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CYTOMETRIC AND ELECTRON MICROSCOPIC STUDIES OF THE DIRECT INTERACTION OF DIVALENT NICKEL WITH INTACT AND CHEMICALLY MODIFIED HUT-78 LYMPHOBLASTS G.I. MALININ, F.J. HORNICEK, H.K. LO, AND T.I. MALININ Physics Department, Georgetown University Washington, DC Mannheimer Foundation Homestead, Florida Department of Orthopaedics and Rehabilitation University of Miami School of Medicine Miami, Florida Cytometric and ultrastructural studies on 24 hr cultures of intact, 1.0 mM H5106, and 0.1 mM SeO2-oxidized HUT-78 lymphoblasts were performed after their direct, 30 min interaction with 1.0 mM NiCl2. Except for moderately depressed cell viability, divalent nickel did not alter the progression of intact and oxidized target cells through the phases of the cell cycle. Although the plasma membrane remained structurally intact, marked distortion of mitochondria structure and increased osmiophilia were an invariable attribute of all nickel-pulsed cells. Moreover, numerous electron-opaque, intracellular depositions were detected in SeO2-oxidized, nickel-pulsed cells. It is concluded that the initial state of plasma membrane, and the interaction of nickel with other trace elements, have jointly determined the response of HUT-78 cells to brief and direct, divalent nickel pulses. INTRODUCTION Although nickel is now recognized as an essential trace element (Ankel-Fuchs and Thauer, 1988), this metal and its complexes can and do exert a wide spectrum of deleterious effects on a multitude of biological systems (DiPaolo and Casto, 1979; Harnett et al., 1982; Sen and Costa, 1985; Sunderman, 1976; Sunderman and Mastromatteo, 1975). Predictably, chemical speciation, concentration, temperature, duration of exposure, and relative susceptibility of biological targets to nickel are major determinants of nickel-mediated biological processes (Drake, 1988).

1. Address all correspondence to: G. I. Malinin, Ph.D., Department of Physics, Georgetown University, Washington, DC 20057-0995. Tel: (202) 687-6038. Fax: (202) 687-7084. 2. Key Words: Lymphoblasts, Nickel, Cytometry, Ultrastructure, Membrane. Cell Biology and Toxicology, Vol. 8, No. 1, pp. 27-41 Copyright © 1992 Princeton Scientific Publishing Co., Inc. ISSN: 0742-2091

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Among the cellular systems known to be transformed or affected by nickel, lymphocytes are especially noteworthy. Owing to the propensity of nickel to initiate delayed-type hypersensitivity reaction, T-cell transformation, production of lymphokines, and cooperative stimulation of B-cells, the role of lymphocytes in nickel-induced contact dermatitis is well recognized (Drake, 1988; Everness et al., 1990; Hutchinson et al., 1972; Nordlind and Henzen, 1984; Nordlind, 1985; Singaglia et al., 1985). Nonetheless, relatively little is known concerning the mechanisms of nickel interaction with lymphocytes and with eukaryotic ceils in general (Drake, 1988). Moreover, this dearth of precise information is compounded further by contradictory data. For instance, it was reported that after traversing plasma membrane, nickel accumulated in the cytoplasm and nuclei of lymphocytes (Nordlind, 1984; Hildebrand et al., 1991) or, alternatively, that no nickel is found in nuclei (Berry et al., 1985). In contrast to the divided opinions on the status of intracellular nickel, there is a virtual unanimity that plasma membrane of eukaryotic cells has a high affinity for nickel, and that mechanisms of transmembrane nickel transport ultimately determine the extent and mode of intracellular nickel disposition (Drake, 1988). It has been shown that the interaction of extracellular nickel with cell membranes in vivo and in vitro is not direct, but is mostly modulated by tertiary albumin-nickel-L-histidine complexes (Abbrachio et al., 1982; Glennon and Sarkar, 1982; Lucassen and Sarkar, 1979; Webb and Weinzierl, 1972). Therefore, it is to be expected that the presence of L-histidine and serum albumin in culture media would also modulate nickel uptake by lymphocytes in vitro, and such modulation has been demonstrated (Nieboer et al., 1984). Conversely, it is reasonable to assume that the consequences of direct and albumin modulated interactions of lymphocytes with nickel will differ from each other. Furthermore, alterations of transmembrane nickel transport and its inlracellular sequestration can be also anticipated in lymphocytes with chemically-modified plasma membrane. To the best of our knowledge, the in vitro studies with nickel were carded out only on lymphocytes with intact plasma membrane, for a relatively long time and in the presence of serum-bound nickel. Similarly, no effects exerted by brief pulses of nickel directly on intact ceils, or on lymphocytes with chemically modified plasma were reported. Because direct and brief interaction of nickel with lymphocytes may prove to be useful for the elucidation of nickel-mediated cellular function, we have evaluated the effect of these factors on the ultrastructure and cell cycle of intact HUT-78 lymphoblasts (Gootenberg et al., 1981) and on the same cells oxidized by periodic acid and by selenium dioxide. MATERIALS AND METHODS Materials

The RPMI-1640 medium, Dulbecco's Ca2+ and Mg2+-free phosphate buffered saline (PBS) and penicillin (1585 U/mg) were from Grand Island Biological Co., Grand Island, NY. Streptomycin and NaHCO3 were from Eli Lilly and Co., Indianapolis, IN, and J.T. Baker Chemical Co., Phillipsburg, NJ. Fetal calf serum (FCS), heat-inactivated (HI) for 30 min at 56°C,

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nickel chloride (NiC12), paraperiodic acid (H5IO6) and selenium dioxide (SeO2) were from Sigma Chemical Co., St. Louis, MO. Fisher Scientific Co., Silver Springs, MD, supplied trypan blue, whereas glutaraldehyde, sodium cacodylate, osmium tetroxide, British Araldite and 4',6-diamidino-2-phenylindole (DAPI) were from Polysciences, Inc., Warrington, PA. Accurate Scientific and Chemical Co., Hicksville, NY provided Nonidet P-40 (NP-40). Methods Culture of HuT-78 cells. Cultures of HUT-78 lymphoblasts (Gootenberg et al., 1981) were grown in RPMI-1640 medium supplemented with 10% v/v HI-FCS, 100 units of penicillin/ml, 100 I.tg/ml of streptomycin and 0.9 mg/ml of NaHCO 3. Cells, growing exponentially in a humidified 5% CO2 atmosphere at 37*C, were harvested, washed in three changes of PBS, resuspended in fresh PBS and their viability determined by trypan blue exclusion. Immediately thereafter, 10 ml aliquots of 5 x 106 viable cells/ml were either: 1. Incubated for 30 min in 25°C PBS or 2. Pulsed for 30 rain with 1.0 mM NiC12 dissolved in 25°C PBS and washed in fresh PBS. Immediately before pulsing of HUT-78 lymphoblasts with 1.0 mM NiC12, their plasma membrane was modified by H5IO6 and SeO2 oxidation reactions as described elsewhere (Malinin et al., 1983). In brief, oxidation of HUT-78 cells was performed as follows: a. b. c.

d.

Lymphoblasts were oxidized for 20 min at 0°C by 1.0 mM H5IO6 dissolved in PBS and then washed with fresh PBS. Oxidation of cells by 1.0 mM SeO2 in PBS was also carried out for 20 min at 0°C and was followed by immediate washing with fresh PBS. Tandem 20 min oxidation of cells by 1.0 mM H5IO6 and by 0.1 mM SeO2 in PBS were performed at 0°C. Oxidized cells were washed with fresh PBS after each oxidation reaction. Immediately thereafter, oxidized lymphoblasts were pulsed for 30 min at 25°C with 1.0 mM NiC12 in PBS and then washed with RPMI-1640 medium.

After the above listed procedures, all cells were cultured for 24 hr as stipulated previously. Thereafter, cell cultures were harvested for an immediate flow cytometric and electron microscopic analysis. Flow Cytometry. The concomitant determinations of the cell cycle progression and viability were performed as described elsewhere (Hornicek et al., 1986). In brief, isolated nuclei were stained with DAPI and analyzed with a PHYWE flow cytometer (currently referred to as a ICP22 pulse cytophotometer; Ortho Instruments, Westwood, MA) interfaced to an IBM-PC (Thornthwaite et al, 1985). The fluorescence of DAPI-stained nuclei was quantified using a UGI excitation, TK 405 dichroic, and LP 395 barrier filters. The percentage of cells in each phase of the cell cycle was determined and after calculating the mean percentages, a Student's t-test was used to determine statistical differences. Viable cells were determined based on their DNA content (Hornicek et al, 1986) and ability to exclude trypan blue.

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Electron Microscopy. Harvested cells were washed with fresh RPMI-1640 medium, pelleted by centrifugation and fixed at 4°C for 2 hr with sucrose (3% w/v)-containing 2.5% v/v glutaraldehyde in 0.05 M cacodylate buffer (pH 7.4). Fixed cells were osmified for 1 hr with 1% w/v osmium tetroxide in 0.1 M cacodylate buffer at pH 7.4, dehydrated in graded ethanol solutions and embedded in British Araldite. Ultrathin sections were cut with a diamond knife on an LKB ultratome III and examined with a Philips 300 electron microscope directly. To preclude possible masking of intracellular nickel accumulation by competing metal ions, osmified sections were not counterstained with any metal cations. RESULTS

Flow Cytometry High proliferation rates and viability were an invariable attribute of intact HUT-78 lymphoblasts, cultured as stated (Table 1). Direct, 30 min pulses with 1.0 mM NiC12 generally decreased the viability of the exponentially growing cells by about 10% (p

Cytometric and electron microscopic studies of the direct interaction of divalent nickel with intact and chemically modified HuT-78 lymphoblasts.

Cytometric and ultrastructural studies on 24 hr cultures of intact, 1.0 mM H5IO6, and 0.1 mM SeO2-oxidized HuT-78 lymphoblasts were performed after th...
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