103

THE EFFECT OF ALUMINUM ON THE STROMAL CELLS (IN VITRO) ON BONE MARROW IN RATS K.

ZAMAN*, M. MUKHTAR**, H. SIDDIQUE†, AND H. MISZTA+ of Pathology of Medicine of Pennsylvania

*Department School

University Philadelphia, **Department

Pennsylvania

of Biosciences and Drexel University

Philadelphia, †The

Biotechnology

Pennsylvania

Wistar Institute

Philadelphia,

Pennsylvania

The +

Laboratory of Toxicology Jagiellonian University Cracow, Poland

The aim of this study was to describe the effects of aluminum on the stromal cells of rat bone marrow using a combination of in vivo exposure and in vitro culture of bone marrow. The toxic effects of aluminum are manifested by a decrease in the erythrocyte count and hemoglobin level in the peripheral blood. Aluminum stimulated an increase in the number offibroblasts while the macrophage count dropped. The number of adipocytes remained unaffected. An increase in mean corpuscular hemoglobin (MCH) was also found in the peripheral blood of experimental animals. .

INTRODUCTION

Aluminum in the most widely distributed element in the earth’s crust, but it is not found in the metallic state. It is most commonly found in combination with such elements as oxygen, fluoride, and silicon. Aluminum is the third most abundant element in the lithosphere, hydrosphere, and atmosphere (after oxygen and silicon) (Abderson, 1978). Aluminum, unlike many other metals, has no known physiological function and is not considered an essential dietary compound (Jones et al., 1986). It does, however, have biological effects. Stemweiss and Gilman (1982) have shown that the activation of the guanine nucleotide binding regulatory component of adenylate 1. Address all correspondence to: Dr. K. Zaman, Department of Pathology, School of of Pennsylvania, 4300 Chestnut Street, 201 F, Philadelphia, PA 19104. 2. Key words: bone marrow, erythrocytes, hemoglobin, aluminum, stromal cells. 3. Abbreviation: MCH, mean corpuscular hemoglobin.

University

Downloaded from tih.sagepub.com at NORTH CAROLINA STATE UNIV on March 30, 2015

Medicine,

104

cyclase by fluoride requires aluminum at levels of 5-10 pM. Jones et al. (1986) reported that aluminum increased 3-H-thymide incorporation into DNA in the presence of other mitogens and by itself caused an increase in cell growth; they also found that aluminum stimulated mitosis in murine cells in tissue culture. Aluminum is

recognized as the cause of several dialysis-related diseases, such as dialysis encephalopathy, vitamin D-resistant osteomalacia, and microcytic anemia (Alfrey et al., 1976; Flendring, 1976; Wills and Savory, 1983; Zaman et al., 1989, 1990a). We previously reported on the effect of aluminum on ALA-D activity and iron levels in rats in vivo and on the heme biosynthesis in rats ill vitro (Zaman et al., 1989, 1990b). We have also shown the effect of aluminum

on

the activities of

acetylcholinesterase, glutathione reductase, and glucose-6-phosphate dehydrogenase in the bone marrow of rats (Zaman et al., 1990c). There is no report concerning the effects of aluminum upon the stromal cells in the bone marrow of rats. Bearing in mind the destructive effect of aluminum on the hemopoietic system and in view of its close link with the stromal cells of the red marrow, we have undertaken this study of the effect of aluminum on the stromal cells of the bone marrow in rats in vin~o.

MATERIAL AND METHODS

experiment was carried out on 20 male rats of the Wistar strain whose average body weight was 150 t 10 g. All animals were housed in steel cages and maintained on a 12 hr light/dark cycle at a room temperature of 22 t 2°C and were allowed free access to deionized water and maintained on standard rat chow (Ralston Purina Co., Chicago, IL). Aluminum chloride (Fischer, Springfield, NJ) and all other chemicals used in this study were reagent grade. All glassware was acid leached and rinsed with deionized water to remove contaminating aluminum and iron. The

Aluminum stock solution was prepared in deionized water, filter sterilized, and stored in the dark at 22° C. Stock solution was stored in tissue grade polystyrene tubes to avoid leaching of metal. The aluminum level in the stock solutions as well as in the blood and culture was verified by flameless atomic absorption spectroscopy, using a Perkin-Elmer 460 atomic absorption spectrophotometer. Prior to proper, samples of blood were taken from the tail veins of animals; a routine erythrocyte count, hemoglobin level measurement according to Drabkin’s method, and mean corpuscular hemoglobin (MCH) were determined directly. divided into two groups: a control group (n=10) and an experimental the rats in the experimental group received intravenous ml of 0.5 aluminum chloride solution, containing 4 mg/kg of body weight. injections the control animals received 0.5 ml of saline solution daily for five Simultaneously, days. On the sixth day, blood samples were taken from the tail veins of the rats in both The rats

were

group (n=10). For 5 days

Downloaded from tih.sagepub.com at NORTH CAROLINA STATE UNIV on March 30, 2015

105

groups and the hemoglobin level, erythrocyte count, and MCH were determined. The tissue cultures of bone marrow stromal cells from the control and experimental animals were maintained in liquid medium using the monolayer method according to Dexter et al. (1983). Animals were killed by cervical dislocation. Femoral bones were removed, cut at their bases, and the bone marrow was washed out using a 1-ml syringe with a 23-gauge needle. The cells were suspended in Eagle’s medium, dispersed, and counted using a hemocytometer. The cells were grown in monolayers in Leighton’s bottles with

glass covering. The glasses covered with layers of growing cells allowed for morphological observations with histochemical tests. The cultures were maintained for 5 days at 37°C in 1.5 ml of Eagle’s medium supplemented with 20% fetal calf serum. The medium was first removed after 24 hours and then replaced by fresh medium every other day.

Moiphological Studies After 24 hours the stromal cells remained on the glass. The unattached cells were removed at media change. Five-day cultures were stained according to the method of Wright’s (Harper) (1974). To study morphological changes, the types of cells (fibroblasts, macrophages, and adipocytes) and their number were determined and

given as percentages.

_

Histochemical Studies The monolayers were fixed with formaldehyde vapor for 5 min at room temperature. In order to identify the kinds of cells (fibroblasts, macrophages, and adipocytes), histochemical reaction according to Bauldry et al. (1985) was carried out for nonspecific esterase (E.C.3.1.1.8) using naphthol AS-D chloracetate. .

Statistical Analysis of Results The differences in the hemoglobin level, erythrocyte counts, and MCH and the differences in the numbers of various types of stromal cells between the experimental and control cultures were tested using Student’s t-test. Standard deviation and average standard errors were also calculated. RESULTS

Hematologicalllldices (Table 1) The hemoglobin level, erythrocyte count, and the MCH in the control and experimental rats before treatment and those indices in the control group prior to and after saline injections did not differ significantly. The hemoglobin level in the control group was 19.80 g/dl. However, in the experimental group the level was X ~ 15.25 g/dl. The difference between these mean values were statistically significant (p < 0.001).

Downloaded from tih.sagepub.com at NORTH CAROLINA STATE UNIV on March 30, 2015

106

TABLE 1. --

Hematological Indices

in

Peripherial

Blood

(S.I. Units)



Erythrocyte Couttt The number of erythrocytes in the control group ways 7 8.12 T/I and x 6.94 T/I in the experimental group. The differences between these mean values were statistically significant (p< 0.001). =

=

Mean Co~puscular Hemoglobin (MCH) In the control group the MCH was X = 23.15 pg; it was X ~ 23.20 pg in the experimental group. The differences between these mean values were not statistically

significant.

Morphological Obsef1Jatiolls (Table 2) Fibroblasts, macrophages, and adipocytes were found in these tissue cultures Fibroblasts. The percentage of fibroblasts on the 5th day of culture was x = 34.85% in the control group. However, in the experimental group it was x = 56.27%. The differences between these mean values were statistically significant (p < 0.001).

Downloaded from tih.sagepub.com at NORTH CAROLINA STATE UNIV on March 30, 2015

107 and Number of Particular Stromal Cells of the Bone Marrow in Rats (in %)

TABLE 2.

Type

Macropjiages. The percentage of macrophages on the 5th day of culture in the control group was X 46.16%; in the experimental group the percentage was x 18.93%. The differences between these mean values were statistically significant (p< 0.001). =

=

Adipocytes. The percentage of adipocytes on the 5th day of culture in the control group was 7 17.82%; however, in the experimental group it was x 20.76%. The differences between these mean values were not statistically significant. =

=

,

DISCUSSION Numerous experiments have demonstrated the link between stromal cells and hematopoicsis (Dexter et al., 1983, 1978; Tavassoli, 1976; Zipori and Bol, 1979). The cells of the adherent layers of bone marrow perfonn a supportive function and produce hemopoiesis regulatory, thus providing the marrow microenvironment. Stromal cells include fibroblasts, macrophages, and adipocytes. Ghio et al. (1977) reported that the fibroblasts released regulators that manifested synergic effects with erythropoietin and were able even in the absence of this hormone to induce the growth of erythroidal colonies. Macrophages might synthesize or accumulate some agents that are responsible for the stimulation of erythropoiesis (Murphy and Urabe, 1978; Rich et al., 1982; Polemacher et al., 1977). Rich and Kubnek (1985) found that macrophages had the capability to stimulate and maintain in culture some early erythroidal precursors BFU-E (burst forming units erythroid) had the capability to maintain in culture some precursors of mixed colonies containing erythroidal cells, neutrophils, macrophages, eosinophils, and megakaryocytes. Responding to external stimuli, e.g., changes in oxygen pressure, the macrophages may affect the composition of the marrow

Downloaded from tih.sagepub.com at NORTH CAROLINA STATE UNIV on March 30, 2015

108

microenvironment and produce agents that directly influence other types of stromal cells and precursors of hemopoiesis (Broxmayer et al., 1984). The third type of stromal cells include fat cells or adipocytes. lu vitr-o adipocytes appear most often at the sites of granulopoiesis. Their definite functions are not known, but it is possible that they perform a trophic function (Tavassoli, 1976; Tavassoli and Friedenstein, 1983). The experiments presented in this paper demonstrate the toxic effect of aluminum that is manifested in the peripheral blood by a reduced erythrocyte count and suppressed hemoglobin level. There was no decrease in MCH; hence, such a response occurs only after chronic intoxication. Aluminum stimulated a numerical increase in fibroblasts and a suppressed number of macrophages. This fact viewed against a background of decrease in erythrocyte count and hemoglobin level seems to confirm the important regulatory role that macrophages exercise in the erythroid line. Aluminum injections did not significantly change the number of adipocytes. Aluminum significantly changes the proportions of cells in the bone marrow stroma. One must take into account the existence of an increased toltrance to heavy metals in rodents where intoxication enhances the production of metallothioneine in the kidneys, helping them to eliminate these metals (Dexter et al., 1983). The effect of aluminum on stromal cells appears to be extremely significant.

REFERENCES ABDERSON, W. (1978). "Aluminum and aluminum alloys." In: Encyclopedia of chemical technology (M. Grayson and D. Eckroth, ed.). Wiley, New York, NY. ALFREY, A.C., LEGENDRE, G.R., and KAEHNY, W.D. (1976). "The dialysis encephalopathy

syndrome: Possible aluminum intoxication." N. Engl. J. Med. 294: 185-188. BAULDRY, S.A., WILSON, F.D., STOMBER, P.C., and ACKERMAN, G.A. (1985). "Stimulation of normal rat bone marrow fibroblasts proliferation by sera from leukemic Fischer rats." Exp. Hematol. 13: 750-759. BROXMAYER, H.E. and PLATZER, E. (1984). "Lactoferrin acts on I-A and I-E/C antigen subpopulations of mouse peritoneal macrophages in the absence of T lymphocytes and other cell types to inhibit production of granulocyte-macrophage colony stimulating factors in vitro." J. Immunol. 133: 1-9. DEXTER, T.M., SPOONER, E., HENDRY, J., and LAJTHA, L.G. (1978). "Stem cells in vitro." In: Hematopoietic cell differentiation (D.W. Golde, M.J. Cline, C.F. Fox, eds.). Academic Press. New York, NY. pp. 163-173. DEXTER, T.M., SPOONER, E., VARGA, J. ALLEN, T.D., and LANOTTE, M. (1983). "Stromal cells and diffusible factors in the regulation of hemopoietic cell development." In: Hemopoietic stem cells (S.V. Killmann, E.P. Cronkite, and C.M. Muller-Berat, eds.). Copenhagen, Denmark. pp. 303-318. FAULKNER, W.R. and KING, J.W. (1970). Manual of clinical laboratory procedures. Chem. Rub. Co., Cleveland, OH. FLENDRING, J.A., MRUIS, H., and DAS, H.A. (1976). "Aluminum and dialysis dementia." Lancet i: 1235. GIO, R., BIANCHI, G., LOWENBERG, B., DICKIE. K.A., and AJMAR, F. (1977). "Effects of fibroblasts on the growth of erythroid progenitor cells in vitro." Exp. Hematol. 5: 341-347. HARPER, T.A. (1974). The peripheral blood film. Butterworth, London, U.K. p. 248. JONES, T.R., ANTONETTI, D.L., and REID, T.W. (1986). "Aluminum ions stimulate mitosis in murine cells in tissue culture." J. Cellular Biochem. 30: 31-39.

Downloaded from tih.sagepub.com at NORTH CAROLINA STATE UNIV on March 30, 2015

109 MURPHY, M.J. and URABE, A. (1978). "Modulatory effects of macrophages on erythropoiesis." In: In vitro aspects of erythropoiesis (M.J. Murphy, ed.). Springer Verlag, New York, NY. POLEMACHER, R.E., VANSOEST, P.L., WAGERMAKER, G.E., and VANT, H.T. (1977). "Particle induced erythropoietin independent effects on erythroid precursors cells in murine bone marrow." Cell Tissue Kinet. 12: 539-550. RICH, J.N., HEIT, W., and KUBANEK, B. (1982). "External erythropoietic production by macrophages." Blood 60: 1007-1017. RICH, J.N. and KUBANEK, B. (1985). "The central role of the macrophage in hemopoietic microenvironmental regulation." In: Hematopoietic stem cell physiology (E.P. Cronkite, N. Dainiak, and R.P. McCaffrey, eds.). Alan R. Liss, New York, NY. pp. 283-298. STERNWEISS, P.C. and GILMAN, A.G. (1982). "Aluminum. A requirement for activation of the regulatory component of adenylate cyclase by fluoride." Proc. Natl. Acad. Sci. 79: 4888-4891. TAVASSOLI, M. (1976). "Marrow adipose cells." Arch. Pathol. Lab. Med. 100: 287. TAVASSOLI, M. and FRIEDENSTEIN, A. (1983). "Hemopoietic stromal microenvironment." Amer. J. Hematol. 15: 195-203. WILLS, M.R. and SAVORY, J. (1983)."Aluminum poisoning. Dialysis encephalopathy, osteomalacia and anemia." Lancet ii: 29-34. ZAMAN, K., DABROWSKI, Z., and MISZTA, H. (1989). "The toxic effects of aluminum on the activity of ALA-D, on the iron level in bone marrow in rats." J. Interdise. Cycle Res. (Holland) 20: 245-246. ZAMAN, K., DABROWSKI, Z., and MISZTA, H. (1990a). "Effects of aluminum on erythroidal cells in bone marrow in rats." Folia Histochem. Cytochem. 28: 60-69. ZAMAN, K., DABROWSKI, Z., MISZTA, H., and SMOLENSKI, O. (1990b). "The effect of aluminum on the heme biosynthesis in vitro in bone marrow cells in rats." Folia Haematol. (Germany) 117: 307-311. ZAMAN, K., MISZTA, H., and DABROWSKI, Z. (1990c). "The effect of aluminum upon the activity of selected bone marrow enzyme in rats." Folia Haematol. (Germany) 117: 447-451. ZIPORI, D. and BOL, S. (1979). "The role of fibroblastoid cells and macrophages from mouse bone marrow in the in vitro growth promotion of hemopoietic tumor cells." Exp. Hematol. 7: 206218.

Downloaded from tih.sagepub.com at NORTH CAROLINA STATE UNIV on March 30, 2015

The effect of aluminum on the stromal cells (in vitro) on bone marrow in rats.

The aim of this study was to describe the effects of aluminum on the stromal cells of rat bone marrow using a combination of in vivo exposure and in v...
375KB Sizes 0 Downloads 0 Views