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Cd2+ responses of cultured human blood cells
69, 214-224(1983)
TOXICOLOCYANDAPPLIEDPHARMACOLOCY
Cd2+ Responses
of Cultured
Human Blood Cells
M. D. ENGER, C. E. HILDEBRAND, University
of California.
Received
Los Alamos
December
National
Laboratory.
18, 1982: accepted
AND C. C. STEWART Los Alamos,
February
New Mexico
87545
16. 1983
Cd2+ Response of Cultured Human Blood Cells. ENGER, M. D., HILDEBRAND, C. E., AND C. C. (1983). Toxicol. Appl. Pharmacol. 69, 2 14-224. Cd’+ cytotoxicity. uptake. and partitioning, and Cd2+-induced metallothioneine synthesis were studied in cultured peripheral human blood cells. Mononuclear cells were found to resist relatively high levels of Cd2+. Few cells were killed below 50 pM Cd2+. Above this value, survival decreased exponentially with dose. The mean LD50 for mononuclear cells cultured in Cd’+ for 40 hr was 100 pM. Polymorphonuclear cells (granulocytes) were found to be more resistant, with a significantly higher threshold and LD50. and a more complex dose response. Most of the Cd2+ incorporated by blood cells was taken up by nucleated cells. Despite their greater resistance, polymorphonuclear cells incorporated more Cd’+ at higher doses (50 to 150 pM) than did mononuclear cells. No Cd’+ was bound to metallothioneine in polymorphonuclear cells following exposure to Cd2+ for even extended periods of time ( 18 hr) at high doses of IWCd2+ (25 PM). Instead Cd2+ appeared in a Sephadex G-75 peak of approximately 60,000 Da, as well as in the void peak. No significant amount of preexisting metallothioneine (MT) or metallothioneine mRNA was found in the mononuclear cells. However, MT synthesis was induced rapidly following exposure to Cd2+. [‘“Cd”]MT appeared within 1 hr following exposure to 50 pM ‘OsCd2+,and MT synthesis rates measured from [3SS]cysteineincorporation were found to be maximal within 4 hr. STEWART,
Few studies have addressedthe metabolism of Cd2+ in blood cells, the role of blood cells of various kinds in Cd2+ transport, and the effects of Cd’+ on blood cell viability and function. The importance of elucidating such Cd’+ responsesin blood cells is underscored by observations that anemia may accompany other toxic responsesfollowing Cd inhalation by humans or chronic ingestion in animals, and that stimulation or inhibition of immune responsemay occur in animals who have ingested sublethal amounts of Cd” (Piscator, 1976; Jones et al., 1971; Prigge et al., 1977). In addition, the blood is the most likely means of Cd’+ dissemination in the body. The kinetics of Cd2+ appearance in and clearance from the blood following acute exposure or during chronic exposure are complex (Frazier, 1980; Suzuki and Taguchi, 1980; Brem0041-008X/83
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Copyright c I983 by Academic Press.Inc All rtghts of reproduclmn m any form reserved.
214
ner, 1979). A number of differing half-lives are shown for clearance of blood Cd2+ following acute exposure (Welinder et al.. 1977; Bremner, 1979). During chronic exposure, blood levels aswell asblood cell/plasma ratios change markedly both during the tissue Cd’+ accumulation phase and following the appearance of toxicity (Kjellstrom and Nordberg, 1978; Lauwerys et al., 1979: Suzuki and Taguchi, 1980). Little is known of the roles of individual types of blood cells in such processes.Since lifetimes of various blood cell types vary greatly, knowledge of their relative importance in carrying the blood Cd’+ burden could help to better our understanding of particular patterns of blood Cd’+ clearance and plasma/blood cell ratios. Further, Cd2+uptake studies in various blood cell types, when combined with determinations of their
Cd2+ UPTAKE
relative Cd2+ resistance and ability to produce thioneine, will delimit their possible roles in Cd*+ detoxification and/or as targets of Cd2+ toxicity. In particular, the roles of nucleated blood cells versus erythrocytes need further definition. The studies of Hildebrand and Cram (1979) showed the importance of such a definition. They noted that when cultured total blood cells were exposed to 2 X lo-’ M ‘09Cd2+ for 3 days, one-fourth of the Cd2+ total burden was found in mononuclear cells. The remainder was in the red cell fraction of the gradient which also contained the granulocytes. Uptake was at least 800-fold greater in the mononuclear cells than in cells derived from the red cell fraction on a per-cell basis. The red cell and mononuclear cell fractions also differed markedly in their metabolism of Cd*+, as indicated by completely different Sephadex G-75 column chromatography distributions of “‘Cd’+ in lysed cell cytoplasms. In mononuclear cell cytoplasm most Cd2+ eluted with metallothioneine (MT). Little Cd’+ eluted with MT in the lysate from the red cell fraction; most appeared in fractions of higher molecular weight, and a prominent peak that eluted as would a protein of -60,000 Da was observed. These observations thus raise a number of questions which we have addressed by defining Cd2+ uptake, sensitivity, and metabolism in resolved monocytes, lymphocytes, and granulocytes.
METHODS Cell Resolution
and Culture
Blood used for most studies was drawn from healthy adult donors (M.D.E., F.A.E., C.E.H.). All experiments involving comparison of differing cell types were repeated with blood from a single donor (M.D.E.). Variation in mononuclear cell response was determined with blood samples drawn by the Mayo Clinic Department of Laboratory Medicine (Rochester, Minn) from the Mayo Clinic’s “normal value” volunteer healthy donor population. These donors have blood chemistry values that in each instance are within normal ranges. Peripheral blood mononuclear cells were resolved from plasma, red cells,
215
IN BLOOD CELLS
and granulocytes by lymphocyte separation medium (LSM, Litton). Freshly drawn blood (with heparin or citrate-dextrose as anticoagulant) were mixed with an equal volume of sterile saline; 8 to 9 ml was layered on 4 ml LSM in a 15-ml centrifuge tube and centrifuged 40 min at 135Og in an IEC CRU-5000 centrifuge at 20°C. The supematant plasma was drawn off to within 0.5 cm of the LSM layer and was discarded. The LSM layer was drawn off to within 0.5 cm of the red cell pellet, diluted 1 to 5 with Hanks’ balanced salt solution (BSS), and centrifuged to collect the cells. These were washed once in Hanks’ BSS and then cultured at 0.5 to I.0 X lo6 cells/ ml in medium (RPM1 medium 1640, Gibco) with 25 mM HEPES. 10% fetal calf serum, and penicillin and strep tomycin. Monocytes were resolved from lymphocytes by plating mononuclear cell suspensions in the absence of serum (Stewart, 1981). All monocytes and some lymphocytes attach. Nonadherent lymphocytes (NAL) were rinsed off and adherent cells cultured 24 hr in the presence of serum. The initially adherent lymphocytes (ANAL) were then rinsed off, leaving monocytes attached. Granulocytes were prepared as follows. Total blood cells were collected by centrifugation (10 min at 200g). The supernatant plasma was discarded, the top one-third of the cell pellet was removed by aspiration and diluted with four volumes of aMOPs (Gibco olMEM buffered with 4 g/liter MOPS, morpholinopropane sulfonic acid, and containing 100 units/ml penicillin, 100 rg/ml streptomycin). Then 10 ml was layered on 5-ml Ficoll-Hypaque in a conical 15-ml tube and centrifuged at 120g for 10 min at 20-22°C. The plasma layer was removed and discarded. The Ficoll-Hypaque layer was removed to within 0.5 cm of the red cell pellet, and the mononuclear cells therein were washed and cultured in RPM1 (over 99% mononuclear cells in this fraction). The red blood cell pellets were pooled, diluted gradually with 20 volumes of 0.83% NH&l, and incubated 20 min at 37°C to promote red cell lysis. Unlysed cells were collected by centrifugation at I5Og for 10 min at 20-22°C. These were washed by suspension and centrifugation two times with 50 ml aMOPs and one time with RPM1 and suspended in RPM1 at 5 X 10’ cells/ml for survival measurements or at 2 X lo6 cells/ml for Cd2+-uptake measurements. This cell preparation was over 90% granulocytes. Cell cultures were maintained in a water-jacketed CO2 (5%) incubator at 37°C.
Measurement
of Cd”
Cytotoxicity
Aliquots (I ml) of 5 X IO5 cells in RPM1 were added to polypropylene tubes containing 0, 5, 7.5, 10, 12.5, 15, 20, and 25 pl 10-l M Cd’+, lo-“ M HCI. (Controls to which these volumes of lOa M HCI were added showed no cell killing). These were incubated 40 to 42 hr at 37°C. The fraction of cells remaining viable was then deter-
216
ENGER. HILDEBRAND.
mined by the Pronase-Cetrimide procedure of Stewart el al. (1975). To each culture was added 0.25 ml of filtered Pronase solution (12.5 mg/ml, frozen immediately upon dissolution in saline and used within 2 hr of thaw). After 15 min at 37°C the digests were added to 10 ml Cetrimide solution (80 g hexadecyltrimethylammonium bromide, 8.5g NaCl, 370 mg NarEDTA per 1 HrO). Nuclei of surviving cells were then enumerated with a Coulter counter. Cd Uptake and Partitioning Solutions of ‘@‘CdClr of known specific activity were added to cultures of 2 X lo6 cells/ml in complete RPM1 in polypropylene tubes. These were incubated at 37°C for the times prescribed. Incorporation was terminated by the addition of cold Hanks’ BSS and centrifugation. The cells were washed in Hanks’ BSS and frozen as suspensions of lo6 to 10’ cells/ml of buffer A (10 mM TrisCl, pH 7.4 at 20°C 10 mM KCI, 1.5 mM MgClr, 0.2 mM Cleland’s reagent). Upon thaw, cells were lysed by the addition of l/10 volume 10% Nonidet P-40 nonionic detergent (NP-40, Shell). After mixing vigorously, standing I5 min in ice, and again mixing, nuclei were removed by centrifugation. (Ahquots were removed before and after centrifugation to measure incorporation into total cells and into cytoplasm.) The cytoplasm was fractionated by chromatography on Sephadex G-75. Radioactivity in cell and chromatograph fractions was determined by liquid scintillation spectrometry (Enger et al., 1981; Hildebrand
et al., 1982). The ‘?d2+ or [“Slcysteine peak eluting between the void (high molecular weight) and low molecular weight peaks was identified with metallothioneine (MT). The material comigrated with authentic MT (prepared by standard protocols) on Sephadex G-75 and on both native and denaturing electrophoretic gels. It also corn&rated with the cell-free translation product of mRNA identified as metallothioneine mRNA by sequencing cloned complementary DNA (unpublished results). Because six Sephadex columns were employed and flow rates differed, the fraction numbers at which the various peaks eluted varied, but MT migrated in a constant position relative to the void and low molecular weight peaks. Measurement of Metallothioneine
Synthesis
Cells. cultured in RPM1 at 2 X IO6 cells/ml, were exposed to [“Slcysteine (10 &/ml) subsequent to exposure to unlabeled CdCI,. Cells were harvested as above, but frozen in buffer B (10 II’IK KCI, 10 mM Tris-Cl, pH 7.4 at 20°C 1.5 mM MgC12, 100 pM CdClr, 20 mM mercapmethanol). Cells were lysed as above, and cytoplasms were chromatographed on Sephadex G-75 to resolve “S incorporated into MT from that in higher molecular weight proteins.
AND STEWART
RESULTS Cd” Cyto.uicity
Mononuclear cells cultured in Cd” for 40 hr showed a dose response for cytotoxicity as illustrated in Fig. 1A. There was little or no cell killing at Cd*+ concentrations below 50 FM. No cytotoxic response was evident in this dose range (up to 150 FM) after 24-hr exposure. Depending on the individual from whom blood was obtained, survival began to decrease rapidly and exponentially as a function of dose between 50 and 70 PM Cd’+. The points at which such rapid dose response began were obtained by extrapolating to 100% survival a line drawn through the plotted values for survival at 75, 100, and 125 PM Cd*+. Such “threshold” values (for the beginning of the rapid exponential dose response) were obtained simultaneously for mononuclear cells cultured from 15 adult humans (Mayo Clinic normal value population). The range of values obtained was 53 to 66 PM. The mean value was 58.1, SD + 5.3. The LD50 values, or doses at which one-half of the cells were killed in 40 hr. were determined also. These ranged from 83 to 114 PM. The mean was 100.1; SD + 8.6. Figure IA shows the dose response of the first individual in the set of fifteen. The dose response shown by monocytes resembled that of total mononuclear cells (-25% monocytes, 75% lymphocytes). There was also a shoulder in the monocyte doseresponse curve, with an extrapolated threshold value of 49 PM, followed by a rapid, exponential decrease in survival as dose was increased (Fig. 1B). The LD50 was 66 PM. Mononuclear cells cultured in small plates, as were the monocytes, responded with a threshold of 53 PM and LD50 of 89 PM. A repeat determination of the monocyte dose response provided values of 67 PM for threshold and 85 PM for an LD50. The response of adherent lymphocytes was determined also. These are lymphocytes that initially adhere to plates when mononuclear cells are cultured in the absence of serum but
Cd’+ UPTAKE
217
IN BLOOD CELLS
50 t s
IO00
El
IIL a w
El
0
.c :
t a ‘II
0 A 50-
\
20
m Q > tt 0 10
0
z
P I-5 t,
\ 20-
:: k
0
\ IO
1 0
50
‘j
too
pM
150
50
Cd++
100
150
/.L M Cd++
IO 0
I
I
I
50
100
150 pM
I 200 Cd+*
I
/
250
300
350
FIG. I. (A) Dose response of cultured mononuclear cells cultured in Cd’+ 40 hr at 37°C. (B) Dose response of cultured monocytes. (C) Dose response of cultured polymorphonuclear cells.
which can be rinsed off (without detaching monocytes) after a 24-hr culture in the presence of serum. In one experiment these had a threshold value of 56 PM and an LD50 of 93 /.LM. Polymorphonuclear cells (granulocytes) were found to be more resistant to Cd” than were mononuclear cells. As seen in Fig. lC, the point at which survival began to decrease rapidly with dose was significantly higher (-90 PM) for these cells. Cd2+, 128 PM, was
required to kill one half of these cells in 40 hr. There was also a significant fraction, 16%, of the population that resisted concentrations as high as 350 PM Cd2+. ‘09Cdz’ Uptake and Intracellular in Mononuclear Cells
Partitioning
Mononuclear cells exposed to a high subtoxic dose (50 PM) of io9Cd2+ take up this isotope as a function of time as shown in Fig.
218
ENGER, HILDEBRAND.
2. Uptake was not linear with time. The slope of the incorporation curve decreased after 2 hr. The initial rate of incorporation as derived from the data presented in Fig. 2 was approximately 6 pg/109 cells/hr. That derived from a repeat experiment was 5 pg/109 cells/ hr. Nuclear concentrations (Fig. 2) plateaued at 6 pg/109 cells or less even though total incorporation continued to increase. The dose response for uptake was determined at the same time that the kinetic data of Fig. 2 were obtained. These data are shown in Fig. 3. They show that uptake in mononuclear cells is proportional to dose when cells are exposed to ‘09Cd2+ for 8 hr in concentrations ranging from 25 to 150 PM. A different pattern of accumulation was observed when the fraction of “‘Cd2’ bound to MT was followed as a function of time that mononuclear cells were exposed to 50 PM lo9Cd2+. As indicated from the data presented in Fig. 4, accumulation of CdMT began approximately 1 hr after addition of Cd*+ (Fig. 4). Accumulation then continued to increase linearly, in spite of the facts that total accumulation (Fig. 2) and total non-MT cytoplasmic Cd2+ accumulation were less rapid after the first 2 hr of exposure. This differential partitioning early and later during Cd2+ accumulation was reflected also in the Sephadex G-75 chromatography profiles of cytoplasm from cells exposed to 50 PM lo9Cd2+ for 1 and 4 hr (Fig. 5).
AND STEWART 70 TOTAL
6o T 5o g m0 4o \, zz 3o :I/” E 11
0 0
o/o
//,/z /O
00
J?25
/O 50 75 pM Cd” (8h
150
100 125 EXPOSURE)
FIG. 3. 8-hr IWCd2+uptake in cultured human mononuclear cells as a function of dose.
Granulocytes The pattern of uptake and intracellular ‘09Cd2+ partitioning which occurs in granulocytes is distinctly different from that seen in mononuclear cells or in human or rodent fibroblasts. As seen in Fig. 6, uptake was significantly greater in these cells than in mononuclear cells in the range of 25 to 150 PM Cd*+. Uptake rates in these cells, as exposed to 50 FM lo9Cd2+ in two different experiments, ranged from 10 to 30 pg/ lo9 cells/hr. These values were never found to be greater than 6 pg/ lo9 cells/hr in mononuclear cells. Thus, granulocytes incorporate substantially more Cd2+ at relevant (compared to cytotoxic reI
I
I
I
w i 30
rnj2:’
,*/No’
0
0
h HO&
HOURS
EXPOSED
T O 5Dpt.4
‘OgCd++
FIG. 2. Total and nuclear ‘09Cd2+uptake in mononuclear cells as a function of time exposed to 50 pM lwCd2+.
EXPOSED
70 50pM
1
J
Cdef4
FIG. 4. Accumulation of ‘OsCdz+bound to metallothioneine as a function of time that mononuclear cells are exposed to 50 jtM ‘@‘Cd*‘.
Cd2+ UPTAKE
\ 2 g 20009 O1 16002
VOID
MT
4h
l200600-
400 -
00-
IO
20
30
FRACTION
40
50
60
NUMBER
FIG. 5. Partitioning of lWCd2+ from cultured mononuclear ceils exposed to 50 pM ‘osCdZ+ for 1 hr (upper) and 4 hr (lower) among void, MT, and low molecular weight fractions resolved by Sephadex G-75 chromatography.
I
I
I
I
1000 1000 -
I
-025 8
sponses) exposure levels (25 to 150 PM) and are more resistant to the cytotoxic effects of Cd’+ than are mononuclear cells. The dose-response curve for Cd2+ uptake (Fig. 7) by granulocytes shows that uptake increased more rapidly with dose above 50 PM Cd2+. Uptake by granulocytes and mononuclear cells during exposure to a low level of Cd2+ was also determined. After 8.5 hr of exposure to 0.2 PM Cd2+, granulocytes and mononuclear cells each contained approximately 0.3 pg Cd’+ per lo9 cells. This concordance in uptake at low but not at high doses reflects the differing dose responses for uptake (Fig. 3 and 7). The most striking difference between granulocytes and mononuclear cells was seen when their cytoplasms were resolved on Sephadex G-75 following exposure to lo9Cd2+. As seen in Fig. 8, neither the low molecular weight nor the MTlo9Cd2+ peaks were evident in resolved cytoplasms from granulocytes that had
I
q 150pM
4
219
IN BLOOD CELLS
I
I
I
I
I
Cd2
pt.4 12
16
20
HOURS
FIG. 6. Kinetics of ‘09Cdz’ uptake in cultured poiymorphonuclear cells.
[Cd2+l,pLM FIG. 7. Dose response for losCd2+ uptake in cultured polymorphonuclear cells (granulocytes).
ENGER. HILDEBRAND,
AND STEWART
toplasms were resolved by Sephadex chromatography. In cytoplasm from cells induced with 50 PM Cd’+ for 4 hr and exposed to [3SS]cysteine the last 2 hr of induction, more 35S was incorporated into MT than into all proteins of the void peak (Fig. 9A). The total void counts/min were 1802, compared with 1986 in the MT peak. When RNA synthesis was inhibited by 5 /*g/ml actinomycin added 10 min before the cells were induced with Cd2+, most of the incorporation into MT was suppressed (Fig. 9B). There were now only about 27 counts/min that could be attributed to MT, versus 1348 in the void. No detectable MT peak was seen in uninduced cells. Incorporation into the void peak was not increased significantly by induction of MT with 50 PM Cd’+. The response was FRACTION NUMBER found to be comparable in nonadherent lymFIG. 8. Partitioning of ‘@%X2+incorporated into cultured phocytes (NAL) to that of the total monopolymorphonuclear cells (granulocytes) in cytoplasmic nuclear cell population. fractions resolved by Sephadex G-75 chromatography. Hb The dose response for Cd’+-induced MT = hemoglobin. synthesis (measured as [35S]MT) is shown in Table 1. This response initially increased rapbeen exposed to 25 PM lo9Cd2+ for 7 and 18 idly with dose, increased less rapidly between hr. Instead, a prominent peak was evidenced about 10 and 50 PM, and did not increase in which elutes before CdMT but after, and is going from 50 to 100 wh4. resolved from, the void peak. The relative The kinetics of the MT response are shown proportion of this peak increased with time in Fig. 10. As suggested by the kinetics of (cf. 18 versus 7 hr profiles). Radiolabel CdMT appearance, it is rapid and maximal ( lo9Cd2+) eluted with this peak even when suf2 to 4 hr after induction with 50 PM Cd’+. ficient cold Cd2+ was added to the lysis buffer Mononuclear cells from adult male and fe( 100 PM) to compete all ‘09Cd2+ from the void male subjects responded similarly. peak. [“*S]Cy.steine Incorporation
DISCUSSION Cd” Cytotoxicit?
As might be expected from their heterochromatic nature, little [35S]cysteine was incorporated into granulocytes. Also, as expected from the lack of cadmium-binding MT peak, a [35S]MT peak was not evident when cytoplasm from these cells, induced with Cd2+ and exposed to [35S]cysteine, was resolved on Sephadex G-75. In contrast, cadmium-induced, [35S]cysteine-labeled mononuclear cells showed pronounced 35S-labeled MT peaks when their cy-
An important aspect of the cytotoxic response of nucleated blood cells is that all have shoulders, indicative of thresholds for cytotoxicity, in their dose-response curves. Little or no cell killing occurred below 50 PM Cd’+. This observation, combined with the fact that blood levels of Cd’+ were reported not to exceed 3 I.LM even in highly exposed individuals (Lauwerys et al., 1979), suggests that nucleated blood cells in vivo are not a significant
Cd2+ UPTAKE
IN BLOOD
221
CELLS I
I
B
-
4000
-
3000
-
2000
-
1000
I
i
t
t
I)
I
VOID
200
1
I
.; 160\ In
5000
-
4000
-
3000
+
I/
I 50
I
IO FZT103NO
-lODo
n D
66
ID
20
30
FRACTION
N”M4BOER
40
50
68
NUMBER
FIG. 9. (A) [‘?3]Cysteine incorporated into cytoplasmic fractions of mononuclear cells during the second to fourth hours exposed to 50 PM Cd2+. (B) Effect of pretreatment with actinomycin D (10 min, 5 &ml) on [‘%i]cysteine incorporation. Void, MT, and low molecular weight fractions of cytoplasms were resolved with Sephadex G-75 chromatography.
target for the cytotoxic effects of Cd*+. That blood cells may be significant targets in terms of altered function is, however, not obviated. Kiremidjian-Schumacher et al., (198 la,b), for example, found significant Cd*+ effects on the ability of lymphocytes to produce macrophage migration inhibition factor (MIF) at lO-‘j M Cd*+, a concentration which does not affect viability. Monocytes and lymphocytes responded similarly. The monocytes were somewhat more sensitive than lymphocytes, but showed the same type of survival curve. Polymorphonuclear cells, or granulocytes, differed significantly in both their (higher) LD50 values and in the nature of their response at higher doses.
Cd2’ Uptake and Partitioning The initial rate of Cd*+ uptake was found to be 5-6 pg/109 cells/hr when mononuclear cells were exposed to 50 PM Cd*+. Based on this rate, intra- and extracellular concentrations would be approximately equal after a 2hr exposure. The dose response for uptake was found to be linear with dose. Thus, we would expect that at low doses the intracellular concentration would approximate the extracellular after 2 hr of exposure. Whether such a situation would be obtained in vivo cannot be stated, since the type and amount 9 b
TABLE
1
DOSE RESPONSE OF Cd’+-INDUCED Cd’+
(/AM)
6.25 12.5 25 50 100
MT
I
I
I
I
I
9400 ADULT 0 ADULT
MALE
MONONUCLEAR
CELLS
SYNTHESIS
Relative MT synthesis’ 40 45 54 79 80
’ Counts/min [35S]cysteine in MT as percentage of counts/min in void.
2
TIME
4 6 EXPOSED
6 T O 50pM
IO Cd++
12
FIG. 10. Time course of Cd2+-induced MT synthesis in cultured human male and female mononuclear cells as a function of time exposed to 50 pM Cd2+.
222
ENGER,
HILDEBRAND,
of serum present affect cellular uptake markedly (Enger et al., 198 1). The kinetics of CdMT appearance indicated that newly synthesized MT appears in cytoplasm about 1 hr following Cd2+ exposure. The amount of MT that existed prior to induction was not significant relative to the induced levels. Protection ascribable to MT is thus induced-as opposed to constitutivein these cells (when they are challenged within 24 hr of sampling). A different pattern of uptake was seen in granulocytes. Although these cells are more resistant to the cytotoxic effects of extracellular Cd2+, they actually incorporate much more Cd2+ when exposed to high doses (50 to 150 PM). Their relative resistance is not ascribable to reduced uptake, nor can it be ascribed to high constitutive or more rapidly induced MT levels, since there was no CdMT in the cytoplasm of these cells. Instead, much of the intracellular Cd2+ was bound to a ligand which chromatographed on Sephadex G-75 as if slightly smaller than hemoglobin (-60 kDa). The nature of this ligand is a subject for further investigation. Whatever its identity, the absence of MT and the relative resistance of granulocytes are worthy of note, since MT has been present and found to play the primary role in cytoprotection in many other Cd2+-resistant cells examined to date. Its absence in these cells may reflect a more general inability of these cells to respond to agents which induce specific or general protein synthesis in other cells. In view of their highly heterochromatic nature, such lack of response capability would not be surprising. The proportion of Cd2+ bound to the “-60 kDa” peak was found to be greater after 18than after 7-hr exposure to 25 pM Cd’+. To say that this signifies induced synthesis of this protein would, however, be premature. More studies need be performed before such a conclusion is warranted. The dose response for Cd2+ uptake by granulocytes differed from that shown by mononuclear cells in that uptake increased disproportionately with dose. Thus, at high doses
AND
STEWART
uptake was much greater in granulocytes than in the mononuclear cells. At low doses however, such as might be found in the blood of highly exposed humans, uptake was similar in granulocytes and mononuclear cells. Exposed in vitro to 0.2 pM Cd2+, both incorporate about 0.3 pg/109 cells during an 8.5hr exposure. There is a significant difference in cell size, however, so that intracellular concentrations are probably not equal. The percell uptake numbers are nevertheless significant in the context of the amount of Cd2+ incorporated by red cells versus nucleated cells. Hildebrand and Cram (1979) observed that, in blood cell cultures exposed to 0.2 PM Cd’+ for 3 days, about one-fourth of the Cd’+ was found in mononuclear cells, and three-fourths was found in the red cell fraction. This latter fraction, however, also contains the granulocytes. There are in peripheral human blood about three times as many granulocytes as mononuclear cells. Since their Cd2+ uptake is about the same as that of mononuclear cells (on a per-cell basis), most of the incorporation into the red cell fraction must be due to the uptake by granulocytes. Without correcting for such nucleated cell uptake, Hildebrand and Cram calculated that red cells exposed to 0.2 pM Cd2+ for 3 days incorporated only 0.01 pg/ IO9 cells, whereas mononuclear cells incorporated 6 pg per IO9 cells. These observations suggest that red cell Cd2’ incorporation (on either a per-cell or total-population basis) is not significant relative to uptake by other nucleated populations. The exact amount of Cd’+, if any, incorporated by red cells exposed to low levels of Cd2+ thus needs to be determined with red cell populations free of nucleated cells before one can ascribe to them any significant role in Cd2+ metabolism in the blood. Early work also reported that Cd2+-binding ligands similar to those we have found to be present in mononuclear cells (MT) and granulocytes (a protein eluting from Sephadex G75 between the void and hemoglobin) were recovered from lysates of erythrocytes isolated from Cd’+-exposed mice (Nordberg et
Cd2+ UPTAKE
al., 197 1). In these studies, however, nucleated cells were present in the erythrocyte preparation, so that most or all of the Cd2+binding proteins recovered could have been derived from nucleated cells.
[3’S]Cy.steine Incorporation
IN BLOOD CELLS
223
not induce by activating or derepressing the translation of preexisting thioneine mRNA. That AM does not directly block the translation process itself in lymphocytes was reported by Cooper and Braverman ( 1980). The dose response for Cd2+-induced MT synthesis suggests that doses well below the maximum subtoxic dose can elicit a significant MT synthesis response in mononuclear cells. Whether this response suffices to protect these cells against Cd2+-induced alterations in their ability to perform their many varied, specialized functions cannot be deduced from these data. The observations that Cd’+ exposure has deleterious effects on immune function, and the fact that these cells rapidly incorporate Cd2+, indicate that possible direct effects of Cd2+ on the functional capabilities of mononuclear cells should continue to be investigated.
The kinetics of CdMT appearance in mononuclear cells suggested that little MT preexists in these cells prior to exposure to Cd2+ and that subsequent to exposure, MT is synthesized relatively rapidly. These suggestions were enforced by the analyses of [35S]cysteine incorporation into MT following exposure to Cd2+. No detectable amount of 35Swas incorporated into MT in cells not exposed to Cd2+. Following addition of 50 I.~M Cd2+, however, [35S]cysteine incorporation increased rapidly. A maximal incorporation ACKNOWLEDGMENTS (MT synthesis) rate was obtained within 4 hr of Cd induction. These kinetics resemble those We gratefully recognize the technical assistance ofJohn seen in Cd2+ resistant hamster lines (HildeL. Hanners and S. J. Stewart. We thank F. A. Enger for brand et al., 1982). The response was specific many blood samples. We thank Dr. Steve Barham and as well as rapid. No significant increase in 35S the Mayo Clinic/Foundation for the normal value blood samples. This work was supported by the U.S. Departincorporation into other proteins (Sephadex ment of Energy. G-75 void peak) followed Cd’+ induction. The rapid appearance of MT synthesis following induction could be explained by either REFERENCES a rapid activation of the thioneine genes, or by depressed translation of preexisting thiBREMNER, I. (1979). Mammalian absorption, transport oneine mRNA. That some protein synthesis and excretion of cadmium. In The Chemiswy, Biuchemistry and Biology of Cadmium. (M. Webb, ed.), in resting human lymphocytes is regulated at pp. 175-193. Elsevier/North-Holland, Amsterdam. the level of translation was shown by Ferrer COOPER, H. L., AND BRAVERMAN, R. (1980). Protein et al. ( 1980), who reported that enhanced prosynthesis in resting and growth-stimulated human petein synthesis following PHA stimulation of ripheral lymphocytes. Exp. Cell Res. 127, 35 I-359. resting lymphocytes does not require RNA ENGER, M. D., FERZKO, L. T., TOBEY, R. A., AND HILDEBRAND,C. E. (I 98 I). Cadmium resistance correlated synthesis, indicating that preexisting mRNAs with cadmium uptake and thionein binding in CHO are being utilized. Further, they reported the cell variants Cd’2OF4 and Cd’ 3OF9. J. Toxicol. and isolation of a ribosome-bound translational Env. Health 7, 675490. inhibitor in resting lymphocytes. It is for these FERRER, M., BURRONE, 0. R., AND ALGRANATI, I. D. reasons that we measured the effect of acti(1980). Inhibition of polypeptide synthesis by a factor isolated from ribosomes of resting human lymphocytes. nomycin D (AM) on Cd’+-stimulated MT FEBSLetl. 121,203-206. synthesis. The fact that pretreatment with AM FRAZIER,J. M. (1980). Cadmium and zinc kinetics in rat blocked most Cd2+-stimulated MT synthesis plasma following intravenous injection. J. Toxicol. and suggests that the response to Cd’+ occurs at Env. Health 6, 503-5 18. the level of transcription. That is, Cd2+ does HILDEBRAND, C. E., AND CRAM, L. S. (1979). Distri-
224
ENGER. HILDEBRAND,
bution of cadmium in human blood cells cultured in low levels of CdC&: Accumulation of Cd in lymphocytesand preferential binding to metallothionein. Proc. Sot. for Exp. Biol.
and Med.
161, 438-443.
HILDELIRAND,C. E., GRIFFITH, J. K., TOBEY, R. A., AND WALTERS, R. A. (1982). Molecular mechanisms of Cd detoxification in Cd-resistant cultured cells: Role of metallothionein and other inducible factors. In The Biological Role of Metallothionein (E. C. Foulkes. ed.), pp. 279-303. Elsevier/North-Holland, Amsterdam. JONES,R. H., WILLIAMS, R. L.. AND JONES.A. M. (197 1). Effects of heavy metal on the immune response. Preliminary findings for cadmium in rats. Proc. Sot. Exp. Biol. Med. 137, 1231-1236. KIREMIDJIAN-SCHUMACHER, L., STOTZKY, G.. DICKSTEIN, R. A., AND SCHWARTZ,J. (198la). Influence of cadmium, lead, and zinc on the ability of guinea pig macrophages to interact with macrophage migration inhibitory factor. Environ. Res. 24, 106-I 16. KIREMIDJIAN-SCHUMACHER. L., STOTZKY, G., LIKHITE, V., SCHWARTZ,J., AND DICKSTEIN, R. A. (198lb). Influence of cadmium, lead, zinc on the ability of sensitized guinea pig lymphocytes to interact with specific antigen and to produce lymphokine. Environ. Res. 24, 96-105. KJELLSTROM, T.. AND NORDBERG, G. F. (1978). A kinetic model of cadmium metabolism in the human being. Environ. Res. 16, 248-269. LAUWERYS, R., ROELS, H.. REGNIERS, M., BUCHET.
AND STEWART J. P., BERNARD, A., AND GORET, A. (1979). Significance of cadmium concentration in blood and in urine in workers exposed to cadmium. Environ. Res. 20, 375391. NORDBERG. G. F., PISCATOR, M., AND NORDBERG. M. ( 197 1). On the distribution of cadmium in blood. Acfa Pharmacol. Toxicol. 30, 189-295. PISCATOR,M. (1976). The chronic toxicity of cadmium. In Trace Elements in Human Health and Disease II, (A. S. Prasad and D. Oberlease, eds.). pp. 432-441, Academic Press, New York. PRIGGE, E., BAUMERT, H. P., AND MUHLE, H. (1977). Effects of dietary and inhalative cadmium on hemoglobin and hematocrit in rats. Bull. of Environ. Contam. Toxicol.
585-590.
STEWART, C. C., CRAMER, S. F.. AND STEWARD, P. G. ( 1975). The response of human peripheral blood lymphocytes to phytohemagglutinin: Determination of cell number. Cell. Immund 16, 237-250. STEWART, C. C. (1981). Obtaining and culturing murine monocytes. In Methods for Studying Mononuclear Phagocyfes. (D. 0. Adams, ed.), pp. 2 l-32, Academic Press, New York. SUZUKI, S., AND TAGUCHI, T. (1980). Retention, organ distribution, and excretory pattern of cadmium orally administered in a single dose to two monkeys. J. Toxicol. Environ.
Health
6, 783-796.
WELINDER, H., SKERFVING, S.. AND HENRIKEN. 0. ( 1977). Cadmium metabolism in man. Brit. J. Ind. Med. 34,221-228.
TOXICOLOCYANDAPPLIEDPHARMACOLOCY
Cd2+ Responses
of Cultured
Human Blood Cells
M. D. ENGER, C. E. HILDEBRAND, University
of California.
Received
Los Alamos
December
National
Laboratory.
18, 1982: accepted
AND C. C. STEWART Los Alamos,
February
New Mexico
87545
16. 1983
Cd2+ Response of Cultured Human Blood Cells. ENGER, M. D., HILDEBRAND, C. E., AND C. C. (1983). Toxicol. Appl. Pharmacol. 69, 2 14-224. Cd’+ cytotoxicity. uptake. and partitioning, and Cd2+-induced metallothioneine synthesis were studied in cultured peripheral human blood cells. Mononuclear cells were found to resist relatively high levels of Cd2+. Few cells were killed below 50 pM Cd2+. Above this value, survival decreased exponentially with dose. The mean LD50 for mononuclear cells cultured in Cd’+ for 40 hr was 100 pM. Polymorphonuclear cells (granulocytes) were found to be more resistant, with a significantly higher threshold and LD50. and a more complex dose response. Most of the Cd2+ incorporated by blood cells was taken up by nucleated cells. Despite their greater resistance, polymorphonuclear cells incorporated more Cd’+ at higher doses (50 to 150 pM) than did mononuclear cells. No Cd’+ was bound to metallothioneine in polymorphonuclear cells following exposure to Cd2+ for even extended periods of time ( 18 hr) at high doses of IWCd2+ (25 PM). Instead Cd2+ appeared in a Sephadex G-75 peak of approximately 60,000 Da, as well as in the void peak. No significant amount of preexisting metallothioneine (MT) or metallothioneine mRNA was found in the mononuclear cells. However, MT synthesis was induced rapidly following exposure to Cd2+. [‘“Cd”]MT appeared within 1 hr following exposure to 50 pM ‘OsCd2+,and MT synthesis rates measured from [3SS]cysteineincorporation were found to be maximal within 4 hr. STEWART,
Few studies have addressedthe metabolism of Cd2+ in blood cells, the role of blood cells of various kinds in Cd2+ transport, and the effects of Cd’+ on blood cell viability and function. The importance of elucidating such Cd’+ responsesin blood cells is underscored by observations that anemia may accompany other toxic responsesfollowing Cd inhalation by humans or chronic ingestion in animals, and that stimulation or inhibition of immune responsemay occur in animals who have ingested sublethal amounts of Cd” (Piscator, 1976; Jones et al., 1971; Prigge et al., 1977). In addition, the blood is the most likely means of Cd’+ dissemination in the body. The kinetics of Cd2+ appearance in and clearance from the blood following acute exposure or during chronic exposure are complex (Frazier, 1980; Suzuki and Taguchi, 1980; Brem0041-008X/83
$3.00
Copyright c I983 by Academic Press.Inc All rtghts of reproduclmn m any form reserved.
214
ner, 1979). A number of differing half-lives are shown for clearance of blood Cd2+ following acute exposure (Welinder et al.. 1977; Bremner, 1979). During chronic exposure, blood levels aswell asblood cell/plasma ratios change markedly both during the tissue Cd’+ accumulation phase and following the appearance of toxicity (Kjellstrom and Nordberg, 1978; Lauwerys et al., 1979: Suzuki and Taguchi, 1980). Little is known of the roles of individual types of blood cells in such processes.Since lifetimes of various blood cell types vary greatly, knowledge of their relative importance in carrying the blood Cd’+ burden could help to better our understanding of particular patterns of blood Cd’+ clearance and plasma/blood cell ratios. Further, Cd2+uptake studies in various blood cell types, when combined with determinations of their
Cd2+ UPTAKE
relative Cd2+ resistance and ability to produce thioneine, will delimit their possible roles in Cd*+ detoxification and/or as targets of Cd2+ toxicity. In particular, the roles of nucleated blood cells versus erythrocytes need further definition. The studies of Hildebrand and Cram (1979) showed the importance of such a definition. They noted that when cultured total blood cells were exposed to 2 X lo-’ M ‘09Cd2+ for 3 days, one-fourth of the Cd2+ total burden was found in mononuclear cells. The remainder was in the red cell fraction of the gradient which also contained the granulocytes. Uptake was at least 800-fold greater in the mononuclear cells than in cells derived from the red cell fraction on a per-cell basis. The red cell and mononuclear cell fractions also differed markedly in their metabolism of Cd*+, as indicated by completely different Sephadex G-75 column chromatography distributions of “‘Cd’+ in lysed cell cytoplasms. In mononuclear cell cytoplasm most Cd2+ eluted with metallothioneine (MT). Little Cd’+ eluted with MT in the lysate from the red cell fraction; most appeared in fractions of higher molecular weight, and a prominent peak that eluted as would a protein of -60,000 Da was observed. These observations thus raise a number of questions which we have addressed by defining Cd2+ uptake, sensitivity, and metabolism in resolved monocytes, lymphocytes, and granulocytes.
METHODS Cell Resolution
and Culture
Blood used for most studies was drawn from healthy adult donors (M.D.E., F.A.E., C.E.H.). All experiments involving comparison of differing cell types were repeated with blood from a single donor (M.D.E.). Variation in mononuclear cell response was determined with blood samples drawn by the Mayo Clinic Department of Laboratory Medicine (Rochester, Minn) from the Mayo Clinic’s “normal value” volunteer healthy donor population. These donors have blood chemistry values that in each instance are within normal ranges. Peripheral blood mononuclear cells were resolved from plasma, red cells,
215
IN BLOOD CELLS
and granulocytes by lymphocyte separation medium (LSM, Litton). Freshly drawn blood (with heparin or citrate-dextrose as anticoagulant) were mixed with an equal volume of sterile saline; 8 to 9 ml was layered on 4 ml LSM in a 15-ml centrifuge tube and centrifuged 40 min at 135Og in an IEC CRU-5000 centrifuge at 20°C. The supematant plasma was drawn off to within 0.5 cm of the LSM layer and was discarded. The LSM layer was drawn off to within 0.5 cm of the red cell pellet, diluted 1 to 5 with Hanks’ balanced salt solution (BSS), and centrifuged to collect the cells. These were washed once in Hanks’ BSS and then cultured at 0.5 to I.0 X lo6 cells/ ml in medium (RPM1 medium 1640, Gibco) with 25 mM HEPES. 10% fetal calf serum, and penicillin and strep tomycin. Monocytes were resolved from lymphocytes by plating mononuclear cell suspensions in the absence of serum (Stewart, 1981). All monocytes and some lymphocytes attach. Nonadherent lymphocytes (NAL) were rinsed off and adherent cells cultured 24 hr in the presence of serum. The initially adherent lymphocytes (ANAL) were then rinsed off, leaving monocytes attached. Granulocytes were prepared as follows. Total blood cells were collected by centrifugation (10 min at 200g). The supernatant plasma was discarded, the top one-third of the cell pellet was removed by aspiration and diluted with four volumes of aMOPs (Gibco olMEM buffered with 4 g/liter MOPS, morpholinopropane sulfonic acid, and containing 100 units/ml penicillin, 100 rg/ml streptomycin). Then 10 ml was layered on 5-ml Ficoll-Hypaque in a conical 15-ml tube and centrifuged at 120g for 10 min at 20-22°C. The plasma layer was removed and discarded. The Ficoll-Hypaque layer was removed to within 0.5 cm of the red cell pellet, and the mononuclear cells therein were washed and cultured in RPM1 (over 99% mononuclear cells in this fraction). The red blood cell pellets were pooled, diluted gradually with 20 volumes of 0.83% NH&l, and incubated 20 min at 37°C to promote red cell lysis. Unlysed cells were collected by centrifugation at I5Og for 10 min at 20-22°C. These were washed by suspension and centrifugation two times with 50 ml aMOPs and one time with RPM1 and suspended in RPM1 at 5 X 10’ cells/ml for survival measurements or at 2 X lo6 cells/ml for Cd2+-uptake measurements. This cell preparation was over 90% granulocytes. Cell cultures were maintained in a water-jacketed CO2 (5%) incubator at 37°C.
Measurement
of Cd”
Cytotoxicity
Aliquots (I ml) of 5 X IO5 cells in RPM1 were added to polypropylene tubes containing 0, 5, 7.5, 10, 12.5, 15, 20, and 25 pl 10-l M Cd’+, lo-“ M HCI. (Controls to which these volumes of lOa M HCI were added showed no cell killing). These were incubated 40 to 42 hr at 37°C. The fraction of cells remaining viable was then deter-
216
ENGER. HILDEBRAND.
mined by the Pronase-Cetrimide procedure of Stewart el al. (1975). To each culture was added 0.25 ml of filtered Pronase solution (12.5 mg/ml, frozen immediately upon dissolution in saline and used within 2 hr of thaw). After 15 min at 37°C the digests were added to 10 ml Cetrimide solution (80 g hexadecyltrimethylammonium bromide, 8.5g NaCl, 370 mg NarEDTA per 1 HrO). Nuclei of surviving cells were then enumerated with a Coulter counter. Cd Uptake and Partitioning Solutions of ‘@‘CdClr of known specific activity were added to cultures of 2 X lo6 cells/ml in complete RPM1 in polypropylene tubes. These were incubated at 37°C for the times prescribed. Incorporation was terminated by the addition of cold Hanks’ BSS and centrifugation. The cells were washed in Hanks’ BSS and frozen as suspensions of lo6 to 10’ cells/ml of buffer A (10 mM TrisCl, pH 7.4 at 20°C 10 mM KCI, 1.5 mM MgClr, 0.2 mM Cleland’s reagent). Upon thaw, cells were lysed by the addition of l/10 volume 10% Nonidet P-40 nonionic detergent (NP-40, Shell). After mixing vigorously, standing I5 min in ice, and again mixing, nuclei were removed by centrifugation. (Ahquots were removed before and after centrifugation to measure incorporation into total cells and into cytoplasm.) The cytoplasm was fractionated by chromatography on Sephadex G-75. Radioactivity in cell and chromatograph fractions was determined by liquid scintillation spectrometry (Enger et al., 1981; Hildebrand
et al., 1982). The ‘?d2+ or [“Slcysteine peak eluting between the void (high molecular weight) and low molecular weight peaks was identified with metallothioneine (MT). The material comigrated with authentic MT (prepared by standard protocols) on Sephadex G-75 and on both native and denaturing electrophoretic gels. It also corn&rated with the cell-free translation product of mRNA identified as metallothioneine mRNA by sequencing cloned complementary DNA (unpublished results). Because six Sephadex columns were employed and flow rates differed, the fraction numbers at which the various peaks eluted varied, but MT migrated in a constant position relative to the void and low molecular weight peaks. Measurement of Metallothioneine
Synthesis
Cells. cultured in RPM1 at 2 X IO6 cells/ml, were exposed to [“Slcysteine (10 &/ml) subsequent to exposure to unlabeled CdCI,. Cells were harvested as above, but frozen in buffer B (10 II’IK KCI, 10 mM Tris-Cl, pH 7.4 at 20°C 1.5 mM MgC12, 100 pM CdClr, 20 mM mercapmethanol). Cells were lysed as above, and cytoplasms were chromatographed on Sephadex G-75 to resolve “S incorporated into MT from that in higher molecular weight proteins.
AND STEWART
RESULTS Cd” Cyto.uicity
Mononuclear cells cultured in Cd” for 40 hr showed a dose response for cytotoxicity as illustrated in Fig. 1A. There was little or no cell killing at Cd*+ concentrations below 50 FM. No cytotoxic response was evident in this dose range (up to 150 FM) after 24-hr exposure. Depending on the individual from whom blood was obtained, survival began to decrease rapidly and exponentially as a function of dose between 50 and 70 PM Cd’+. The points at which such rapid dose response began were obtained by extrapolating to 100% survival a line drawn through the plotted values for survival at 75, 100, and 125 PM Cd*+. Such “threshold” values (for the beginning of the rapid exponential dose response) were obtained simultaneously for mononuclear cells cultured from 15 adult humans (Mayo Clinic normal value population). The range of values obtained was 53 to 66 PM. The mean value was 58.1, SD + 5.3. The LD50 values, or doses at which one-half of the cells were killed in 40 hr. were determined also. These ranged from 83 to 114 PM. The mean was 100.1; SD + 8.6. Figure IA shows the dose response of the first individual in the set of fifteen. The dose response shown by monocytes resembled that of total mononuclear cells (-25% monocytes, 75% lymphocytes). There was also a shoulder in the monocyte doseresponse curve, with an extrapolated threshold value of 49 PM, followed by a rapid, exponential decrease in survival as dose was increased (Fig. 1B). The LD50 was 66 PM. Mononuclear cells cultured in small plates, as were the monocytes, responded with a threshold of 53 PM and LD50 of 89 PM. A repeat determination of the monocyte dose response provided values of 67 PM for threshold and 85 PM for an LD50. The response of adherent lymphocytes was determined also. These are lymphocytes that initially adhere to plates when mononuclear cells are cultured in the absence of serum but
Cd’+ UPTAKE
217
IN BLOOD CELLS
50 t s
IO00
El
IIL a w
El
0
.c :
t a ‘II
0 A 50-
\
20
m Q > tt 0 10
0
z
P I-5 t,
\ 20-
:: k
0
\ IO
1 0
50
‘j
too
pM
150
50
Cd++
100
150
/.L M Cd++
IO 0
I
I
I
50
100
150 pM
I 200 Cd+*
I
/
250
300
350
FIG. I. (A) Dose response of cultured mononuclear cells cultured in Cd’+ 40 hr at 37°C. (B) Dose response of cultured monocytes. (C) Dose response of cultured polymorphonuclear cells.
which can be rinsed off (without detaching monocytes) after a 24-hr culture in the presence of serum. In one experiment these had a threshold value of 56 PM and an LD50 of 93 /.LM. Polymorphonuclear cells (granulocytes) were found to be more resistant to Cd” than were mononuclear cells. As seen in Fig. lC, the point at which survival began to decrease rapidly with dose was significantly higher (-90 PM) for these cells. Cd2+, 128 PM, was
required to kill one half of these cells in 40 hr. There was also a significant fraction, 16%, of the population that resisted concentrations as high as 350 PM Cd2+. ‘09Cdz’ Uptake and Intracellular in Mononuclear Cells
Partitioning
Mononuclear cells exposed to a high subtoxic dose (50 PM) of io9Cd2+ take up this isotope as a function of time as shown in Fig.
218
ENGER, HILDEBRAND.
2. Uptake was not linear with time. The slope of the incorporation curve decreased after 2 hr. The initial rate of incorporation as derived from the data presented in Fig. 2 was approximately 6 pg/109 cells/hr. That derived from a repeat experiment was 5 pg/109 cells/ hr. Nuclear concentrations (Fig. 2) plateaued at 6 pg/109 cells or less even though total incorporation continued to increase. The dose response for uptake was determined at the same time that the kinetic data of Fig. 2 were obtained. These data are shown in Fig. 3. They show that uptake in mononuclear cells is proportional to dose when cells are exposed to ‘09Cd2+ for 8 hr in concentrations ranging from 25 to 150 PM. A different pattern of accumulation was observed when the fraction of “‘Cd2’ bound to MT was followed as a function of time that mononuclear cells were exposed to 50 PM lo9Cd2+. As indicated from the data presented in Fig. 4, accumulation of CdMT began approximately 1 hr after addition of Cd*+ (Fig. 4). Accumulation then continued to increase linearly, in spite of the facts that total accumulation (Fig. 2) and total non-MT cytoplasmic Cd2+ accumulation were less rapid after the first 2 hr of exposure. This differential partitioning early and later during Cd2+ accumulation was reflected also in the Sephadex G-75 chromatography profiles of cytoplasm from cells exposed to 50 PM lo9Cd2+ for 1 and 4 hr (Fig. 5).
AND STEWART 70 TOTAL
6o T 5o g m0 4o \, zz 3o :I/” E 11
0 0
o/o
//,/z /O
00
J?25
/O 50 75 pM Cd” (8h
150
100 125 EXPOSURE)
FIG. 3. 8-hr IWCd2+uptake in cultured human mononuclear cells as a function of dose.
Granulocytes The pattern of uptake and intracellular ‘09Cd2+ partitioning which occurs in granulocytes is distinctly different from that seen in mononuclear cells or in human or rodent fibroblasts. As seen in Fig. 6, uptake was significantly greater in these cells than in mononuclear cells in the range of 25 to 150 PM Cd*+. Uptake rates in these cells, as exposed to 50 FM lo9Cd2+ in two different experiments, ranged from 10 to 30 pg/ lo9 cells/hr. These values were never found to be greater than 6 pg/ lo9 cells/hr in mononuclear cells. Thus, granulocytes incorporate substantially more Cd2+ at relevant (compared to cytotoxic reI
I
I
I
w i 30
rnj2:’
,*/No’
0
0
h HO&
HOURS
EXPOSED
T O 5Dpt.4
‘OgCd++
FIG. 2. Total and nuclear ‘09Cd2+uptake in mononuclear cells as a function of time exposed to 50 pM lwCd2+.
EXPOSED
70 50pM
1
J
Cdef4
FIG. 4. Accumulation of ‘OsCdz+bound to metallothioneine as a function of time that mononuclear cells are exposed to 50 jtM ‘@‘Cd*‘.
Cd2+ UPTAKE
\ 2 g 20009 O1 16002
VOID
MT
4h
l200600-
400 -
00-
IO
20
30
FRACTION
40
50
60
NUMBER
FIG. 5. Partitioning of lWCd2+ from cultured mononuclear ceils exposed to 50 pM ‘osCdZ+ for 1 hr (upper) and 4 hr (lower) among void, MT, and low molecular weight fractions resolved by Sephadex G-75 chromatography.
I
I
I
I
1000 1000 -
I
-025 8
sponses) exposure levels (25 to 150 PM) and are more resistant to the cytotoxic effects of Cd’+ than are mononuclear cells. The dose-response curve for Cd2+ uptake (Fig. 7) by granulocytes shows that uptake increased more rapidly with dose above 50 PM Cd2+. Uptake by granulocytes and mononuclear cells during exposure to a low level of Cd2+ was also determined. After 8.5 hr of exposure to 0.2 PM Cd2+, granulocytes and mononuclear cells each contained approximately 0.3 pg Cd’+ per lo9 cells. This concordance in uptake at low but not at high doses reflects the differing dose responses for uptake (Fig. 3 and 7). The most striking difference between granulocytes and mononuclear cells was seen when their cytoplasms were resolved on Sephadex G-75 following exposure to lo9Cd2+. As seen in Fig. 8, neither the low molecular weight nor the MTlo9Cd2+ peaks were evident in resolved cytoplasms from granulocytes that had
I
q 150pM
4
219
IN BLOOD CELLS
I
I
I
I
I
Cd2
pt.4 12
16
20
HOURS
FIG. 6. Kinetics of ‘09Cdz’ uptake in cultured poiymorphonuclear cells.
[Cd2+l,pLM FIG. 7. Dose response for losCd2+ uptake in cultured polymorphonuclear cells (granulocytes).
ENGER. HILDEBRAND,
AND STEWART
toplasms were resolved by Sephadex chromatography. In cytoplasm from cells induced with 50 PM Cd’+ for 4 hr and exposed to [3SS]cysteine the last 2 hr of induction, more 35S was incorporated into MT than into all proteins of the void peak (Fig. 9A). The total void counts/min were 1802, compared with 1986 in the MT peak. When RNA synthesis was inhibited by 5 /*g/ml actinomycin added 10 min before the cells were induced with Cd2+, most of the incorporation into MT was suppressed (Fig. 9B). There were now only about 27 counts/min that could be attributed to MT, versus 1348 in the void. No detectable MT peak was seen in uninduced cells. Incorporation into the void peak was not increased significantly by induction of MT with 50 PM Cd’+. The response was FRACTION NUMBER found to be comparable in nonadherent lymFIG. 8. Partitioning of ‘@%X2+incorporated into cultured phocytes (NAL) to that of the total monopolymorphonuclear cells (granulocytes) in cytoplasmic nuclear cell population. fractions resolved by Sephadex G-75 chromatography. Hb The dose response for Cd’+-induced MT = hemoglobin. synthesis (measured as [35S]MT) is shown in Table 1. This response initially increased rapbeen exposed to 25 PM lo9Cd2+ for 7 and 18 idly with dose, increased less rapidly between hr. Instead, a prominent peak was evidenced about 10 and 50 PM, and did not increase in which elutes before CdMT but after, and is going from 50 to 100 wh4. resolved from, the void peak. The relative The kinetics of the MT response are shown proportion of this peak increased with time in Fig. 10. As suggested by the kinetics of (cf. 18 versus 7 hr profiles). Radiolabel CdMT appearance, it is rapid and maximal ( lo9Cd2+) eluted with this peak even when suf2 to 4 hr after induction with 50 PM Cd’+. ficient cold Cd2+ was added to the lysis buffer Mononuclear cells from adult male and fe( 100 PM) to compete all ‘09Cd2+ from the void male subjects responded similarly. peak. [“*S]Cy.steine Incorporation
DISCUSSION Cd” Cytotoxicit?
As might be expected from their heterochromatic nature, little [35S]cysteine was incorporated into granulocytes. Also, as expected from the lack of cadmium-binding MT peak, a [35S]MT peak was not evident when cytoplasm from these cells, induced with Cd2+ and exposed to [35S]cysteine, was resolved on Sephadex G-75. In contrast, cadmium-induced, [35S]cysteine-labeled mononuclear cells showed pronounced 35S-labeled MT peaks when their cy-
An important aspect of the cytotoxic response of nucleated blood cells is that all have shoulders, indicative of thresholds for cytotoxicity, in their dose-response curves. Little or no cell killing occurred below 50 PM Cd’+. This observation, combined with the fact that blood levels of Cd’+ were reported not to exceed 3 I.LM even in highly exposed individuals (Lauwerys et al., 1979), suggests that nucleated blood cells in vivo are not a significant
Cd2+ UPTAKE
IN BLOOD
221
CELLS I
I
B
-
4000
-
3000
-
2000
-
1000
I
i
t
t
I)
I
VOID
200
1
I
.; 160\ In
5000
-
4000
-
3000
+
I/
I 50
I
IO FZT103NO
-lODo
n D
66
ID
20
30
FRACTION
N”M4BOER
40
50
68
NUMBER
FIG. 9. (A) [‘?3]Cysteine incorporated into cytoplasmic fractions of mononuclear cells during the second to fourth hours exposed to 50 PM Cd2+. (B) Effect of pretreatment with actinomycin D (10 min, 5 &ml) on [‘%i]cysteine incorporation. Void, MT, and low molecular weight fractions of cytoplasms were resolved with Sephadex G-75 chromatography.
target for the cytotoxic effects of Cd*+. That blood cells may be significant targets in terms of altered function is, however, not obviated. Kiremidjian-Schumacher et al., (198 la,b), for example, found significant Cd*+ effects on the ability of lymphocytes to produce macrophage migration inhibition factor (MIF) at lO-‘j M Cd*+, a concentration which does not affect viability. Monocytes and lymphocytes responded similarly. The monocytes were somewhat more sensitive than lymphocytes, but showed the same type of survival curve. Polymorphonuclear cells, or granulocytes, differed significantly in both their (higher) LD50 values and in the nature of their response at higher doses.
Cd2’ Uptake and Partitioning The initial rate of Cd*+ uptake was found to be 5-6 pg/109 cells/hr when mononuclear cells were exposed to 50 PM Cd*+. Based on this rate, intra- and extracellular concentrations would be approximately equal after a 2hr exposure. The dose response for uptake was found to be linear with dose. Thus, we would expect that at low doses the intracellular concentration would approximate the extracellular after 2 hr of exposure. Whether such a situation would be obtained in vivo cannot be stated, since the type and amount 9 b
TABLE
1
DOSE RESPONSE OF Cd’+-INDUCED Cd’+
(/AM)
6.25 12.5 25 50 100
MT
I
I
I
I
I
9400 ADULT 0 ADULT
MALE
MONONUCLEAR
CELLS
SYNTHESIS
Relative MT synthesis’ 40 45 54 79 80
’ Counts/min [35S]cysteine in MT as percentage of counts/min in void.
2
TIME
4 6 EXPOSED
6 T O 50pM
IO Cd++
12
FIG. 10. Time course of Cd2+-induced MT synthesis in cultured human male and female mononuclear cells as a function of time exposed to 50 pM Cd2+.
222
ENGER,
HILDEBRAND,
of serum present affect cellular uptake markedly (Enger et al., 198 1). The kinetics of CdMT appearance indicated that newly synthesized MT appears in cytoplasm about 1 hr following Cd2+ exposure. The amount of MT that existed prior to induction was not significant relative to the induced levels. Protection ascribable to MT is thus induced-as opposed to constitutivein these cells (when they are challenged within 24 hr of sampling). A different pattern of uptake was seen in granulocytes. Although these cells are more resistant to the cytotoxic effects of extracellular Cd2+, they actually incorporate much more Cd2+ when exposed to high doses (50 to 150 PM). Their relative resistance is not ascribable to reduced uptake, nor can it be ascribed to high constitutive or more rapidly induced MT levels, since there was no CdMT in the cytoplasm of these cells. Instead, much of the intracellular Cd2+ was bound to a ligand which chromatographed on Sephadex G-75 as if slightly smaller than hemoglobin (-60 kDa). The nature of this ligand is a subject for further investigation. Whatever its identity, the absence of MT and the relative resistance of granulocytes are worthy of note, since MT has been present and found to play the primary role in cytoprotection in many other Cd2+-resistant cells examined to date. Its absence in these cells may reflect a more general inability of these cells to respond to agents which induce specific or general protein synthesis in other cells. In view of their highly heterochromatic nature, such lack of response capability would not be surprising. The proportion of Cd2+ bound to the “-60 kDa” peak was found to be greater after 18than after 7-hr exposure to 25 pM Cd’+. To say that this signifies induced synthesis of this protein would, however, be premature. More studies need be performed before such a conclusion is warranted. The dose response for Cd2+ uptake by granulocytes differed from that shown by mononuclear cells in that uptake increased disproportionately with dose. Thus, at high doses
AND
STEWART
uptake was much greater in granulocytes than in the mononuclear cells. At low doses however, such as might be found in the blood of highly exposed humans, uptake was similar in granulocytes and mononuclear cells. Exposed in vitro to 0.2 pM Cd2+, both incorporate about 0.3 pg/109 cells during an 8.5hr exposure. There is a significant difference in cell size, however, so that intracellular concentrations are probably not equal. The percell uptake numbers are nevertheless significant in the context of the amount of Cd2+ incorporated by red cells versus nucleated cells. Hildebrand and Cram (1979) observed that, in blood cell cultures exposed to 0.2 PM Cd’+ for 3 days, about one-fourth of the Cd’+ was found in mononuclear cells, and three-fourths was found in the red cell fraction. This latter fraction, however, also contains the granulocytes. There are in peripheral human blood about three times as many granulocytes as mononuclear cells. Since their Cd2+ uptake is about the same as that of mononuclear cells (on a per-cell basis), most of the incorporation into the red cell fraction must be due to the uptake by granulocytes. Without correcting for such nucleated cell uptake, Hildebrand and Cram calculated that red cells exposed to 0.2 pM Cd2+ for 3 days incorporated only 0.01 pg/ IO9 cells, whereas mononuclear cells incorporated 6 pg per IO9 cells. These observations suggest that red cell Cd2’ incorporation (on either a per-cell or total-population basis) is not significant relative to uptake by other nucleated populations. The exact amount of Cd’+, if any, incorporated by red cells exposed to low levels of Cd2+ thus needs to be determined with red cell populations free of nucleated cells before one can ascribe to them any significant role in Cd2+ metabolism in the blood. Early work also reported that Cd2+-binding ligands similar to those we have found to be present in mononuclear cells (MT) and granulocytes (a protein eluting from Sephadex G75 between the void and hemoglobin) were recovered from lysates of erythrocytes isolated from Cd’+-exposed mice (Nordberg et
Cd2+ UPTAKE
al., 197 1). In these studies, however, nucleated cells were present in the erythrocyte preparation, so that most or all of the Cd2+binding proteins recovered could have been derived from nucleated cells.
[3’S]Cy.steine Incorporation
IN BLOOD CELLS
223
not induce by activating or derepressing the translation of preexisting thioneine mRNA. That AM does not directly block the translation process itself in lymphocytes was reported by Cooper and Braverman ( 1980). The dose response for Cd2+-induced MT synthesis suggests that doses well below the maximum subtoxic dose can elicit a significant MT synthesis response in mononuclear cells. Whether this response suffices to protect these cells against Cd2+-induced alterations in their ability to perform their many varied, specialized functions cannot be deduced from these data. The observations that Cd’+ exposure has deleterious effects on immune function, and the fact that these cells rapidly incorporate Cd2+, indicate that possible direct effects of Cd2+ on the functional capabilities of mononuclear cells should continue to be investigated.
The kinetics of CdMT appearance in mononuclear cells suggested that little MT preexists in these cells prior to exposure to Cd2+ and that subsequent to exposure, MT is synthesized relatively rapidly. These suggestions were enforced by the analyses of [35S]cysteine incorporation into MT following exposure to Cd2+. No detectable amount of 35Swas incorporated into MT in cells not exposed to Cd2+. Following addition of 50 I.~M Cd2+, however, [35S]cysteine incorporation increased rapidly. A maximal incorporation ACKNOWLEDGMENTS (MT synthesis) rate was obtained within 4 hr of Cd induction. These kinetics resemble those We gratefully recognize the technical assistance ofJohn seen in Cd2+ resistant hamster lines (HildeL. Hanners and S. J. Stewart. We thank F. A. Enger for brand et al., 1982). The response was specific many blood samples. We thank Dr. Steve Barham and as well as rapid. No significant increase in 35S the Mayo Clinic/Foundation for the normal value blood samples. This work was supported by the U.S. Departincorporation into other proteins (Sephadex ment of Energy. G-75 void peak) followed Cd’+ induction. The rapid appearance of MT synthesis following induction could be explained by either REFERENCES a rapid activation of the thioneine genes, or by depressed translation of preexisting thiBREMNER, I. (1979). Mammalian absorption, transport oneine mRNA. That some protein synthesis and excretion of cadmium. In The Chemiswy, Biuchemistry and Biology of Cadmium. (M. Webb, ed.), in resting human lymphocytes is regulated at pp. 175-193. Elsevier/North-Holland, Amsterdam. the level of translation was shown by Ferrer COOPER, H. L., AND BRAVERMAN, R. (1980). Protein et al. ( 1980), who reported that enhanced prosynthesis in resting and growth-stimulated human petein synthesis following PHA stimulation of ripheral lymphocytes. Exp. Cell Res. 127, 35 I-359. resting lymphocytes does not require RNA ENGER, M. D., FERZKO, L. T., TOBEY, R. A., AND HILDEBRAND,C. E. (I 98 I). Cadmium resistance correlated synthesis, indicating that preexisting mRNAs with cadmium uptake and thionein binding in CHO are being utilized. Further, they reported the cell variants Cd’2OF4 and Cd’ 3OF9. J. Toxicol. and isolation of a ribosome-bound translational Env. Health 7, 675490. inhibitor in resting lymphocytes. It is for these FERRER, M., BURRONE, 0. R., AND ALGRANATI, I. D. reasons that we measured the effect of acti(1980). Inhibition of polypeptide synthesis by a factor isolated from ribosomes of resting human lymphocytes. nomycin D (AM) on Cd’+-stimulated MT FEBSLetl. 121,203-206. synthesis. The fact that pretreatment with AM FRAZIER,J. M. (1980). Cadmium and zinc kinetics in rat blocked most Cd2+-stimulated MT synthesis plasma following intravenous injection. J. Toxicol. and suggests that the response to Cd’+ occurs at Env. Health 6, 503-5 18. the level of transcription. That is, Cd2+ does HILDEBRAND, C. E., AND CRAM, L. S. (1979). Distri-
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