ECOTOXICOLOGY

AND

ENVIRONMENTAL

SAFETY

24,328-337

(t 992)

Individual and Combined Cytotoxic Effects of Cadmium, Copper, and Nickel on Brown Cells of Mercenaria mercenaria’ G.IAROOGIAN,*

SANDERSON,? ANDR.A.VOYER*

*United States Environmental Protection Agency, Environmental Research Laboratory, 27 Tarzwell Drive, Narragansetl. Rhode Island 02882; and TSAIC, Narragansett, Rhode Island 02882 Received

November

25, 1991

An assaybased on the lysosomal incorporation of neutral red dye by brown cells of Mercenaria (Bivalvia) was used to measure the cytotoxicity of Cd’+, Cu’+, and Ni*+ singly and in combination. Cytotoxicity was a linear function of Cd2+ concentration O.l- 1.5 mA4 and CL?+ concentration between 10 and 100 pA4. Nickel was not cytotoxic at concentrations as high as 10 mM. The presence of Cu’+ lessened the cytotoxic effect of Cd’+. Ni’+ did not affect cytotoxicity in combination with either Cu2+ or Cd’+. Ni’+ inflated estimates of cell survival by the neutral red assay in this study. Cells exposed to Ni2+ yielded measured quantities of neutral red dye in excess of those measured in cells from control treatments. 0 1992 Academic Press. Inc. mercenaria

INTRODUCTION The potential toxic effects of individual heavy metals on aquatic species have been studied extensively (Bryan, 1984). Virtually all such investigations have been conducted using whole animal testing procedures. Alternatively, In vitro tests have been employed as a means of screening the possible ecotoxicity of chemical agents (Rachlin and Perlmutter, 1968; Kocan et al., 1979). Use of these tests is supported by the good correlation found between results of such assays and those of standard lethality tests (Babich and Borenfruend, 1990a; Dierickx and Van De Vyver, 1991). Dierickx and Van De Vyver (199 l), in addition, also suggested that in vitro methods are easier, quicker, and less expensive to use than in vivo test procedures. In vitro toxicity screening programs to date have generally used cell lines from fresh water species, particularly those of fishes (Babich et al., 1986b). The neutral red (NR) assay is a specific in vitro assay that has proven to be useful in evaluating the toxicity of chemical agents (Babich and Borenfruend, 1990b). Its use is based on the uptake and accumulation of the vital dye, neutral red, in lysosomes of viable cells. Brown cells of the estuarine mollusc, Mercenaria mercenaria, are rich in lysosomes and are of potential use in conjunction with the NR assay (Zaroogian et al., 1989). The NR assay of Babich and Borenfruend (1987a,b) was thus adapted to permit use of these cells in toxicity screening programs (Zaroogian, unpublished). The intent of this project was to apply the NR assay using these brown cells in assessing the cytotoxicity of Cu2+, Cd2+, and Ni2+ singly and combined in binary mixtures. The working hypothesis of this effort was that each metal in a mixture acts independently and that their combined effects are additive. Since toxicants exert their effects through chemical reactions with biochemical substances in cells, toxicological ’ ERLN Contribution No. 1340. 0147-6513192 $5.00 Copyright 0 1992 by Academic Press. Inc. A,, rights of reproduction I” any form reserved

328

CYTOTOXlC

EFFECTS

OF

Cd.

Cu.

AND

Ni ON

BROWN

CELLS

studies at the cellular level can yield useful information on cellular function anism of toxic response, in addition to providing estimates of toxicity. MATERIALS

AND

329 and mech-

METHODS

Brown Cell Isolates Brown cells are collected from red glands of two M. mercenaria for each experiment. Red glands are excised, pooled, teased, and digested in 5 ml of medium containing 0.1% pronase (Bowheinfwe, Mannheim) and 0.1% collagenase (CLS @, Worthington) in a buffered salt solution (NaCl, 0.4 A4; KCl, 9.4 mM; K2HP04, 0.4 mM; NaHC03. 2.4 mM; CaCl, . 2H20, 7.5 mM; Hepes, 0.1 IV; pH 7.5). Red glands are incubated at 5°C overnight and then 1 hr at 37°C. Brown cells are filtered through a 120~pm Nytex screen into 15-ml polyallomer tapered centrifuge tubes following digestion of red glands. Cell suspensions are brought to 10 ml of basal salt solution (BSS) (buffered salt solution excluding Hepes), centrifuged at 50g for 10 min at 15°C and washed three times, Compacted cells are resuspended in 2.0 ml of BSS and counted on a hemocytometer. Cell viability was >93%, as determined by trypan blue exclusion.

In L’itro CJ’toto.uicity Testing One million brown cells are added to individual 1.5 polyethylene microfuge tubes and appropriate concentrations of metals and BSS added to yield a final volume of 0.5 ml. Cells are then incubated at 20°C with intermittent shaking to maintain cells in suspension. After 2.5 hr, cells are centrifuged at 3000g for 5 sec. resuspended in 1.O ml BSS washing solution containing 60 pg/ml of NR, and incubated at 20°C for 15 min. Centrifugation at 3000g for 5 set is repeated a second time. The dye solution is removed. Cells are washed and fixed rapidly with 0.5 ml of 0.4% formalin-l .O% CaClz solution and then centrifuged at 3000g for 5 sec. To extract the NR incorporated in lysosomes of viable cells, the resulting pellet is resuspended in 0.5 ml of a 1.0% acetic acid-50% ethanol solution and incubated at 20°C for 30 min. Cells are centrifuged at 85OOg for 60 set, and 0.4 ml of supernatant from each microfuge tube is transferred to a separate microcuvette and mixed with 0.5 ml of extraction medium. NR concentration of each solution is estimated from light absorbance at 540 pm.

E.xperirnental lkrign Relationships between brown cell viability and metal concentrations in binary mixtures of Cu’+. Cd”, and Ni’+ were determined using response surface techniques in the first series of tests (Myers, 1976). The range of Cu’+ concentrations was 0 to 100 phi and the range of Cd’+ and Ni” concentrations was 0 to 2.0 mM. Experimental combinations of metal concentrations in a mixture were predicted on a ‘-factor central composite design, and three observations were prepared per combination. The experimental treatment representing the design center was replicated four times, with three observations per replicate. In the test with Cd2+ X CuZt the entire experiment was completed as a single unit, whereas in tests with CL?+ X Ni’+ and with Cd” X Ni’+ the various treatments were examined as blocks. Brown cells were also exposed to a control treatment without metal additions which consisted of three observations.

330

ZAROOGIAN,

ANDERSON,

AND

VOYER

Cytotoxic effects of metal mixtures were estimated as percentage of NR incorporated by cells in the control treatment. These estimates were based on averaged measured absorbance values of multiple observations at each mixture treatment. Results were analyzed using the model F=

bo + b,x, + 62x2 + b,,x: + b&

+ b,2x,x2,

where P = estimated percentage brown cell viability, xl, x2 = metal concentrations, b. = a calculated constant, b, , b2, b,, , b12 = estimated regression coefficients for linear, quadratic, and interactive effects, respectively (Myers, 1976). In the second series of tests conducted, the effect of the presence of a metal on cell survival was assessedusing a 22 factorial arrangement of treatments. Mixtures of Cd2’ X Cu’+ and of Cd2+ X Ni2+ were examined in this series. Absence of metal in the mixture constituted the low treatment level. The high treatment level of each metal was chosen as the equivalent of the concentration of the respective metal at the design center in the initial series of experiments. Each of the four treatment combinations in this second series was replicated five times. Measured absorbance values representing NR concentrations were analyzed by analysis of variance to determine the significance of main and interactive effects of metals on brown cell viability. RESULTS The cytotoxicity was a linear function of Cu2+ concentration between 10 and 100 pm (Fig. 1) and a linear function of Cd2+ concentration between 0.1 and 1.5 mM (Fig. 2). Ni2+ was not toxic to brown cells at concentrations as high as 10 mA4. Thus the order of in vitro metal toxicity to brown cells was Cu2+ > Cd2+ > Ni’+. Survival of brown cells exposed to mixtures of Cu2+ and Cd2+ was predominantly influenced by linear and quadratic effects of copper (P < 0.01) and to a lesser degree by the linear effect of Cd’+ (P < 0.05). Statistical analysis also suggested significant (P

y = -0.42x

20

+ 91.54

R* = 0.99

00 0

25 CONCENTRATION

50

75

100

Cu (pM)

brown FIG. I. Cytotoxic effect of various concentrations of Cu2+ on M. mercenaria represents the mean of three independent determinations by the neutral red assay.

cells. Each data point

CYTOTOXIC

EFFECTS

OF

Cd.

Cu.

y = -36.14x 20

R’

AND

Ni ON

BROWN

331

CELLS

+ 88.76 = 0.97

1

1

O-I0.0 FIG. 2. Cytotoxic represents the mean

0.5

1.0

CONCENTRATION

Cd (mM)

1.5

effect of various concentrations of Cd’+ on M. I~W~MUIU brown of three independent determinations by the neutral red assay.

cells.

Each

data

point

< 0.05) influence of an interaction between the two metals. These influences on the overall pattern of survival are indicated by the predictive equation, r = 46.09 OS7[Cu”] + 1 1.09[Cd2+] + O.O03[Cu’+]’ - O.O3[Cd’+][Cu’+] - 2.75[Cd’+]’ and its graphical presentation (Fig. 3). Maximum survival ranged from about 45 to 53%’ over the range of Cu*+ and Cd’+ concentrations examined. Cell survival progressively decreased to a minimum of about 20% of the control value, as Cu2+ increased to ap-

53

+

22

FIG. 3. Survival

of M

IW~L‘~~~UI’I~I

brown

cells

in various

combinations

of Cd’+

and

Cu”.

332

ZAROOGIAN,

ANDERSON,

AND VOYER

proximately 65 WV. This surface also implies that for a given concentration of Cu*+, addition of Cd2+ resulted in an enhancement of cell survival. This toxic effect of Cu2+ is further illustrated by results of the second experiment in which the presence of metal ion was examined (Fig. 4). Results presented in this latter figure reveal toxicity attributable to Cd*+, as well. A 50% decrease in cell survival is implied to have occurred in a treatment of 1.O mM Cd2+ and 0 Cu’+. Likewise, a 60% reduction in absorbance was observed in the treatment combination of 0 mM Cd2+ and 50 PM Cu*+. These two results, together with the 62% reduction in survival at 1.O mM Cd*+ and 50 PM Cu2+, yielded two nonparallel dose-response curves. This relationship suggests a combined effect of the two metals in which the toxicity of Cd*+ was lessened in the presence of Cu2+. It further suggests the existence of the interaction indicated by analysis of the initial experiment. Moreover, results of both experiments together demonstrate that this interrelationship persists over a range of Cd2+ concentrations and that the effects of Cu2+ on Cd*+ toxicity increase with each increase in the concentration of the latter metal (Fig. 5). The survival of brown cells in mixtures of Cd*+ and Ni*’ is summarized by an estimating equation, r = 88.44 - 21.19[Cd2+] + 23.46[Ni2+] + 1.68[Cd2+]* 2.55[Cd’+][Ni’+] - 7.82[Ni2+12. The response surface shows that brown cell survival was affected by Cd*+ only and that survival decreased linearly from 100% to about 60%, as Cd2’ concentration increased from 0 to 2.0 mM (Fig. 6). A slightly greater resistance of brown cells to Cd*+ in the presence of Ni*+ is also implied. These interpretations are supported by results of the second experiment in which only Cd2+ exerted toxicity, as measured by changes in absorbance (Fig. 7). Decreases in brown cell survival in mixtures of Cu*+ and Ni*+ correlate with additions of Cu*+ but not Ni*+ (Figs. 8 and 9). Graphical presentation of the predictive equation, P = 88.43 - l.l6[Cu*+] + 3.22[Ni2+] + O.O07[Cu*+]* + 0.0 1[Ni2’][Cu2’] - l.27[Ni2+]*, shows cytotoxicity increased about 50% as Cu*+ concentration increased from 0 to 65

1mYCd

00 50

0 CONCENTRATION

Cu (PM)

FIG. 4. Concentration of neutral red in brown cells of hf. mercenaria either alone or together.

in the presence of Cu2+ and Cd*+,

CYTOTOXIC

EFFECTS

0

OF

Cd,

(*) and estimated

(0) survival

AND

Ni

ON

50

15 CONCENTRATION

FIG. 5. Observed of Cu” and Cd’+.

Cu.

BROWN

333

CELLS

a5

Cu (pk.4)

of brown

cells of .hL mcwmuria

exposed

to combinations

@f, with no additional toxicity evident beyond this concentration (Fig. 8). As in the above experiment with Ct.?+, the overall pattern ofchanges in cell survival is curvilinear. In each instance, R* values were at least 0.92. The quadratic model thus accounted for virtually all of the experimental variability observed and provided a useful means of relating cell survival to each metal mixture. DISCUSSION Mammalian and fish cell lines of freshwater species have been used in cytotoxicity testing (Babich and Borenfreund, 1987a.b: 1990b). Previously, in virro tests using cells

FIG. 6. Survival

of brown

cells of M. mercmariu

exposed

to various

combinations

of Ni*+ and Cd”

334

ZAROOGIAN.

0'

ANDERSON,

of neutral

VOYER

(

I

0

1 CONCENTRATION

FIG. 7. Concentration singly and together.

AND

red in brown

Cd (mM)

cells of M. mc~tvurirc

in the presence

of Cd’+ and Ni’+

of estuarine species have not been developed. The adaptation of the existing NR assay developed by Babich and Borenfruend (1987a,b) that the authors present here thus represents a new and potentially useful method of assessing hazards associated with discharge of waste materials into near-coastal areas. However, as a method, it is significant to note the inordinate uptake and binding of NR in brown cells, in the presence of Ni2+ relative to that in the control treatment. For example, brown cells treated with

FIG. 8. Survival

of brown

cells of M. wwcenaria

in various

combinations

of Ni*+ and CL?‘.

CYTOTOXIC

EFFECTS

OF

Cd.

Cu.

AND

Ni ON

BROWN

335

CELLS

OmM Ni

01-7 50

0 CONCENTRATION FIG. 9. Concentration singly

of neutral

red

in brown

cells

Cu (j&i) of ,ZI

merwmmr

in the

presence

of Cu”

and

Ni”

or in combination.

I.0 mM Ni” and those treated with 6.0 mA4 Ni”+ retained 8 and 20% more NR, respectively. than did cells from the control treatment. One explanation is that this result is an anomaly of the assay. Ni’+ is known to bind at the y-glutamyl linkage of the glutathione molecule. a strong nucleophile, and not at the sulfhydryl linkage (Martin and Edsall, 1959; Chou ef al.. 1975). Conversely, NR is a weakly cationic (electrophile) dye which readily diffuses through the plasma membrane by non-ionic diffusion. Thus. if Ni” were to induce increased production of glutathione, additional binding sites for NR would be available and result in increased uptake and retention of the dye in Ni2+-treated cells. Since NR incorporation was the measure of cell survival, one possible consequence of this scenario would be a masking of Cd”+ and CL?’ toxic effects. The overall pattern of brown cell survival to Cu’+, though, was not altered by addition of Ni’+, as evidenced by comparison of response surfaces representing mixture effects with CU’+. Regardless, caution should be exercised when interpreting results of the NR assay. In contrast to the findings presented here, there are reports of Ni’+ ameliorating Cd’+ toxicity, both in viva and in vitro. For instance, Ni’+ inhibited Cd’+ toxicity in cultured mouse cells (Borenfreund and Puerner, 1986). Pretreatment of rats with Ni” protected against nephro- and hepatotoxicity (Tandon cf al.. 1984). And finally. the toxicity of Cd’+ to a bacterium and fungus was lessened in the presence of Ni’+ (Babich el al., 1986a). The authors rejected the null hypothesis of independent metal effects only in the case of mixtures of CL? and Cd’+. At the outset. it was anticipated that. since Cd” increases the stability of lysosomal membranes and Cu7+ decreases it (Sternlieb and Goldfischer, 1976) the effect of Cd’+ would be to neutralize that of Cu’+. This supposition was proved incorrect in that the presence of Cu7+ diminished the toxic effect of Cd’+. Other results indicate that uptake of Cd’+ into brown cells decreases in the presence of Cult and glutathione (GSH) inhibitors in a dose-dependent manner (Zaroogian et al.. 1992, manuscript). Thus, the lessening effect of Cu’ ‘- on Cd’* toxicity

336

ZAROOGIAN,

ANDERSON,

AND

VOYER

the authors report may be related to the availability of Cd” at the site of toxic action. The tighter binding capacity of Cu*+, as compared to that of Cd*+, at the sulfhydryl linkage of the GSH molecule (Ahrland et al., 1958) and the possible replacement of Cd’+ by Cu*+ at this site, as suggested by Bourcier et al. ( 1983), offers a second possible explanation for the less-than-additive action of these two metals the authors observed. Third, cells in viva and in vitro apparently possess mechanisms which defend against cadmium toxicity and the toxicity of cadmium may be modified by trace metals, e.g., Cu*+ (Fischer, 1985). Meshitsuka et al. ( 1987) also found that addition of Cu2+ diminished Cd*+ uptake in mammalian cell cultures. However, these scientists reported an additive joint effect of Cu2+ and Cd*+. These results contrast with the present data and suggest the possibility that the mode of toxic action of metals in mammalian and invertebrate cells differ. If so, then the use of mammalian cells to assessthe hazard associated with the discharge of wastes into estuarine areas may be inappropriate. CONCLUSIONS Results indicate that the order of cytotoxicity to brown cells was Cu*+ > Cd*+ > Ni2+. They also demonstrate that primary cultures of brown cells are useful in evaluating toxicity of metals. Results also illustrate the general utility of linear modeling techniques in assessing possible interactions between toxicants. The neutral red assay was highly reproducible as adapted for use in this series of experiments. Finally, it is clear that caution should be exercised when interpreting results of this assay, since observed responses, as in the case of Ni*+, could be a result of the assay and not a measure of toxicity. ACKNOWLEDGMENTS It is a pleasure to acknowledge J. Heltshe. S. Nelson, and S. Ferraro anonymous reviewers for their helpful criticism and suggestions.

for their

reviews.

Also,

thanks

to

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CYTOTOXIC

EFFECTS OF Cd, Cu, AND Ni ON BROWN CELLS

337

BRYAN. G. W. (1984). Pollution due to heavy metals and their compounds. In Murk, &&),q~~ (0. Kinnc, Ed.), Vol. 5. part 3. p. 1289. Wiley, New York. CHOU, S. T., MCAULIFFE. C. A., AND SAYLE, B. J. (1975). Reactions of the tripeptide. glutathione. with divalent cobalt, nickel, copper and palladium salts. J. Inorg. Nucl. C/lctn. 37, 45 l-454. DIERICKX. P. J.. AND VAN DE VYVER, I. E. (I 99 I). Correlation of the neutral red uptake inhibition assay of cultured fathead minnow fish cells with fish lethality tests. &r/l Environ. Conram. Ttrricol. 46, 649653.

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2, 409-4

14.

STERNLIEB. D.. AND GOLDFISCHER.S. ( 1976). Heavy metals and lysosomes. In L~:\o~~nc>.r irr B~o/og~‘ and Pulho/~!?)~(J. T. Dingle and R. T. Dean, Eds.). Vol. 5. pp. 185-200. North Holland-American Elsevier. Amsterdam/Oxford/New York. TANDON. S. K., KHANDELWAL. S., MATHUR. A. K.. AND ASHQUIN. M. (1984). Preventive effectsof nickel in cadmium hepatotoxicity and nephrotoxicity. .dnn. Clrrz. Lab. Sci. 14, 390-396. ZAROOGIAN, G., YEVICH. P.. AND ANDERSON. S. (1992). Effect of selected inhibitors on cadmium. nickel and benzo(a)pyrene uptake into brown cells of hfercmnriu mercmaria. Mur. Environ. Re.s.. manuscript. ZAROOC~IAN.G.. YEVICH. P.. AND PAVICNANO, S. ( 1989). The role of the red gland in hfcrwnariu muwtwru in detoxiiication. AILw. Emiron. RCS 28, 447-450.