Effects of Cadmium on Osteoclast Formation and Activityin Vitro

TOXICOLOGY AND APPLIED PHARMACOLOGY 140, 451–460 (1996)ARTICLE NO. 0242

Effects of Cadmium on Osteoclast Formation and Activity in Vitro1

ALLISON K. WILSON, ELIZABETH A. CERNY, B. DAVID SMITH,2 ARATI WAGH,2 AND MARYKA H. BHATTACHARYYA

Center for Mechanistic Biology and Biotechnology, Argonne National Laboratory, Argonne, Illinois 60439-4833

Received February 1, 1996; accepted May 30, 1996

tion (OSHA) lowered its permissible exposure level for air-Effects of Cadmium on Osteoclast Formation and Activity in borne Cd (OSHA, 1992). The current OSHA action level,

Vitro. WILSON, A. K., CERNY, E. A., SMITH, B. D., WAGH, A., at which increased biological monitoring of workers mustAND BHATTACHARYYA, M. H. (1996). Toxicol. Appl. Pharmacol.

be conducted, is 5 mg Cd/liter whole blood. Studies of occu-140, 451–460.pational exposure to Cd report a range of mean blood Cd

Chronic exposure to cadmium has been linked to bone loss, low levels from 0.31 to 22.2 mg/liter (Hassler et al., 1983; Thunbone mass, and increased incidence of fracture. To determine if et al., 1989; Chia et al., 1989; Kawada et al., 1990; Sacco-Cd could directly increase the formation of cells responsible for Gibson et al., submitted for publication). In the environmen-bone resorption, we cultured normal canine bone marrow cells tal setting, exposure to Cd from tobacco increases with thecontaining the progenitor cells for osteoclasts. Cultures were evalu- number of cigarettes smoked (Elinder, 1983a,b). Medianated for the number of multinucleate osteoclast-like cells (MNOCs)

blood Cd concentration for those who smoke§20 cigarettesformed. Exposure to Cd (10–100 nM) increased the number ofper day is 2 mg/liter compared to 0.2 mg/liter for nonsmokers.MNOCs formed over control values when cultured in the presenceAlthough Cd accumulates in the liver and kidney and pro-but not in the absence of a bone wafer. The MNOCs formed werelonged exposure can cause proximal renal tubule damagefunctional, evidenced by pits excavated on the bone wafers in-and impaired vitamin D metabolism above threshold levelscluded in the cultures. By 12 days, MNOCs formed in the presence

of 50 nM Cd excavated significantly more pits and a greater pit (200 mg of Cd per gram of renal cortex) (Friberg et al.,area than did untreated MNOCs. By 14 days, the control values 1986), recent data indicate that bone is a target organ forwere similar to those of the Cd-exposed MNOCs, but pit formation Cd damage at lower exposure levels (2 –5 mg/liter in blood)was enhanced by Cd in that the ratio of pit complexes to single and in a shorter time frame (Ando et al., 1978; Bhattacharyyapits was increased twofold over that for untreated cultures. Mature et al., 1988a,b; Ogoshi et al., 1992; Sacco-Gibson et al.,osteoclasts, isolated from the long bones of rat neonates and cul-

1992; Wang and Bhattacharyya, 1993). Chronic exposure totured for 1–3 days on bone slices, provided a direct method toCd has been linked to bone loss, low bone mass, and in-assess the effect of Cd on osteoclast activity. Exposure of osteoclastcreased incidence of fractures (Adams et al., 1969; Tsuchiya,cultures to 100 nM Cd increased the number of osteoclasts present1969; Daniell, 1976; Kazantzis, 1979; Nogawa, 1981;over that for untreated osteoclasts by a factor of 1.7 { 0.1, theElinder et al., 1983a,b; Aloia et al., 1985; Slemenda et al.,number of pits excavated by 2.8 { 0.6, the area excavated by 3.2

{ 0.8, and the area excavated per osteoclast by 1.8 { 0.4 (mean 1989).{ SE; nÅ six experiments). These data suggest that Cd accelerates The functions of osteoblasts and osteoclasts, the bone-the differentiation of new osteoclasts from their progenitor cells associated cells that form and resorb bone, respectively, in-and activates or increases the activity of mature osteoclasts. clude growth, modeling, repair, and remodeling of the skele-q 1996 Academic Press, Inc.

ton (Vaananen, 1993; Bloom and Fawcett, 1994). The adultskeleton is surprisingly dynamic, with bone remodeling oc-curring in response to mechanical and metabolic demands.

Exposure to cadmium is a concern in the increasingly Remodeling is initiated by activation, a process of preparingregulated workplace and for the smoking population. In the bone for resorption, recruiting preosteoclasts that fuse1992, the U.S. Occupational Safety and Health Administra- into mature osteoclasts, and signaling the mature osteoclasts

to initiate bone resorption. Once the bone is resorbed, osteo-blasts follow behind, secreting collagen matrix for subse-1 The U.S. Government’s right to retain a nonexclusive royalty-free li-quent mineralization. Uncoupling of the remodeling pro-cense in and to the copyright covering this paper, for governmental pur-

poses, is acknowledged. cesses of bone resorption and formation can lead to a net2 Work performed by B. D. Smith and A. Wagh was conducted under a loss of bone.

Student Research Participant Program administered by Argonne Department How Cd acts to induce bone loss at a cellular level is notof Educational Programs and supported by the U.S. Department of Energy,known. Under comparable incubation conditions in organOffice of Energy Research, through its University/National Laboratory Co-

operative Program. and cell culture systems, bone resorption appears to be more

451 0041-008X/96 $18.00Copyright q 1996 by Academic Press, Inc.

All rights of reproduction in any form reserved.

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452 WILSON ET AL.

incubated with antibody for 30 min at room temperature in PBS–HS andsensitive than bone formation to Cd exposure (Miyahara etwashed three times in cold PBS-HS, and positive cells were visualized byal., 1988, 1991, 1992; Suzuki et al., 1989, 1990; Iwami andusing the Vectastain ABC kit (Vector Labs, Burlingame, CA) and counter-

Moriyama, 1993). Because low-level exposure to Cd is more stained with 1% methyl green. The MNOCs were defined as TRAP-positivecommon in our society than higher-level exposure, we chose or 23c6-positive cells with three or more nuclei (see Figs. 1A and 1B forto study the effects of low-level Cd exposure on bone resorp- examples). In each experiment, four bone wafers were cultured for each

treatment.tion. Our laboratory, in a study in which ovariectomizedand sham-operated dogs were exposed to Cd at low levels, Osteoclast isolation. Pregnant Sprague–Dawley rats (Charles River,

Wilmington, MA) were used as a source of rat neonates. Mature osteoclastsdemonstrated that Cd at 2–5 mg/liter blood began to induceand preosteoclasts were isolated from the long bones of 2- to 4-day-oldbone loss, as measured by 45Ca release from the skeletonmale and female rat neonates essentially as described (Chambers and Mag-without renal dysfunction or calciotropic hormone interac-nus, 1982; Boyde et al., 1984). The femora, tibiae, and humeri were placed

tion (Sacco-Gibson et al., 1992). To extend these findings in a-MEM on ice until dissected free of soft tissue, curetted in roomto a cellular level, we cultured normal canine marrow in temperature media, and agitated to release bone-associated cells. The dis-the presence of Cd to determine if low-level Cd exposure persed cell solution was plated into wells containing bovine bone wafers

in a-MEM and was incubated in 5% CO2 at 377C for 25 min to facilitatestimulated the differentiation of osteoclasts as indicated byattachment of the osteoclasts to the bone wafer. Time from euthanizationincreased numbers of MNOCs formed. In addition, isolatedto initial plating was approximately 10 min. Nonadherent cells were washedosteoclasts from neonatal rat long bones were cultured tooff the bone wafer by dipping into media, and the wafer was placed in

determine if Cd increased the activity of mature osteoclasts. fresh a-MEM containing 10% fetal bovine serum, 1% of a penicillin–Primary cell culture systems were used to study osteoclasts streptomycin combination, and the experimental treatment. An example of

a typical preparation is illustrated in Figs. 1C and 1D. After a 4- to 72-hrin vitro because of the terminally differentiated nature of theincubation period, the cells on the bones were fixed and histochemicallyosteoclast.stained for alkaline phosphatase activity and TRAP activity by using com-mercial kits (Sigma Chemical Co.). After quantitation of the osteoclasts

METHODS and osteoblasts present, the cells were wiped off the bone wafers, and thewafers were soaked for 20 min in 1 M ammonium hydroxide, mounted in

Bone marrow cultures. Two female beagle dogs, 1 year old, wereglycerol, and examined for pit formation.

purchased from Ridgeland Farms (Mt. Horeb, WI) and housed in a facilityQuantitation of pits. The pits excavated by osteoclasts were viewedinspected and approved by the American Association for Accreditation of

via bright-field microscopy, with the condenser adjusted to maximize con-Laboratory Animal Care. The experimental protocol was approved by thetrasting planes of focus. Pits were classified into six categories by sizeInstitutional Animal Care and Use Committee. By using a 20-gauge spinal(small, medium, or large) and shape (single or complex). Size classificationneedle, bone marrow (5–10 ml) was aspirated aseptically from the humeruswas aided by the use of an ocular grid. A single pit was a rounded areaor iliac crest of the anesthetized dog (Thiamylal, 5 mg/kg) into a syringe

containing heparin (250 units/ml of marrow). Aspirations were alternated apparently excavated by a one-time attachment of an osteoclast. A complexbetween dogs and sites to prevent fibrotic tissue accumulation. pit was made up of two or more rounded areas attached to one another,

Mononuclear bone marrow cells, composed of a heterogeneous popula- apparently made by multiple attachments of a migrating osteoclast (Fig. 2).tion of progenitor cells for numerous blood and bone cells, were isolated The counting-by-category (CBC) method was used to evaluate total pitby centrifugation through a Histopaque column (1.077 g/ml; Sigma Chemi- area. For this method, the mean area of 10–20 pits of each size–shapecal Co., St. Louis, MO) and were cultured overnight in a-minimal essential category was determined from several representative bone wafers using amedia (a-MEM) containing 15% fetal bovine serum, 1% of a penicillin– Bioquant image analysis system (R&M Biometrics Inc., Nashville, TN).streptomycin combination, and 200 pg granulocyte–macrophage colony- For routine pit area determinations, the number of pits was counted forstimulating factor (R&D Systems, Minneapolis, MN) per milliliter. Unat- each size –shape category. The total area for each category was calculatedtached cells were further cultured in four-well chamber slides (Nunc, Naper- by multiplying the number of pits by the mean pit area for that category.ville, IL) at a density of 0.75 1 106 cells per well in a-MEM containing The total area on the given bone wafer was calculated by summing the20% horse serum and 1% of a penicillin–streptomycin combination. A areas for each pit category (six categories).bone wafer was included in some cultures to test for osteoclastic resorptive The total pit area of four bones was determined by the CBC method andactivity. Shafts of bovine or canine long bone were cut into approximately compared to direct measurement by the image analysis method (IA) to100-mm-thick slices using a custom-made hydraulic precision bone saw ensure a correspondence between the two methods. The CBC method re-(Argonne National Laboratory). The bone slices were cut into wafers of sulted in areas somewhat higher than the IA method but the two methodsuniform area (20 mm2). Cadmium chloride or the osteotropic hormone, were comparable (CBC/IA area ratio, 1.31 { 0.13, mean { SE, n Å 4).1,25-(OH)2-vitamin D3 (ICN, Costa Mesa, CA) was added at the time of

Statistics. Means { SE were calculated for each experimental conditionplating and was replenished every 3–4 days when half of the medium from(n Å 3–4 for each experiment). The significances of differences due to Cdeach well was replaced. Cultures were incubated for up to 21 days at 377Cwere determined by Student’s t test for those cases in which a singlein a humidified atmosphere of 5% CO2 in air. Cells were fixed, stained forexperimental treatment group was compared to a control. One-tailed t teststhe presence of tartrate-resistant acid phosphatase (TRAP) at a tartratewere used because values were expected to increase according to publishedconcentration of 50 mM (TRAP kit; Sigma Chemical Co.), and counter-results (1,25-(OH)2-vitamin D3 effects) or according to the current hypothe-stained with Mayer’s hematoxylin. Alternatively, cultures were stained forsis (Cd effects). For multiple treatment groups, differences were tested forMNOCs with 23c6, an osteoclast-reacting monoclonal antibody directedsignificance by ANOVA followed by the least significant differences (LSD)against the a-chain of the human vitronectin receptor, as described (Hortontest. Data from several individual experiments are presented to illustrateet al., 1985; Kukita et al., 1989; Athanasou et al., 1990). Cells were fixedthe absolute differences observed between experiments. In addition, thein 2% formaldehyde in phosphate-buffered saline (PBS) for 2 min, extractedcombined results of multiple experiments are presented as the ratios of thewith 100% methanol for 1 min at room temperature, and washed three

times in cold PBS containing 1% horse serum (PBS–HS). The cells were Cd-treated to control values.


453CADMIUM AND OSTEOCLAST FORMATION AND ACTIVITY

RESULTS (Fig. 3B) and by fewer numbers of cells overall after thefirst week of culture for both conditions. Table 1 illustrates

Effect of Cd on MNOC Differentiation the effects of low-level cadmium exposure on MNOC forma-tion and function. By Day 12, Cd at 50 nM in the cultureBone marrow contains progenitor and precursor cells formedia significantly increased the number of MNOCs locatedmany different cell types including osteoclasts and osteo-on the bone wafer, the total number of pits, and the excavatedblasts. In our system, canine bone marrow cells were cul-pit area. This effect of Cd was transient because at Day 14,tured under conditions that induced the differentiation ofthe number of MNOCs on the bone in untreated culturesosteoclast-like cells. Osteoclasts are characterized by thehad increased and was no longer significantly lower thanpresence of many nuclei, expression of TRAP activity, andthat in the Cd-exposed cultures. At Day 21, the decreasedthe vitronectin receptor. Tartrate-resistant acid phosphatasenumbers of osteoclasts indicated that the cultures were pastis specific for osteoclasts in bone and marrow tissue, al-their optimal growth period and resorptive activity. Thethough TRAP is also expressed in other differentiated cellsnumber and area of pits excavated by MNOCs were depen-of the monocytic lineage located elsewhere (Athanasou etdent on the number of MNOCs present and increased fromal., 1990). Figure 1A illustrates an example of an MNOCDays 12 to 14 for both the control and Cd-exposed cultures.cultured on a bone wafer and stained for TRAP activity. TheAlthough MNOCs in the control cultures had excavated theMNOCs formed in these cultures also cross-reacted withsame pit areas as those in the Cd-exposed cultures by Day 14,the 23c6 antibody directed against the human vitronectinthe pattern of resorption was visually different; Cd-exposedreceptor (Fig. 1B). The vitronectin receptor is a cell surfacecultures exhibited more extensive pit complexes at all timeprotein present on phagocytes that recognizes the RGD se-points than control cultures did. This pattern was indicatedquence common to extracellular matrix glycoproteins. Theby a twofold increase in the ratio of pit complexes to singleRGD sequence is important in mediating cellular adhesion,pits (Culture Days 12–21, Cd vs control, mean { SE; 4.1localization, and migration—functions required for osteo-{ 0.7 (n Å 8) vs 1.9 { 0.5 (n Å 5), p õ 0.05). The boneclast function (Baron et al., 1993). Because the 23c6 anti-marrow data indicate that Cd exposure may speed the pro-body cross-reacts with the canine vitronectin receptor, itcess of osteoclast differentiation. The data also suggest thatprovides an additional method for identifying osteoclasts orCd may increase the bone-resorbing activity of osteoclasts, aosteoclast-like cells in canine tissue. Bone resorption, theprocess more easily studied in a short-term culture of isolatedfunctional property of osteoclasts, was observed in culturesmature osteoclasts.that contained bone wafers (Fig. 2). Differentiation of

MNOCs in canine marrow cultures was responsive to theosteotropic hormone, 1,25-(OH)2-vitamin D3 (Fig. 3), a char- Effects of Cd on Isolated Osteoclastsacteristic demonstrated in human (MacDonald et al., 1987;Kurihara et al., 1990) and mouse cultures (Takahashi et al., Mature osteoclasts and their immediate precursors were

isolated from the long bones of neonatal rats. The cultures1988; Shinar et al., 1990).Continuous Cd exposure (10–1000 nM) did not increase were highly enriched for osteoclasts but were heterogeneous

and included preosteoclasts, osteoblasts, and occasional stro-MNOC formation when marrow was cultured on a plasticsubstrate (Fig. 3A); however, MNOCs could be induced to mal cells (Figs. 1C and 1D). The osteoclasts exhibited TRAP

activity and were activated to increase pit formation by 1,25-differentiate from the bone marrow in response to Cd whena devitalized bone wafer was included in the culture (Fig. (OH)2-vitamin D3 (Table 2). The ratio of the number of

complex to single pits increased in response to 1,25-(OH)2-3B). Toxicity was observed at 500–1000 nM Cd, indicatedby fewer MNOCs formed in the presence of the bone wafer vitamin D3 as did the area excavated per osteoclast. 1,25-

FIG. 1. Typical MNOC and isolated osteoclast preparations. MNOCs were stimulated to differentiate from canine bone marrow, or osteoclasts wereisolated from the long bones of neonatal rats and were cultured on bone wafers as described under Methods. Cultures were stained for TRAP activity(A, C, D) or with 23c6 antibody (B) to identify osteoclasts (red) and for alkaline phosphatase activity (C, D) to identify osteoblasts (blue). (A) Exampleof a TRAP-positive MNOC formed in a culture of bone marrow cells. Note the background topography of the bone wafer and the many TRAP-negativemononuclear cells. (B) Example of bone marrow culture stained with the osteoclast-specific monoclonal antibody, 23c6. The large multinucleated cellstained positive for the vitronectin receptor whereas the smaller mono- and binucleated cells were negative. (C) A typical isolated osteoclast preparationviewed under low magnification. Note that the cell population included osteoblasts, osteoclasts (multinucleate TRAP-positive cells), and preosteoclasts(mononucleate or binucleate TRAP-positive cells). (D) Clusters of mononucleate cells in isolated osteoclast preparations at 4 hr stained for TRAP activityand viewed under higher magnification. Two examples are illustrated that appear to be fusing to form osteoclasts.

FIG. 2. Pit complex excavated by MNOCs. The MNOCs were stimulated to differentiate in culture on bone wafers as described under Methods. (A)Pit complex, probably excavated by the MNOC shown, histochemically stained for TRAP activity. (B) Same pit complex as in A after the MNOC wasremoved. Note the rounded pit at the top end of the complex that was probably being excavated by the MNOC shown in A. The pit image is out offocus because the focal plane of the pit complex is different than that of the bone surface.


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456 WILSON ET AL.

mium increased the number of pits excavated and, conse-quently, the area resorbed. The increase in complex pit for-mation due to Cd exposure observed in the marrow cultures(Table 1) was not observed in the isolated osteoclast cultures(Table 3). When six separate experiments were normalizedto the extent of the Cd effect, Cd at 100 nM increased thenumber of osteoclasts and the area excavated per osteoclastby approximately twofold and increased the number and areaof pits by approximately threefold (Fig. 4). Toxicity wasobserved in a number of cultures at higher concentrationsof Cd. Osteoblasts appeared to be more sensitive to Cd expo-sure than osteoclasts. With 200 nM Cd, osteoblastic morphol-ogy began to change from wellspread to spindleshaped, andthe cells were often clumped. Cellular debris also becameapparent. The numbers of osteoblasts decreased significantlyat 500 nM Cd. The number of osteoclasts was not affectedat concentrations up to 200 nM Cd. At 500 nM Cd or greater,obvious osteoclast toxicity was often observed, indicated bya condensed morphology and fewer osteoclasts present overtime.

DISCUSSION

Cadmium was first associated with bone disease after en-vironmental exposure in Japan, leading to identification ofItai-Itai disease (Takeuchi, 1978; Nogawa et al., 1981), andafter occupational exposure among cadmium workers inFrance (Nicaud et al., 1942) and Sweden (Friberg, 1950).In these chronic exposure situations, bone disease was con-comitant with kidney damage. Patients were treated withlarge doses of vitamin D with varying degrees of success toovercome the lack of vitamin D activation by the damagedkidney. However, Cd can directly induce bone loss. Underconditions of Cd exposure that did not cause kidney dysfunc-tion, bone loss was demonstrated by decreased bone mineraldensities in mice (Bhattacharyya et al., 1988a,b) and in dogs

FIG. 3. Effect of Cd and 1,25-(OH)2-vitamin D3 on MNOC formation. (Bhattacharyya et al., 1992) and decreased bone strength inCanine bone marrow was cultured as described under Methods. Cells were rats (Ogoshi et al., 1992). Cd-induced bone loss may be dueexposed to Cd (10–1000 nM) or 1,25(OH)2 –vitamin D3 (10 nM) during the to decreased bone formation as measured by a decreasedentire course of the experiment. A representative experiment illustrating

number of active bone formation sites in beagles (Andersonmarrow cultured on plastic substrate (A) and marrow cultured on plasticand Danylchuk, 1978, 1979) or by increased bone resorptionsubstrate in the presence of a bone wafer (B). Cultures were fixed and

stained at the optimal timepoint for MNOC formation in each instance. as measured by 45Ca release from the prelabeled skeletonData are expressed as means { SE (n Å 3–4). Values for Cd treatments (Ando et al., 1978; Bhattacharyya et al., 1988a,b; Sacco-marked by asterisks are significantly different (ANOVA / LSD; *p õ Gibson et al., 1992; Wang and Bhattacharyya, 1993).0.01) from control values. Values for 1,25-(OH)2-vitamin D3 treatment are

When in vivo studies were extended to the in vitro culturesignificantly different (Student’s t test; *p õ 0.001) from control values.of bone cells reported here, MNOC formation and resorp-tive activity were both stimulated at low concentrations(10 –100 nM) in media containing 10– 20% serum. Because(OH)2-vitamin D3 did not increase the number of osteoclasts

in the 3-day culture. Cd rapidly binds to albumin and metallothionein in serum-containing media, the concentrations of free Cd ion wereContinuous Cd exposure at 100 nM increased the number

of osteoclasts in culture (Table 3), probably by fusion of well below 10– 100 nM, indicating that bone cells are highlysensitive to Cd. This sensitivity is confirmed by our obser-immediate preosteoclasts (Fig. 1D) because the time was

insufficient for differentiation from progenitor cells. Cad- vation of toxic responses in the 200– 500 nM concentration



TABLE 1Effect of Cadmium on MNOC Formation and Function in Dog Bone Marrow Cultures a

No. of TRAP-positive 1003 1 pit areaCondition MNOCs on bone No. of pits (mm2)

Day 12Control 9 { 4 (4) 6 { 3 (4) 18 { 13 (4)50 nM Cd 62 { 29* (2)b 70 { 14** (4) 282 { 39** (4)

Day 14Control 80 { 38 (4) 121 { 58 (4) 773 { 423 (4)50 nM Cd 75 { 45 (3) 99 { 59 (3) 725 { 366 (3)

Day 21Control 11 { 6 (3) 91 { 55 (3) 377 { 216 (3)50 nM Cd 8 { 4 (3) 140 { 38 (3) 892 { 302 (3)

a Values are means { SE for the number of bone wafers shown in parentheses. Values marked by asterisk are significantly different (*p õ 0.025 and**p õ 0.002 by one-tailed Student’s t test) from control values at the corresponding time point.

b Cells could be quantitated on only two of the four bone wafers due to microbial growth after fixation. Quantitation of pit formation on these waferswas not affected.

range, expressed as changes in cell morphology or de- Because the remodeling process is very tightly regulated,any bone resorption that occurs is closely followed by bonecreases in osteoblast and/or osteoclast cell number (or

both). Significant decreases in alkaline phosphatase activ- formation, resulting in no net bone loss. Cadmium may beacting to uncouple this process by initially stimulating osteo-ity, an osteoblastic cell marker, were also observed in other

cultures of osteoblast-like cells at Cd concentrations as low clastic activity while concomitantly decreasing osteoblasticactivity. The levels of Cd exposure required to stimulateas 100 nM (Iwami and Moriyama, 1993), while overt toxic-

ity, as measured histologically by a low cell density and osteoclast activity, however, appear to be lower than thoserequired to decrease osteoblast activity (Figs. 3B and 4, Ta-cell debris in intercellular spaces, occurred at 2.7 mM Cd

(Miyahara et al., 1988). Our in vivo results indicate that bles 1– 3; Miyahara et al., 1988, 1991, 1992; Suzuki et al.,1989, 1990; Iwami and Moriyama, 1993), making this mech-Cd begins to cause a release of calcium from bone when

blood Cd concentrations are 2 –5 mg/liter or 20– 50 nM in anism unlikely at low exposure levels. Alternatively, Cd maybe limiting (but not decreasing) the naturally induced bonewhole blood (Sacco-Gibson et al., 1992). Because approxi-

mately 90% of the Cd in whole blood is bound to blood formation response of osteoblasts to signals released duringincreased bone resorption, yielding a net bone loss. Thiscells, in vivo Cd concentrations in serum are probably in

the range of 2– 5 nM, not much lower than 10 nM, a concen- possibility has not been tested directly.A direct effect of Cd on the bone resorbing cells has beentration found to stimulate bone demineralization in serum-

containing cultures (Bhattacharyya et al., 1988b). These difficult to establish. Our dog bone marrow data (Table 1,Fig. 3) support the data of Miyahara and co-workers (1991),results indicate that the bone resorption responses in mar-

row cells and primary osteoclast cultures reported here are where Cd exposure (60– 90 nM) increased MNOC formationin mouse marrow cultures. We were not successful in dupli-highly relevant to in vivo bone responses to Cd.

TABLE 2Effect of 1,25-(OH)2-Vitamin D3 (10 nM) on Osteoclastic Parametersa

1003 1 areaNo. of 1003 1 pit area (mm2) per Ratio of pits

Condition osteoclasts No. of pits (mm2) osteoclast (complex/single)

Experiment 1Control 34 { 3 66 { 19 80.9 { 24.4 2.3 { 0.7 0.67 { 0.061,25-(OH)2-vitamin D3 35 { 3 146 { 26* 460.9 { 115.9** 14.3 { 4.7* 2.52 { 0.50*

Experiment 2Control 23 { 3 78 { 24 105.6 { 35.1 5.7 { 2.2 1.41 { 0.251,25-(OH)2-vitamin D3 22 { 3 295 { 34** 681.3 { 115.0** 34.3 { 7.5* 6.42 { 1.67*

a Isolated osteoclast preparations were cultured for 3 days as described under Methods. Values are means { SE; n Å 3–4 bone wafers. Values markedby asterisks are significantly different (*p õ 0.025 and **p õ 0.01 by one-tailed Student’s t test).


458 WILSON ET AL.

TABLE 3Effect of Cadmium (100 nM) Exposure on Osteoclastic Parametersa

1003 1 areaNo. of 1003 1 pit area (mm2) per Ratio of pits

Condition osteoclasts No. of pits (mm2) osteoclast (complex/single)

Experiment 1Control 34 { 3 66 { 19 81 { 24 2.3 { 0.7 0.67 { 0.06Cd 64 { 7*** 151 { 20** 269 { 35*** 3.3 { 0.3 0.89 { 0.02***

Experiment 2Control 1 { 1 6 { 2 5.9 { 1.0 —b — c

Cd 24 { 9** 29 { 5** 13.0 { 3.0** 1.0 { 0.1 0.17 { 0.05Experiment 3

Control 68 { 12 94 { 12 203 { 11 3.1 { 0.5 1.47 { 0.22Cd 108 { 9* 382 { 66*** 1082 { 190*** 10.2 { 2.1** 1.99 { 0.05*

a Isolated osteoclast preparations were cultured for 3 days as described under Methods. Values are means { SE; n Å 3–4 bone wafers. Values markedby asterisks are significantly different from the control value (*, p õ 0.050; **, p õ 0.025; and ***, p õ 0.010 by one-tailed Student’s t test).

b This ratio is uncertain when the number of osteoclasts is low (Column 1).c This ratio is uncertain when the total number of pits is low (n õ 10; Column 2).

cating the data of Iwami and Moriyama, where Cd (100– portant to the effect of Cd on the formation of MNOCs. In1000 nM) inhibited 1,25-(OH)2-vitamin D3-induced MNOC cultures without bone wafers, Cd did not stimulate MNOCformation in a mouse marrow culture system. Neither of formation (Fig. 3A), although 1,25-(OH)2-vitamin D3 didthese studies included bone wafers to assay the resorptive induce MNOC differentiation. Alternatively, 10– 100 nM Cdactivity of MNOCs formed. Interestingly, we found that the increased the number of MNOCs in cultures that containedpresence of bone wafers in bone marrow cultures was im- bone wafers (Fig. 3B) but in a transient manner (Table 1).

The importance of bone as opposed to plastic as a substratefor osteoclast differentiation and functionality is not unprec-edented. The morphology and biochemical properties of os-teoclasts grown on plastic differ from those grown on bone(Moonga et al., 1990); in other culture systems, the extracel-lular matrix is important for proper cell differentiation andfunction (Lin and Bissel, 1993).

In addition to the transient increase in the number ofMNOCs formed, Cd transiently increased the number andarea of pits excavated by MNOCs that were formed in cul-ture (Table 1). The areas resorbed by the Cd-exposed andcontrol MNOCs were similar by Day 14, but the greaterextent of complexes formed due to Cd exposure suggestedthat Cd may have altered the resorption process of MNOCs.Thus, we have now shown that the concentration of Cdfound to stimulate MNOC formation (50– 100 nM) (Table1; Miyahara et al., 1991) also affects the bone-resorbingactivity of the MNOCs formed in culture. In calvarial bonecultures, Miyahara et al. (1992) and Suzuki et al. (1989)demonstrated that prostaglandin E2 produced by osteoblastsincreased in response to Cd exposure and appeared to playFIG. 4. Effect of Cd on osteoclast number, activity, and pit formation.a role in bone demineralization stimulated by Cd exposure.Osteoclasts were isolated, cultured on bone wafers, and exposed for 3 days

to Cd at 100 nM. Six separate experiments were normalized to the extent Thus, the stimulatory effect on osteoclast activity may beof the Cd effect and the values combined. A Cd/C ratio of 1.0 represents paracrine via osteoblasts.no Cd effect. Individual data points show results from separate experiments. Osteoclast function is more easily studied by isolatingThe means { SE for the six experiments are given below each parameter

bone cells from neonatal long bones, a population enrichedand are plotted on the graph (wide horizontal bar with vertical uncertaintybars). with osteoclasts because the skeleton of neonates is actively



Aloia, J. F., Cohn, S. H., Vaswani, A., Yeh, J. K., Yven, K., and Ellis, K.growing and modeling. Exposure to Cd (100 nM) increased(1985). Risk factors for postmenopausal osteoporosis. Am. J. Med. 78,the number of pits and the extent of pit formation over95–100.

control levels (Table 3; Fig. 4). The numbers of osteoclastsAnderson, C., and Danylchuk, K. D. (1978). Effect of chronic low-level

increased in response to Cd exposure, presumably from fu- cadmium intoxication on the haversian remodeling system in dogs. Calcif.sion of preosteoclasts (Fig. 1D). It is clear from Table 3 Tissue Res. 26, 143 –148.and illustrated in Fig. 4, where individual values for each Anderson, C., and Danylchuk, K. D. (1979). Effect of chronic low-levelexperiment are shown, that the primary osteoclast cultures cadmium intoxication on the haversian remodeling system in dogs: A

reversible phenomenon. Calcif. Tissue Int. 27, 121 –126.varied from one preparation to another. One factor that in-Ando, M., Sayato, Y., and Osawa, T. (1978). Studies on the disposition offluenced the extent of the stimulatory effects of Cd on osteo-

calcium in bones of rats after continuous oral administration of cadmium.clast formation, activation, and activity was the serum used.Toxicol. Appl. Pharmacol. 46, 625–632.Some sera contained unknown factors that diminished the

Athanasou, N. A., Quinn, J., Horton, M. A., and McGee, J. O’D. (1990).effects of Cd, either by maximally stimulating osteoclastNew sites of cellular vitronectin receptor immunoreactivity detected with

activity in control cultures such that the Cd effect was not osteoclast-reacting monoclonal antibodies 13C2 and 23C6. Bone Miner.significant or by failing to support pit formation by osteo- 8, 7–22.clasts whether or not the cultures were exposed to Cd. Baron, R., Ravesloot, J-H., Neff, L., Chakraborty, M., Chatterjee, D., Lomri,

A., and Horne, W. (1993). Cellular and molecular biology of the osteo-Because the numbers of osteoclasts did not increase inclast. In Cellular and Molecular Biology of Bone (M. Noda, Ed.), pp.response to 1,25-(OH)2-vitamin D3 (Table 2), the mechanism445–49. Academic Press, San Diego.for the increased Cd-induced pit formation is not the same

Bhattacharyya, M. H., Whelton, B. D., Peterson, D. P., Carnes, B. A.,as that for the osteotropic hormone. Cadmium also did notMoretti, E. S., Toomey, J. M., and Williams, L. L. (1988a). Skeletal

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rat limb bones in culture. Proc. Natl. Acad. Sci. USA 85, 8761–8765.probably increased the activity of osteoclasts; however,Bhattacharyya, M. H., Sacco-Gibson, N. A., and Peterson, D. P. (1992).whether Cd increased the activation of osteoclasts or if Cd

Cadmium-induced bone loss: Increased susceptibility in female beaglesincreased osteoclast activity cannot be determined fromafter ovariectomy. In Cadmium in the Human Environment (G. F. Nordb-these data. The average activity for each osteoclast can be erg, R. F. M. Herber, and L. Alessio, Eds.), pp. 279–286. International

calculated (Table 3; Fig. 4), but this value is not indicative Agency for Research on Cancer, Lyon.of whether the treatment increased the number of osteoclasts Bloom, W., and Fawcett, D. (1994). Bone. In Textbook of Histology, pp.in each culture that became activated to resorb bone or in- 194–233. Chapman & Hall, New York.creased the activity of just a portion or all of the osteoclasts. Boyde, A., Ali, N. N., and Jones, S. J. (1984). Resorption of dentin by

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Elinder, C-G., Kjellstrom, T., Lind, B., Linnman, L., Piscator, M., andACKNOWLEDGMENTSSundstedt, K. (1983b). Cadmium exposure from smoking cigarettes: Vari-ations with time and country where purchased. Environ. Res. 32, 220–This work was supported in part by National Institutes of Health Grant227.ES04816 and by the U.S. Department of Energy, Office of Health and

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Effects of Cadmium on Osteoclast Formation and Activityin Vitro