Exposure of scorpionfish (Scorpaena guttata) to cadmium: biochemical effects of chronic exposure

Aquatic Toxicology, 16 (1990) 311-320 Elsevier

311

AQT 00394

Exposure of scorpionfish (Scorpaenaguttata) to cadmium: biochemical effects of chronic exposure Steven M. Bay, Darrin J. Greenstein, Peter Szalay* and David A. Brown* Southern California Coastal Water Research Project, Long Beach, CA. U.S.A. (Received 3 April 1989; revision received 23 October 1989; accepted 24 October 1989)

Scorpionfish (Scorpaena guttata) were exposed to sublethal levels of cadmium in seawater (10 and 20 mg/l) for 4 wk. Measurements of enzyme activities were conducted in order to examine the relationship between toxic effects and the subcellular distribution of Cd, Cu and Zn. Liver, kidney, intestine and gill tissue were analyzed for the activity of the enzymes alkaline phosphatase, succinate dehydrogenase, glyceraldehyde phosphate dehydrogenase and Cu Zn-superoxide dismutase (SOD). Exposure to Cd resulted in the inhibition of only Cu-Zn SOD activity in the intestine. The activity of SOD was reduced by sixfold in intestines at the 20-mg Cd/1 exposure level. Intestine SOD activity was also correlated positively with the concentration of Cu in the high-molecular-weight fraction of the cytosol (ENZ). The sulfhydryl-containing enzymes glyceraldehyde phosphate dehydrogenase and succinate dehydrogenase were not affected by Cd exposure, suggesting that the amounts of Cd found in the ENZ pool after the exposures examined here were insufficient to have a direct impact on enzyme activities. The mechanism of reduction of SOD activity in these fish appeared to be related instead to Cd-induced alterations of Cu and/or Zn metabolism. Key words: Cadmium; Scorpionfish; Enzyme activity; Toxicity

INTRODUCTION T h e p u r p o s e o f this s t u d y was to d e t e r m i n e if m e a s u r i n g the effectiveness o f t r a c e m e t a l b i n d i n g by m e t a l l o t h i o n e i n is a useful i n d i c a t o r o f the t o x i c effects o f m e t a l bioaccumulation

resulting from chronic exposure.

The protein

metallothionein,

f o u n d in t h e s o l u b l e p o r t i o n o f cell e x t r a c t s (cytosol), has a s t r o n g b i n d i n g affinity

*Present address." Division of Immunology, Beckman Research Institute of the City of Hope, 1500 E. Duarte Rd., Duarte, CA 91010, U.S.A. Correspondence to." S.M. Bay, Southern California Coastal Water Research Project, 646 West Pacific Coast Highway, Long Beach, CA 90806, U.S.A. 0378-4274/90/S 3.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)

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for both essential (Zn, Cu) and nonessential (Cd, Hg) trace metals (Kagi and Nordberg, 1979). In acute laboratory exposures of animals to Cd or HG, toxic effects are usually not found as long as the accumulated metal is bound to metallothionein (Winge et al., 1974; Brown and Parsons, 1978; Brown et al., 1984; Pruell and Englehardt, 1980). The occurrence of toxicity in these experiments was found to correspond to an overwhelming of metallothionein's capacity to bind newly accumulated metal and subsequent metal binding to macromolecules, such as cytosolic enzymes. Most previous studies documenting the relationship between the subcellular distribution of Cd and toxic effects in fish have used exposures near acutely lethal levels which resulted in severe effects, such as mortality and tissue necrosis (Brown et al., 1984: Benson and Birge, 1985). The nature of the relationship between subcellular Cd distribution and toxicity resulting from chronic exposure has not been well documented. Results from our studies with scorpionfish (Scorpaena guttata) indicate that chronic Cd exposure produces very different subcellular distribution patterns compared with acute exposure conditions (Brown et al., 1990). In this paper, we examine the relationship between the subcellular Cd distribution pattern produced by chronic sublethal exposure and toxic effects on selected enzymes. Data are presented that (i) confirm that the partitioning of trace metals between cytosolic pools is a useful indicator of sublethal toxic effects, (ii) document the significance of Cd effects on Zn and Cu metabolism, and (iii) determine the relative sensitivity of different fish tissues to Cd toxicity. MATERIALS AND METHODS

Details of the fish exposure conditions, tissue sampling and fractionation of cytosol into metallothionein-containing (MT) and enzyme-containing (ENZ) pools are given in the preceding paper (Brown et al., 1990). Briefly, scorpionfish (S. guttata) were exposed to Cd in seawater in laboratory aquaria for a period of 4 wk. Cadmium was added from a stock solution of CdCI2 to achieve nominal concentrations of 0, 10, and 20 mg Cd/l. These Cd levels corresponded to 0.16 and 0.32 of the 4 d LC50 for scorpionfish. One 312-1 aquarium containing I 1 scorpionfish was used for each exposure level. Aquaria were provided with continuous biological filtration and the water was changed weekly. Five fish from each exposure level were dissected at the end of the 4-wk exposure. Samples of liver, kidney, gill, and intestine were removed and stored at - 8 0 ° C until analyzed for enzyme activities. Tissue samples were homogenized in 0.05 M Tris (pH 8.0) using a glass/Teflon homogenizer. Homogenates were centrifuged at 1 400 × g to remove tissue debris and then subsampled for later analysis of alkaline phosphatase and glyceraldehyde phosphate dehydrogenase activity. Cu-Zn-superoxide dismutase (SOD) activity measurements were made on subsamples of the high molecular weight fraction of cytosol (ENZ pool) prepared for trace metal analyses (Brown et al., 1990).

313 Separate tissue samples were used to prepare mitochondria for succinate dehydrogenase activity measurements. These samples were homogenized in 0.25 M sucrose10 mM HEPES (pH 7.5) and centrifuged at ! 400 x g. Mitochondria-containing pellets were then collected by centrifugation at 14000 x g (10 min), washed once, and resuspended in homogenization buffer.

Enzyme activity AIkaline phosphatase Alkaline phosphatase activity was determined by measuring the rate of conversion of p-nitrophenyl phosphate to p-nitrophenol at 405 nm (Walter and Schutt, 1974). Measurements were made at 25°C and pH 9.4. One unit of alkaline phosphatase activity was defined as the amount of enzyme needed to hydrolyze 1.0/~mol of substrate/min.

Glyceraldehyde phosphate dehydrogenase Glyceraldehyde phosphate dehydrogenase (GAPDH) activity measurements were made using glyceraldehyde-3-phosphate as the substrate. Oxidation of this compound by the enzyme was measured by following the change in light absorbance (340 nm) caused by the conversion of NAD to N A D H (Duggleby and Dennis, 1974). Measurements were made at 25°C and pH 8.7. The data were expressed as units of enzyme activity, where one unit represented a substrate conversion rate of 1 pmol/ min.

Cu- Zn-Superoxide dismutase Cu- and Zn-containing superoxide dismutase (Cu-Zn-SOD) activity was determined by a modification of the method of Heikkila and Cabbat (1976). The inhibitory effect of superoxide dismutase upon the superoxide mediated autooxidation of 6-hydroxydopamine was observed at 490 nm. One unit of superoxide dismutase activity was defined as that concentration of enzyme causing 50% inhibition of the autooxidation rate of 0. I mM of 6-hydroxydopamine at 25°C and pH 8.4.

Succinate dehydrogenase Succinate dehydrogenase activity measurements were made using a modification of the phenazine methosulfate procedure of Ackrell et al. (1978). Assays were performed at 30°C and a pH of 7.8 with succinate as the substrate. Cytochrome C was used as the terminal electron acceptor. Enzyme activity was measured as the change in cytochrome C absorbance observed at 550 nm. One unit of succinate dehydrogenase activity was defined as the amount of enzyme required to oxidize 1.0/.tmol of substrate/min. Activity was expressed in terms of mitochondrial pellet protein concentration, which was measured using the Coomassie Blue assay (Bradford, 1976).

314

Data analysis Analysis of variance (ANOVA) was used to test the significance of changes in enzyme activity related to exposure concentration or time. ANOVAs indicating significant effects were further examined by the Ryan-Einot-Gabriel-Welsch multiple F test to determine which treatment groups were significantly different (SAS Institute, Inc., 1982). The relationship of changes in enzyme activity with variations in ENZ pool metals was examined by calculating Spearman rank correlation coefficients between the enzyme and metals data. RESULTS AND DISCUSSION

Subcellular Cd distribution The concentrations of Cd, Cu, and Zn in the cytosolic high-molecular-weight (ENZ) pool of scorpionfish tissues exposed to 10 and 20 mg Cd/l for 4 wk are described in Brown et al. (1990). Statistically significant increases of ENZ-Cd were found in all four tissues (liver, kidney, gill, and intestine) of fish exposed to either 10 or 20 mg Cd/l, suggesting the potential for toxic effects by Cd in each organ. Elevation of ENZ-Cd was greatest in gill tissue, which had Cd concentrations of up to 23 times the control value. Maximum ENZ-Cd concentrations in the other tissues ranged between five and eight times their respective control values. A consistent trend towards decreased ENZ-Cu and -Zn concentrations was found in fish exposed to cadmium, although these changes were not statistically significant. ENZ-Cu concentrations were most affected in gill and intestine (38 and 67% of controls, respectively), while the greatest alterations in ENZ-Zn concentrations were found in the intestine and kidney (66 and 75% of controls).

Enzyme activio' Chronic Cd exposure did not result in activity changes for either G A P D H or succinate dehydrogenase, as shown in Table I. These enzymes were considered to be likely targets of Cd toxicity because they both contain sulfhydryl groups which are readily bound by alkylating agents such as Cd (White et al., 1973). Correlation coefficients between enzyme activity and subcellular metal concentration in each fish were calculated and indicated the presence of several positive associations between G A P D H and ENZ-Cd (liver and kidney), ENZ-Cu (liver), and ENZZn (liver and gill), as shown in Table II. The finding of correlations between G A P D H and ENZ-Cu or -Zn was unexpected since these metals are not known to be essential to the function of G A P D H . No significant correlations between succinate dehydrogenase activity and ENZ pool metals were found (Table II). Alkaline phosphatase and C u - Z n - S O D , unlike G A P D H and succinate dehydrogenase, contain zinc and copper (for SOD) as essential components of their active structures. These enzymes were used as indicators of toxicity resulting from Cd-in-

315

TABLE I

Enzyme activities in liver, kidney, gill, and intestine of scorpionfish exposed to Cd for 4 wk. Values are mean _+ SE, N = 5. Activity is expressed as U/g tissue except for succinate dehydrogenase, where the units are U/rag mitochondrial protein. Tissue/exposure mg Cd/I

Succinate dehydrogenase

GAPDH

Alkaline phosphatase

SOD

Liver control 10 20

28 + 3 27+2 36+6

190 + 20 194+20 200+__ 18

16 + 2 14+3 14+3

1 330 + 23 I 9 2 0 + 115 1037+285

K i d n e y control 10 20

69 + 7 66+7 64-,-6

84 + 6 97+9 100+2

17 + 4 16+2 16+ I

1 329 + 319 1265+83 975+73

Gill control

56 + 6 5 8 + I0 67+7

55 + 9 53+8 64+2

7 -1- 1 5+ I 5+0

552 + 86 3 9 4 + 130 378+43

72 + 14 51 + 6 50+8

42 + 1 55+3 64+3

6+ I 7+ 1 7+ 1

840 + 144 5 5 5 + 255 150+40 ~

10 20

Intestine control 10 20

aSignificant difference from control (19 < 0.05. A N O V A ) .

duced perturbations of Cu or Zn levels in the cytosol. Alkaline phosphatase activities were unaffected by exposure to 10 and 20 mg Cd/I (Table I). There was only one correlation between alkaline phosphatase activity and enzyme pool metal concentration: a positive relationship with ENZ-Zn in gill tissue (Table II). Exposure of scorpionfish to 20 mg Cd/l resulted in a sixfold reduction in the activity of intestine Cu-Zn-SOD (Table I). Trends (not statistically significant) of reduced SOD activity in exposed scorpionfish were also noted for the other tissues, with the next greatest response occurring in gill tissue. Significant correlations between SOD activity and ENZ-Cd were not found, but there were significant positive correlations between SOD activity and ENZ-Cu and/or ENZ-Zn for each of the four tissues (Table II). Other investigators have found decreased heart Cu-Zn-SOD activity following exposure of rats to Cd (Jamall and Smith, 1985). These authors attributed decreased Cu-Zn-SOD activity to decreases of Cu, which is essential for the normal functioning of this enzyme; Zn was not measured in their study. In the present investigation, evidence of reduced ENZ-Cu and -Zn concentrations was found in gill, kidney and intestine tissue after exposure to 10 and 20 mg Cd/I (Brown et al., 1990). Reductions in ENZ-Cu were correlated with elevated ENZ-Cd levels in gills and nearly so (p = 0.08) in intestines at the 10- and 20-mg Cd/l exposure levels. These data indicate

316

T A B L E I1

Spearman correlation coefficients of enzyme activity versus enzyme pool metal concentration. ENZ-Cd Succinate dehydrogenase liver kidney gill

ENZ-Zn

0.16

0.08

0.03

- 0.12

- 0.40

- 0.42

0.10

intestine

ENZ-Cu

- 0.31

-0.09

0.50

0.12

[).51

GAPDH

liver kidney

0.59" 0.57"

0.64 ~

0.56"

- 0.00

0.00

gill

0.16

intestine

0.38

- 0.16

0.28

0.45

0.26

0.26

0.18

- 0.00

Alkaline

- 0.23

0.62 - 0.45

phosphatase

liver kidney gill

-0.22

intestine

0.15

0.14 - 0.31

0.57" - 0.09

SOD liver

0.04

0.59"

0.73"

kidney

- 0.06

0.80'

0.80 ~

gill

-0.23

0.21

intestine

- 0.49

0.78 ~

"Signiticant correlation

0.62 ~ - 0.10

(p ~< 0.05, r = 0 . 5 2 l .

that the reduced Cu-Zn-SOD activities found in scorpionfish exposed to 10 and 20 mg Cd/l may have been the result of displacement of Cu and/or Zn from the ENZ pool by Cd. Cu-Zn SOD, together with selenium-dependent glutatione peroxidase function in mammalian systems to protect cells from superoxide anion radicals produced during oxidative metabolism. Jamall and Smith (1985) attribute the cardiotoxicity resulting from exposure to Cd as a consequence of peroxidative damage caused by reductions in Cu-Zn-SOD and glutathione peroxidase activities. Since measurements of lipid peroxidation were not conducted in the present study, the importance of this mechanism of toxic action to fish exposed to Cd cannot be determined. It is recommended that such measurements be included in future studies. The predicted correspondence between ENZ-Cd concentration and enzyme activity was not found in this study. Enzyme effects were greatest in the intestine, even though gill tissue had much higher concentrations of ENZ-Cd. Multiple factors probably contributed to this lack of correspondence. First, enzyme effects appeared

317

to be more related to alterations in cytosolic Cu and Zn concentrations than to cytosolic Cd concentration. Therefore, effects due to Cu or Zn displacement may not be well correlated with Cd concentration in a given tissue. For example, scorpionfish ENZ-Cu concentration was most affected in gill tissue, while ENZ-Zn was most affected in the intestine (Brown et al., 1990). In addition, the composition of the ENZ pool may be too heterogeneous to permit accurate generalizations regarding toxicity based on analyses of metal content. This pool includes the void volume produced by Sephadex G-75 chromatography and includes all cytosolic components having molecular weights of 20 000 or greater. MetalIoenzymes such as C u - Z n - S O D comprise a small fraction of this pool and the components of this pool (along with their metal-binding characteristics) probably vary greatly between different tissue types. Consequently, equivalent changes in metal content of ENZ pools from different tissues may not always produce the same effects on specific proteins. CONCLUSIONS

The relationship between cytosolic Cd distribution and toxic effects observed in this study was not as well defined as that which has been demonstrated under acute Cd exposure conditions (Goering and Klassen, 1983; Pruell and Englehardt, 1980; and Winge et al., 1974). The chronic Cd exposures used in this study produced relative ly few toxic effects, as indicated by enzyme activity changes. Other indicators of sublethal toxicity (histopathology, blood chemistry) have also been investigated on these fish, and indicate a similar lack of severe effects (Perkins and Rosenthal, 1984; Bay et al., 1984). This lack of effects may have been due in part to the influence of factors other than Cd on these parameters (e.g., diet, hormones, other metals). Inhibition of the cytosolic enzyme C u - Z n - S O D was found to be the most useful indicator of Cd toxicity in this study. No effects on the membrane-bound enzymes alkaline phosphatase and succinate dehydrogenase were found, although much evidence indicates that membrane systems of the cell, particularly those of mitochondria, are important sites of Cd toxicity (Byczkowski and Sorenson, 1984; Webb, 1977). The Cu Zn SOD activity measurements indicated the approximate degree of impact of chronic Cd exposure on scorpionfish tissues to be intestine > kidney = gill > liver. This is similar to the order of sensitivity predicted on the basis of the distribution of Cd between MT and ENZ pools in scorpionfish exposed to 0.1-20 mg Cd/! (Brown et al., 1990). These results indicate that measurement of the cytosolic distribution of metals may be a sensitive indicator of the potential for toxicity. The predicted direct correlation between enzyme activity and ENZ-Cd was not found in this study. The results of this study indicate that Cd exposure produced toxic effects not as a result of direct enzyme inhibition (e.g. inhibition of sulfhydryl enzymes), but rather via a disruption of Cu and Zn homeostasis. The observation

318 of positive correlations between enzyme activity a n d E N Z - C u or - Z n in this study underscores the i m p o r t a n t role these metals play in the regulation of cellular m e t a b o lism. F o r instance, Z n is k n o w n to play a role in genetic transcription a n d thus changes in its availability could affect enzyme synthesis (Slater et al., 1971; R i o r d a n , 1977). The relative sensitivity of the intestine to Cd exposure is of particular interest since the liver, kidney a n d testis are usually considered to be the principal sites of chronic Cd toxicity ( D u d l e y et al., 1985; Christie and Costa, 1984). The gastrointestinal tract m a y also be the most sensitive tissue for other types of toxicants. F o r instance, exposure to radiation has its m a j o r effect o n the gastrointestinal tract of mice (Lamb, 1977). The gastrointestinal tract is also the site o f the greatest frequency of cancers unrelated to s m o k i n g by men (American C a n c e r Society, 1985). Thus, toxicological studies which do not examine the intestine may miss an i m p o r t a n t site o f effects. ACKNOWLEDGEMENT This study was supported by g r a n t n u m b e r R-810248-01 from the U n i t e d States E n v i r o n m e n t a l Protection Agency. REFERENCES Ackrell, B.A.C., E.B. Kearney, and T.P. Singer, 1978. Mammalian succinate dehydrogenase. In: Methods in enzymology, Vol. LIII, Part D, pp. 466-483. American Cancer Society, 1985. Cancer facts and figures, 1984, American Cancer Society, Washington, D.C., 10 pp. Bay, S.M., D.J. Greenstein, P. Szalay and D.A. Brown, 1984. Biologicaleffects ofcadmium detoxification. In: Southern California Coastal Water Research Project, 1983-1984 Biennial Report, edited by W. Bascom, Southern California Coastal Water Research Project, Long Beach, Calif., pp. 269-285. Benson, W.H. and W.J. Birge, 1985. Heavy metal tolerance and metallothionein induction in fathead minnows: results from field and laboratory investigations. Environ. Toxicol. Chem. 4. 209-217. Bradford, M.M., 1976. A rapid and sensitivemethod for the quantitation of microgram quantities of protein utilizing the principle of protein~lye binding. Anal. Biochem. 72, 248--254. Brown, D.A. and T.R. Parsons, 1978. Relationship betweencytoplasmicdistribution of mercury and toxic effects on zooplankton and chum salmon (Oncorhynchus keta) exposed to mercury in a controlled ecosystem. J. Fish. Res. Board Can. 35, 880-884. Brown, D.A., S.M. Bay, J.F. Alfafara, G.P. Hershelman and K.D. Rosenthal, 1984. Detoxification/toxification of cadmium in scorpionfish (Scorpaena guttata): acute exposure. Aquat. Toxicol. 5, 93--107. Brown, D.A., S.M. Bay, G.P. Hershelman and E.M. Perkins, 1989. Exposure of scorpionfish (Scorpaena guttata) to cadmium: effects of acute and chronic exposures on the subcellular distribution of cadmium, copper and zinc. Aquat. Toxicol. 16, 295-310. Byczkowski, J.Z. and J.R.J. Sorenson, 1984. Effects of metal compounds on mitochondrial function: a review. Sci. Total Environ. 37, 133-162. Christie, N.T. and M. Costa, 1984. In vitro assessment of the toxicity of metal compounds IV. Disposition of metals in cells: interactions with membranes, glutathione, metallothionein and DNA. Biol. Trace Element Res. 6, 139 158. Dudley, R.E., L.M. Gammal and C.D. Klassen, 1985. Cadmium-induced hepatic and renal injury in

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