Biological Trace Element Research - American Chemical Society

Chapter 26

Trace Element-Induced Toxicity in Cultured Heart Cells Craig C. Freudenrich, Shi Liu, and Melvyn Lieberman

Downloaded by COLUMBIA UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: December 26, 1991 | doi: 10.1021/bk-1991-0445.ch026

Department of Cell Biology, Division of Physiology, Duke University Medical Center, Durham, NC 27710

We have undertaken a multidisciplinary approach to study the toxic effects of several trace elements (e.g. Sb, Cd, Co, Pb, Hg, Zn) on the structure and function of cardiac cells. Although the basic mechanisms underlying these toxic effects remain unknown, altered membrane ion transport has been proposed as a major determinant in the onset of cell injury. By applying an array of techniques including light microscopy, atomic absorption spectroscopy, electrophysiology, and microspectrofluorometry, we show that cultured embryonic chick heart cells can serve as a useful model to study how trace elements affect the transport mechanisms that maintain intracellular ionic homeostasis. We present examples of how this model can be used to characterize the basic effects on ion homeostasis, to identify the mode of uptake of trace elements, and to quantitate the effects of trace elements on identified ion transport mechanisms. With this approach it should be feasible to elucidate the basic mechanisms underlying trace element-induced cardiotoxicity.

Several trace elements (e.g. Sb, Cd, Co, Pb, Hg, Zn) have been shown to induce a variety of cardiotoxic effects such as arrhythmias, conduction abnormalities, reduced contractility, cardiac swelling, myofibril degeneration, and sudden death (1-6), but the mechanisms underlying these effects remain unknown. One hypothesis suggests that alterations of ion transport mechanisms are implicated in the initiation and maintenance of cell injury (7-10). However, this hypothesis has been difficult to evaluate for the following reasons: (a) studies involving animal models have revealed trace element-induced tissue damage, but have not detected the early physiological and biochemical disturbances in heart muscle (11) and (b) studies with tissue culture systems have often been used to screen toxic agents based on observations of morphologic changes and/or reduced viability (12,13), but have not provided data that permit conclusions as to the cellular mechanisms of injury. Thus,

0097-6156/91/0445-0332$06.00/0 © 1991 American Chemical Society

In Biological Trace Element Research; Subramanian, K., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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Trace Element-Induced Toxicity in Heart Cells

the progress in this area can be adequately summarized by Iyengar's comment that "the lack of a multidisciplinary approach has been the Achilles heel of biological trace element research" (14). We agree that such an approach is necessary to elucidate the mechanisms of trace element-induced cardiotoxicity. Studies in our laboratory over the past 20 years have established that cultured embryonic chick heart cells can provide an ideal model to study mechanisms of ion transport (15). The cells lack significant diffusional barriers, contract spontaneously, and have well-defined voltage-dependent ion channels as well as co- and countertransport mechanisms (e.g. Na/K pump, Na/H exchange, Na/Ca exchange, Na-dependent C1/HC0 exchange, Na+K+2C1 cotransport). Furthermore, the cells can be grown in a variety of configurations suitable for study with biochemical, microscopic, spectroscopic, and electrophysiological techniques. In this chapter, we will present examples of how the cultured chick heart cell model can be used to characterize the toxic effects of trace elements on ion transport, to identify the mode of uptake of trace elements, and to quantitate the effects of trace elements on specific ion transport mechanisms. This information will be necessary to assess the role of membrane transport in trace element-induced cardiotoxicity.


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Characterization of Cardiotoxic Effects of Trace Elements on Ion Transport Hearts from 11 day old chick embryos were disaggregated and the cells were cultured in antibiotic-free medium (15) either as monolayers, aggregates, or poly strands. The cells were noted to contract spontaneously within 1-2 days of culture. All experiments were conducted at 37 °C and cells were incubated or supervised with a balanced salt solution (HTBSS), pH 7.4 composed of the following (inmM): NaCl, 142.2; KC1, 5.4; MgS0 , 0.8; CaCl , 1.0; NaH P0 , 1.0; glucose, 5.6; Hepes + Tris, 10; bovine serum albumin, 1 g/L; in some cases, the phosphate and sulfate were removed to prevent precipitation of any of the trace elements. Heart cell monolayers in culture for 3 days were incubated for 1-4 h in a HTBSS containing either 100 μΜ C o d , 25 μΜ PbCl , 100 μΜ CdCl , or 500 μΜ ZnS0 . Phase-contrast microscopic observations of contractile activity at 1 and 4 h revealed that cells exposed to Co and Pb became arrhythmic, whereas those exposed to Cd and Zn were quiescent. These contractile changes were accompanied by morphologic changes as shown in Figure 1. In all cases, intercellular spaces were increased, indicative of cell shrinkage. Also, cells exposed to Cd and Zn became rounded and contained vacuoles, densities, and membrane blebs, indicative of cell damage. Cell monolayers exposed to Co, Pb, Cd, and Zn for 1-4 h wererinsedfirst with ice cold, isotonic choline chloride solution to remove extracellular ions and then processed for Κ and Na analyses by atomic absorption spectroscopy (16). In most cases, cells exposed to the trace elements lost Κ at 1 and 4 h (Table I). At 1 h, cells exposed to Co, Pb, and Cd lost Na; whereas, cells exposed to Zn gained Na by 4-fold. By 4 h, cells exposed to all of the trace elements, except Co, gained Na. These data indicate that changes in cell volume and in the regulatory processes for Κ and Na (e.g. Na and Κ conductances, Na/K pump) occurred in the presence of trace elements. 4

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Figure 1. Phase-contrast micrographs of cultured chick heart cells after 4 h incubation at 37 °C in medium + trace elements: (a) Control (b) 100 μΜ C o Q (c) 25 μΜ PbCl (d) 100 μΜ CdCl (e) 500 μΜ ZnS0 . The horizontal bar = 50 urn. 2

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Table I. Effects of Trace Elements on Κ and Na Content in Cultured Heart Cells

Ion Content (nmol/mg protein) Trace Element

(μΜ)

Κ

Na 1 hour

Control CoCl PbCl CdCl ZnS0 2

2


2

4

100 25 100 500

1312 1172 1153 1214 534

± ± ± ± ±

131 9 27 60 89

100 25 100 500

1056 1102 1078 816 38

± ± ± ± ±

13 35 58 55 25

139 ± 7 114 ± 1 123 ± 12 105 ± 2 526 ± 89 4 hours

Control CoCl PbCl CdCl ZnS0 2

2

2

4

118 ± 5 115 ± 3 141 ± 19 631 ± 39 992 ± 68

Data are mean ± SE (3)

Transmembrane potentials were recorded from aggregates of spontaneously contracting cells according to Liu et al (17) and the cells were exposed to 100 μΜ concentrations of CdQ , CoCl , ZnS0 , PbCl , Sb (tartar emetic), and HgCl as shown in Figure 2. In all cases, except Co, the beating rate of the cells was increased prior to becoming quiescent (depolarized to between -35 and -40 mV); these events usually occurred within 3 min. Examination of the action potentials prior to the cessation of beating revealed that the amplitude and plateau phase were decreased and that the configuration of the action potential was altered. These results can be related to the changes in contractile activity described previously and suggested that the basic ionic processes underlying generation of the cardiac action potential were altered by acute exposure to the aforementioned trace elements. We have used ion-selective microelectrodes (ISME) to measure intracellular activities of Na, K, CI, and Η in cultured chick heart cells (17). Although trace elements could possibly interfere with the ISME resins, we found no evidence of interference when Na-, K-, Cl-, and pH-ISMEs were calibrated in the presence of 100 μΜ Sb, Cd, Co, Pb, Hg, or Zn. Therefore, it is feasible to use these ISMEs in experiments involving trace elements. In preliminary experiments to determine the effects of trace elements on ion regulation, we monitored intracellular pH in polystrands by pH-ISME that were made with the Fluka proton cocktail in a manner similar to that described previously (17). The cells were exposed to either 100 μΜ PbCl or 100 μΜ ZnS0 . As shown in Figure 3, exposure to Pb increased intracellular pH by about 0.1 units within 5 min. In contrast, exposure to Zn decreased intracellular pH by approximately 0.2 units. These results indicated that Pb and Zn can alter the regulation of Η in heart 2

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Figure 2. Effects of trace elements on cardiac action potentials (AP). Left: 100 μ Μ trace element was added (up arrow) and removed (down arrow); horizontal bar = 1 min. Right: A P (dashed line, element; solid line, control); horizontal bar = 50 msec and is located at 0 mV; vertical bar = 50 mV for both sides.




26. F R E U D E N R I C H E T A L .

Figure 3. Effects of 100 μΜ PbCl (A) and 100 μΜ ZnS0 (B) on intracellular pH as measured using a pH-ISME. 2

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cells by affecting one or more pH regulatory mechanisms (e.g. Na/H exchange, Nadependent C1/HC0 exchange, pH buffering capacity, Ca/H interactions). Future studies will be directed toward assessing the effects of trace elements on specific pH regulatory mechanisms and toward characterizing their effects on the regulation of other ions using ISME techniques. 3


Identification of the Mode of Uptake of Cardiotoxic Trace Elements To resolve mechanisms associated with trace element-induced cardiotoxicity, it will be necessary to determine whether the trace elements can act from the outer sarcolemmal surface and/or the cytoplasm. The uptake of trace elements by cells can be measured by conventional methods (i.e. radioisotope tracer flux, atomic absorption spectroscopy) that are quantitative and sensitive to low concentrations; however, these methods are limited because they (a) cannot distinguish between trace elements bound to the cell surface from those that have permeated the sarcolemma (b) may have variable responses due to heterogeneity of the cell population (c) require processing of samples and data and, therefore, do not provide information in real time. Although radioisotopic tracer flux and atomic absorption spectroscopic techniques can be applied to the cultured chick heart cells (15), we have developed a new approach to detect trace element uptake in single, living cells in real time by using the Ca-sensitive fluorescent dye, fura2 (18). The method takes advantage of the fact that many of the trace elements can alter, by direct binding, the fluorescent properties of fura2. To demonstrate these effects, solutions of 10 μΜ fura2 were prepared with a 140 mM KC1, 10raMMops buffer (pH 7.2) that had been passed through a Chelex (Bio-Rad) column to remove all divalent cations. The solutions were placed in a chamber on the heated (37 °C) stage of a microscope attached to a Spex spectrofluorometer (19). Fluorescence excitation spectra (emission = 510 nm) were acquired before and after the addition of various trace elements (3-1000 μΜ final concentrations). Figure 4 shows the spectra for Cd, Co, Pb, Mn, Hg, and Zn (100 μΜ) normalized to the respective trace element-free spectrum. Note that all trace elements, except Pb, had maximal effects at concentrations < 100 μΜ. The type of changes in the fluorescent properties of fura2 evoked by trace elements were variable. Cd and Zn enhanced fluorescence at all wavelengths; whereas, Hg, Co, Mn, and Pb quenched fluorescence (Pb predominantly quenched the fluorescence, but at higher concentrations). By measuring thefluorescenceat the Ca-insensitive excitation wavelength (360 nm), we determined the half-maximal concentrations (μΜ): Cd, 4.7; Zn, 23; Hg, 31; Co, 2.8; Mn, 3.6; and Pb, 447. Although the effects on fura2 fluorescence would interfere with measurements of cytosolic Ca , they provide a convenient means to detect the permeation of trace elements into single, living cells. Cell monolayers were grown on glass coverslips, single cells were microinjected with fura2, and the cells were perfused with HTBSS on the stage of the microspectrofluorometer (19). Fluorescence (excitation, 350 and 360 nm; emission, 510 nm) was measured from single cells as shown in Figure 5. The fluorescence of cells exposed to 0.5 mM Cd increased; whereas, the fluorescence of those exposed to 1 mM Co decreased. In these examples, the fura2 fluorescence changed as predicted from the spectra (Figure 4) and confirmed that both Cd and Zn had permeated the sarcolemma. 2+


FREUDENRICH ET AL.


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We have utilized this method to follow Mn influx in cultured chick heart cells (20). We found that Mn quenched the intracellular fura2fluorescenceas predicted from the spectra in Figure 4 and that the rate of quenching, which is related to the rate of influx, was proportional to the extracellular Mn concentration. The quenched signal evoked by 50 μΜ Mn was not affected by perfusing the cells with impermeant metal chelators, 3 mM ethylene-diaminetetraacetic acid (EDTA) and 0.2 mM diethylenetriaminepentaacetic acid (DTPA), but was reversed by perfusion with a permeant metal chelator, 20 μΜ N,N,N',N -tetrakis(2-pyridylmethyl)ethylenediamine (TPEN); these results confirmed that the quenched signal was actually due to Mn entry. In addition, the rate of quenching of intracellular fura2 by 0.1 mM Mn was reduced by about 90% when the cells were treated with 10 μΜ verapamil and 10 μΜ nifedipine; these results indicate that a major pathway of Mn influx is through voltage-dependent Ca channels. This approach has also been used to detect Cd influx in smooth muscle cells (21) and La influx in heart cells (22). The advantages of using fura2fluorescenceto monitor trace element influx into cells are as follows: (a) trace elements in the cvtosol can be detected because of complexation with fura2 that was microinjected into the cell (b) fura2 is sufficiently sensitive to detect micromolar concentrations of trace elements in the cytosol (c) the method provides data inrealtime in living single cells and, thus, can be used to test many experimental conditions. The major disadvantage of thisfluorescenceassay is that it does not allow for quantitation of the absolute amount of trace element uptake. However, when used in conjunction with conventional radioisotopic or atomic absorption methods, the fluorescence method can provide useful information about rates, amounts, and mechanisms of toxic trace element uptake in heart cells.


,

Quantitation of Toxic Effects of Trace Elements on Defined Ion Transport Mechanisms As mentioned earler, cultured chick heart cells have many defined ion transport mechanisms that can be characterized by a variety of electrophysiological techniques. For example, Stimers et al (23) showed that Na/K pump current can be quantitated in aggregates of cultured chick heart cells under voltage-clamp conditions by exposing the cells to K-free solution or to 1 mM ouabain; this has also been accomplished in single heart cells using the whole-cell patch voltage-clamp technique (24). Using this approach, we examined the effect of 100 μΜ ZnS0 on the Na/K pump current of a single cell. As shown in Figure 6, exposure to Zn for 1 min reduced the Na/K pump current by approximately 16%; these results were consistent with the changes in Na and Κ content upon exposure to Zn (Table I). This preliminary result demonstrates the feasibility of this approach to determine the effects of trace elements on specific electrogenic transport mechanisms. 4

Summary The cultured chick heart cell model offers several advantages to study trace element-induced cardiotoxicity: (a) the cells are in a physiological state (e.g. spontaneously contracting) and lack any diffusional barriers so that the direct effects of trace elements on cardiac cells can be assessed (b) the cells can be grown in a variety of configurations that are well suited to characterize the toxic effects of trace



FREUDENRICH ET AL.

Trace Element-Induced Toxicity in Heart CeUs

Kp-free

Figure 6. Effect of 100 μΜ ZnS0 on the Na/K pump current in a single heart cell. 4


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elements on cell structure and function by various microscopic, biochemical and biophysical methods (c) the cells are suitable for determining trace element uptake by radioisotope tracer flux, atomic absorption spectroscopy and a novel fluorescence method (d) the cells have many well-defined ion transport mechanisms that can be quantitated using biophysical methods so that the effects of toxic trace elements on specific ion transport mechanisms can be assessed. The types of information that can be obtained from this model will be necessary to elucidate the mechanisms underlying trace element-induced cardiotoxicity.


Acknowledgments We thank Kathleen Mitchell and Meei Liu for their work in preparing the cell cultures and performing the ion content experiments. This work was supported in part by National Institutes of Health Grants HL-07101, HL-17670, and HL-27105 to M.L. and C.C.F. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

16. 17.

Van Stee, E. W. In Cardiovascular Toxicology; Van Stee, E. W., Ed.; Raven Press: New York, 1982; p 1. Carson, B. L.; Ellis, Η. V.; McCann, J. L. Toxicology and Biological Monitoring of Elements in Humans; Lewis Publishers, Inc.: Michigan, 1986. Van Fleet, J. F.; Ferrans, V. J. Am. J. Pathol 1986, 124, 98. Hurst, J.W. The Heart; McGraw-Hill Book Co: New York, 1986, Sixth Edition. Kopp, J. Handbook of Experimental Pharmacology 1986, 80, 195. Braunwald, E. Heart Disease; W. B. Saunders Co.: Philadelphia, 1988, Third Edition. Trump, B. F.; Berezesky, I. Current Topics in Membranes and Transport; Academic Press, Inc.: New York, 1985; Vol. 25, p 279. Bridges, J.W.; Benford, D. J.; Hubbard, S. A. Ann. New York Acad. Sci. 1983, 407, 42. Buja, L. M.; Hagler, Κ. K.; Willerson, J.T. Cell Calcium 1988, 9, 205. Ferrans, V.J. In Physiology and Pathophysiology of the Heart; Sperelakis, N., Ed.; Kluwer Acad. Publ., 1989, Second Edition, p 691. Zbinden, G. Cardiac Toxicology; CRC Press: Boca Raton, FL, 1981; Vol. 3, p 8. Thelstam, M . ; Mollby, R. Toxicology 1980, 17, 189. Wenzel, D. G.; Cosma, G. N. Toxicology 1984, 33, 117. Iyengar, G. V. Elemental Analysis of Biological Systems; CRC Press: Boca Raton, FL, 1989; Vol. 1, p 1. Horres, C. R.; Wheeler, D. M . ; Piwnica-Worms, D.; Lieberman, M . In The Heart Cell in Culture; Pinson, Α., Ed.; CRC Press: Boca Raton, FL;1987, Vol. 1, p 77. Murphy, E.; Aiton, J. F.; Horres, C.R.; Lieberman, M . Am. J. Physiol. 1983, 245, C316. Liu, S.; Jacob, R.; Piwnica-Worms, D.; Lieberman, M . Am. J. Physiol. 1987, 253, C721.


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Grynkiewicz, G.; Poenie, M.; Tsien, R.Y. J. Biol. Chem. 1985, 260, 3440. Freudenrich, C.C.;Lieberman, M. Spex Biomedical Application Note B-3; Spex Industries, Inc.: Edison, NJ, 08820, 1989. Freudenrich, C. C.; Lieberman, M . Biophysical J., 1989, 47a. Smith, J. B.; Dwyer, S. D.; Smith, L. J. Biol. Chem., 1989, 264, 7115. Peeters, G. Α.; Kohmoto, O.; Barry, W. H. Am. J. Physiol., 1989, 256, C351. Stimers, J. R.; Shigeto, N.; Lieberman, M . J. Gen. Physiol., 1990, 95, 61. Stimers, J. R.; Liu, S.; Lieberman, M . FASEB J., 1990, 4, A295. July 16, 1990


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