors also can influence (increase) apoptosis. Retinoids (vitamin A and retinoic acids), which are prominent morphogenetic molecules, promote apoptosis (5-7) and influence the effect of other apoptosis inducers (8,9) in different experimental systems and in normal and pathological (cancerous) cells alike. The first encounter between the developing hormone receptor and the appropriate hormones perinatally- develops the hormonal imprinting which adjusts the binding capacity of the receptor and the response of the cell to the hormone, for life (10-12). In the absence of the ,,target” hormone the receptor fails to develop normally (13), however, if molecules different from the appropriate hormone but able to bind to the maturing receptor, are present in this critical period, faulty imprinting is provoked, which leads to the life-long abnormal binding *Corresponding author: G.Csaba MD PhD., Department of Genetics, Cell and Immunobiology, Semmelweis University of Medicine, H-1445 Budapest, POB 370, Hungary. Fax: (36-l) 210-2950; e-mail: [email protected]
PL-340
Apoptosis after Neonatal Vii
A Treatment
Vol. 61, No. 23, 1997
capacity of receptor and abnormal response of the receptor bearing cell and its progeny generations (12). Perinatal treatment with synthetic steroid hormones or benzpyrene can change in this way the binding capacity of their own and other steroid receptors (14,15) what is followed by disturbances in other (biochemical and morphological) parameters (16-20). Retinoids, which also have receptors in the steroid-thyroid receptor superfamily (21) can disturb the development of steroid receptors and -as a consequence of this- adult sexual behavior and hormone levels alike (22,23). In the present experiment the prolonged effect of vitamin A imprinting (neonatal treatment) was studied on the spontaneous and dexamethasone induced apoptosis of blood lymphocytes in adults. Methods N’ewborn l’cmale Wistar rats of our closed breed were treated subcutaneously with 3 mg retinol dissolved in sunflover seed oil. Controls received the vehicle only. When the animals were four months old blood was taken by cardial puncture in ether narcosis into heparinized tubes. Sepurution und treatment ofthe lymphocytes One sample (about 8-10 ml) was diluted with RPM1 cultivation medium and was centrifuged to a Ficoll-paque (Pharmacia, Lund, Sweden) for 20 min at 20 ‘C with 2000 rpm. After that the lymphocyte ring was collected to RPM1 medium and centrifuged again with 1000 rpm for 5 min at 20 “C The sediment was resuspended in serum containing medium. The cells were plated (cell count 2x106/ml) and were incubated or not incubated with Concanavalin A (ConA. 10-j g/ml) for 48 h. After that in some of the groups dexamethasone treatment was done in 1(I” M concentration. By this treatments 8 groups were formed: Neonatal]), not treated (control, groups l-4), and neonatally retinol treated (groups S-8) animals: 1 and 5: without any further treatment; 2 and 6: Con-A treatment; 3 and 7: dexamethasone treatment; 4 and 8: Con-A and dexamethasone treatment.
After thorough washing in PBS the cells were centrifuged (1800 fixed in -20 “C 70% ethyl-alcohol, stored at 4 “C for 1 hour and supplemented with 0.5 mg/ml RNAse A (Sigma, St Louis, Miss. -37 (‘C for 30 min, the cells were stained with 50 ug/ml propidium for I5 min.
rpm, 5 min) the pellet was resuspended in 0.5 ml PBS USA). After incubation at iodide at room temperature
The analysis was performed on a FACStar flow cytometer (Becton-Dickinson, Mountain Vie\v. California, USA) in conjunction with a HP-300 computer and Consort 30 data acquisition package. Excitation was measured at 488 nm, emission at 585142 nm. Approximately lo4 cells per sample were scanned using low flow rates (100 to 200/set) to maximiLe resolution. Significance was evaluated by Student t-test. For light microscopic control of apoptosis Giemsa staining and Apop-Tag reaction was used. Results and Discussion Apoptosis is a special form of cell death, regulating cell number and eliminating pathological cells from the population. Members of the immune system, first of all lymphocytes are subjected to apoptosis in case of development and overpopulation alike (24,25). These cells
Vol. 61, No. 23, 1997
Apoptosis after Neonatal Vitaok
A Treatment
PL341
express glucocorticoid receptors, the presence of whose is needed for the apoptosis promoting effect of dexamethasone (1). Concanavalin A (Con-A) also can promote apoptosis by binding to cell surface (receptor) carbohydrates (26). Vitamin A (retinol) can increase the number of apoptotic cells in adult animals (5,6,27). In our experiments retinol was used neonatally and adult animals were studied for apoptosis. This means, that the acute effect of retinol was excluded and only the changes caused by the neonatal treatment (and inherited from cell to cell) could be evaluated at adult age. This neonatal influence caused the significant elevation of the number of spontaneously (without further treatment) apoptotic cells (Table. 1). This elevation is only 15%, however significant (p
1
of untreated and neonatally
group control (c) c+Con-A c+dexameth.(d) c+Con-A+d ROH treated(R) R+Con-A R+d R+Con-A+d
% 37.37 36.34 40.75 38.01 42.79 39.22 45.09 43.39
retinoi treated rats. SE 2.04 2.15 2.77 2.12 1.36 1.90 1.98 2.38
Significances between: 1 and 5 = ~~0.05; 1 and 7 = ~~0.05; 2 and 5 = ~~0.05; 2 and 7 = ~~0.02 n = number of measurements The retinol + dexamethasone combination caused higher elevation in apoptosis, than retinol alone. Considering that the elevation caused by dexamethasone alone was not significant and retinol and dexamethasone caused significant elevation to control, this means that the neonatal retinol treatment potentiated the apoptosis of lymphocytes induced by dexamethasone, and this would be accepted as an effect of neonatal imprinting. Con-A was used for stimulating cell proliferation prior to dexamethasone treatment. However, according to the literary data Con-A can influence apoptosis, too (26). In our experiment ConA neither influenced significantly apoptosis alone nor in combination with dexamethasone. Nevertheless the spontaneous apoptosis of neonatally retinol treated animals was significantly higher (~~0.05) than that of the Con-A treated control animals. Summarizing the results, the experiments call attention to the prolonged effect of perinatal vitamin A treatment in a new index: apoptosis. This means that a developmental process can be influenced by a morphogenetic molecule months after the direct intervention done after the completion of the intrauterine life Considering the use of vitamin A in prevention of hypovitaminosis in lactating mothers or in infants, the experiments have some human aspects.
PL342
Apoptosis after Neonatal Vitamin A Treatment
Vol. 61, No. 23, 1997
Acknowledgements
This work was supported by the National 17849, Hungary
Research Fund (OTKA) T-017775,
T-17722. T-
References
1. J.A.CIDLOWSKI, K.L. KING. R.B. EVANS-STORMS, J.W. MONTAGUE. C.D. BORTNER and F.M. HUGHES, Recent Progr. Horm. Res. 51 457-490 (1996). 2. J. GRUBER, R. SGONC, Y.H. HU, H. BEUG and G. WICK, Eur. J. Immunol. 24 11IS1121 (1994). 3. T.W. McCLOSKEY, N. OYAZU, M. CORONESI and S. PAHWA, Clin. Immunol. Immunopathol. 71 14-18 (1994). 4. D. SCHULER, B. SZENDE, J.D. BORSI, T MARTON, J. BOCSI, E. MAGYAROSSY. R. KOOS and M. CSOKA, Pediatr. Hematol. Oncol. 11 641-649 (1994). 5. ~i‘,.i‘.WANG and J.M. PHANG. Cancer Lett. 107 65-71 (1996). 6. L. NAGY. V.A. THOMAZY, R.A. CHANDRARATNA. R.A. HEYMAN and P.S. DAVIES. (1996) Leuk. Res. 20. 499-505. 7. M. PONZONI, P. BOCCA. V. CHIESA. A. DECENSI, V. PISTOIA, L.RAFFAGHELLO. C. ROZZO and P.G. MONTALDO, Cancer Res. 55 853-861 (1995). 8. R.P. BISSONETTE, T. BRUNNER, S.B. LAZARCHIK, N.J. YOO, M.F. BOEHM. D.R.GREEN and R.A. HEYMAN, Mol. Cell. Biol. 15 5576-5585 (1995). 9. Y. YANG, S. MINUCCI. K. OZATO, R.A. HEYMAN and J.D. ASHWELL. J. Biol. Chem. 270 18672-18677 (1993). 10. G. CSABA, Biol. Rev. 55 47-63 (1980). 11, G.CSABA, Experientia 42 750-759 (1986). 12. G. CXABA, Zool. Sci. 8 813-825 (1991). 13. G.CSABA and S.U. NAGY, 1. Endocrinol. Invest. 8 557-560 (1985). 14. G. CSABA, A. INCZEFI-GONDA and 0. DOBOZY, Acta Physiol. Hung. 67 207-212 15. A. INCZEFI-GONDA, G.CSABA and 0. DOBOZY, Acta Physiol. Hung. 67 27-29 ( 1986). 16. 1. IGUCHI. P.L. OSTRANDER. K.‘l‘. MILLS and H.A. BERN, Med. Sci. Res. 15,489490 (1987). 17. H.A. BERN. M. EDERY, K.T. MILLS. A.T. KAHRMAN, T. MAR1 and L.H.LASSO, Cancer Res. 47 4165-4172 (1987). 18. D.F.C. GIBSON, D.A. ROBERTS and S. EVANS Eur. J. Cancer 27 3133-3137 (1991). 19. AN. TCHERNITCHIN and N. TCHERNITCHIN, Med. Sci. 20 391-397 (1992) 20. S. MIRZAHOSSEINI, CS. KARABPLYOS, 0. DOBOZY and G. CSABA, Hum. Exp. Toxicol 15 573-576 (1996). 2 1. D.J. MANGELSDORF, Nutr. Rev. 52 532-544 (1994). 22. A. GALL and G. CSABA, Endocrin. Metab. 4 115-l 19 (1997). 23. G. CSABA and A. GAAL, Hum. Exp. Toxicol. 16 193-I 97 (1997). 24. T.G. COTTER, S.V. LENNON, J.G. GLYNN AND S.J. MARTIN, Anticancer. Res. 10 1153-1159(1959). 25. T.G. COTTER, Semin. Immunol. 4 399-405 (1992). 26. G.V. KULKARNI and C.A. McCULLOCH. J. Cell Physiol. 165 119-l 33 (1995). 27. M.B. GLOZAK and M.B. ROGERS, Dev. Biol. 179 458-470 (1996). 28. S.A. AHMED and N. SRIRANGANATHAN. Immunopharmacology 28 55-66 (1994).
PI1 soo24.3205(97)oo94o-5
PlL4RM4COLOGYLET AccelenafedCommunicatian INCREASED APOPTOSIS OF ADULT RAT LYMPHOCYTES AFTER SINGLE NEONATAL VITAMIN A TREATMENT (HORMONAL IMPRINTING). A FLOW CYTOMETRIC ANALYSIS. Annamtia Gail’,
J. Bocsi*, A. Falus’, B. Szende’ and G. Csaba”
‘Department of Genetics, Cell and Immunobialogy and *lst Department of Pathology and Experimental Cancer Research, Semmelweis University of Medicine, H-l 445 Budapest, POB 370, Hungary (Submitted May 29, 1997,accepted June 20, 1997; receivedin final form September11,1!297)
Abstract. Newborn rats were treated with a single dose of vitamin A (retinol) and apoptosis of peripheral lymphocytes was studied by flow cytometry in adult age. Vitamin A treatment (hormonal imprinting) caused a moderate, however significant elevation in the number of apoptotic lymphocytes after three months. Dexamethasone or Concanavalin-A alone did not influence apoptosis significantly. However, in the neonatally retinol treated rats dexamathasone significantly elevated the quantity of apoptotic lymphocytes related to the control or Concanavalin-A treated control cells. The results call attention to the prolonged effect of hormonal imprinting in a new index and to the possible dangerous effects in human, neonatally treated with vitamin A. o 1997Elsevier Science Inc.
Key Words: apoptasis, programmed cell death, hormonal imprinting, retinoids, vitaminA Introduction Apoptosis participates in many developmental processes during the intrauterine life as well, as in adults. Morphogenetic hormones as regulators of the development can influence apoptosis as this is done by dexamethasone, a synthetic glucocorticoid (l-4). However, nonhormone molecules having hormone-like effects and intracellular receptors also can influence (increase) apoptosis. Retinoids (vitamin A and retinoic acids), which are prominent morphogenetic molecules, promote apoptosis (5-7) and influence the effect of other apoptosis inducers (8,9) in different experimental systems and in normal and pathological (cancerous) cells alike. The first encounter between the developing hormone receptor and the appropriate hormones perinatally- develops the hormonal imprinting which adjusts the binding capacity of the receptor and the response of the cell to the hormone, for life (10-12). In the absence of the ,,target” hormone the receptor fails to develop normally (13), however, if molecules different from the appropriate hormone but able to bind to the maturing receptor, are present in this critical period, faulty imprinting is provoked, which leads to the life-long abnormal binding *Corresponding author: G.Csaba MD PhD., Department of Genetics, Cell and Immunobiology, Semmelweis University of Medicine, H-1445 Budapest, POB 370, Hungary. Fax: (36-l) 210-2950; e-mail: [email protected]
PL-340
Apoptosis after Neonatal Vii
A Treatment
Vol. 61, No. 23, 1997
capacity of receptor and abnormal response of the receptor bearing cell and its progeny generations (12). Perinatal treatment with synthetic steroid hormones or benzpyrene can change in this way the binding capacity of their own and other steroid receptors (14,15) what is followed by disturbances in other (biochemical and morphological) parameters (16-20). Retinoids, which also have receptors in the steroid-thyroid receptor superfamily (21) can disturb the development of steroid receptors and -as a consequence of this- adult sexual behavior and hormone levels alike (22,23). In the present experiment the prolonged effect of vitamin A imprinting (neonatal treatment) was studied on the spontaneous and dexamethasone induced apoptosis of blood lymphocytes in adults. Methods N’ewborn l’cmale Wistar rats of our closed breed were treated subcutaneously with 3 mg retinol dissolved in sunflover seed oil. Controls received the vehicle only. When the animals were four months old blood was taken by cardial puncture in ether narcosis into heparinized tubes. Sepurution und treatment ofthe lymphocytes One sample (about 8-10 ml) was diluted with RPM1 cultivation medium and was centrifuged to a Ficoll-paque (Pharmacia, Lund, Sweden) for 20 min at 20 ‘C with 2000 rpm. After that the lymphocyte ring was collected to RPM1 medium and centrifuged again with 1000 rpm for 5 min at 20 “C The sediment was resuspended in serum containing medium. The cells were plated (cell count 2x106/ml) and were incubated or not incubated with Concanavalin A (ConA. 10-j g/ml) for 48 h. After that in some of the groups dexamethasone treatment was done in 1(I” M concentration. By this treatments 8 groups were formed: Neonatal]), not treated (control, groups l-4), and neonatally retinol treated (groups S-8) animals: 1 and 5: without any further treatment; 2 and 6: Con-A treatment; 3 and 7: dexamethasone treatment; 4 and 8: Con-A and dexamethasone treatment.
After thorough washing in PBS the cells were centrifuged (1800 fixed in -20 “C 70% ethyl-alcohol, stored at 4 “C for 1 hour and supplemented with 0.5 mg/ml RNAse A (Sigma, St Louis, Miss. -37 (‘C for 30 min, the cells were stained with 50 ug/ml propidium for I5 min.
rpm, 5 min) the pellet was resuspended in 0.5 ml PBS USA). After incubation at iodide at room temperature
The analysis was performed on a FACStar flow cytometer (Becton-Dickinson, Mountain Vie\v. California, USA) in conjunction with a HP-300 computer and Consort 30 data acquisition package. Excitation was measured at 488 nm, emission at 585142 nm. Approximately lo4 cells per sample were scanned using low flow rates (100 to 200/set) to maximiLe resolution. Significance was evaluated by Student t-test. For light microscopic control of apoptosis Giemsa staining and Apop-Tag reaction was used. Results and Discussion Apoptosis is a special form of cell death, regulating cell number and eliminating pathological cells from the population. Members of the immune system, first of all lymphocytes are subjected to apoptosis in case of development and overpopulation alike (24,25). These cells
Vol. 61, No. 23, 1997
Apoptosis after Neonatal Vitaok
A Treatment
PL341
express glucocorticoid receptors, the presence of whose is needed for the apoptosis promoting effect of dexamethasone (1). Concanavalin A (Con-A) also can promote apoptosis by binding to cell surface (receptor) carbohydrates (26). Vitamin A (retinol) can increase the number of apoptotic cells in adult animals (5,6,27). In our experiments retinol was used neonatally and adult animals were studied for apoptosis. This means, that the acute effect of retinol was excluded and only the changes caused by the neonatal treatment (and inherited from cell to cell) could be evaluated at adult age. This neonatal influence caused the significant elevation of the number of spontaneously (without further treatment) apoptotic cells (Table. 1). This elevation is only 15%, however significant (p
1
of untreated and neonatally
group control (c) c+Con-A c+dexameth.(d) c+Con-A+d ROH treated(R) R+Con-A R+d R+Con-A+d
% 37.37 36.34 40.75 38.01 42.79 39.22 45.09 43.39
retinoi treated rats. SE 2.04 2.15 2.77 2.12 1.36 1.90 1.98 2.38
Significances between: 1 and 5 = ~~0.05; 1 and 7 = ~~0.05; 2 and 5 = ~~0.05; 2 and 7 = ~~0.02 n = number of measurements The retinol + dexamethasone combination caused higher elevation in apoptosis, than retinol alone. Considering that the elevation caused by dexamethasone alone was not significant and retinol and dexamethasone caused significant elevation to control, this means that the neonatal retinol treatment potentiated the apoptosis of lymphocytes induced by dexamethasone, and this would be accepted as an effect of neonatal imprinting. Con-A was used for stimulating cell proliferation prior to dexamethasone treatment. However, according to the literary data Con-A can influence apoptosis, too (26). In our experiment ConA neither influenced significantly apoptosis alone nor in combination with dexamethasone. Nevertheless the spontaneous apoptosis of neonatally retinol treated animals was significantly higher (~~0.05) than that of the Con-A treated control animals. Summarizing the results, the experiments call attention to the prolonged effect of perinatal vitamin A treatment in a new index: apoptosis. This means that a developmental process can be influenced by a morphogenetic molecule months after the direct intervention done after the completion of the intrauterine life Considering the use of vitamin A in prevention of hypovitaminosis in lactating mothers or in infants, the experiments have some human aspects.
PL342
Apoptosis after Neonatal Vitamin A Treatment
Vol. 61, No. 23, 1997
Acknowledgements
This work was supported by the National 17849, Hungary
Research Fund (OTKA) T-017775,
T-17722. T-
References
1. J.A.CIDLOWSKI, K.L. KING. R.B. EVANS-STORMS, J.W. MONTAGUE. C.D. BORTNER and F.M. HUGHES, Recent Progr. Horm. Res. 51 457-490 (1996). 2. J. GRUBER, R. SGONC, Y.H. HU, H. BEUG and G. WICK, Eur. J. Immunol. 24 11IS1121 (1994). 3. T.W. McCLOSKEY, N. OYAZU, M. CORONESI and S. PAHWA, Clin. Immunol. Immunopathol. 71 14-18 (1994). 4. D. SCHULER, B. SZENDE, J.D. BORSI, T MARTON, J. BOCSI, E. MAGYAROSSY. R. KOOS and M. CSOKA, Pediatr. Hematol. Oncol. 11 641-649 (1994). 5. ~i‘,.i‘.WANG and J.M. PHANG. Cancer Lett. 107 65-71 (1996). 6. L. NAGY. V.A. THOMAZY, R.A. CHANDRARATNA. R.A. HEYMAN and P.S. DAVIES. (1996) Leuk. Res. 20. 499-505. 7. M. PONZONI, P. BOCCA. V. CHIESA. A. DECENSI, V. PISTOIA, L.RAFFAGHELLO. C. ROZZO and P.G. MONTALDO, Cancer Res. 55 853-861 (1995). 8. R.P. BISSONETTE, T. BRUNNER, S.B. LAZARCHIK, N.J. YOO, M.F. BOEHM. D.R.GREEN and R.A. HEYMAN, Mol. Cell. Biol. 15 5576-5585 (1995). 9. Y. YANG, S. MINUCCI. K. OZATO, R.A. HEYMAN and J.D. ASHWELL. J. Biol. Chem. 270 18672-18677 (1993). 10. G. CSABA, Biol. Rev. 55 47-63 (1980). 11, G.CSABA, Experientia 42 750-759 (1986). 12. G. CXABA, Zool. Sci. 8 813-825 (1991). 13. G.CSABA and S.U. NAGY, 1. Endocrinol. Invest. 8 557-560 (1985). 14. G. CSABA, A. INCZEFI-GONDA and 0. DOBOZY, Acta Physiol. Hung. 67 207-212 15. A. INCZEFI-GONDA, G.CSABA and 0. DOBOZY, Acta Physiol. Hung. 67 27-29 ( 1986). 16. 1. IGUCHI. P.L. OSTRANDER. K.‘l‘. MILLS and H.A. BERN, Med. Sci. Res. 15,489490 (1987). 17. H.A. BERN. M. EDERY, K.T. MILLS. A.T. KAHRMAN, T. MAR1 and L.H.LASSO, Cancer Res. 47 4165-4172 (1987). 18. D.F.C. GIBSON, D.A. ROBERTS and S. EVANS Eur. J. Cancer 27 3133-3137 (1991). 19. AN. TCHERNITCHIN and N. TCHERNITCHIN, Med. Sci. 20 391-397 (1992) 20. S. MIRZAHOSSEINI, CS. KARABPLYOS, 0. DOBOZY and G. CSABA, Hum. Exp. Toxicol 15 573-576 (1996). 2 1. D.J. MANGELSDORF, Nutr. Rev. 52 532-544 (1994). 22. A. GALL and G. CSABA, Endocrin. Metab. 4 115-l 19 (1997). 23. G. CSABA and A. GAAL, Hum. Exp. Toxicol. 16 193-I 97 (1997). 24. T.G. COTTER, S.V. LENNON, J.G. GLYNN AND S.J. MARTIN, Anticancer. Res. 10 1153-1159(1959). 25. T.G. COTTER, Semin. Immunol. 4 399-405 (1992). 26. G.V. KULKARNI and C.A. McCULLOCH. J. Cell Physiol. 165 119-l 33 (1995). 27. M.B. GLOZAK and M.B. ROGERS, Dev. Biol. 179 458-470 (1996). 28. S.A. AHMED and N. SRIRANGANATHAN. Immunopharmacology 28 55-66 (1994).