Cadmium (Cd2+) disrupts intercellular junctions and actin filaments in LLC-PK1 cells*1

TOXICOLOGY AND APPLIED PHARMACOLOGY 107,8 1-97 (199 1)

Cadmium (Cd2’) Disrupts intercellular Junctions and Actin Filaments in LLC-PK, Cells’

WALTER C. PROZIALECK* AND ROBERT J. NIEWENHUIS~

*Department of Physiology and Pharmacology, and TDepartment of Anatomy, Philadelphia College of Osteopathic Medicine, Philadelphia, Pennsylvania 19131

Received May 29, 1990; accepted September 17, 1990

Cadmium (Cd2’) Disrupts Intercellular Junctions and Actin Filaments in LLC-PK, Cells. PRO-

ZIALECK, W. C., AND NIEWENHUIS, R. J. (1991). Toxicol. Appl. Pharmacol. 107, 81-97. Studies reported in the literature suggest that cadmium (Cd”) may disrupt the junctions between cells in some tissues and cell culture systems. In order to examine this possibility in more detail, we have studied the effects of Cd2+ on the integrity of intercellular junctions in the established porcine renal epithelial cell line, LLC-PK, . Junctional integrity was assessed by monitoring the collapse of domes and by measuring changes in the transepithelial electrical resistance in confluent cell monolayers. Exposure to Cd*+ caused a rapid decrease in transepithelial resistance and the con- comitant collapse of domes. These effects occurred at Cd*’ concentrations (20-60 PM) and durations

of exposure (as little as 1 hr) that did not alter levels of ATP or kill the cells. Electron microscopic studies showed that Cd*+ caused time-dependent changes in adhering and occluding junctional complexes, which eventually resulted in the complete separation of the cells. Additional studies, in which rhodamine-coupled phalloidin was used to visualize F-actin, showed that Cd*+ altered the structure of actin filaments in the cells; there was a significant reduction in the amount of junction-associated F-actin and in the number of stress fibers. These results indicate that Cd’+ has relatively specific damaging effects on the adhering and occluding junctions between LLC- PK, cells and that these effects may involve the disruption of cytoskeletal actin filaments. o 1991

Academic Press, Inc.

Cadmium (Cd2’) is an important industrial and environmental pollutant that has been shown to cause severe damage to a variety of organ systems (for reviews see Flick et al., 1971; Foulkes, 1986; Friberg et al., 1986) and to have teratogenic, mutagenic, and carcinogenic activities (Chernoff, 1973; Degraeve, 1981; Elinder and Kjellstrom, 1986; Ferm, 197 1). Although the overt toxic effects of Cd2+ have been fairly well-characterized, the specific mechanisms underlying many of these effects have yet to be elucidated. Cd2’ has been shown

’ Preliminary results of these studies were presented at the 1988 meeting of the American Society for Pharma- cology and Experimental Therapeutics and the 1989 and 1990 meetings of the Federation of American Societies for Experimental Biology.

81

to alter a wide variety of cellular and biochemical processes (Jamall and Smith, 1985; Muller and Ohnesorge, 1984; Nath et al., 1984; Vallee and Ulmer, 1972). However, the cause- effect relationships between these biochemical changes and the overt toxic effects of Cd2+ have not been established. In this regard, the findings suggesting that Cd2+ can disrupt the junctions between cells in certain epithelial or endothelial surfaces may be particularly important.

Cells of transporting epithelia and vascular endothelia are attached to each other by spe- cialized junctional complexes that are neces- sary for the restriction of permeability and the normal movement of materials across the cell monolayer. These junctional complexes,

0041-008X/91 $3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

82 PROZIALECK AND NIEWENHUlS

which include the zonula occludens (tight or occluding junctions), the zonula adherens (belt-like desmosomes), the macula adherens (spot-like desmosomes), and gap junctions, are composed of a variety of junction-associated proteins and are closely associated with the cytoskeletal elements of the individual cells (Alberts et al.. 1989; Cereijido et al., 1988; Meza et al., 1980).

In surveying the literature concerning the toxic effects of Cd*+, we noticed that many effects appeared to involve the disruption of junctions between cells in endothelial or epithelial surfaces. For example, in the testis, Cd*+ disrupts the junctions between the endothelial cells of the capillaries and venules, resulting in an increase in vascular permeability, which is followed by edema, hemorrhage, and ne- crosis (Aoki and Hoffer, 1978; Elinder, 1986a; Fende and Niewenhuis, 1977; Gunn and Gould, 1970). Similar Cd2+-induced alterations in endothelial integrity also occur in other tissues including: the lung, placenta, uterus, ovary, and the developing nervous system (Bus et al., 1978; Elinder, 1986b; Nolan and Shaikh, 1986). In the kidney, Cd*+ acts on the epithelium of the proximal convoluted tubule to increase the renal excretion of fluid, electrolytes, sugars, amino acids, and low-molecular weight proteins (Kjellstriim, 1986; Pis- cator, 1986). Although there are no reports concerning the effects of Cd2+ on intercellular junctions in the renal tubular epithelium per se, the Cd*‘-induced functional changes suggest that it may disrupt transport processes that depend on the integrity of the tight junctions between tubular epithelial cells. Additional evidence that Cd’+ may disrupt intercellular junctions stems from the finding that Cd2+ can affect components of the cytoskeleton that are functionally coupled to the junctional complexes. For example, Cd’+ causes the disassembly of microtubules in Swiss 3T3 cells (Penino and Chou, 1986) and the breakdown of actin filaments in cultured MDCK cells (Mills and Ferm, 1989).

In order to examine the effects of Cd’+ on intercellular junctions in more detail, we have studied the effects of Cd2+ on the integrity of occluding and adhering junctions in the established porcine renal epithelial line, LLC- PK, . This cell line was chosen for these studies because it possesses some of the morphologic and functional properties of the proximal tubular epithelium (Hull et al., 1976; Mullin et al., 1980: Rabito. 1986), which is one of the main sites of Cd’+ toxicity in vivo. In culture, LLC-PKr cells form tight junctions with each other, leading to the development of a polar- ized monolayer sheet with functionally distinct apical and basolateral cell surfaces. Confluent monolayers of LLC-PK, cells form character- istic dome-like structures and have a transepithelial electrical resistance of more than 100 Q. cm2 (Rabito, 1986). The integrity of the junctions between the cells can be assessed by monitoring the number of domes in the monolayer and the transepithelial electrical resistance (Mullin and O’Brien, 1986). In the studies reported here, we present evidence showing that Cd’+ has a relatively specific damaging effect on the adhering and occluding junctions between LLC-PK, cells and that this effect does not result from alterations in cellular levels of ATP or cell death.

METHODS

Growth of LLC-PK, cells in culture. Seed cultures of LLC-PK, cells were generously provided by Dr. James Mullin, of the Lankenau Medical Research Center (Phil- adelphia, PA). The cells (passages 187-200) were grown in monolayer cultures in Alpha minimum essential medium (Hazelton Research Products, Lenexa, KS) supple- mented with 10% calf serum, 2 mM glutamine, and Pen- icillin-Streptomycin (5 units-5 *g/ml). For most of the routine morphologic and biochemical studies, the cells were grown in 24-well polystyrene flasks (Costar Model 3424 Mark II). The cells were usually seeded at a density of 50,000 cells/well in I ml of medium. The cultures were maintained at 37°C in a humidified atmosphere of 5% C02:95% air. Under these conditions, the cells usually reached confluency in 4-5 days. All cultures used in these studies appeared to be morphologically normal and showed no evidence of contamination by bacteria or mycoplasma.

Cd*+ DISRUPTS INTERCELLULAR JUNCTIONS 83

The cells were exposed to Cd*+ by adding concentrated solutions of CdClz in Mops-buffered saline (pH 7.4) directly to the culture medium to achieve the desired final concentration.

Evaluation of the integrity of intercellular junctions. The integrity of the junctions between LLC-PK, cells was assessed by monitoring the collapse of domes in the monolayers and by measuring changes in the transepithelial electrical resistance. To examine the effects of Cd2+ on domes, the cells were grown to confluency in 24well plastic flasks, treated with Cd2+, and then viewed with a phase- contrast microscope at low power (63X). The domes present in randomly selected 63X fields were then counted by a trained observer who was not aware of the treatments the cells had received. To examine the effects of Cd’+ on transepithelial electrical resistance, 250,000-300,000 cells were seeded into 12-mm diameter Millicell-HA inserts (Millipore Corp., Bedford, MA). Each insert contained 0.5 ml medium and was placed inside a sample well having a diameter of 22 mm that contained 2 ml medium. The cells were fed on the day after seeding and the electrical resistance measurements were made on the third day. Cells were exposed to Cd2’ by adding 20X concentrated solutions to the medium on both sides of the cell monolayer. Transepithelial electrical resistance was measured at 37°C with an EVOM volt-ohm meter and “chopstick” elec- trodes (World Precision Instruments, New Haven, CT).

Detachment of cells from growing surface. To quantify the Cd2+-induced detachment of cells from the growing surface, confluent cell monolayers in standard 24-well flasks were treated with Cd2+ as described above. The culture medium and detached cells were aspirated and dis- carded. The cells that remained on the growing surface of each well were then fixed and stained for l/2 hr with 0.5 ml of 0.1% methylene blue in 50% ethanol (Ford et al.. 1989). Excess stain was decanted, and the cells were washed by rapidly immersing the flasks three times in 5 liters of cold tap water. The flasks were air-dried, and the stained cells in each well were solubilized with 2 ml of 1% sodium dodecyl sulfate (SDS) in 50 mM Tris-HCI buffer (pH 7.0). The optical density of the solution was then determined at a wavelength of 660 nm. Preliminary studies showed that the absorbance at 660 nm was directly related to the number of cells on the growing surface.

Determination of ATP levels. Levels of cellular ATP were determined by the firefly luciferase method (Lundin et al., 1986). Confluent monolayers of LLC-PK, cells in 24-well flasks were treated with Cd2+ or other agents as described above. At the end of the incubation period, the medium was quickly drawn off. The cells remaining on the growing surface were extracted with 0.5 ml/well of 10 mM Tris buffer (pH 7.5) containing 5 mM EDTA and 0.1% Triton X- 100. Twenty-five microliters of the extract was then diluted in 75 pl deionized water and assayed for ATP by reaction with firefly luciferin-lucifemse (Sigma Chemical Co., St. Louis, MO), in a Turner Designs Model

20 luminescence photometer (Turner Designs, Mountain View, CA). Each assay included appropriate blanks and internal standards containing known amounts of ATP.

Evaluation of cell viability. The effects of Cd’+ on the viability of LLC-PK, cells were assessed by harvesting the Cd’+-treated cells and attempting to grow them in Cd’+- free media. In these studies, the cells that remained on the growing surface after treatment with Cd2+ were freed by treatment with trypsin, reseeded into standard 24-well flasks at a density of 50,000 cells/well, and then grown in Cd’+-free media for 48 hr. The cells on the growing surfaces were fixed, and stained with methylene blue, as described above. The stained cells in each well were solubilized in 2 ml 1% SDS and the absorbance was determined at 660 nm. Cell viability was defined as the ratio of the absorbance of the cells that had been treated with Cd’+ to the absorbance of nontreated controls.

In addition, cell viability was measured by the trypan blue exclusion method (Medzihradsky and Marks, 1975) and by monitoring the release of lactate dehydrogenase into the medium (Schaeffer and Stevens, 1987). Similar results were obtained with each of these procedures.

Electron microscopic studies. LLC-PK, cells were grown on a thin layer of EMbed 812 (Electron Microscopic Sci- ences, Ft. Washington, PA) in polystyrene tissue culture flasks, treated with Cd’+, and then fixed in glutaraldehyde and osmium using standard procedures. For transmission electron microscopy, the cells were stained with lead citrate and uranyl acetate and then embedded by covering the cells with EMbed 8 12 taken from the same batch as the bottom layer. Small blocks containing the embedded monolayer of cells were removed, sectioned with a diamond knife, and examined with a JEOL IOOC electron microscope. For scanning electron microscopy, the cells were fixed with glutaraldehyde and then postfixed with a modified OsO,-thio- carbohydiazide-OsO., (O-T-O-T-O) method, which allows the cells to be air-dried from hexamethyldisilazone and examined without gold coating. The samples were examined with a JSM-5OA scanning electron microscope.

Visualization of actin filaments. Monolayers of LLC-PK, cells were grown on glass coverslips and treated with Cd2+. The treated cells were washed in phosphate-buffered saline and fixed in 4.0% formaldehyde. The cell membranes were permeabiiizied by placing the coverslips in -20°F acetone for 1-3 min. The samples were air-dried and covered with a solution of rhodamine-phaUoidin (Molecular Probes, Inc., Eugene, OR) for 20 min at mom temperature. After washing in phosphate-buffered saline, the coverslips were mounted on glass slides with a water soluble mounting medium and examined with standard rhodamine filters on a Nikon Op tiphot microscope with epifluorescence attachment.

Uptake and binding of Cd’+ by LLC-PK] cells. Con- fluent monolayers of LLC-PK, cells in standard 24-well flasks containing I ml medium per well were incubated at 37°C in the presence of 40 pM CdC12 and 0.25 &i/well rosCd2+ (New England Nuclear Corp., Boston, MA) for

84 PROZIALECK AND NIEWENHUIS

FIG. 1. Phase-contrast micrographs of normal and Cd*+-treated LLC-PK, cells. Confluent cells were treated with 60 pM Cd*+ for 8 hr as described under Methods. (A) Normal LLC-PK, cells. 420X; (B) Cd’+-treated cells, 420X; (C) Cd-treated cells. 950X.


FIG. I-Continued

varying lengths of time. At the end of the incubation period, the medium was drawn off, and the cells in each well were washed twice with I ml Mops-buffered saline. The cells were then extracted with 0.5 ml 1 M NaOH. Portions of the extract were then assayed for protein content or counted for radioactivity in a liquid scintillation counter.

RESULTS

Eflects of Cd2+ on the General Morphology of LLC-PK, Cells

Figure 1A shows a low power phase-contrast micrograph of normal LLC-PK, cells. Note that the irregularly shaped cells are very closely packed and form a confluent monolayer. A typical dome can be seen as the round, out of focus group of cells in the center of the photo. These domes form when groups of cells are lifted above the main monolayer as fluid and electrolytes are transported into the space be-

tween the cells and the growing surface, Figure 1B shows the appearance of the cells after 24 hr of exposure to 40 PM Cd*+. Note that no domes are visible and that the cells seem to be detaching from each other as evidenced by the bright borders around the individual cells. These Cd*+-induced changes were even more apparent at higher magnification (Fig 1C). It should be noted that although the cells appear to be separating from each other, only a few cells actually detached from the growing surface. A variety of other cytotoxic agents, including Hg*+, Pb2+, Al’+, and CN failed to produce this type of effect even when they were present at lethal concentrations (data not shown).

Eflectts of Cd2+ on Domes and Transepithelial Electrical Resistance

As may be seen in Fig. 2, exposure to Cd*+ caused a pronounced decrease in the number


0 4 8 12 16 20 24 2%

TIME (hours)

FIG. 2. Cd*+-induced collapse of domes. Confluent cells in 24-well flasks were treated with varied amounts of Cd’+ for up to 24 hr. The domes present in randomly selected 63X fields were counted by a trained observer who was not aware of the treatments the cells had received. The area of the viewing field was approximately 6.2 mm’. Each point represents the mean of the dome counts from four to eight fields from separate wells. The SD for the individual points ranged from I to 3.

of domes in confluent monolayers of LLC- PK, cells. The extent of this decrease was dependent on the concentration of Cd2+ and the duration of exposure. At Cd*+ concentrations of 40 PM or more, the number of domes decreased quite rapidly; only a few domes were present after 8 hr of exposure, and no domes were present after 24 hr. The Cd’+-induced collapse of domes appeared to be reversible, since the domes reappeared when these cells were incubated in Cd*+-free media for 24 hr.

Figure 3 shows that Cd*+ caused a marked drop in the electrical resistance across the cell monolayer. The time and concentration dependence for the decrease in electrical resistance were similar to those for the Cd2+-induced collapse of domes. At Cd*+ concentrations of 40 FM or more, resistance was significantly decreased after only 1 hr and was reduced to near zero after 8 hr of exposure.

Detachment of Cells from the Growing Surface

Figure 4 shows the concentration and time dependence for the Cd2+-induced detachment

0 4 6 12 16 20 24 20

TIME (hours)

FIG. 3. Cd*‘-induced decrease in transepithelial electrical resistance. Confluent cells in 12-mm Millicell-HA inserts were treated with Cd*+, and the transepithelial resistance was determined as described under Methods. Each point represents the mean 4 SE of four to eight samples.

of LLC-PKr cells from their growing surface. Note that the concentrations of Cd*+ and times of exposure that were needed to cause detachment of the cells were much greater than those that caused the collapse of domes and the decrease in transepithelial resistance. For example, exposure to 40 gM Cd*+ for 24 hr caused little or no detachment of cells from the growing surface (Fig. 4) whereas the same

5 o.oooJ 0 4 6 12 16 20 24

TIME (hours)

-! 26

FIG. 4. Cd*+-induced detachment of cells from their growing surface. Confluent cells in 24-well flasks were treated with varied amounts of Cd*+ for up to 24 hr. The cells remaining on the growing surface were fixed, stained with methylene blue, and solubilized as described under Methods. The methylene blue absorbance was directly related to the number of cells remaining on the growing surface. Each point represents the mean of the absorbance values from four wells. For no point was the SD greater than 15% of the mean.

Cd’+ DISRUPTS INTERCELLULAR JUNCTIONS 87

concentration of Cd’+ caused a significant decrease in transepithelial resistance and the collapse of domes after only 2 hr of exposure (Figs. 2 and 3).

Efects of Cd2+ on A XP Levels and Cell Via- bility

The results of the studies described above show that Cd’+ causes LLC-PK, cells to separate from each other without detaching from their growing surface, suggesting that Cd2+ disrupts the junctional complexes between cells. However, from these studies it is not clear whether the Cd2+-induced disruption of intercellular junctions represents a primary effect of Cd*+ or occurs secondarily to the general metabolic derangement or death of the cells. To address this issue, we examined the effects of Cd*+ on ATP levels and cell viability in relation to the disruption of intercellular junctions.

The top graph in Fig. 5 shows the effects of 40 PM Cd*+ on domes, transepithelial resistance, ATP levels, and cell viability, as a function of time. Note that the Cd’+-induced decrease in resistance and collapse of domes be- gan to occur after only l-2 hr of exposure and became quite pronounced after 4-6 hr. By contrast, no changes in the levels of ATP or cell viability were evident even after 24 hr of Cd’+ exposure. Thus, it appears that the disruption of intercellular junctions by Cd*+ did not result from the general metabolic derangement or death of the cells.

These effects of Cd2+ were qualitatively different from those produced by other cytotoxic agents such as HgZf and CN-. At concentrations of 120 PM or less, Hg*+ had little effect on LLC-PK, cells under these test conditions. At higher concentrations, Hg2’ did cause the collapse of domes and a decrease in transepithelial resistance. However, unlike Cd2+, Hg2’ did not cause the individual cells to separate from each other, but rather caused large sheets

110.. Cd’+@0 IrM) A

o-o ncsswaf

, 12 10 20 24 TiNtC(hwn)

0 4 8 12 16 20 24

‘-T 1

n 01 %+s : 1 0 4 0 12 10 20 24 20

WE (h-4

FIG. 5. Comparison of the effects of Cd’+, Hg’+, and CN- on LLC-PK, cells. The various parameters were measured by the procedures described under Methods. The results are expressed as percentages of control values. Each point represents the mean value of four to eight rep- licate samples.

particular interest is the finding that the concentrations of Hg2+ and times of exposure that produced these effects also decreased the levels of ATP and actually killed the cells (Fig. 5, middle graph). The effects of CN- differed from those of both Cd2’ and Hg2+. As may be seen in the graph at the bottom of Fig.. 5, CN- caused a pronounced decrease in the number of domes but had little effect on transepithelial

of cells to detach from the growing surface. Of resistance. The CN--induced collapse of


FIG. 6. Scanning electron micrographs showing the effects of Cd’+ on LLC-PK, cells. Ceils were treated with Cd2+ and processed for scanning electron microscopy as described under Methods. (A) Normal LLC- PK, cells; (B) Cells treated with 50 FM Cd2+ for 1 hr; (C) Cells treated with 50 PM Cd2+ for 4 hr, 1038X.


FIG. 6-Continued

domes coincided with a sharp decrease in ATP levels.

Efects of Cd2+ on the Ultrastructure of LLC- PK, Cells

Examination of the Cd2+-treated cells by electron microscopy revealed time- and concentration-dependent changes in the shape of the cells and the ultrastructure of the intercellular junctional complexes. The Cd*+%- duced changes in cell shape were especially apparent when the cells were viewed with the scanning electron microscope. As may be seen in Fig. 6A, normal LLC-PK, cells were closely attached to each other and showed a typical squamous appearance. By contrast, cells that were treated with sublethal concentrations of Cd2’ appeared to contract, round up, and separate from each other (Figs. 6B and 6C). The

transmission electron micrographs in Fig. 7 also show the Cd*+-induced changes in cell shape as well as specific changes in the adhering and occluding junctions. Figure 7A shows the appearance of normal LLC-PK, cells. Note that the cells lie in close proximity to each other and that distinct occluding junctions (zo) and adhering junctions (za) are present. Figure 7B shows the appearance of the cells after exposure to 50 pM Cd’+ for 1 hr. Note that there is a large fluid-filled space between the cells and that the membrane-associated plaques of adhering junctions (arrows) appear to show decreased electron density when compared to controls. However, occluding junctions (zo) appear to remain relatively intact. Figure 7C shows the appearance of cells that were exposed to 50 pM Cd2+ for 4 hr. Note that the cell processes are very thin or attenuated and that the cells are separating from each other. No occluding or adhering junctional com-

PROZIALECK AND NIEWENHUIS

FIG. 7. Transmission electron micrographs showing the effects of Cd’+ on junctional complexes in LLC- PK, cells. Cells were treated with Cd” and processed for transmission electron microscopy as described under Methods. (A) Normal LLC-PK, cells showing distinct occluding (20) and adhering junctions (za). 36,000X; (B) Cells treated with 60 pM Cd” for 1 hr. Adhering junctions show decreased electron density (arrows) but occluding junctions (20) remain intact, 23.000X: (C) Cells treated with 60 pM Cd’+ for 8 hr. Both the adhering and occluding junctions have broken down and the cells appear to be in the process of separating from each other, 30.000X.

Cd2+ DISRUPTS INTERCELLULAR JUNCTIONS 91

C

FIG. ‘I-Continued

plexes are present. The breakdown of junc- (Fig. 8B). There appeared to be a marked re- tions was not uniform and varied from one duction in the number of stress fibers and in cell to another, as well as from one area to the junction-associated actin around the bor- another. The electron microscopic studies ders of the cells. Some of the cells appeared to showed no evidence of Cd’+-induced damage be separating and pulling away from each to the mitochondria or cell membrane, which other. would be indicative of general oxidative damage to the cells (Scott et al., 1989).

Uptake and Binding of Cd’+ by LLC-PK, Cells

Efects of Cd’+ on Actin Filaments

Figure 8 shows that Cd*+ caused changes in the structure of actin filaments in the cells, which coincided with the disruption of cell- cell junctions. Large bundles of actin filaments were present within the untreated cells. Stress fibers were attached to the cell membrane, and the borders of each cell were highlighted by bright bands of junction-associated actin (Fig. 8A). Note that fewer filaments were present in the Cd2+-treated cells and that the ones that were visible showed a washed out appearance

Table 1 shows the amount of Cd’+ that was associated with the cells after varying times of exposure. The values represent the total amount of Cd*+ that was bound to the cell surface and taken up by the cells. Note that the accumulation of Cd2+ by the cells proceded rather slowly. Interestingly, only small amounts of Cd*+ were associated with the cells at the time the changes in cell-cell junction were occurring (2-4 hr) suggesting that the intercellular junctions may be one of the initial sites of Cd*+ toxicity in LLC-PK, cells.


FIG. 8. Effects of Cd*+ on F-a&n in LLC-PK, cells. Cells were treated with Cd2’, and F-actin was visualized by using rhodamine-coupled phalloidin as described under Methods (A). Normal LLC-PK, cells (B). Cells treated with 60 jtM Cd’+ for 8 hr, 900X.


TABLE 1

UPTAKE AND BINDING OF Cd*+ BY LLC-PK, CELLS

Cell associated Cd*+ Time of exposure (hr) (pmol/mg protein)

1 51 8 2 16t 4 4 21+ 8 8 49+ 6

24 229 f 70

NoIe. Confluent cells in 24-well flasks were treated with CdClr and losCd2+, solubilized, and counted for radioactivity as described under Methods. The results represent the mean + SD of four to six samples from different wells.

DISCUSSION

These results show that exposure to micro- molar concentrations of Cd*+ for relatively short periods of time causes the disruption of the junctions between LLC-PK, cells. The disruptive effects of Cd*+ on intercellular junctions were evidenced by a rapid decrease in transepithelial resistance, the collapse of domes in the cell monolayer, and morphologic changes in the junctional complexes them- selves. Results of electron microscopic and histofluorescent studies showed that Cd*+ produced time- and concentration-dependent changes in the structure of both adhering and occluding junctional elements, which coincided with changes in the structure of cytoskeletal actin filaments.

Although we have not attempted to establish the cause-effect relationship between the changes in F-actin and the disruption of the junctional complexes, there is evidence to suggest that the two phenomena may be related. Actin microfilaments are major structural constituents of the adhering junctions and junction-associated contractile rings (Cereijido et al., 1988; Madam 1987). In addition, large bundles of microfilaments, known as stress fibers, attach to the cell membrane near the base of the cells (Geiger, 1983; Mills and Ferm, 1989). Recent studies have shown

that actin filaments may also be associated with the occluding junctional complexes in some cells (Madara, 1987) and that the functional integrity of the occluding junctions may be regulated by the contraction of cytoskeletal F-actin (Madara et al., 1987, 1988; Meza et al., 1984). Interestingly, a variety of agents that are known to disrupt F-actin, such as cyto- chalasins B and D (Madara et al., 1987, 1988; Meza et al., 1980; 1984) and certain tumor- promoting phorbol esters (Ben-Ze’ev, 1986; Mullin and O’Brien, 1986; Ojakian, 1981) have been shown to increase the permeability of epithelial occluding junctions in a manner similar to that which we observed for Cd*+.

The disruption of intercellular junctions and cytoskeletal actin filaments appears to represent fairly specific toxic effects of Cd*’ since they occurred at Cd*+ concentrations (20-60 PM) and durations of exposure (l-8 hr) that did not cause the cells to detach from their growing surface, alter levels of ATP, or kill the cells. However, it is not yet clear whether this apparent specificity of effects is the result of a truly selective toxic action of Cd*+ on intercellular junctions, or whether the specificity simply indicates that Cd*’ has easier access to junctional sites than to other possible sites of toxicity. There is evidence to support each of these possibilities. For example, the fact that Cd’+ is able to disrupt the intracellular actin filaments indicates that it does enter the cells and, therefore, is probably available to a variety of potential sites of toxicity. On the other hand, the disruption of cell-cell junctions occurs at a time when the cells have taken up relatively small amounts of Cd*‘. Further- more, we have recently found that the disruption of intercellular junctions by Cd*+ seems to be more pronounced when Cd*+ is added to the basolateral cell surface, rather than to the apical surface (data not shown), indicating that the rate of access of Cd2’ to sites on the basolateral cell surface may be an important determinant of activity.

There are several possible molecular mechanisms to account for the junction- and actin-


perturbing effects of Cd2+. One possibility is that Cd2+ might interact directly with the molecular components of the junctional complexes or actin filaments. In support of this hypothesis is the finding by Kunimoto and Miura (1985) that Cd’+ can bind to isolated cytoskeletal elements from erythrocyte ghosts. Another possibility is that Cd*+ might act by disrupting the biochemical systems that reg- ulate junctional and cytoskeletal function. In this regard, the findings that Cd2+ can interfere with the normal physiologic actions of Ca2+ may be especially relevant, since Cal+ plays a key role in the formation. maintenance, and regulation of occluding junctions, adhering junctions, and actin filaments (Bennett and Weeds, 1986; Meza et al., 1980: Takeichi, 1988; and Volberg et al., 1986). These effects of Ca2+ involve a direct action on various structural proteins. such as the cadherin class of cell adhesion molecules, and an indirect action through the calmodulin and protein kinase C signaling systems (Bennett and Weeds, 1986; Takeichi, 1988). Several studies have shown that Cd’+ can interfere with the normal actions of Ca2+ in these systems. Cd*+ has been shown to either activate or inhibit various calmodulin-dependent processes ( Akerman et al., 1985; Cheung, 1984; Chao et al., 1984; Cox and Harrison, 1983: Flik et al., 1987; Mills and Johnson, 1985) and to biphasically activate and inhibit protein kinase C (Mazzei et al., 1984). Since both calmodulin and protein kinase C appear to play key roles in regulating the structure and function of junctional complexes, the disruption of these regulatory systems by Cd’+ could cause profound alterations in junctional integrity. The fact that the phorbol esters, which activate protein kinase C, produce effects on tight junctions and actin filaments (Ben Ze’ev, 1986; Mullin and O’Brien, 1986; Ojakian, 1981; Schliwa et al., 1984) that are similar to those of Cd’+ suggests that Cd2+, too, might be producing its junctional effects via the activation of protein kinase C.

The selective disruption of intercellular junctions and cytoskeletal F-actin occurred at Cd2+ concentrations (20-60 pM) that may be toxicologically relevant. Although these concentrations are somewhat higher than the reported plasma concentrations of Cd*+, they are well below the concentrations that are reached in various tissues during Cd’+ poisoning (for review see Nordberg et al.. 1986). It should be noted that in our studies, the medium in which the cells were grown and exposed to Cd2+ contained 10% calf serum. Since Cd*+ binds extensively to serum proteins (Nordberg et al., 1986) the concentrations of free Cd’+ in the medium were probably much lower than the concentrations of Cd2+ that were added. Preliminary results (not shown) indicate that Cd2+ is 2-5 times more potent when it is added to cells in serum-free medium or buffered saline, although the effects under these conditions are qualitatively identical to those produced when serum is present.

Although Cd2+ has been shown to produce a variety of cytotoxic effects on renal epithelial cells in culture (Boogaard et al., 1989; Cherian. 1980, 1985; Hazen-Martin et al., 1989a,b), to the best of our knowledge, this is the first study showing that Cd2’ can cause specific pertur- bations in the structure and function of the junctions between such cells. This finding raises the possibility that Cd2+ might produce some of its nephrotoxic effects by disrupting the tight junctions between cells in the proximal tubular epithelium. However. several is- sues must be resolved before this hypothesis can be confirmed. For example, nephrotoxicity is commonly associated with chronic exposure to Cd2+ (Kjellstrom, 1986: Piscator, 1986) whereas our studies dealt with the effects of acute Cd2+ exposure. Furthermore, most of the Cd’+ that is presented to the renal tubular epithelium in vivo is conjugated to metallothioneins or other low-molecular weight proteins (Elinder and Nordberg, 1986; Webb, 1986), whereas our studies focussed on the effects of Cd2+ itself. Whether Cd’+-metallothionein conjugates can produce similar


effects in the LLC-PK, system remains to be seen.

In spite of these caveats, we feel that the disruption of intercellular junctions in certain tissues could account for some of the toxic effects of Cd2+ in vivo. Acute exposure to sys- temically administered Cd2+ has been shown to increase the permeability of vascular beds in a variety of tissues, eventually leading to secondary necrotic changes (Nolan and Shaikh, 1986). Such increases in microvascular permeability could result from the selective disruption of endothelial cell-cell junctions by Cd2+. In addition, the ability of Cd2+ to disrupt intercellular junctions and cytoskeletal actin filaments could have important implications concerning the mechanisms underlying the carcinogenic and teratogenic effects of Cd’+.

ACKNOWLEDGMENTS

The authors gratefully acknowledge the excellent tech- nical assistance of Brian Kahan, Michael Fiorina, and John Pellegrino. In addition we thank Scott Cosmi for his assistance with the electron microscopy and Chris Donovan for her help with the manuscript.

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Cadmium (Cd2+) disrupts intercellular junctions and actin filaments in LLC-PK1 cells*1