Effect of Zinc on Cells and Biomembranes

Symposium on Trace Elements

Effect of Zinc on Cells and Biomembranes Milos Chvapil, MD., Ph.D., D.Se.

It is becoming increasingly evident that zinc ions as an integral part of tissues and biologic fluids are one of many homeostatic mechanisms regulating the reactivity of tissues and cells. Various aspects of the role of zinc in preserving the integrity of the cells and tissues were reviewed by us recently.6, 7 In this report, I would like to concentrate on the effect of zinc on cells as documented in the literature or by our experiments aimed at the analysis of the mechanisms of the mode of action of this cation at the cellular level. Finally, I would like to point to some perspectives of this field of research and discuss briefly a few clinical implications of the data presented. The available literature on the effect of zinc (often compared with the effect of other metals) on various cells points in general to two differing effects as related to the type of cells: . 1. In lymphocytes, zinc acts as a nonspecific mitogen. 2 , 3, 5, 24, 34, 37 Within a rather narrow range of Zn++ concentration in the culture medium (1.5 to 4.5 x 10-4 M) the blastogenic transformation of lymphocytes as well as their mitosis was highly increased, comparable to the effect of phytohemagglutinin.24 ,34 The kinetics of the effect of zinc ions on cultured lymphocytes indicate that this metal is a rather weak mitogen, as both effects are mostly expressed after 6 days of continuous contact of Zn++ with lymphocytes. 34 Surprisingly, only zinc and mercury ions3 ,5 were stimulatory; Ca++ and Mg++ did not affect DNA synthesis in this culture system, while Mn++, Co++, Cd++, Cu++, and Ni++ at concentrations 10-3 to 10-7 M were inhibitory. In review of recent findings that both DNA polymerase37 and reverse transcriptase2 are zinc-containing enzymes, it seems plausible to assume that enhanced mitosis of lymphocytes by zinc relates to increased activity of these enzymes, involved in cell mitosis. The rate of DNA synthesis in cultures of chick embryo cells "Professor, Department of Surgery, College of Medicine, University of Arizona, Tucson, Arizona Supported in part by NIH Grants ES 00790, HL 16385 and AM 16489. Co-workers participating in the reported research are C. F. Zukoski, L. Stankova, E. Carlson, B. Hattler, J. Ludwig, J. Campbell, D. Montgomery, P. Weldy, and D. S. Clark.

Medical Clinics of North America- Vol. 60, No. 4, July 1976

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MILOS CHVAPIL

were inhibited by depleting zinc content in the medium or reducing the concentration of serum. 33 The essential role of Zn++ in DNA replication in PHA-stimulated human lymphocytes was demonstrated by adding a chelator with high affinity for Zn++ or Ni++ to the culture medium. Addition of only Zn++ or Ni++ to the medium prevented the inhibition of thymidine incorporation by lymphocytes. 41 Other evidence indicating the importance of Zn++ for the synthesis of DNA was presented by Hattler, Chvapil, and Zukoskp4 who found that lymphocytes isolated from zincsupplemented animals showed a significantly higher mitotic index when exposed to PHA as compared to lymphocytes from animals fed control diets. A similar finding was reported by Phillips and Azari. 3I They showed that zinc transferrin, when added to serum-free culture of PHA-stimulated human lymphocytes, causes an increase in DNA synthesis over that seen with PHA alone. As other zinc complexes were ineffective, the authors conclude that zinc-transferrin complex has a special role in lymphocyte activity and zinc metabolism. Later we will show that the same may be true for the effect of zinc on other cells. Although everything points to a direct effect of Zn++ in DNA polymerase activity, our finding that Zn:8-hydroxyquinoline unsaturated complexes (1: 1, 1 :2), which do not permeate cell membranes,t enhance lymphocyte mitosis as well, indicates the possible membrane-related effectY So far, lymphocytes seem to be the only cell activated by supplementation of zinc. 2. In most cells studied the elevated content of zinc in in vitro as well as in vivo systems inhibits various functions of the cell. Terminology such as functional immobilization, inactivation, and paralysis of cells was proposeds,13,42 to describe such an inhibitory effect of zinc. This type of effect was demonstrated so far on mast cells, platelets, macrophages, polymorphonuclear leukocytes, and with spermatozoa.

Mast Cells As early as 1960 Hogberg and Uvnas,t8 and quite recently Kazimierczak and Maslinski,22 demonstrated the inhibitory effect of zinc ions on the disruption of mast cells of rat mesentery induced by compound 48/80, lecithinase A, or by antigen-antibody reaction. Pb++ and Cu++ also inhibited disruption of mast cells at 10-7 to 10-5 M concentrations. Cd++, Co++, Mn++, and Ni++ were only moderately inhibitory at 10-5 to 10-3 concentrations. ls Still, biologically important is the fact that only zinc can be administered in vivo without risk of toxic side effects. In fact, the Polish authors administered zinc to rats intraperitoneally for 10 to 20 days (4 mg of Zn++ per kg per day) and showed the inhibition of the spontaneous release of histamine from lung tissue as well as that induced by a low dose of compound 48/80. The protective effect of zinc on histamine release by mast cells was also evident after only a few injections of zinc to animals. Based on the finding that unsaturated Zn:8-HQ (1 :1) complex was even more potent inhibitor of histamine release from isolated mast cells than zinc alone, Kazimierczak and MaslinskpI proposed that zinc acts on the cell membrane. This is in essence a conclu,sion which is similar to the suggested mchanism by which zinc stabi-

801

EFFECT OF ZINC ON CELLS

Table 1. Fibrinogen is Essential for Protective Effect of Zinc on Platelets SAMPLE Platelets Platelets Platelets Platelets Platelets

+ zinc + collagen + plasma + zinc + collagen + fibrinogen + zinc + collagen + albumin + zinc + collagen + globulin + zinc + collagen

RELEASED CB-SEROTONIN (in per cent of platelets radioactivity) 78.9 ± 45.4 ± 44.2 ± 78.6 ± 60.9 ±

1.3 2.9 2.0 0.3 2.0

Concentration of zinc was 15 /LM, plasma formed 30 per cent of the final volume, proteins formed 0.6 per cent of the final concentration.

lizes the membrane of liver lysosomal vacuoles.u As zinc is preventing the induced histamine release from mast cells only when added simultaneously with drug 48/80 to the medium, it seems that zinc ions compete with compound 48/80 for receptor binding sitesP

Platelets In many aspects similar to mast cells, platelets are also affected in their function by zinc ions. The importance of divalent cations in the function of platelets was stressed by several authors. For instance, calcium is required for contraction and release mechanisms. 17 Manganese ions inhibit in vitro platelet aggregation and especially release reaction, possibly by interfering with the role of calcium.35 While calcium is important for platelet aggregation, Mg++ is necessary for platelet deaggregation and inhibits aggregation in the presence of Ca++. 4 Although several studies were presented on the effect of divalent ions on various aspects of platelet functions, we have not found any information on the effect of zinc. Our studies on the effect of zinc and other metals on platelets 13 can be summarized as follows: Collagen-induced aggregation of dog platelets and collagen- or epinephrine-induced release of C14-serotonin was significantly inhibited by zinc ions. An optimal range of concentrations of zinc inhibiting collagen-induced aggregation or release reaction was Table 2. Zinc Protects Platelets Only in the Presence of Serum Proteins SAMPLE Platelets + epinephrine Platelets + zinc + epinephrine Platelets + plasma + epinephrine Platelets + plasma + zinc + epinephrine

RELEASED CB-SEROTONIN (in per cent of platelets radioactivity) 29.4 ± 41.5 ± 12.3 ± 9.6 ±

1.8 1.9 0.2 0.7

Zinc concentration was 15 /LM, and epinephrine concentration was 5 /LM. Plasma formed 20 per cent of the total volume.

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MILOS CHVAPIL

narrow, between 10 and 15 JLM zinc; effective concentration of zinc inhibiting epinephrine-induced release reaction was also 15 JLM, although a wider range of concentrations was also effective (1 to 25 JLM). The presence of plasma in the incubating medium was essential for the inhibitory effect of zinc; only fibrinogen and not albumin or globulin substituted for plasma (Table 1). Without plasma in the medium, addition of zinc enhanced the release reaction induced by either epinephrine or collagen (Table 2). Among various divalent cations studied (Zn++, Mn++, Cd++, Pb++, Co++, Ca++) at 15 and 150 JLM concentrations, only zinc ions were inhibitory (Fig. i). Thus, in our testing system and using dog platelets, we did not find inhibition of serotonin release by Mn++, as reported by Sachetti et al. 21 Our preliminary data indicate that the uptake of Ca45 by platelets was inhibited by zinc ions. As will be discussed later, Ca++ is an essential metal for secretory activity of both platelets and mast cells as well as other cells. The most exciting part of the studies on zinc-platelets interaction is the evidence that supplementation of zinc in dogs effectively decreased aggregability of platelets as well as the magnitude of 14C-serotonin

Q) 0. E

40


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C',......

12 x

E

30

c.

~ 1::l (J)

U)


20

(J)

~ c

'2

~

10

(J)

U)

",'

J:

0

0

Zn

Mn

Cd

Cu

Co

Ca

(15 )JM)

Mg

Figure 1. The effect of various divalent cations on the H3-serotonin release from dog platelets. All metals tested were used as chlorides, dissolved in Tris-buffered saline. The final concentrations of metals in the medium with platelets were 15 I'M and 150 I'M. In the graph only the 15 I'M effects are presented. The results at 150 I'M were similar. The H3- serotonin labeled platelets were suspended in plasma and Tris-buffered saline, 1: 1. Platelets in 36 per cent plasma and metals were preincubated for 10 minutes at 37 C. The collagen was added (501'1, 1 mg per ml stock solution). After 5 minutes the samples were centrifuged (1260 x g, 15 minutes) and the supernate counted for released H'-serotonin. (P. Weldy and M. Chvapil, unpublished results.) 0

803

EFFECT OF ZINC ON CELLS

PLASMA ZINC LEVEL

COLLAGEN INDUCED AGGREGATION 100%

COLLAGEN INDUCED H3-SEROTONIN RELEASE

37.1 16.6

D

CONTROL

~

ZINC TREATED

Figure 2. Protective effect of zinc administered to dogs in vivo on platelet aggregability and release reaction. Mongrel dogs, 25 kg in weight, received intraperitoneal injections of 40 ml of 1.6 mM zinc sulfate in Tris-buffered saline, pH 6.9, 22 and 6 hours before the blood was sampled. Aggregability, tested in platelet-rich plasma, was induced by a 100 j.tg suspension of microcrystalline collagen CAviten, Fort Worth, Texas). Release reaction was tested in the presence of 10 per cent plasma and was also induced by collagen. Zinc content in the plasma was measured by atomic absorption spectrometry. CL. Stankova and M. Chvapil, unpublished results.)

release (Fig. 2). Thus, platelet is another cell, the reactivity of which is affected not only in vitro situations, but also in vivo by zinc.

Macrophages In the course of our studies on the role of zinc in the structure and function of various biomembranes6 ,7 we found that the known cytotoxic effect of silica particles (1 /L size) on peritoneal macrophages is decreased when the animals are treated with zinc. 2 ,4 We showed that the significantly increased viability of these macrophages corresponds to an inhibition of phagocytosis by these cells. This finding initiated, in our Division, extensive research on the effect of supplemented zinc, in in vivo as well as in in vitro situations, on various functions of macrophages and polymorphonuclear leukocytes. Although most of these studies are still in progress, we have already accumulated enough experimental evidence to believe that zinc supplementation in in vitro or in vivo situations results in the inhibition of several functions of macrophages and polymorphonuclear leukocytes. The second general conclusion we derived from our experiments suggests that zinc ions are one of the factors regulating the functional activity of macrophages, polymorphonuclear leukocytes and possibly some other cells. The evidence favoring both statements is briefly presented below. 8 ,42

804

MILOS CHVAPIL

A Figure 3. Morphology of peritoneal macro phages from a control guinea pig , fed a diet containing 40 ppm zinc , and animals fed a diet containing 2000 ppm zinc. Note the multiple shapes of macrophages from the control animal and the rounded macrophages from the animals fed diets high in zinc content. The picture was taken 24 hours after incubation of harvested peritoneal macrophages in 15 per cent autologous serum. (Chvapil, Carlson, and Campbell: unpublished results.)

There seems to exist a close relation between nutritional zinc status and at least mobility of macrophages or activity of their plasma membrane. Macrophages harvested from the peritoneal cavity of animals (rat, guinea pig) fed diets containing 2000 ppm zinc or injected with zinc sulfate remain round for 24 hours in a medium with 15 per cent autologous serum, and show minimal cytoplasmic elongations and extrusions (Fig. 3A). Their mobility is limited as shown by testing their migration capacity. Another evidence indicating immobilization of membrane activity of these cells is the inhibition of phagocytosis of Staph. albus. 20 All these findings contrasted with those for macrophages harvested from animals fed a control diet containing 40 ppm zinc. These cells displayed multiple forms of plasma membrane with elongations, cytoplasmic connections, active migration, and phagocytosis (Fig. 3B). It was rather surprising to find entranced migratory activity of macrophages harvested from the peritoneal cavity of Zinc-deficient guinea pigs (Table 3). If zinc supplementation inhibits migration and other activities of macrophages and eventually of polymorphonuclear leukocytes, then in-

805

EFFECT OF ZINC ON CELLS

Migration and Viability of Stimulated Peritoneal Macrophages from Guinea Pigs Fed Various Zinc Diets

Table 3.

SERUM Zn

ZINC DIETt (no. of animals)

(I-'g%)

MIGRATION TEST (mm'/24 hours)

VIABILITY (%) FINAL

275

142 ± 21

90.0

105

231 ± 11

82.0

92

317 ± 15

71.0

2000 ppm (5)

***

40 ppm (control) (6)

**

0.5 ppm (6)

**

*,:' *

**

***

tGuinea pigs weighing 80 gm were fed diets containing 40 and 2000 ppm zinc for 14 days.

A diet containing 0.5 ppm zinc was adIninistered for 3 weeks. Four days before sacrifice

paraffin oil was injected intraperitoneally. The medium contained 15 per cent autologous serum during 24 hours of in vitro activation. The area of migration was measured planimetrically. Viability was determined by trypan blue exclusion test. Variability is given as mean ± S.E. Number of asterisks refer to statistical significance, ** - p < 0.01, *** - p < 0.001, adapted according to reference 42.

duction of a sterile inflammatory reaction, for instance by intraperitoneal injection of mineral oil to animals treated with zinc, should result in less cellular infiltration of peritoneal cavity by either type of inflammatory cell. This was indeed the result of our experiments shown in Table 4. The amount of cells harvested 16 hours or 4 days after injection of the stimulant into the peritoneal cavity was significantly lower than in control animals. By increasing serum zinc level of these animals to 200 fLg per 100 ml, the mobilization of both polymorphonuclear leukocytes (16 hours) and macrophages (4 day intervals) was inhibited. Other evidence of the impaired function of phagocytic cells in zinc-treated rats was the limited transport of injected mineral oil from the cavity. The susceptibility of macrophages and polymorphonuclear leukocytes to zinc inhibition appears to differ. In experiments in which zinc was added to the medium with "normal" macrophages (peritoneal exudate) or polymorphonuclear leukocytes (peripheral blood), maximum in-

Inhibition of Mobilization and Phagocytosis of Polymorphonuclear Leukocytes in Peritoneal Cavity by Zinc Supplementation to Rats*

Table 4.

GROUP Control Zinc treated**

SERUM ZINC (I-'g/ml) 1.08 2.26

MOBILIZATION OF CELLSt (106 x cell count/cavity) 110.4 13.6

PHAGOCYTOSIS OF MINERAL OIL § (in % of total)

71

27

*500 gm body weight, male, Sprangue-Dawley rats, 5 in each group were injected Lp. with 15 ml of mineral oil 16 hours before kill. *':'Zinc sulfate, 0.5 mg per 100 gm of body weight per 12 hours was injected for 3 days before administering mineral oil. t Refers to total cell counts harvested from peritoneal cavity. § Indicates how much mineral oil was removed from the cavity. (L. Stankova and M. Chvapil, unpublished results).

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MILOS CHV APIL

Table 5. Effect of Zn++ on Oxygen Uptake in Resting and Activated Polymorphonuclear Leukocytes" 02-UPTAKE ZN++ ADDITION TO MEDIUM

(I-'M)

0 15.4 30.6 61.5 106.7 153.0

(I-'moles/hr/10' cells) PER CENT OF

Resting

Latex Activated

ORIGINAL ACTIVITY

1.3 1.3 1.2 1.3 1.4 1.2

7.2 7.4 6.5 4.5 3.4 2.4

100 103 91 62 47 33

"PMN leukocytes were isolated by dextran sedimentation from venous blood of a dog. Remaining Rbc were lysed by 0.87% ammonium chloride. Oxygen uptake was measured in 1 ml cell using Clark electrode. The medium consisted of 132 mM NaCl, 10 mM Tris buffer, pH 7.4; 4.92 mM KCl, 1.23 mM MgSO, and 5.6 mM glucose. Particle size of latex was 0.81 1-'. (L. Stankova and M. Chvapil, unpublished results).

hibition of macrophage migrating activity was obtained at 15 /LM of zinc; latex-activated polymorphonuclear leukocytes showed comparable inhibition of oxygen-consumption at a concentration of zinc almost three times higher. It should be stressed that no serum proteins were added to the medium. The presence of plasma proteins in the incubation medium seems to play a definite role in the magnitude of the inhibitory, effect of zinc on either cell type. Research along these lines, however, needs more investigation. Zinc inhibits oxygen-consumption of activated leukocytes only; resting cells are not affected by zinc (Table 5). There are more reasons to believe that zinc regulates even in vivo the function and eventually the mobilization of neutrophils. Probably the most convincing evidence was presented by Lennard et al. 26 who studied the relation of zinc content in polymorphonuclear leukocytes and functional capacity of these cells in burn patients. Their work suggests a parabolic correlation between phagocytosis of neutrophils and zinc content either in cells or in the serum. The peak of phagocytic activity seemed to coincide with slightly subnormal levels of serum zinc. (Note that mobilization and migratory activity of peritoneal macrophages was maximal in guinea pigs fed a zinc-deficient diet resulting in decreased serum zinc content. B ) Beitsel found that within 7 hours after induction of an infection in men or animals, serum zinc levels decrease and this coincides with enhancement of some functions of neutrophils. 30 Kampschmidt and Pulliam19 reported recently an increase in total counts of peripheral blood neutrophils paralleled with the decrease in serum zinc, all being part of a delayed hypersensitivity reaction. All these studies seem to point to the importance of zinc in the regulation of functional activity of neutrophils. The "normal" serum zinc level does not correspond with maximal cell activity; it is rather the slightly "subnormal" zinc content which coincides with enhanced activity of neutrophils. There is another possible biologic effect of zinc on cell activity. Our findings on the inhibitory effect of zinc ions on some functions of

807

EFFECT OF ZINC ON CELLS

Table 6. Effect of Prostatic Fluid on Oxygen Consumption by Polymorphon uclear Leukocytes FINAL CONCENTRATION OF PROSTATIC FLUID IN THE MEDIUM (PER CENT)

o o

7.14 7.69 14.28 15.38 21.42

02-UPTAKE

(",moles/hr/10' cells) PER CENT OF

Resting

Latex-Activated

ORIGINAL ACTIVITY

1.28 1.20 1.34 1.32 1.34 1.51 1.31

7.06 7.69 3.30 3.28 2.61 2.33 1.71

100 43 42 33 30 22

Human prostatic fluid containing 200 ppm zinc was added to medium (see legend to Table 5).

polymorphonuclear leukocytes led us to investigate the following clinical problem. An inadequate phagocytosis and killing reaction of polymorphonuclear leukocytes has been observed by urologists in several bacterial inflammatory infections of the prostate. This was considered in fact a possible reason for chronic nature of most bacterial inflammations of the prostate. Prostatic fluid, seminal fluid, and prostate tissue itself are known to contain almost the highest concentration of zinc in biologic fluids or tissues. 15 , 23 It occurred to us that the high content of zinc in these structures may be responsible for the impaired function of polymorphonuclear leukocytes. Polymorphonuclear leukocytes from peripheral blood were incubated at different concentrations of human prostatic fluid, and oxygen-consumption and phagocytosis were studied (Table 6). Five-fold dilution of prostatic fluid (20 per cent concentration) completely inhibited oxygen-consumption and phagocytosis of latex particles by neutrophils. Zinc is not, however, the only characteristic constituent of prostatic fluid. High levels of polyamines in prostatic fluid were reported by several authors. 29 , 38 In our further experiments aimed to analyze the mechanism of inhibition of polymorphonuclear leukocytes by prostatic fluid, we modified the fluid as indicated in Table 7.

Table 7. Effects of Modifications of Prostatic Fluid on its Inhibition of Polymorphonuclear Leukocytes EFFECT ON 02-UPTAKE TREATMENT OF PROSTATIC FLUID

Intact, no treatment Dialysis against buffer Dialysis against CaEDTA Direct addition of CaEDTA Heating (65°, 30 min)

BY

PMN

LEUKOCYTES

inhibits inhibits no inhibition no inhibition inhibits

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MILOS CHVAPIL

While heated prostatic fluid was still inhibitory, dialysis of the fluid against a chelating agent with high affinity to zinc (CaNa2 -EDTA) completely removed the inhibitory effect of the fluid on polymorphonuclear leukocyte oxygen-consumption. Furthermore, addition of CaNa 2 -EDTA to the prostatic fluid in a concentration approximately 10 times higher than the actual content of zinc in the prostatic fluid-medium system, also blocked the inhibitory effect of the fluid. Finally, we have not found any effect of various concentrations of some polyamines, such as putrescine, spermine, or spermidine, on phagocytosis and oxygen-consumption of activated polymorphonuclear leukocytes. We believe, therefore, that inhibition of phagocytes by prostatic fluid is related to the high content zinc in this system.

Proposed Modes of Action of Zinc on Various Cells In spite of a definite genetic, morphologic, and functional heterogeneity of discussed cells, certain common mechanisms of zinc effect can be proposed, as indicated in Figure 4. It has to be stressed that only a few of the many indicated mechanisms were already supported by experimental data. Thus, the following discussion still remains highly speculative. In principle, at least three basic sites of zinc effect at the cell membrane level are suggested: 1. Interaction of zinc with some functional groups of intrinsic components of the plasma membrane. This refers to formation of mercaptides with thiol groups of proteins, possible linking to the phosphate moiety of phospholipids or interaction with carboxyl groups of sialic acid or proteins. This type of reaction may result in change of fluidity of the membrane, or may stabilize the membrane, as already shown for plasma membrane of fibroblasts 39 and for lysosomal membrane of hepatocytesY The direct incorporation of supplemented zinc into biomembranes is shown by the finding that zinc content almost doubles in

Figure 4.

Proposed mechanisms for the effect of zinc on various cells.

809

EFFECT OF ZINC ON CELLS

Table 8. Zinc and ATPase in Alveolar Macrophage

SAMPLE Intact Homogenate 400 x g pellet 400 x g supernate

ATP-ASE ACTIVITY (/Lmoles Pj/mg prot./60 min.) Activated" Activated + 0.5 mM Zn++ § 0.11 0.14 0.32 0.12

o o o o

"Freshly isolated dog pulmonary alveolar macrophages were activated at 37" by exposing them to E. coli for 30 minutes at the cell/E. Coli ratio 1/10. §Zinc ions were added after the activation during the next 30-minute assay for ATPase. Modified from (17).

purified lysosomal membrane of hepatocytes from liver of animals fed high zinc diet (unpublished data). 2. There are, however, several enzymes attached to the plasma membrane controlling the structure and function of the membrane. In this respect, information exists that zinc inhibits various forms of ATPases. s Table 8 is derived from our experiments with rabbit lung alveolar macrophages, and shows complete inhibition of ATPase by zinc. Phospholipase A 2 was also shown to be inhibited by zinc ions. 40 Inhibition of either enzyme by zinc may explain immobilization of energydependent activity of plasma membrane or increased integrity of the membrane structure. The direct correlation between a certain enzyme inhibition by zinc and function of the cell membrane is, however, difficult to make because of the complexity of the reaction system. 3. Finally, several receptors at the plasmatic membrane level have been postulated to function as a gate for transmitting the information to intracellular space. As indicated in the case of mast cells, histamine and serotonin releasing agents seem to work through specific receptors at the membrane. Masking of such receptor site by membrane impermeable Zn:8-HQ complex would thus explain the inhibition of the release reaction. 21 In this brief review, I would like to mention three other mechanisms, possibly operating intracellularly. We have already shown that zinc inhibits oxidation of NADPH in liver microsomes 12 and in the granular fraction of rabbit lung alveolar macrophages.s In the case of macrophages, monocytes, or polymorphonuclear leukocytes, this effect may represent the actual mechanism by which zinc inactivates various functions of these cells. As the inhibition of NADPH oxidation occurs at rather low concentrations of zinc, NADPH oxidation is crucial to production of H 2 0 2, and thus to bacteriocidal activity;2s. 32 it is linked to the function of hexosemonophosphate shunt. If, however, Zn++ is bound to NADPH through phosphate residue (J. Ludwig, personal communication), the turnover of this pyridine nucleotide may be impaired. The role of Ca++ in the function of cell micro skeleton, represented by microtubules and by microfilament, has been well documented. The contractile elements of this system are in some way responsible for the

810

MILOS CHVAPIL

mobility of micro-organelles, transport of granules to the membrane, as well as excitability of the plasma membrane itself. Chelation of Ca++ interferes with the function of cell microskeleton. 16 • 25 In excitable membranes, phosphatidyl serine seems to function as a binding site for calcium. 27 • 36 It was proposed that displacement of calcium may result in a change in the molecular structure of the plasma membrane. If zinc displaces or inhibits uptake of Ca++ by the cell, making this ion limiting to various biostructures, a deterioration of the structure and related function may result. Evidence has already been presented on the affinity of Zn++ (and Cd++) to the microtubules. 6 • 7 Another line of thought and speculation relates to possible effect of zinc on the function of enzymes such as superoxide dismutase or glutathione peroxidase. While the first is zinc metaloenzyme, the second is selenium metaloenzyme. Both enzymes serve the function of protective mechanisms against peroxidative deterioration of cellular structures. Decreased peroxidability of fatty acids in the liver of rats fed a diet with high content of zinc has been shown;9. 10 however, the mechanism still remains to be elucidated. Our present view is that both enzymatic as well as nonenzymatic lipid peroxidation is inhibited by feeding a diet with high content of zincP Further Perspectives in the Research on the Effect of Zinc on Cells I would like to indicate three basic topics for future research. 1. The already established effect of zinc on macrophage, mast cell, and platelet suggests that some other cells will also be affected by zinc because of origin, structural, and functional similarities existing among various cells. Thus, monocytes, basophils and possibly the activity of the reticuloendothelial system might be the most likely candidates for zinc control. 2. There exists a broad spectrum of pathologic events, related in their manifestation to the presence and involvement of the above discussed cells. The role of macrophages, basophils and mast cells in a delayed type of hypersensitivity reaction has been actively studied and is much better understood than it was a few years ago. Since these reactions form the basis for the rejection of transplanted organs, it may well be that by zinc interference with the cells involved in the rejection phenomenon the fate of the transplant may be improved. I certainly do not expect that zinc will be "a miraculous drug" which will drastically change the prognosis in organ transplantation. It may well be one of the many factors protecting the functional integrity of a biological system, thus preventing the tissue destruction seen in immunologic rejection and related to labilization of membranes in macrophages, followed by the release of lysosomal enzymes, etc. Another example where zinc treatment may be of some practical importance refers to the role of macrophages (eventually mast cells) in the dynamics of fibroproliferative inflammation with fibrosis as the final outcome. Activation of polymorphonuclear leukocytes and macrophages is an integral part of any inflammation. The involvement of these cells in phagocytosis of some toxic particles, such as silica dust, is rather er-

811

EFFECT OF ZINC ON CELLS

roneous, as macrophage is a permanent loss - the cytotoxicity of silica shortly kills the cell, and accumulating necrotic tissue induces more fibrotic collagenous structures to be formed by a rather complicated chain of reactions. 3. There is not enough knowledge of how to increase the content of zinc in biological fluids and in some tissue above the "normal" values. To establish the eventual role of zinc as pharmacological modality will require detailed knowledge of modes of zinc supplementation. At this time we have definite problems of increasing in a predictable manner the content of zinc in animals by nutritional means. It seems to take a long time to observe the first elevation of serum zinc above "normal" values, even when feeding the diet containing 2000 ppm zinc, i.e., almost 100 times more than the zinc content in a standard diet. Administration of zinc by the parenteral route also creates several difficulties, as we still do not know enough about optimal form, dose, and timing of zinc administration, and the available data are quite conflicting. In conclusion I would like to stress that despite a definite gap in the knowledge of the effect of zinc on cells, the available information supports the view that zinc ions regulate the function of several cells.

REFERENCES 1. Albert, A.: Selective Toxicity. New York, John Wiley and Sons, 3rd ed., 1965, pp. 222-269. 2. Auld, D. A., Livingston, D. M., and Vallee, B. L.: RNA-dependent DNA polymerase (reverse transcriptase) from avian myeloblastosis virus: A zinc metalloenzyme. Proc. Nat. Acad. Sci., 71 :2091-2095, 1974. 3. Berger, N. A., and Skinner, A. M.: Characterization of lymphocyte transformation induced by zinc ions. J. Cell BioI., 61 :45-55, 1974. 4. Bottecchia, D., and Fantin, G.: Platelets and clot retraction effect of divalent cations and several drugs. Thrombosis et diathesis haemorrhagica, 30:567-576, 1973. 5. Caron, G. A., Poutala, S., and Provost, T. T.: Lymphocyte transformation induced by inorganic and organic mercury. Internat. Arch. Allergy, 37:76-87,1970. 6. Chvapil, M.: New aspects in the biologic role of zinc: A stabilizer of macromolecules and biological membranes. Life Sci., 13:1041-1049, 1973. 7. Chvapil, M., Elias, S. L., Ryan, J. N., et al.: Pathophysiology of Zinc. In International Review of Neurobiology. Supplement 1. New York, Academic Press, 1972, pp. 105124. 8. Stankova, L., Drach, G. W., Hicks, T., et al.: Regulation of some functions of granulocytes by zinc of the prostatic fluid and prostate tissue. J. Lab. Clin. Med. (in press). 9. Chvapil, M., Peng, Y. M., Aronson, A. L., et al.: Effect of zinc on lipid peroxidation and metal content in some tissues of rats. J. Nutrition, 4:434-443, 1974. 10. Chvapil, M., Ryan, J. N., Elias, S. L., et al.: Protective effect of zinc on carbon tetrachloride-induced liver injury in rats. Exper. Molec. Path., 19:186-196,1973. 11. Chvapil, M., Ryan, J. N., and Zukoski, C. F.: The effect of zinc and other metals on the stability of lysosomes. Proc. Soc. Exper. BioI. Med., 140 :642-646, 1972. 12. Chvapil, M., Sipes, I. G., Ludwig, J. C., et al.: Inhibition of NADPH oxidation and oxidative'metabolism of drugs in liver microsomes by zinc. Biochem. Pharmacol., 24:1-3, 1975. 13. Chvapil, M., Weldy, P. L., Stankova, L., et al.: Inhibitory effect of zinc ions on platelet aggregation and serotonin release reaction. Life Sci., 16 :561-572,1975. 14. Chvapil, M., Zukoski, C. F., Hattler, B. G., et al.: Zinc and activity of cell membranes. In Prasad, A. S., ed.: Trace Elements and Human Disease Symposium. Detroit, 1974 (in press). 15. Gonic, P., Oberlease, D., Knechtges, T., et al.: Atomic absorption spectrophotometric determination of zinc in the prostate. Invest. Urol., 6:345-349, 1969. 16. Grosman, N., and Diamant, B.: Studies on the role of calcium in the anaphylactic histamine release from isolated rat mast cells. Acta Pharmacol. Toxicol., 35:284-292, 1974.

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