The susceptibility of superoxide dismutase in Lemna minor to systemic copper concentrated from wastewater

~

Wat. Res. Vol. 28, No. 12, pp. 2469-2476, 1994

Pergamon

0043-1354(94)E0085-K

Copyright © 1994ElsevierScienceLtd Printed in Great Britain.All rights reserved 0043-1354/94$7.00+ 0.00

THE SUSCEPTIBILITY OF SUPEROXIDE DISMUTASE IN L E M N A M I N O R TO SYSTEMIC COPPER CONCENTRATED FROM WASTEWATER JAMESA. BUCKLEY* ~ Department of Civil Engineering, University of Washington, Seattle, WA 98195, U.S.A. (First received June 1993; accepted in revised form March 1994) Abstraet--Lemna minor (duckweed) was grown for 7 days in secondary-treated domestic wastewater containing additions of total Cu from 0.024--0.220mg/1. Starch gel electrophoresis of plant extracts followed by enzyme staining showed that activity of one of four isozymes, probably Mn-SOD, was inhibited in Lemna containing 408 #g Cu/g (dry wt) but not in plants containing 215 ~g Cu/g (dry wt) or less. Measurement of SOD activity in a reaction mixture showed significant (P < 0.05) reduction in activity in extracts made from plants containing 408 #g Cu/g (dry wt) but no reduction in activity when plants contained 215 #g Cu/g (dry wt) or less. The four isozymes found in Lemna were tentatively identified by CN-sensitivity as two each of Cu,Zn-SOD and Mn-SOD. The activity of SOD in Lemna grown in defined medium was measured at 12 U/mg total protein. Key words--starch gel electrophoresis, duckweed, Lemna minor, superoxide dismutase, SOD, copper,

wastewater

INTRODUCTION An important issue in water pollution research is identifying the chronic effects and associated modes of action of pollutants. Metals are especially common pollutants found in wastewater and are readily taken up by aquatic organisms so that systemic levels may greatly exceed levels in the surrounding water. Cu is one such metal and has been found in excess of dietary needs in aquatic organisms exposed to contaminated waters. High systemic levels of Cu in plants are often associated with chlorosis resulting from disfunctional iron metabolism but another important area of cell chemistry with which Cu may interfere is the activity of the enzyme Superoxide Dismutase (SOD). The SOD found in higher plants contains either Cu and Zn or Mn (Fridovich, 1978) and is composed of a number of isozymes that catalyse the dismutation of the superoxide radical, 0 2 . The Cu,Zn-SOD enzyme, for example, is composed of two active subunits each with Cu at the active site facing the solvent front and coordinated to four histidine residues (Fridovich, 1978). In this position, Cu alternates between Cu 2+ and Cu ÷ during a catalytic cycle: SOD-Cu 2+ + 0 2 ---*SOD-Cu ÷ + 02 SOD-Cu + + 0 2 + 2H + ~ SOD-Cu 2+ +

(1)

H202. (2)

*Present address: Metro Environmental Laboratory, 322 West Ewing Street, Seattle, WA 98119-1507, U.S.A.

Because Cu is a cofactor of the enzyme itself, there are interesting relationships between exongenous Cu and SOD. Free Cu 2+ will follow the same redox cycle and mimic SOD activity. In fact, the aqua complex is a more efficient catalyst than Cu,Zn-SOD at pH values where hydrolysis of Cu 2÷ does not occur (Brigelius et al., 1974; Czapski and Goldstein, 1990). In spinach chloroplasts, Cu 2÷ mimics SOD as follows (Sandmann and Boger, 1980): Cu 2+ + O~- --, Cu ÷ + 02

(3)

A Cu-penicillamine chelate with Cu2+-Cu + mixed valence had superoxide dismutating ability when applied to embryonic leaves of flax (Youngman et al., 1979). A concentration of 50 nM in 3 mi was equivalent to 50 U of SOD. In addition, there are a number of Cu-amino acid chelates as well as other potential ligands in the plant cell milieu which may mimic SOD (Lapluye, 1990). The Cu + formed when Cu 2+ oxidizes the superoxide anion (reactions 1 and 3) competes with catalase for H2 O2 and produces the hydroxyl radical OH" via a Fenton-type reaction: Cu + + H202 --~ Cu 2+ + O H - + OH"

(4)

The radical is powerful oxidant that initiates lipid peroxidation in photosynthetic membranes. In marine algae, Cu 2+ and the Cu-l,10-phenanthroline complex both mimicked the activity of SOD in producing H202 which resulted in increased levels of intracellular OH' and subsequent decreased rate of growth (Florence and Stauber, 1986).

2469

2470

J A ~ S A . BUCKLEY

Exogenously applied C u 2+ c a n n o t only mimic S O D but it can activate the S O D a p o p r o t e i n ( M c C o r d a n d Fridovich, 1969). It has been reported t h a t in yeast, Cu t r e a t m e n t induces enzymes related to the m e t a b o l i s m of oxygen, including S O D a n d catalase, a n d t h a t C u acts as a regulator o f biosynthesis o f these enzymes t h r o u g h a direct influence on gene expression (Galiazzo et al., 1988). However, Cu has been reported to inhibit the activity o f catalase in m a r i n e d i a t o m s (Florence a n d Stauber, 1986). T h e aquatic p l a n t L e m n a minor (duckweed) can be f o u n d growing in waters which m a y be c o n t a m i n a t e d with Cu such as near sewage outfalls (Hillman, 1961). In fact, it has been used in biological t r e a t m e n t o f wastewaters ( O r o n et al., 1988). Despite early interest in the use o f L e m n a in experimental work (Hillman, 1961) a n d m o r e recently in A q u a t i c Toxicology (Wang, 1990), published i n f o r m a t i o n on S O D in duckweed is lacking. T h e p u r p o s e o f this work is to identify the relationship (if any) between levels of systemic Cu a n d the activity of S O D in Lemna. L e m n a was g r o w n in wastewater c o n t a i n i n g different levels o f total C u a n d then S O D isozymes in the treated plants were identified by starch gel electrophoresis. S O D activity in the different Cu t r e a t m e n t s was m e a s u r e d by b o t h staining intensity following electrophoresis a n d reaction mixture assay. W h e n used together these two techniques are complimentary by p r o v i d i n g a picture a n d a measure, respectively, o f S O D activity. The study findings have application in the areas o f bioavailability o f Cu in wastewater, relating tissue levels o f C u to chronic effects a n d m o d e s o f action o f exogenous Cu.

METHODS

Experimental procedures

Domestic wastewater was selected as a growth medium because it occurs in waters inhabited by Lemna (Hillman, 1961), provides abundant growth (Buckley, 1993) and Lemna is used in the biological treatment of wastewaters (Oron et al., 1988). In addition, the chemical complexity of wastewater provides a multitude of ligands for Cu with the result that a variety of Cu species are formed that differ in their bioavailability (Buckley, 1993). Final effluent was collected by 24-h composite or by grab sampling from a plant treating primarily domestic wastewater by the activated-sludge process. The sample was dechlorinated, filtered through a 0.45 # m membrane and stored in polythene containers at 3.8-6.5°C for up to 14 days. The macronutrient content and other selected water quality factors in the collected samples is shown in Table 1. Lemna was cultured at 25°C in Hoagland's medium (ASTM, 1988) modified by the omission of sucrose, yeast extract and bactotryptone. Cultures were maintained axenic by treatment with HOC1 (Hillman, 1961). Wastewater was prepared for 7-day static-renewal experiments (ASTM, 1988) by adjusting the pH to 7.0 with HCI, aliquoting 100 ml to 250 ml beakers and adding CuSO4 to each aliquot to achieve an array of total Cu concentrations from 0.024 (control, no added Cu) to 0.220 rag/1. These concentrations were selected to span a growth response from no effect to slight inhibition (Buckley, 1993). The beakers containing 6 plants each were incubated at 25 + 0.5°C with a 24-h light period of 3100-3700 Lx and under a headspace

Table I. The content of selected nutrients, metals, total alkalinity, conductivity and total hardness of secondary-treated wastewater used in the experiments (mg/I or as noted) Sample Measurementa 1 2 NO3-N <0.01 0.09 (NH~ + NH4)-N 26.2 20.3 NH3-Nb 0.15 0. l 1 Total PO4 -3.0 T.R. PO~ 3.2 3.0 Cd <0.002 <0.003 Cr <0.005 <0.005 Cu 0.029 0.024 Ni <0.01 <0.01 Pb <0.03 <0.03 Zn 0.048 0.030 T. Alkalinityd 134 130 T. Hardnessd 67 66 Conductivitye 567 560 aValues (except NH3-N ) from treatment plant records. bUnionized ammonia based on pH 7.0. CTotal Reactive Phosphorus. dmg/l as CaCO3. e#mhos/cm.

with elevated level of CO 2 to maintain a pH of about 7.0 in the experimental solutions. Every 24-h all plants were transferred to freshly-prepared experimental solutions. Starch gel electrophoresis Lemna prepared for analysis by electrophoresis and enzyme staining was rinsed in deionized water (DW), l0 -2 M EDTA and DW, then blotted dry, weighed and processed as follows:

1. The plants were ground to fine powder in liquid nitrogen. 2. The powder was mixed with 2 volumes of ice cold 0.5M, pH7.0 phosphate buffer containing 5% (w:v) Polyvinylpolypyrrolidone (Sigma Chemical Co.). 3. The slurry was filtered through a No. 123 cotton filter sub (Schleicher & Schuell, Inc.) and collected in an ice-cold test tube. 4. The filtrate was centrifuged at 15,000g for 20 min at 4°C. 5. The supernatant was removed with a chilled pipet, placed in a cold cryotube and stored in liquid nitrogen. The Lemna extracts were thawed in an ice bath, mixed and diluted with 0.5 M pH 7.0 phosphate buffer to a uniform protein content and then 35-42/~1 was applied to wicks in horizontal starch gels and electrophoresis was conducted as outlined by Aebersold et al. (1987). The gel was then cut into slices about 1 mm thick and stained for activity of superoxide dismutase (SOD) using the agar overlay method (Aebersold et al., 1987). Egg albumin (J. T. Baker Co.) at 0.2 mg/wick was the negative-staining control. Bovine erythrocyte SOD (Sigma Chemical Co.) in 0.05M, pH7.8 phosphate buffer was applied at 12-75 U/wick as a standard and positive-staining control and for spiking the Lemna extract to check for staining interferences. Cu,Zn-SOD was differentiated .from MnSOD by including 3 mM NaCN in the staining mixture to inhibit its activity (Galiazzo et al., 1988). The protein content of the Lemna extracts was measured by a modification of the micro-Lowry method (Sigma Chemical Co.). Superoxide dismutase assay

SOD activity was measured by the inhibition of a superoxide radical-dependant reaction in which hydroxylamine is

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Sensitivity o f superoxide dismutase to Cu Table 2. Intensity of staining on a scale of I to 25 of SOD isozymes separated by electrophoresis of extracts from Lemna grown for 7 days in the indicated mean (range) concentrations of Cu in wastewater Zemna

Cu treatment (mg/L) 0.027 (0.005)

0.077 (0.0o5)

O.122 (0.0o5)

0.220 (0.0o5)

Cu content (#g/g) Isozymea Position in gel 1 2 2a 3 3a 4

(I~ (J) (M) (K, N) (P) (L)

43 (19-75) d

134 (62-239)

220 413 (103-397) (199-761)

10 3

10 3

10 3

3-5 3

1

1

1

1

1

1

1

1

Spikeb

Standard¢

10 3 5 15 20

5 15 25

al is fastest and 4 is slowest in migration toward the anode. bStandard added to the 0.027 mg/1 Cu treatment (control). cSOD purified from bovine erythrocytes. °95% Prediction Interval. eLetters correspond to positions of isozymes in Fig. 1. Typical condition for Electrophoresis: Lemna Extract: 3.4 mg protein/mL Each wick: 35 #1 of Lemna extract. Each wick: 0.12 mg Lemna protein. Standard and spike: 45 Units SOD/wick. Gel buffer: Tris/citric acid, pH 8.7. Electrode buffer: LiOH/Boric acid, pH 8.0. Run: 250 V, 5 h, 1-5°C. Stain method: agar overlay, 37°C, dark. oxidized to nitrite which is measured colorimetrically by absorbanee at 530rim (Elstner and Heupel, 1976). The reaction mixture contained: 1.0 ml of 65 m M PO4 buffer of p H 7.8; 0.5 ml o f Lemna extract diluted with PO 4 buffer; 0.1 ml of xanthine (0.7#mol); 0.1ml of hydroxylamine (1.0/~mol); 0 . 3 m l of X O D ( 6 5 # g protein) for a total volume o f 2.0 mi~ The mixture was incubated for 20 min at 25°C and the reaction was terminated by pipeting 1.0 ml into a test tube in an ice bath. C u , Z n - S O D was inhibited by the addition to the reaction mixture o f 0.02 ml o f N a C N (I/~mol) in place o f 0.02 ml of the buffer. Three nitrite blanks and standards and 4 SOD standards were included with every batch o f samples. Measurement o f total copper The wastewater and wastewater spiked with C u was prepared for determination o f total Cu according to standard E P A methodology (Anon, 1983) and then analyzed by ICP. D u e to a limited a m o u n t o f tissue available for analysis, the Cu content o f the Lemna analyzed for SOD activity was determined from total C u analysis o f plants grown in replicate experiments. F r o m the latter, a relationship between C u uptake (/tg/g, dry wt) and total C u in the experimental solutions (mg/l) was developed. The values for Cu uptake reported here were obtained from the resulting regression equation [P <0.001, R e ( a d j ) = 9 3 . 5 % ] using measured values for total Cu in the experimental solutions.

A B C D E F G IOOOOO OOC(X) ooooo OOOOO OOO OO OO J

. . . . . . . . . . . . . . . . . . . . . . . . . . . .

K .........................

.

co

.

.

M

OON

(DP L ........................... A. B. C. D.

Extract from Lemna grown in 0.024 m g total Cu/L (control, no Cu added) containing 39 ~g Cu/g. Extractfrom Lemna grown in 0.074 m g total Cu/L containing 130 Stg Cu/g. Extract from Lemna grown in 0.I 19 m g total Cu/L containing 215 ~tg Cu/g. Extract from Lemna grown in 0.217 m g total Cu/L containing 408 l.tgCu/g.

E.

Replicate for A.

F.

Replicate for B.

G.

Extract A spiked with S O D standard with 12 U of activity.

H.

S O D standard from bovine erythrocytes, 12 U of

I.

activity. Position of fastest migrating isozyme, isozyme 1 (Table 2).

Water quality measurements Total residual chlorine, measured amperometrically by the back titration method and other water quality factors were measured according to A P H A (1985).

H

J.

Position of isozyme 2.

Statistical analysis

K.

Position of isozyme 3.

The absorbance values obtained in the SOD assay were arcsin-transformed and the EC50, or concentration o f extract (containing active SOD) that diminishes the nitrite forming response by 50%, was obtained by simple linear regression followed by inverse prediction (Netter et aL, 1985). The EC50s for the bovine erythrocyte SOD standards were measured similarly and expressed in Units/ml. The EC50 o f the extract was expressed in Units (U) o f

L.

Position of slowest migrating isozyme, isozymc 4.

M.

Position of isozyme 2a of the SOD standard.

N.

Position of isozyme 3 of the SOD standard.

P.

Position of isozyme 3a of the SOD standard.

Fig. 1. Diagram of gel in Fig. 2 labeled to identify stained features in the gel.

2472

JAMESA. BUCKLEY

activity/ml by means of Parallel Slope Analysis (Eldred and Hoffert, 1981) and then converted to U/mg of total protein in the extract. RESULTS

(H), showing that the extract matrix did not inhibit SOD activity. Addition of 3 mM CN to the stain mixture (Fig. 3) complexes Cu 2÷ at the active site of Cu,Zn-SOD and thereby inactivates the enzyme as shown by the following reaction (Stiff, 1971):

Bioconcentration of copper

The concentration of Cu in Lemna averaged 43 (control), 134, 220 and 413/lg/g (dry wt) when grown for 7 days in wastewater containing total Cu concentrations that averaged 0.027 (control), 0.077, 0.122 and 0.220mg/I, respectively (Table 2). Steady-state uptake conditions were reached within 5 days (Buckley, 1993). Eleetrophoresis and superoxide dismutase stain

Extracts of the Lemna plants with different levels of systemic Cu produced the banding patterns of SOD diagramed in Fig. I and shown in Fig. 2. The symbols in Fig. 1 represent the positions of the isozymes of SOD that were revealed by staining reactions in the gel photographed in Fig. 2. Results from the stained gels indicate that extracts prepared from Lemna containing approximately 408 pg Cu/g exhibited a 50-70% reduction in activity of the fastest migrating SOD isozyme (row I, column D, Fig. 1) as estimated from staining intensity. Conversely, contents of 130 and 215/~g/g did not appear to diminish the staining intensity of that isozyme (row I, column B and C, Fig. 1), relative to the control (row I, column A, Fig. 1). The staining intensity of the spiked extract and a SOD standard were similar (G and H in Fig. 1) with respect to the isozymes present in the standard

Cu 2+ + 3CN- ~ Cu(CN)2 + I(CN)2.

(5)

As shown in Fig. 3, there was loss of activity in the J and L bands (Fig. 1) from all Lemna extracts. In addition, bands M, N, and P in the spike (G) and the SOD standard (H) show diminished stain intensity. The reduction in stain intensity upon addition of CN identifies these bands as isozymes of Cu,Zn-SOD. The remaining stained bands (I and K), which represent most of the activity in the gel, must be Mn-SOD which is the other form of SOD in higher plants and is resistant to treatment with CN. Table 2 summarizes the results of two experiments in which extracts were electrophoresed on starch gels in seven seperate runs (including the run shown in Fig. 2) and then stained for the presence of SOD. This process resolved four isozymes of SOD that had varying rates of migration as shown by the positions in the gel and varying intensities of staining as indicated by the numbers in the table. The numbers are on a scale of from 1 (least) to 25 (most) and show a reduction in stain intensity at the highest level of systemic Cu for the isozyme in position 1 which was identified as Mn-SOD. Egg albumin, which has no SOD activity, stained negative for SOD and showed three major and two minor bands under a general stain for protein.

Fig. 2. Starch gel stained for SOD following electrophoresis of extracts from Lemna grown in wastewater with added Cu (see Fig. 1). Direction of migration is up the page.

Sensitivity of superoxide dismutase to Cu

2473

Fig. 3. A slice of the same gel shown in Fig. 2. The gel was stained for SOD but with the addition of 3 mM CN- to the stain mixture.

Superoxide dismutase assay

The SOD assays were conducted to complement the results from electrophoresis and SOD-selective staining and to quantify the activity of SOD according to the concentration of Cu in the plants. Figure 4(a) shows a standard curve for bovine erythrocyte SOD with a range of activities from low (high values of absorbance at 530 n m (A530) to high (low values of A530) as measured by chemical assay. The more SOD present as units (U) of activity, the fewer superoxide radicals present to oxidize hydroxylamine to nitrite and less nitrite to be measured colorimetrically by absorbance at 530nm. Data points in the linear region of the curve were used to calculate an EC50 of 0.087 U/ml [Fig. 4(b)]. An extract was prepared from Lemna grown in Hoagland's culture medium. Measurement of the extract for SOD activity resulted in a dose-response curve [Fig. 5(a)] that resembled that for the SOD standard and a transformed curve that yielded and EC50 of 12 U/rag total protein [Fig. 5(b)] as calculated from the SOD standard. The dose-response curves for the standard (Fig. 4) and for Lemna extract (Fig. 5) are similar in shape indicating a similarity in response from SOD from either source. This favorable comparison and reports of use of the assay with other plants, including flax (Youngman et al., 1979) and peas (Elstner and Heupel, 1976), provided the rationale for its use with Lemna. The relative contribution of Cu,Zn-SOD and (presumed) Mn-SOD to the activity shown in Fig. 5

was determined by selecting 4 volumes of duckweed extract estimated to border the EC50. These were assayed with and without 0.5 mM CN in the reaction mixture. The EC50s for both treatments were again 12 U/mg total protein (Fig. 6) which indicates that the activity of Lemna SOD was not measurably affected by treatment with CN. This finding is consistent with the earlier conclusion with stained gels that Mn-SOD was probably the principle isozyme in Lemna SOD because treatment with CN resulted in the loss of only minor staining bands. Results from the analysis by starch gel electrophoresis and SOD staining of extracts from Lemna containing 39, 130, 215 and 408 pg Cu/g, shown in Fig. 2 were compared with measurements on the same extracts by the SOD assay (Table 3). For Lemna containing 408/1g Cu/g the mean EC50 was decreased to 14.2 U/mg total protein compared to the control of EC50 of 19.6 U/mg total protein. The SOD standards for the two assays had EC50s of 0.096 and 0.092 U/ml which showed good reproducibility. These results were consistent with findings from gel electrophoresis and SOD staining in which activity appeared to be reduced from 50-70% in extracts from Lemna grown in the highest treatment of Cu. However, the reduction in activity for these extracts as measured in the assay was only about 30%.

DISCUSSION

Domestic wastewater is a complex mixture of proteins, carbohydrates, lipids, humic substances plus

2474

JAMES A. BUCKLEY 0.30

m

(a) 0.20

0.25

_

0

0

0.15

0.20

<

0.15

i

0.10

0.10 0.05 0.05

I

0

I

I

-2

-1

(b)

1.5 -(b) 1.0

--

0

1.0 .=.

8<

.~ 0 . 5 <

0.5

o -2

-1

,

,

0

1

o

I -4

Log SOD (U/ml)

-3

Log extract (ml)

Fig. 4. (a) Measurement of activity of a SOD standard by inhibition of nitrite formation as measured by absorbance (As~o). (b) Transformation of the As30 values in (a) for calculation of an ECS0 (arrow).

Fig. 5. (a) Measurement of activity of SOD from Lemna by inhibition of nitrite formation as measured by absorbance (As30). (b) The A53o values were transformed for calculation of an EC50 (arrow).

many inorganic compounds. The bioavailability of metals in this matrix is determined by their chemical form, exchanges with call surface ligands and by

uptake and depuration rates. The chronic effects of metals can be assessed by an analysis of body residue and comparison with changes in important metabolic functions.

1.0 -

v

0.5 --

//

Table 3. The mean (_+ 1 SD, n = 2) EC50 expressed as units of SOD activity/rag of total protein (U/rag) in extracts prepared from Lemna grown for 7 days in the indicated concentrations of Cu added to wastewater Cu (mg/L)

.o <

0 -4

I

I

I

-3

-2

-1

Log extract (ml) Fig. 6. The ECS0 (arrow) for SOD measured in extracts prepared from Lemna in which 0.5 mM CN was absent ( O ) or present ( D ) in the reaction mixture.

Cu (#g/g)

0.024B 39 (I 7-66)c 0.074 130 (59-228) 0.119 215 (101-386) 0.217 408 (196-750) Standard l 2

EC50 (U/rag)

Regression coefficienff

Correlation coefficienP

19.6 _+0.6 23.0_+3.2 17.9_+2.7 14.2b + 1.3 U/ml 0.096 0.092

0.529,0.490 0.499,0.402 0.539,0.496 0.552, 0.478

0.996,0.990 0.994,0.989 0.997,0.999 0.999,0.994

0.617

0.997 0.490

0.984

aControl, intrinsic Cu, no added Cu. Values correspond to the Cu treatments for extracts A to D in Fig. 1. bSignificantly (P < 0.05) different vs control. CSIope of regression line used in parallel slope analysis. dFrom EC50 calculations. c95% prediction interval.

Sensitivity of superoxide dismutase to Cu In this study, when Cu uptake in Lemna was 413 pg/g (dry wt), the stain intensity of the fastest migrating isozyme (multiple molecular form of an enzyme in a single organism), apparently Mn-SOD, was diminished by a factor of two or three below that of the other Cu treatments while the activity of Cu,Zn-SOD was unaffected (Table 2). Mn-SOD could be decreased because it is not being induced by its substrate, the superoxide radical (Fridovich, 1978), which is being removed by Cu-containing SOD mimics. However, to be consistent, it seems that Cu,Zn-SOD should likewise be lower. Alternatively, the arrangement of SODs in the plant cell may facilitate differential effects on the two SODs. Mn-SOD occurs in the mitochondria and Cu,ZnSOD in the cytosol and chloroplast stroma (Elstner, 1987), and bioavailable forms of Cu may unevenly target the mitochondrial expression of Mn-SOD. The latter would be most likely with lipid-soluble Cu complexes. The action could be a direct effect on mitochondrial D N A through destabilization and cross-linking of strands by Cu (Sissoeff et al., 1976). The effect may also be indirect, resulting from mitochondrial membrane peroxidation leading to inhibition of oxidative phosphorylation in ATP synthesis (Viarengo, 1985). Exposure of yeast to 0.025-6.35 mg Cu/l had the opposite effect on SOD isozymes. The activity of Cu,Zn-SOD was increased, whereas activity of Mn-SOD was unaffected (Galiazzo et al., 1988). It was concluded that Cu had a general inducing effect on enzymes and activation of an apoprotein was not involved. As stated above, increasing levels of Cu treatment failed to increase Cu,Zn-SOD activity in Lemna (Fig. 2) either through enzyme induction or activation of apo-SOD. In both cases this could be due to failure of forms of systemic Cu in the plant to be effective, or in the second case, lack of a significant pool of apo-SOD. The decrease in SOD activity may occur through inactivation of SOD or formation of nonfuctional SOD molecules at the level of gene expression or beyond. The failure of a mutant type of yeast to form functional Cu,Zn-SOD has been attributed to the lack of Cu at the active site, perhaps due to conformational changes in the protein resulting from a mutant polypeptide (Chang et al., 1991). The effect of excess Cu on genetic expression or Cu kinetics in Lemna apparently has not been investigated to that level of detail, however, Cu and other metals have been hypothesized to express toxicity when they exceed homeostatic controls, such as sequestering by phytochelatins (Reddy and Prasad, 1990) and binding to enzymes (Lepp, 1981) or to D N A (Sissoeff et al., 1976). The reduction in activity of SOD with increased systemic Cu may also result from fewer superoxide anions formed as photoexcitation of chlorophyl diminishes in concert with growth or a diminished requirement for SOD owing to the presence of Cu 2+

2475

and other redox-active molecular forms of Cu that mimic SOD. The latter interpretation raises the possibility that there are bioavailable redox-active Cu complexes in domestic wastewater that can mimic SOD. The endogenous levels of total Cu typical of secondary-treated wastewater, such as the 0.0240.029 mg/l measured here (Table 1), are below the range (0.122-0.220 mg/1) eliciting a chronic response from Lemna in this work. However, as Lemna finds use in treatment of wastes with higher levels of Cu, and higher levels of systemic Cu occur, the likelihood increases for chronic effects such as inhibition of SOD activity. When extracts prepared from Lemna grown in four concentrations of Cu in wastewater were electrophoresed and stained for SOD activity, four isozymes appeared in each extract (Fig. 2). The four were tentatively identified by cyanide-sensitivity testing as two isozymes each of Cu,Zn-SOD and MnSOD. No reports of SOD isozymes in Lemna were available for comparison to these findings. However, rice leaves and rice seed embryos contain four isozymes of Cu,Zn-SOD and two isozymes of MnSOD (Kanematsu and Asada, 1989), and the bluegreen alga Spirulina and spinach contain two Mn-SOD and two Cu,Zn-SOD isozymes, respectively (Lumsden and Hall, 1974). These reports support the prospect that Lemna has multiple isozymes of the Cu,Zn-SOD and Mn-SOD enzymes common to higher plants. In this work, the activity of SOD extracted from Lemna grown in defined medium was 12 U/mg total protein as defined by the bovine erythrocyte SOD standard. No published values for the activity of Lemna SOD were available for comparison. However, estimates of expected values for Lemna can be made indirectly: 1. The amount of SOD in plants has been reported to be 2.5% of the total protein content (Leshem, 1988) and one unit of activity was contained in about 2 gg of SOD purified from green peas (Elstner and Heupel, 1976). Therefore: 1 U/0.002 mg SOD * 2.5 mg SOD/100 mg total protein = 13 U/mg total protein. 2. SOD is present in most tissues at 10-SM (Fridovich, 1978), the ground tissue was diluted 1 + 2 (0.33) with buffer, the molecular weight of green pea SOD is about 31,500 da (Sawada et al., 1972), with activity of 500 U/rag SOD protein (Elstner and Heupel, 1976) and the protein content of the Lemna extract was 3.7 mg/ml (this work). Therefore: 0.33 • 10-Smol/l extract* 31,500g/mol = 0.104g SOD/I extract. 0.104mg SOD/ml e x t r a c t , 500 U/rag SOD • 1 ml/3.7 mg total protein = 14 U/mg total protein. Given the broad assumptions on which they are based, the estimates of 13 and 14 U/mg total protein

JAMESA. BUCKLEY

2476

are close to the 12 U / m g total protein measured here for L e m n a grown in defined medium. These estimates support the reported 12 U / m g total protein.

REFERENCES

Aebersold P. B., Winans G. A., Teen D. J., Miner G. B. and Utter F. M. (1987). Manual for starch gel electrophoresis: a method for the detection of genetic variation. National Oceanic and Atmospheric Administration Technical Report, National Marine Fisheries Service, No. 61. Anon (1983) Methods for chemical analysis of water and wastes. Environmental Protection Agency, 600/4-79-020. Revised March. APHA (1985) Stan&~rd Methods for the Examination of Water and Wastewater, 16th edn. American Public Health Association, American Water Works Association and Water Pollution Control Federation, Washington, D.C. ASTM (1988) American Society for Testing and Materials, Proposed new standard guide for conducting static toxicity tests with duckweed. Draft No. 7. Brigelius R., Spottl R., Bors W., Lengfelder E., Saran M. and Weser U. (1974). Superoxide dismutase activity of low molecular weight Cu2+-chelates studied by pulse radiolysis. FEBS Lett 47, 72-75. Buckley J. A. (1993) Biologically available Cu in wastewater: Estimation by the response of the aquatic macrophyte Lemna (Duckweeed). Ph.D. dissertation, Univ. of Washington, Seattle, Wash. Chang E. C., Crawford B. F., Hong Z., Bilinski T. and Kosman D. J. (1991). Genetic and biochemical characterization of Cu, Zn superoxide dismutase in Saccharomyces cerevisiae. J. biol. Chem. 266, 4417-4424. Czapski G. and Goldstein S. (1990). Superoxide scavengers and SOD or SOD mimics. Adv. exp. Med. Biol. 264, 45-50. Eldred G. E. and Hoffert J. R. (1981) A test for endogenous interferences in superoxide dismutase assays. Anal. Biochem. 110, 137-143. Elstner E. F. (1987) Metabolism of activated oxygen species. In The Biochemistry o f Plants: A Comprehensive Treatise. (Edited by Stumpf P. K. and Conn E. E.), Vol. II., Biochemistry of Metabolism (Edited by Davis D. D.), Academic Press, New York. Elstner E. F. and Heupel A. (1976) Inhibition of nitrite formation from hydroxylammoniumchloride: a simple assay for superoxide dismutase. Anal. Biochem. 70, 616-620. Florence T. M. and Stauber J. L. (1986) Toxicity of copper complexes to the marine diatom Nitzsehia closterium. Aquat. Toxicol. 8, 11-26. Fridovich I. (1978) The biology of oxygen radicals. Science, N.Y. 201, 875-880.

Galiazzo F., Schiesser A. and Rotilio G. (1988) Oxygenindependent induction of enzyme activities related to oxygen metabolism in yeast by copper. Biochimica biophysica Acta 965, 46-51. Hillman W. S. (1961) The Lemnaceae, or duckweeds: A review of the descriptive and experimental literature. Botan. Rev. 27, 221-287. Kanematsu S. and Asada K. (1989) CuZn-superoxide dismutases in rice: Occurrence of an active, monomeric enzyme and two types of isozyme in leaf and nonphotosynthetic tissues. Plant Cell PhysioL 30, 381-391. Lapluye G. (1990) SOD mimicking properties of copper(lI) complexes: Health side effects. In Emerit et al. eds., Antioxidants in Therapy and Preventive Medicine. (Edited by Emerit et al.,) Plenum Press, New York. pp. 59-68. Lepp N. W. (1981) Copper. In Lepp N. W. ed., Effect of heavy metal pollution on plants., Vol. 1., Effects of Trace Metals on Plant Function. (Edited by Lepp N. W.), pp. 111-143. Applied Science Publishers, London. Leshem Y. Y. (1988) Plant senescence processes and free radicals. Free rad. Biol. Med. 5, 39-49. Lumsden J. and Hall D. O. (1974) Soluble and membranebound superoxide dismutases in a blue-green alga (Spirulina) and spinach. Biochem. biophys. Res. Commun. 58, 35-41. McCord J. M. and Fridovich I. (1969) Superoxide dismutase. An enzymatic function for erythrocuprein (hemocuprein). J. biol. Chem. 244, 6049-6055, Netter J., Wasserman W. and Kutner M. H. (1985) Applied Linear Statistical Models., 2nd edn. Irwin, Homewood, IU. Oron G., De-Vegt A. and Porath D. (1988) Nitrogen removal and conversion by duckweed grown on wastewater. Wat. Res. 22, 179-184. Reddy G. N. and Prasad M. N. V. (1990) Heavy metalbinding proteins/peptides: occurrence, structure, synthesis and functions. A review. Envir. exp. Bot. 30, 251-264. Sandmann G. and Boger P. (1980) Copper-mediated lipid peroxidati0n processes in photosynthetic membranes. Plant Physiol. 66, 797-800. Sawada S., Ohyama T. and Yamazaki I. (1972) Preparation and physicochemical properties of green pea superoxide dismutase. Biochim. biophys. Acta 268, 305-312. Sissoeff I., Grisvard J. and Guille E. (1976) Studies on metal ions-DNA interactions: Specific behavior of reiterative DNA sequences. Prog. Biophys. Molec. Biol. 31,165-199. Stiff M. J. (1971) The chemical states of copper in polluted fresh water and a scheme to differentiate them. Wat. Res. 5, 585-599. Viarengo A. (1985) Biochemical effects of trace metals. Mar. Pollut. Bull. 16, 153-158. Wang W. (1990) Literature review on duckweed toxicity testing. Envir. Res. 52, 7-22. Youngman R. J., Dodge A. D., Lengfelder E. and Elstner E. F. (1979) Inhibition of paraquat phytotoxity by a novel copper chelate with superoxide dismutating activity. Experientia 35, 1295-1296.