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Morphologic and metabolic changes in rat osteoblast cultures during the dark reaction with 8-methoxypsoralen
~-aioiooica la~oetkmt ELSEVIER S('IENCFI IRELAND
Chemico-Biological Interactions 90 (1994) 185-193
Morphologic and metabolic changes in rat osteoblast cultures during the dark reaction with 8-methoxypsoralen Francis J. Hornicek *a, George I. Malinin b, Kresimir Banovac a, Theodore I. Malinin a aDepartment of Orthopaedics and Rehabilitation, R-12, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, USA hDepartment of Physics, Georgetown University, Washington. DC 20057. USA (Received 20 April 1993; revision received 10 August 1993; accepted 11 August 1993)
Abstract The dark reaction of 8-methoxypsoralen (8-MOP) with cultured rat osteoblasts did not cause significant changes in cellular replication rates or in the synthesis of RNA and proteins. Microscopic examination, however, revealed that the dark reaction resulted in massive accumulation of perinuclear lipids and in the statistically significant enhancement of alkaline phosphatase activity. A sharp, and statistically significant, upsurge of lipid synthesis in osteoblasts preceded microscopically detectable accumulation of lipids and occurred only during the initial, but not during the subsequent stages of the dark reaction. These results suggest that in the course of the dark reaction the plasma membrane of osteoblasts is a target of psoralen.
Key words: 8-Methoxypsoralen; Osteoblasts; Lipids; Dark reaction; Plasma membrane
I. Introduction Over the last two decades photosensitizing furocoumarins (psoralens) became the focus of an ever-increasing interest [1]. Furocoumarin derivatives, such as 8-meth* Corresponding author. Abbreviations: BGJb medium, Biggers, Gwatkin, Judah tissue culture medium for bone and cartilage; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; EtOH, ethanol; FCS, fetal calf serum; [3H]leu, tritiated leucine; SBB, Sudan black B; [3HITdR, tritiated thymidine; [3HJUdR. tritiated uridine; UV, ultraviolet. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0009-2797(93)03228-M
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oxypsoralen (8-MOP), and trimethylpsoralen (TMP), became widely used photochemical agents in dermatology [2], and were shown to be valuable structural probes of nucleic acids [3,4]. In parallel with the advances in studies of biological effects of psoralen in the presence of ultraviolet light [1], it became apparent that the interaction of furocoumarins with biological targets prior to the UV irradiation is also a factor in the photomodification of cells and their macromolecular components. Indeed, these so called dark reactions of psoralens were reported to occur with DNA [5], RNA [6], plasma membranes [7-9], and with isolated and cellular lipids [10]. Owing to the usage of psoralens primarily in photochemotherapy of skin and certain blood diseases [11], the studies of the interaction of furocoumarins with skin and blood cells predominated, whereas much less is known regarding effects of psoralen on the other cell types. For example, it was reported that 8-MOP is incorporated by cartilage and synovial fluid in vivo [12], however, there is no information regarding the effects of psoralen on bone matrix or bone cell function, It is almost certain that at least a fraction of bone in the subdermal periosteum should be accessible to light during the therapeutic whole body irradiation. Concurrently, the bone cells shielded from light are nevertheless a target of the dark reaction with systemic psoralens and with the products of the photo-oxidized photosensitizer [13]. Moreover, psoralens are ubiquitous in many edible plants [14], thus, constituting pervasive dietary factors, probably affecting at least some aspects of bone metabolism. Therefore, in this investigation we have assessed certain consequences of the dark 8-MOP interaction with primary cultures of rat osteoblasts. 2. Materials and methods
2.1. Materials Penicillin and fetal calf serum (FCS) were obtained from Grand Island Biological Co. (GIBCO), Grand Island, NY, while streptomycin sulfate was bought from Eli Lilly and Co., Indianapolis, IN. Sodium bicarbonate was obtained from J,T. Baker, Phillipsburg, NJ. Psoralen (8-MOP) was a gift from Paul B. Elder, Co., Byran, OH, and because of its poor solubility in water, was prepared as a stock solution in ethanol at a concentration of 1 mg/ml and kept in the dark. [Methyl-3H]thymidine (1.7 Ci/mmol), [5-3H]uridine (15.2 Ci/mmol) and [l14C]sodium acetate (18.6 mCi/mmol) were obtained from Sigma Chemical Co., St Louis, MO. Tritiated [2,3,4,5-3H]leucine (120 Ci/mmol) was purchased from ICN, Irvine, CA, and L°[2,3-3H]proline (22.7 Ci/mmol) was bought from New England Nuclear, Boston, MA. Trypan blue dye, dissolved in phosphate buffered saline (PBS) at a concentration 0.4'7,, w/v was bought from Fisher Scientific, Pittsburgh, PA. Sudan black B (George T. Gurr, Ltd,, London, England) was prepared as 0.1% w/v solution in 70% v/v EtOH. All plasticware came from Coming, the 96-well plates were from GIBCO and the Lab-Tek tissue culture chamber slides were from Miles Lab Inc., Naperville, IL. Collagenase type II (EC 3.4.24.3), bovine type l trypsin (EC 3.4.21.4), the alkaline phosphatase kit (Sigma No. 86) and EDTA were purchased from Sigma. BGJb medium with the Fitton-Jackson modification (cat. No. B6644) with L-glutamine was also purchased from Sigma.
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2.2. Methods Cell cultures. Primary cultures of osteoblasts were established as described by Luben et al. [15]. In brief, calvariae from 1-day-old Wistar rats were dissected free from the adherent connective tissue, minced, and then digested for specified intervals in a PBS solution, containing 2000 units of collagenase II/ml, 0.35 mg trypsin/ml, and 4 mM EDTA. The first fraction of cells released during the first 20 min of incubation was discarded, whereas the subsequent four cell fractions were pooled, their viability determined by trypan blue exclusion and cultured as follows: Isolated cells (5-32 × 103/well) were inoculated in 96-well plates at 37°C in BGJb medium, supplemented with 10% v/v FCS, and grown in a 5% CO2 humidified atmosphere. Psoralen solution (200 t~l) was added to each well/chamber in a working concentration ranging from 2 ng/well to 2 #g/well. Cells incubated in the presence of 0.001% v/v to 1% v/v ethanol served as controls for alcoholic psoralen. After 48 h of incubation, the BGJb medium was replaced with serum free-BGJb medium, and the incubation extended for an additional 24-96 h. During the last 12 h of culture 1 #Ci of labeled thymidine, uridine, leucine, proline, or acetate [16] in 20 IA of BGJb medium were added to each well. At specified times ranging from 12 to 96 h, the cultured cells were harvested onto glass fiber sheets and their radioactivity determined in triplicate samples, as a minimum.
2.3. Alkaline phosphatase activity determination Alkaline phosphatase activity in osteoblasts was determined directly on the culture dishes as described previously [15]. In brief, rat osteoblasts in BGJb medium containing 10% v/v FCS were seeded into 24-well plates at 1.5 × 105 cell/well. At approximately 70% confluence, the cell cultures were washed with serum-free medium and then incubated for 24 h in the presence of indicated concentrations of 8-MOP. Thereafter, the culture medium was aspirated, the wells were washed with PBS and 1 ml of the reaction mixture, containing 2 mg ofp-nitrophenyl phosphate in alkaline buffer (Sigma No. 221), was added to each well. After 30 min incubation at 37°C, the reaction was terminated by 0.5 ml addition of Na3PO4; the reaction mixture was transferred to glass tubes. After the dilution with 0.05 M NaOH, the absorbance of the reaction product was measured at 410 nm. The standards were prepared as directed by Sigma. Microscopy. Control and experimental cell cultures were grown on Lab-Tek tissue culture slides for the microscopic surveillance and for the assay of alkaline phosphatase activity. To ascertain whether isolated cells were indeed osteoblasts [17], they were stained for alkaline phosphatase using a Sigma kit. At the end of 48 h of cultivation all experimental and control cultures were assayed again for the presence of alkaline phosphatase activity. The viable cultures were monitored visually with phase contrast optics or fixed alternatively for 1 h in 10% v/v formalin containing 1% w/v CaCI 2. Fixed cells were then stained briefly with 0.1% w/v SBB in 70% ethanol and mounted in glycerol for the subsequent phase-contrast and conventional microscopy. Statistics. Student's t-test was used to determine the statistical significance of the Quantitative data.
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3. Results The morphology of intact osteoblasts (Fig. 1), grown as a monolayer, was influenced by cell density. In confluent cultures, osteoblasts varied in shape from elongated, spindle-like cells to large, ovoid cells with small nuclei containing one or
Fig. 1. (A) Phase contrast microphotograph of confluent monolayer of intact (control) osteoblasts. The perinuclear areas are devoid of visible particles and droplets. Phase contrast (× 500). (B) A monolayer of osteoblasts, cultured for 48 h in the presence of 500 ng/well of 8-MOP. As compared to the intact cells (panel A) the nuclei of these cells are enclosed by a dense ring of lipid droplets. Phase contrast ( × 500t. (C) Formol-fixed control osteoblasts stained with 0.1% w/v Sudan Black B. The nuclei are surrounded by a ring of diffuse Sudan Black B-positive material. Phase contrast ( × 800). (Dt Osteoblasts cultured for 48 h in the presence of 500 ng/ml 8-MOP, fixed in formol, and then stained with Sudan Black B. The nuclei of most cells are enclosed by a ring of granular sudanophilic material whereas the remainder of the cytoplasm contains only scattered sudanophilic depositions. Phase contrast ( × 800). (El Formol-fixed control osteoblast stained with 0.1% w/v Sudan Black B ( x 800). (F and G) Formol-fixed osteoblasts grown in the presence of 8-MOP (500 ng/ml). Note the dense ring of perinuclear sudanophilic material (panel F / a n d characteristically granular nature of the perinuclear lipids. (F, x 800, G, x 2000).
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two small nucleoli (panel A). Multinucleated or binucleated cells were absent in either confluent, or in nearly-confluent cultures. Osteoblasts, in nearly-confluent cultures, were cells with extensive, irregularly-shaped cytoplasm and round or ovoid nuclei (panel C). When not in direct contact with each other, the cells were interconnected by a number of cytoplasmic bridges. The nuclei, as a rule, were circumscribed by a ring of very small SBB-positive grains, plainly visible with phase contrast (panel C), but not with the conventional optics (panel E). Cells cultured either for 24 or 48 h in the presence of 8-MOP (500 ng/ml) invariably contained enlarged nuclei surrounded by a dense ring of lipids (panel B). When stained with SBB these perinuclear lipids were intensely sudanophilic, whereas the remainder of the cytoplasm was essentially devoid of the sudanophilic material (panels D, F and G). Again, in contrast to the intact osteoblasts, the intercellular cytoplasmic bridges were absent (panel D). In general, the above-noted microscopic alterations were elicited by all tested concentrations of 8-MOP, but were expressed most vividly at the highest concentration. However, no morphologic deviations from control osteoblasts were noted in cells, cultured in the presence of the indicated amounts of ethanol. The intact cells, when stained for alkaline phosphatase, were 75 + 5% positive. After the incubation in the presence of 8-MOP, approximately the same number of cells remained alkaline phosphatase positive, although the amount of precipitated azo dye exceeded control results. The colorimetric determination of alkaline phosphatase in osteoblast cultures (Table !) was consistent with the microscopic observation and has confirmed that psoralen induces the increased levels of this enzyme in target cells. In response to 8-MOP, the proliferation rate of osteoblasts tended to increase in a dose-dependent manner as assessed by thymidine incorporation. Nonetheless, the differences in the rates of [3H]TdR incorporation by the experimental and control cell cultures were statistically insignificant. The uptake of radioactivity by control osteoblasts (520 4- 158 counts/min) was 0.6 times that of cells exposed to 2000 ng of 8-MOP. For uridine incorporation, the rate of radioactivity uptake by psoralentreated osteoblasts was 0.66 times that of the control (6396 4- 518 counts/min). Statistically, this inhibition of the R N A synthesis was likewise not significant.
Table 1 Alkaline phosphatase activity in osteoblasts after the dark reaction with 8-MOP for 24 h 8-MOP (ng/well)
Alkaline phosphatase activity (units/ml)
0 200 2000
3.5 4- 0.4 3.5 4- 0.3 4.3 ~: 0.2
P-value
N.S.* <0.03
Alkaline phosphatase activity was determined directly in culture plates as described in the methods section. Results are expressed as a mean ± S.D. of quadruplicate samples. The P-value is for experimental versus control (zero concentration 8-MOP): Student's t-test. * N.S., not statistically significant.
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In the tested concentration range (2-2000 ng 8-MOP/well), psoralen failed to either enhance or to depress synthesis of proteins by osteoblasts. The rate of proline incorporation, an indicator of collagen synthesis, and the rate of incorporation of leucine were not significantly altered by the presence of 8-MOP. The rates of proline incorporation were 662 4- 137 counts/min in control and 675 ± 70 counts/min in experimental cell cultures (2000 ng 8-MOP). For leucine incorporation, the rates of uptake were 1122 4- 110 counts/min in control and 888 4- 74 counts/min in 2000 ng 8-MOP-exposed cell cultures. By contrast with the above, psoralen induced an almost immediate upsurge in the incorporation of acetate (P < 0.05). This change was noted by 5 h of culture in the presence of 8-MOP. This critical response was followed by a gradual, dose-dependent decline of acetate incorporation. Control osteoblasts, however, continued to incorporate acetate at increasing rate during the first 24 h which by 48 h was significantly greater than in psoralen-treated cultures (P < 0.001). These data are summarized (Fig. 2). 4. Discussion In undertaking this study we were aware that the consequences of psoralen interaction with osteoblasts were not delineated previously and that their expression may
5000
4000 '
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i
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1000
i
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40
i
60
i
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time Fig. 2. Effects of 8-MOP on the kinetics of lipid synthesis by rat osteoblast cells as assessed by the incorporation of [14C]acetate. Radioactivity (mean counts/rain ± standard deviationl is shown for control (A), and psoralen 2000 ng/well 8-MOP (UI), 200 ng/well 8-MOP (B) exposed rat osteoblast cultures. Time axis is in hour increments.
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be registered at any level of the cellular organization and function. The response of osteoblasts to psoralen was therefore assessed in terms of the demonstrable morphologic changes, cellular replication rates and the alterations in synthesis of proteins and lipids. As measured by the [3Hlthymidine incorporation rates, the dark reaction of psoralen with osteoblasts did not affect their proliferation, and these results are in agreement with the previously published data [7]. During the dark reaction of psoralen with osteoblasts the rate of thymidine incorporation tended to increase in a dose-dependent manner. Although statistically not significant, the increase in thymidine uptake was analogous to the transient increase of iododeoxyuridine incorporation by human iymphoblasts during the dark reaction with psoralen [7]. As was the psoralen effect on the rate of DNA synthesis, the rate of RNA synthesis was not statistically affected by the presence of 8-MOP. However, a tendency towards depression of RNA synthesis with rising 8-MOP concentrations was noted. This could be the result of psoralen complexation with RNA. In our study the dark reaction of psoralen with osteoblasts did not result in statistically significant differences in the incorporation of leucine and proline. These data suggest that under stated conditions psoralen does not exert a detectable effect on the total proteins and collagen synthesis by cultured rat osteoblasts. In contrast to the indicators of nucleic acids and protein synthesis, psoralen has induced an increase of acetate incorporation and the stimulation of alkaline phosphatase activity. The exact mechanisms responsible for the increase of lipid synthesis and the stimulation of alkaline phosphatase activity during the dark reaction of osteoblasts with psoralen cannot be fully accounted for at this time. Nevertheless, possible pathways of psoralen action on osteoblasts can be appraised in the light of the existing evidence and supporting conjectures. It was shown that many cells possess plasma membrane and intracytoplasmic high-affinity psoralen receptors [18], and that the saturation of these receptors with psoralen results in the corresponding decline of EGF binding. Since the EGF receptors are endowed with an intrinsic tyrosine kinase activity [19], it was suggested that binding of 8-MOP by EGF receptors alters the signal transduction pathways in target cells [20]. The binding of psoralen by protein and lipid components of the cell membrane receptors was postulated to be an initial step in the alteration of signalling pathways during the dark reaction and upon subsequent UV-light irradiation [21]. The formation of psoralen complexes with unsaturated [10] and saturated [22] phospholipids strongly supports this conjecture and implies possible inhibition of phospholipases by psoralens. The inhibition of catabolic function of phospholipases by psoralen-lipid complexes could account for the accumulation of sudanophilic lipids by osteoblasts. Recently, by employing staurosporine a potent alkaloid inhibitor of protein kinases, including tyrosine kinase, Yang et al. [23] have elicited a response in rat osteoblasts, in many respects, identical to the effect exerted by psoralen. As was the case with psoralen, staurosporine also induced rapid accumulation of perinuclear lipid depositions and a marked enhancement of alkaline phosphatase activity. Taken together, these data and our results suggest that in osteoblasts, psoralen alters signal
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transduction pathways, probably regulated by protein kinases, and that these alterat i o n s a r e e x p r e s s e d p r i m a r i l y in t r a n s i e n t i n c r e a s e o f lipid s y n t h e s i s a n d s e e m i n g l y l o n g - l a s t i n g a c c u m u l a t i o n o f p e r i n u c l e a r lipids. T h e l a t t e r m a y r e p r e s e n t a u n i q u e response of osteoblasts since no increase of lipid synthesis or accumulation was r e g i s t e r e d b y H U T 102 l y m p h o b l a s t s in t h e c o u r s e o f t h e d a r k r e a c t i o n w i t h 8 - M O P UO].
5. References 1 D. Averbeck, Recent advances in psoralen phototoxicity mechanism, Photochem. Photobiol., 50 (1989) 859-882. 2 T.F. Anderson and J.J. Voorhees, Psoralen. Photochemotherapy of cutaneous disorders, Annu. Rev. Pharmacol. Toxicol., 20 (1980) 235-257. 3 J.E. Hearst, S.T. lsacs, D. Kanne, H. Rapport and K. Straub, The reaction of psoralens with deoxyribonucleic acid, Q. Rev. Biophys., 17 (1984) 1-44. 4 G.D. Cimino, H,B. Gamper, S.T. Isacs and J.E. Hearst, Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry and biochemistry, Annu. Rev. Biochem., 54 (1985) 1151-1193. 5 F. Dall'Aqua, M. Terbojevich, S. Marciani, D. Vedaldi and M. Recher, Investigation on the dark reaction between furocoumarins and DNA, Chem.-Biol. Interact., 21 (1978) 103-115. 6 S.E. Lipson and J.E. Hearst, Psoralen cross-linking of ribosomal RNA, in: H.F. Noller, Jr. and K. Maldove (Eds.), Methods in Enzymology, Academic Press, San Diego, CA, Vol. 164, 1988, pp. 319-329. 7 F.J. Hornicek, T.I. Malinin, H. Gratzner and G.I. Malinin, Cytometric analysis of the proliferative capacity of HUT 102 lymphoblasts exposed to long-wave UV light and psoralen, J. Invest. Dermatol., 93 (1989) 96-99. 8 G.I. Malinin, H.K. Lo and F.J. Hornicek, Structural photomodification of erythrocyte ghosts by long-wave UV light and psoralen, Photobiochem. Photobiophys., 13 (1986) 145-153. 9 G.I. Malinin, H.K. Lo, F.J. Hornicek and T.1. Malinin, Ultrastructural modification of the plasma membrane in HUT 102 lymphoblasts by long-wave ultraviolet light, psoralen and PUVA, J. Invest. Dermatol., 95 (1990) 97-103. 10 F.J. Hornicek, G.I. Malinin, T.I. Malinin and H.K. Lo, Turnover ofphospholipids in HUT 102 lymphoblasts and chromatographic characterization of purified lecithins after their exposure to longwave UV light, psoralen, and UV light and psoralen. J. Photochem. Photobiol. B. Biol., 12 (1992) 359-374. 11 H. H6nigsmann, Psoralen photochemotherapy: mechanisms, drugs, toxicity, Curt. Prob. Dermatol., 15 (1986) 52-66. 12 G.I. Malinin, W.B. Glew, W.P. Roberts, L. Townsend and T.P. Nigra, Kinetics of (14C-5)8methoxypsoralen distribution in rabbits, Arch. Dermatol. Res., 273 (1982) 319-326. 13 A.Y. Potapenko, L.N. Bezdetnaya, L.P. Lysenko, S.N. Akhtyamov, S.K. Tomashaeva and V.L. Sukhorukov, Hypothesis of the induction of psoralen phototoxic effects through the stage of photooxidized psoralen formation. Model studies of erythrocytes. Stud. Biophys., 124 (1988) 205-223. 14 A.M. EIMofty, in: Vitiligo and Psoralen, Pergamon Press, Oxford, 1976, pp. 1-15. 15 R. Luben, G. Wong and D. Cohn, Biochemical characterization with parathormone and calcitonin of isolated bone ceils. Endocrinology, 99 (1976) 526-534. 16 B.V. Howard, Acetate as a carbon source for lipid synthesis in cultured cells. Biochim. Biophys. Acta, 488 (1977) 145-151. 17 M, Owen, Origin of bone ceils, Int. Rev. Cytol., 28 (1970) 213-238. 18 J.D. Laskin and D.L. Laskin, Role ofpsoralen receptors in cell growth regulation, in: F.P. Gasparro (Ed.), Psoralen DNA Photobiology, CRC Press, Boca Raton, FL, Vol. 2, 1988, pp. 135-148. 19 G. Carpenter, Receptors for epidermal growth factor and other polypeptide mitogens. Annu. Rev. Biochem., 56 (1987) 881-914.
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X.-Y. Yang, Z.A. Ronai, R.M. Santella and I.B. Weinstein, Effect of 8-methoxypsoralen and ultraviolet light A on EGF receptor (HER-I) expression, Biochem. Biophys. Res. Commun., 157 (1988) 590-596. 21 W.R. Midden, Chemical mechanisms of the bioeffects of furocoumarins: the role of reactions with proteins, lipids and other cellular constituents, in: F.P. Gasparro (Ed.), Psoralen DNA Photobiology, CRC Press, Boca Raton, FL, Vol. 2, 1988, pp. 1-50. 22 T. Pali, B. Ebert and L.I. Horvath, Structural modification of lipid vesicles by 8-methoxypsoralen, J. Photochem. Photobiol. Biol., 3 (1989) 359-367. 23 R.-S. Yang, K.-S. Lu, W.-M. Fu, T.-K. Liu and S.-Y. Lin-Shiau, Staurosporine-induced morphological changes in the rat osteoblasts, Cell Biol. Int., 17 (1993) 75-82.
Chemico-Biological Interactions 90 (1994) 185-193
Morphologic and metabolic changes in rat osteoblast cultures during the dark reaction with 8-methoxypsoralen Francis J. Hornicek *a, George I. Malinin b, Kresimir Banovac a, Theodore I. Malinin a aDepartment of Orthopaedics and Rehabilitation, R-12, University of Miami School of Medicine, P.O. Box 016960, Miami, FL 33101, USA hDepartment of Physics, Georgetown University, Washington. DC 20057. USA (Received 20 April 1993; revision received 10 August 1993; accepted 11 August 1993)
Abstract The dark reaction of 8-methoxypsoralen (8-MOP) with cultured rat osteoblasts did not cause significant changes in cellular replication rates or in the synthesis of RNA and proteins. Microscopic examination, however, revealed that the dark reaction resulted in massive accumulation of perinuclear lipids and in the statistically significant enhancement of alkaline phosphatase activity. A sharp, and statistically significant, upsurge of lipid synthesis in osteoblasts preceded microscopically detectable accumulation of lipids and occurred only during the initial, but not during the subsequent stages of the dark reaction. These results suggest that in the course of the dark reaction the plasma membrane of osteoblasts is a target of psoralen.
Key words: 8-Methoxypsoralen; Osteoblasts; Lipids; Dark reaction; Plasma membrane
I. Introduction Over the last two decades photosensitizing furocoumarins (psoralens) became the focus of an ever-increasing interest [1]. Furocoumarin derivatives, such as 8-meth* Corresponding author. Abbreviations: BGJb medium, Biggers, Gwatkin, Judah tissue culture medium for bone and cartilage; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; EtOH, ethanol; FCS, fetal calf serum; [3H]leu, tritiated leucine; SBB, Sudan black B; [3HITdR, tritiated thymidine; [3HJUdR. tritiated uridine; UV, ultraviolet. 0009-2797/94/$07.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved. SSDI 0009-2797(93)03228-M
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oxypsoralen (8-MOP), and trimethylpsoralen (TMP), became widely used photochemical agents in dermatology [2], and were shown to be valuable structural probes of nucleic acids [3,4]. In parallel with the advances in studies of biological effects of psoralen in the presence of ultraviolet light [1], it became apparent that the interaction of furocoumarins with biological targets prior to the UV irradiation is also a factor in the photomodification of cells and their macromolecular components. Indeed, these so called dark reactions of psoralens were reported to occur with DNA [5], RNA [6], plasma membranes [7-9], and with isolated and cellular lipids [10]. Owing to the usage of psoralens primarily in photochemotherapy of skin and certain blood diseases [11], the studies of the interaction of furocoumarins with skin and blood cells predominated, whereas much less is known regarding effects of psoralen on the other cell types. For example, it was reported that 8-MOP is incorporated by cartilage and synovial fluid in vivo [12], however, there is no information regarding the effects of psoralen on bone matrix or bone cell function, It is almost certain that at least a fraction of bone in the subdermal periosteum should be accessible to light during the therapeutic whole body irradiation. Concurrently, the bone cells shielded from light are nevertheless a target of the dark reaction with systemic psoralens and with the products of the photo-oxidized photosensitizer [13]. Moreover, psoralens are ubiquitous in many edible plants [14], thus, constituting pervasive dietary factors, probably affecting at least some aspects of bone metabolism. Therefore, in this investigation we have assessed certain consequences of the dark 8-MOP interaction with primary cultures of rat osteoblasts. 2. Materials and methods
2.1. Materials Penicillin and fetal calf serum (FCS) were obtained from Grand Island Biological Co. (GIBCO), Grand Island, NY, while streptomycin sulfate was bought from Eli Lilly and Co., Indianapolis, IN. Sodium bicarbonate was obtained from J,T. Baker, Phillipsburg, NJ. Psoralen (8-MOP) was a gift from Paul B. Elder, Co., Byran, OH, and because of its poor solubility in water, was prepared as a stock solution in ethanol at a concentration of 1 mg/ml and kept in the dark. [Methyl-3H]thymidine (1.7 Ci/mmol), [5-3H]uridine (15.2 Ci/mmol) and [l14C]sodium acetate (18.6 mCi/mmol) were obtained from Sigma Chemical Co., St Louis, MO. Tritiated [2,3,4,5-3H]leucine (120 Ci/mmol) was purchased from ICN, Irvine, CA, and L°[2,3-3H]proline (22.7 Ci/mmol) was bought from New England Nuclear, Boston, MA. Trypan blue dye, dissolved in phosphate buffered saline (PBS) at a concentration 0.4'7,, w/v was bought from Fisher Scientific, Pittsburgh, PA. Sudan black B (George T. Gurr, Ltd,, London, England) was prepared as 0.1% w/v solution in 70% v/v EtOH. All plasticware came from Coming, the 96-well plates were from GIBCO and the Lab-Tek tissue culture chamber slides were from Miles Lab Inc., Naperville, IL. Collagenase type II (EC 3.4.24.3), bovine type l trypsin (EC 3.4.21.4), the alkaline phosphatase kit (Sigma No. 86) and EDTA were purchased from Sigma. BGJb medium with the Fitton-Jackson modification (cat. No. B6644) with L-glutamine was also purchased from Sigma.
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2.2. Methods Cell cultures. Primary cultures of osteoblasts were established as described by Luben et al. [15]. In brief, calvariae from 1-day-old Wistar rats were dissected free from the adherent connective tissue, minced, and then digested for specified intervals in a PBS solution, containing 2000 units of collagenase II/ml, 0.35 mg trypsin/ml, and 4 mM EDTA. The first fraction of cells released during the first 20 min of incubation was discarded, whereas the subsequent four cell fractions were pooled, their viability determined by trypan blue exclusion and cultured as follows: Isolated cells (5-32 × 103/well) were inoculated in 96-well plates at 37°C in BGJb medium, supplemented with 10% v/v FCS, and grown in a 5% CO2 humidified atmosphere. Psoralen solution (200 t~l) was added to each well/chamber in a working concentration ranging from 2 ng/well to 2 #g/well. Cells incubated in the presence of 0.001% v/v to 1% v/v ethanol served as controls for alcoholic psoralen. After 48 h of incubation, the BGJb medium was replaced with serum free-BGJb medium, and the incubation extended for an additional 24-96 h. During the last 12 h of culture 1 #Ci of labeled thymidine, uridine, leucine, proline, or acetate [16] in 20 IA of BGJb medium were added to each well. At specified times ranging from 12 to 96 h, the cultured cells were harvested onto glass fiber sheets and their radioactivity determined in triplicate samples, as a minimum.
2.3. Alkaline phosphatase activity determination Alkaline phosphatase activity in osteoblasts was determined directly on the culture dishes as described previously [15]. In brief, rat osteoblasts in BGJb medium containing 10% v/v FCS were seeded into 24-well plates at 1.5 × 105 cell/well. At approximately 70% confluence, the cell cultures were washed with serum-free medium and then incubated for 24 h in the presence of indicated concentrations of 8-MOP. Thereafter, the culture medium was aspirated, the wells were washed with PBS and 1 ml of the reaction mixture, containing 2 mg ofp-nitrophenyl phosphate in alkaline buffer (Sigma No. 221), was added to each well. After 30 min incubation at 37°C, the reaction was terminated by 0.5 ml addition of Na3PO4; the reaction mixture was transferred to glass tubes. After the dilution with 0.05 M NaOH, the absorbance of the reaction product was measured at 410 nm. The standards were prepared as directed by Sigma. Microscopy. Control and experimental cell cultures were grown on Lab-Tek tissue culture slides for the microscopic surveillance and for the assay of alkaline phosphatase activity. To ascertain whether isolated cells were indeed osteoblasts [17], they were stained for alkaline phosphatase using a Sigma kit. At the end of 48 h of cultivation all experimental and control cultures were assayed again for the presence of alkaline phosphatase activity. The viable cultures were monitored visually with phase contrast optics or fixed alternatively for 1 h in 10% v/v formalin containing 1% w/v CaCI 2. Fixed cells were then stained briefly with 0.1% w/v SBB in 70% ethanol and mounted in glycerol for the subsequent phase-contrast and conventional microscopy. Statistics. Student's t-test was used to determine the statistical significance of the Quantitative data.
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3. Results The morphology of intact osteoblasts (Fig. 1), grown as a monolayer, was influenced by cell density. In confluent cultures, osteoblasts varied in shape from elongated, spindle-like cells to large, ovoid cells with small nuclei containing one or
Fig. 1. (A) Phase contrast microphotograph of confluent monolayer of intact (control) osteoblasts. The perinuclear areas are devoid of visible particles and droplets. Phase contrast (× 500). (B) A monolayer of osteoblasts, cultured for 48 h in the presence of 500 ng/well of 8-MOP. As compared to the intact cells (panel A) the nuclei of these cells are enclosed by a dense ring of lipid droplets. Phase contrast ( × 500t. (C) Formol-fixed control osteoblasts stained with 0.1% w/v Sudan Black B. The nuclei are surrounded by a ring of diffuse Sudan Black B-positive material. Phase contrast ( × 800). (Dt Osteoblasts cultured for 48 h in the presence of 500 ng/ml 8-MOP, fixed in formol, and then stained with Sudan Black B. The nuclei of most cells are enclosed by a ring of granular sudanophilic material whereas the remainder of the cytoplasm contains only scattered sudanophilic depositions. Phase contrast ( × 800). (El Formol-fixed control osteoblast stained with 0.1% w/v Sudan Black B ( x 800). (F and G) Formol-fixed osteoblasts grown in the presence of 8-MOP (500 ng/ml). Note the dense ring of perinuclear sudanophilic material (panel F / a n d characteristically granular nature of the perinuclear lipids. (F, x 800, G, x 2000).
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two small nucleoli (panel A). Multinucleated or binucleated cells were absent in either confluent, or in nearly-confluent cultures. Osteoblasts, in nearly-confluent cultures, were cells with extensive, irregularly-shaped cytoplasm and round or ovoid nuclei (panel C). When not in direct contact with each other, the cells were interconnected by a number of cytoplasmic bridges. The nuclei, as a rule, were circumscribed by a ring of very small SBB-positive grains, plainly visible with phase contrast (panel C), but not with the conventional optics (panel E). Cells cultured either for 24 or 48 h in the presence of 8-MOP (500 ng/ml) invariably contained enlarged nuclei surrounded by a dense ring of lipids (panel B). When stained with SBB these perinuclear lipids were intensely sudanophilic, whereas the remainder of the cytoplasm was essentially devoid of the sudanophilic material (panels D, F and G). Again, in contrast to the intact osteoblasts, the intercellular cytoplasmic bridges were absent (panel D). In general, the above-noted microscopic alterations were elicited by all tested concentrations of 8-MOP, but were expressed most vividly at the highest concentration. However, no morphologic deviations from control osteoblasts were noted in cells, cultured in the presence of the indicated amounts of ethanol. The intact cells, when stained for alkaline phosphatase, were 75 + 5% positive. After the incubation in the presence of 8-MOP, approximately the same number of cells remained alkaline phosphatase positive, although the amount of precipitated azo dye exceeded control results. The colorimetric determination of alkaline phosphatase in osteoblast cultures (Table !) was consistent with the microscopic observation and has confirmed that psoralen induces the increased levels of this enzyme in target cells. In response to 8-MOP, the proliferation rate of osteoblasts tended to increase in a dose-dependent manner as assessed by thymidine incorporation. Nonetheless, the differences in the rates of [3H]TdR incorporation by the experimental and control cell cultures were statistically insignificant. The uptake of radioactivity by control osteoblasts (520 4- 158 counts/min) was 0.6 times that of cells exposed to 2000 ng of 8-MOP. For uridine incorporation, the rate of radioactivity uptake by psoralentreated osteoblasts was 0.66 times that of the control (6396 4- 518 counts/min). Statistically, this inhibition of the R N A synthesis was likewise not significant.
Table 1 Alkaline phosphatase activity in osteoblasts after the dark reaction with 8-MOP for 24 h 8-MOP (ng/well)
Alkaline phosphatase activity (units/ml)
0 200 2000
3.5 4- 0.4 3.5 4- 0.3 4.3 ~: 0.2
P-value
N.S.* <0.03
Alkaline phosphatase activity was determined directly in culture plates as described in the methods section. Results are expressed as a mean ± S.D. of quadruplicate samples. The P-value is for experimental versus control (zero concentration 8-MOP): Student's t-test. * N.S., not statistically significant.
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In the tested concentration range (2-2000 ng 8-MOP/well), psoralen failed to either enhance or to depress synthesis of proteins by osteoblasts. The rate of proline incorporation, an indicator of collagen synthesis, and the rate of incorporation of leucine were not significantly altered by the presence of 8-MOP. The rates of proline incorporation were 662 4- 137 counts/min in control and 675 ± 70 counts/min in experimental cell cultures (2000 ng 8-MOP). For leucine incorporation, the rates of uptake were 1122 4- 110 counts/min in control and 888 4- 74 counts/min in 2000 ng 8-MOP-exposed cell cultures. By contrast with the above, psoralen induced an almost immediate upsurge in the incorporation of acetate (P < 0.05). This change was noted by 5 h of culture in the presence of 8-MOP. This critical response was followed by a gradual, dose-dependent decline of acetate incorporation. Control osteoblasts, however, continued to incorporate acetate at increasing rate during the first 24 h which by 48 h was significantly greater than in psoralen-treated cultures (P < 0.001). These data are summarized (Fig. 2). 4. Discussion In undertaking this study we were aware that the consequences of psoralen interaction with osteoblasts were not delineated previously and that their expression may
5000
4000 '
3000'
i
2000
,q,
1000
i
0
40
i
60
i
80
100
time Fig. 2. Effects of 8-MOP on the kinetics of lipid synthesis by rat osteoblast cells as assessed by the incorporation of [14C]acetate. Radioactivity (mean counts/rain ± standard deviationl is shown for control (A), and psoralen 2000 ng/well 8-MOP (UI), 200 ng/well 8-MOP (B) exposed rat osteoblast cultures. Time axis is in hour increments.
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be registered at any level of the cellular organization and function. The response of osteoblasts to psoralen was therefore assessed in terms of the demonstrable morphologic changes, cellular replication rates and the alterations in synthesis of proteins and lipids. As measured by the [3Hlthymidine incorporation rates, the dark reaction of psoralen with osteoblasts did not affect their proliferation, and these results are in agreement with the previously published data [7]. During the dark reaction of psoralen with osteoblasts the rate of thymidine incorporation tended to increase in a dose-dependent manner. Although statistically not significant, the increase in thymidine uptake was analogous to the transient increase of iododeoxyuridine incorporation by human iymphoblasts during the dark reaction with psoralen [7]. As was the psoralen effect on the rate of DNA synthesis, the rate of RNA synthesis was not statistically affected by the presence of 8-MOP. However, a tendency towards depression of RNA synthesis with rising 8-MOP concentrations was noted. This could be the result of psoralen complexation with RNA. In our study the dark reaction of psoralen with osteoblasts did not result in statistically significant differences in the incorporation of leucine and proline. These data suggest that under stated conditions psoralen does not exert a detectable effect on the total proteins and collagen synthesis by cultured rat osteoblasts. In contrast to the indicators of nucleic acids and protein synthesis, psoralen has induced an increase of acetate incorporation and the stimulation of alkaline phosphatase activity. The exact mechanisms responsible for the increase of lipid synthesis and the stimulation of alkaline phosphatase activity during the dark reaction of osteoblasts with psoralen cannot be fully accounted for at this time. Nevertheless, possible pathways of psoralen action on osteoblasts can be appraised in the light of the existing evidence and supporting conjectures. It was shown that many cells possess plasma membrane and intracytoplasmic high-affinity psoralen receptors [18], and that the saturation of these receptors with psoralen results in the corresponding decline of EGF binding. Since the EGF receptors are endowed with an intrinsic tyrosine kinase activity [19], it was suggested that binding of 8-MOP by EGF receptors alters the signal transduction pathways in target cells [20]. The binding of psoralen by protein and lipid components of the cell membrane receptors was postulated to be an initial step in the alteration of signalling pathways during the dark reaction and upon subsequent UV-light irradiation [21]. The formation of psoralen complexes with unsaturated [10] and saturated [22] phospholipids strongly supports this conjecture and implies possible inhibition of phospholipases by psoralens. The inhibition of catabolic function of phospholipases by psoralen-lipid complexes could account for the accumulation of sudanophilic lipids by osteoblasts. Recently, by employing staurosporine a potent alkaloid inhibitor of protein kinases, including tyrosine kinase, Yang et al. [23] have elicited a response in rat osteoblasts, in many respects, identical to the effect exerted by psoralen. As was the case with psoralen, staurosporine also induced rapid accumulation of perinuclear lipid depositions and a marked enhancement of alkaline phosphatase activity. Taken together, these data and our results suggest that in osteoblasts, psoralen alters signal
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transduction pathways, probably regulated by protein kinases, and that these alterat i o n s a r e e x p r e s s e d p r i m a r i l y in t r a n s i e n t i n c r e a s e o f lipid s y n t h e s i s a n d s e e m i n g l y l o n g - l a s t i n g a c c u m u l a t i o n o f p e r i n u c l e a r lipids. T h e l a t t e r m a y r e p r e s e n t a u n i q u e response of osteoblasts since no increase of lipid synthesis or accumulation was r e g i s t e r e d b y H U T 102 l y m p h o b l a s t s in t h e c o u r s e o f t h e d a r k r e a c t i o n w i t h 8 - M O P UO].
5. References 1 D. Averbeck, Recent advances in psoralen phototoxicity mechanism, Photochem. Photobiol., 50 (1989) 859-882. 2 T.F. Anderson and J.J. Voorhees, Psoralen. Photochemotherapy of cutaneous disorders, Annu. Rev. Pharmacol. Toxicol., 20 (1980) 235-257. 3 J.E. Hearst, S.T. lsacs, D. Kanne, H. Rapport and K. Straub, The reaction of psoralens with deoxyribonucleic acid, Q. Rev. Biophys., 17 (1984) 1-44. 4 G.D. Cimino, H,B. Gamper, S.T. Isacs and J.E. Hearst, Psoralens as photoactive probes of nucleic acid structure and function: organic chemistry, photochemistry and biochemistry, Annu. Rev. Biochem., 54 (1985) 1151-1193. 5 F. Dall'Aqua, M. Terbojevich, S. Marciani, D. Vedaldi and M. Recher, Investigation on the dark reaction between furocoumarins and DNA, Chem.-Biol. Interact., 21 (1978) 103-115. 6 S.E. Lipson and J.E. Hearst, Psoralen cross-linking of ribosomal RNA, in: H.F. Noller, Jr. and K. Maldove (Eds.), Methods in Enzymology, Academic Press, San Diego, CA, Vol. 164, 1988, pp. 319-329. 7 F.J. Hornicek, T.I. Malinin, H. Gratzner and G.I. Malinin, Cytometric analysis of the proliferative capacity of HUT 102 lymphoblasts exposed to long-wave UV light and psoralen, J. Invest. Dermatol., 93 (1989) 96-99. 8 G.I. Malinin, H.K. Lo and F.J. Hornicek, Structural photomodification of erythrocyte ghosts by long-wave UV light and psoralen, Photobiochem. Photobiophys., 13 (1986) 145-153. 9 G.I. Malinin, H.K. Lo, F.J. Hornicek and T.1. Malinin, Ultrastructural modification of the plasma membrane in HUT 102 lymphoblasts by long-wave ultraviolet light, psoralen and PUVA, J. Invest. Dermatol., 95 (1990) 97-103. 10 F.J. Hornicek, G.I. Malinin, T.I. Malinin and H.K. Lo, Turnover ofphospholipids in HUT 102 lymphoblasts and chromatographic characterization of purified lecithins after their exposure to longwave UV light, psoralen, and UV light and psoralen. J. Photochem. Photobiol. B. Biol., 12 (1992) 359-374. 11 H. H6nigsmann, Psoralen photochemotherapy: mechanisms, drugs, toxicity, Curt. Prob. Dermatol., 15 (1986) 52-66. 12 G.I. Malinin, W.B. Glew, W.P. Roberts, L. Townsend and T.P. Nigra, Kinetics of (14C-5)8methoxypsoralen distribution in rabbits, Arch. Dermatol. Res., 273 (1982) 319-326. 13 A.Y. Potapenko, L.N. Bezdetnaya, L.P. Lysenko, S.N. Akhtyamov, S.K. Tomashaeva and V.L. Sukhorukov, Hypothesis of the induction of psoralen phototoxic effects through the stage of photooxidized psoralen formation. Model studies of erythrocytes. Stud. Biophys., 124 (1988) 205-223. 14 A.M. EIMofty, in: Vitiligo and Psoralen, Pergamon Press, Oxford, 1976, pp. 1-15. 15 R. Luben, G. Wong and D. Cohn, Biochemical characterization with parathormone and calcitonin of isolated bone ceils. Endocrinology, 99 (1976) 526-534. 16 B.V. Howard, Acetate as a carbon source for lipid synthesis in cultured cells. Biochim. Biophys. Acta, 488 (1977) 145-151. 17 M, Owen, Origin of bone ceils, Int. Rev. Cytol., 28 (1970) 213-238. 18 J.D. Laskin and D.L. Laskin, Role ofpsoralen receptors in cell growth regulation, in: F.P. Gasparro (Ed.), Psoralen DNA Photobiology, CRC Press, Boca Raton, FL, Vol. 2, 1988, pp. 135-148. 19 G. Carpenter, Receptors for epidermal growth factor and other polypeptide mitogens. Annu. Rev. Biochem., 56 (1987) 881-914.
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X.-Y. Yang, Z.A. Ronai, R.M. Santella and I.B. Weinstein, Effect of 8-methoxypsoralen and ultraviolet light A on EGF receptor (HER-I) expression, Biochem. Biophys. Res. Commun., 157 (1988) 590-596. 21 W.R. Midden, Chemical mechanisms of the bioeffects of furocoumarins: the role of reactions with proteins, lipids and other cellular constituents, in: F.P. Gasparro (Ed.), Psoralen DNA Photobiology, CRC Press, Boca Raton, FL, Vol. 2, 1988, pp. 1-50. 22 T. Pali, B. Ebert and L.I. Horvath, Structural modification of lipid vesicles by 8-methoxypsoralen, J. Photochem. Photobiol. Biol., 3 (1989) 359-367. 23 R.-S. Yang, K.-S. Lu, W.-M. Fu, T.-K. Liu and S.-Y. Lin-Shiau, Staurosporine-induced morphological changes in the rat osteoblasts, Cell Biol. Int., 17 (1993) 75-82.