Aquatic Toxicology, 12 (1988) 157-170 157 Elsevier

AQT 00269

Oxidant-mediated biochemical effects of paraquat in the ribbed mussel, Geukensia demissa

Richard J. Wenning*, Richard T. Di Giulio and Evan P. Gallagher

Ecotoxicology Laboratory, School of Forestry and Environmental Studies, Duke University, Durham, North Carolina, U.S.A.

(Received 30 March 1987; revision received 10 September 1987; accepted 11 September 1987)

The role of reduction/oxidation cycling in the toxic effects of the bipyridyl herbicide paraquat was ex- amined in the hepatopancreas of the ribbed mussel, Geukensia demissa. In vitro studies indicated a dose- dependent increase in the rate of superoxide anion (O2-) generation, as measured by the reduction of cytochrome c, in the microsomal fraction of the hepatopancreas. The highest concentration of paraquat employed (4 mM) elicited an 81°70 increase in 02- production above controls, which was inhibited by the addition of superoxide dismutase (SOD). For in vivo studies, a single application of paraquat (0.5, 1.0, or 2.0 mM) was added to aerated salt-water aquaria containing mussels. Biochemical analyses of antioxidant enzymes, reduced glutathione (GSH), and lipid peroxidation were performed in hepatopan- creatic tissue of mussels after exposures of 6, 12, 24, and 36 h. Catalase activities were significantly elevated (/9<0.05) in all treatment groups above controls at 6 h, in the 0.5 and 1.0 mM treatment groups at 12 h, and in the 0.5 mM group at 36 h. Total SOD activities were nearly doubled from controls in each treatment group at 12 h. However, SOD activities in exposed mussels were generally similar to or below controls at other times. GSH concentrations were generally higher in exposed mussels than in con- trois at all exposure periods. Lipid peroxidation in most treatment groups was significantly higher than in controls at 6, 12, and 24 h. The results support the hypotheses that these bivalves can activate redox cycling compounds and demonstrate in vivo responses typical of oxidative stress as observed in other organisms.

Key words: Paraquat; Lipid peroxidation; Antioxidant enzymes; Glutathione; Superoxide anion produc- tion; Mussels

* Present address: EnviroLogic Data, 4 Milk Street, Portland, ME 04101, U.S.A.

Correspondence to: R.T. DiGiulio, Ecotoxicology Laboratory, School of Forestry and Environmental Studies, Duke University, Durham, NC 27706, U.S.A.

0166-445X/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

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INTRODUCTION

It is becoming increasingly evident that a diverse array of compounds can undergo reduction/oxidation (redox) cycling in cells and that the activated oxygen species (such as superoxide anion, hydrogen peroxide, and hydroxyl radical) associated with this activity are often important in the toxicity of these compounds (Hassan and Fridovich, 1979; Bus, 1982; Mason and Chignell, 1982). Many redox cycling com- pounds, such as phenolics, quinones, nitroaromatics, azo dyes, and bipyridilium ca- tions comprise important contaminants in aquatic systems. This study was undertaken in order to examine activated oxygen, or 'free radical', mediated effects of a model redox cycling compound, paraquat, in the ribbed mussel (Geukensia demissa). While bivalves have been used extensively as monitors for environmental pollution based upon tissue residues (Goldberg et al., 1978; Popham and D'Auria, 1983; Rosas et al., 1983), relatively little work has been reported on biochemical, particularly free radical-mediated, responses of these organisms to pollutants.

Paraquat (methyl viologen, 1,1-dimethyl-4,4'-bipyridilium ion) is a widely used, broad spectrum, herbicide and perhaps the most studied redox cycling compound. The mechanism of paraquat poisoning in animals is generally thought to be an ox- ygen free radical-mediated toxicity that is analogous to the herbicidal mode of ac- tion (Halliwell and Gutteridge, 1985). Microsomal fractions from several mammalian tissues, particularly lung and liver, have been observed to reduce para- quat in the presence of sufficient reducing equivalents, notably NADPH; the resulting paraquat radical can then react with molecular oxygen (O2) to generate the superoxide radical (O2-) and regenerate an oxidized paraquat molecule which can repeat this cycle (Gage, 1968; Bus et al., 1974, 1976a). The importance of oxygen free radicals, generated by the redox cycling of paraquat, is supported by several lines of evidence. For example, the toxicity of paraquat can be increased by ex- posure to elevated oxygen tensions (Fisher et al., 1973) and decreased by the ad- ministration of superoxide dismutase (SOD) (Autor, 1974; Trush et al., 1982). In addition, several studies have demonstrated the ability of paraquat to induce ac- tivities of antioxidant enzymes, including SOD in E. coli (Hassan and Fridovich, 1977) and maize (Matters and Scandalios, 1986), glutathione peroxidase in houseflies (Allen et al., 1984), and catalase and glutathione peroxidase in mice (Matkovics et al., 1980). Furthermore, Frank (1981) demonstrated that pre- exposure of rats to elevated concentrations of O2 resulted in increased activities of SOD, catalase, and glutathione peroxidase in lung tissue; such pre-exposed rats also demonstrated markedly reduced sensitivity to an acute dosage of paraquat. Current- ly, there is debate concerning the primary locus of the cytotoxicity produced by paraquat-generated free radicals. Peroxidation of membrane lipids and oxidations of key reductants such as NADPH and reduced glutathione (GSH) have been the principal subjects of this debate (Bus et al., 1974, 1976b; Talcott et al., 1979; Shu et al., 1979; Stefen et al., 1980; Brigelius et al., 1981; Keeling and Smith, 1982;

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Trush et al., 1982; Sandy et al., 1986). However, both of these effects are consistent with the well-established ability of paraquat to redox cycle in the presence of 02.

Among piscine species, paraquat was shown to enhance the activation of antioxi- dant enzymes and to increase lipid peroxidation in erythrocytes of two carp species (Cyprinus carpio and Carrasius carrasius) by Gabryelak and Klekot (1985). Matkovics et al. (1984) observed increased activities of SOD and glutathione perox- idase and increased lipid peroxidation in livers of carp (Hypophthalmichthys molitrix) exposed to 0.1 ppm paraquat for 24 hours. At higher paraquat concentra- tions (1.0 and 10.0 ppm), these responses were generally diminished.

We are unaware of any reports concerning free radical-mediated responses of bivalve molluscs to paraquat or other redox cycling compounds. However, a few studies have indicated the presence of an antioxidant system in bivalves. Blum and Fridovich (1984) measured SOD, catalase, and glutathione peroxidase in the large clam (Calyptogena magnifica) and the quahog (Mercenaria mercenaria). The pro- perties of glutathione reductase from the hepatopancreas of the blue mussel (Mytilus edulis) were examined by Ramos-Martinez et al. (1983). And decades ago, Marks and Fox (1937) quantified the activity and nature of catalase in the California mussel (Mytilus californianus). Together these studies suggest that bivalves are capable of biochemically responding to paraquat-generated free radicals. The objec- tives of this study were to determine the ability of paraquat to generate O2- in the ribbed mussel (Geukensia demissa) in vitro and to elicit responses in vivo consistent with oxygen free radical generation. These responses included inductions of antioxi- dant enzymes (SOD and catalase), alterations in GSH concentrations, and enhance- ment of lipid peroxidation.

MATERIALS AND METHODS

Chemicals Chemicals, including paraquat, horse heart cytochrome c, bovine liver SOD, dithiothreitol (DTT), phenylmethylsulphonyl fluoride (PMSF), and n- butanol pyridine, were obtained from Sigma Chemical Co. (St. Louis, MO, U.S.A.). Reduced ~-nicotinamide adenine dinucleotide phosphate (NADPH) and xanthine oxidase were obtained from U.S. Biochemical Co. (Cleveland, OH, U.S.A.).

Animals Ribbed mussels (G. demissa) between 6 and 8 cm in shell length were collected between the communities of Beaufort and Atlantic along the central North Carolina coast. Mussels were acclimated for at least 4 days prior to experimentation in 7-1 static, aerated aquaria with 30 parts per thousand (ppt) artificial seawater (In- stant Ocean) at 22°C. The shell surfaces were cleaned of muddy debris and the bisal hairs trimmed prior to paraquat exposures.

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In vitro study

Preparation ofmicrosomes For the in vitro analysis of superoxide production in hepatopancreas microsomes by paraquat , untreated mussels were sacrificed and

the hepatopancreas excised. The crystalline style was removed from the organ im- mediately upon dissection. The hepatopancreae of two individuals were pooled for each sample without regard to sex. Tissues were homogenized in a 1:4 w/v ratio with a teflon pestle-type motorized tissue grinder (A.C. Thomas Co., Philadelphia, PA, U.S.A.) in ice-cold 20 mM Tris-HCl , (pH 7.6), containing 1 mM EDTA, 1 mM DTT, 0.5 M sucrose, 0.15 M KCI, and 100 txM PMSF (added to the buffer f rom a stock solution in isopropanol just before homogenization to prevent its aqueous inactivation; James, 1978) as described by Livingstone and Farrar (1984). The microsomal fraction was obtained by differential centrifugation of the homogenate at 500 x g for 10 min (nuclear), 12000 x g for 30 min (heavy mitochondrial), and 100000 x g for 90 min (microsomal). The microsomal pellet was washed in homogenizing buffer , spun at 100000 x g for 90 min, and resuspended in 20 mM Tris-HC1 buffer (pH 7.6) containing 1 mM EDTA, 1 mM DTT, and 2007o w/v glycerol to yield a concentration of between 5 and 10 mg prote in/ml .

Biochemical assays Cytochrome c reduction in the presence of 0.5, 1.0, 2.0, or 4.0 mM paraquat , which suggests 0 2 - production, was assayed at room temperature in a reaction mixture of 0.175 mM N A D P H , 0.05 mM KCN, 0.125 mM horse heart cytochrome c, and 27 #g microsomal protein in 0.2 M potassium phosphate buffer (pH 7.4) by a modification of Stegeman (1985). The inhibition by SOD of paraquat-st imulated cytochrome c reduction, indicative of O z - generation, was determined by the addition of 0.4-10.0/~g SOD (from a stock solution in dis- tilled, deionized water) to the reaction mixture.

In vivo study

Range finding test for exposures Six individuals were placed in each of eight aerated, 2-1 beakers with 30 ppt artificial seawater. A single dose of 0.4, 1.6, 4.0, 6.4, or 12 mM paraquat , f rom a stock solution in distilled, deionized water, was ad- ministered to each beaker and survival was noted over a one-week period. Failure to close the shell prompt ly upon prodding was considered an indication of mortali- ty. One hundred percent mortali ty was observed in treatment groups exposed to 6.4 m M and 12.0 mM paraquat after one week; mortali ty was not observed in groups exposed to 0.4 mM, 1.6 mM, or 4.0 mM paraquat . On the basis of this test, para- quat concentrations below lethal concentrations were selected for the subsequent in vivo biochemical experiment.

Exposure and Tissue Preparation Sublethal paraquat concentrations were added to three aquaria to achieve initial concentrations of 0.5, 1.0, and 2.0 mM. A fourth aquarium was not dosed and served as a control. At 6, 12, 24, and 36 h

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post-exposure, eight mussels were removed from each treatment and the hepatopan- creae excised immediately. The crystalline style was removed from the organ im- mediately upon dissection. The hepatopancreae of two mussels were pooled for each sample without regard to sex. Tissues were homogenized with a teflon pestle-type motorized tissue grinder in a 1:3 w/v ratio of ice-cold M150 phosphate buffer (pH 7.4). GSH and malondialdehyde were measured immediately after sacrifice. Sample aliquots for determinations of protein concentrations, and activities of SOD and catalase were stored at -70°C and assayed not later than 3 days after sacrifice. Paraquat concentrations in the aquaria were measured at each sampling period col- orimetrically as described by Hassan (1984).

Biochemical assays Spectrophotometric measurements were determined with a Shimadzu (Kyoto, Japan) model UV-260 spectrophotometer. All of the assays were carried out at room temperature. Protein content was determined by the method of Lowry et al. (1951). Catalase activity was determined in the cytosolic fraction at 240 nm according to the method of Luck (1963). GSH levels were determined in whole homogenates treated with 4% sulfosalicyclic acid by the method of Jollow et al. (1974). SOD activity was determined by the xanthine-xanthine oxidase 02 - generating system described by McCord and Fridovich (1969) based upon a defini- tion of one unit of SOD as the amount of enzyme that inhibits the reduction of cytochrome c by 50°70. Lipid peroxidation was determined in whole homogenates by the method of Tanizawa et al. (1981) and the fluorescent intensity was measured with a Perkin-Elmer (Oak Brook, IL, U.S.A.) LS-3 spectrofluorometer with excita- tion and emission wavelengths of 515 nm and 553 nm, respectively.

Statistics For the in vitro analysis, differences among treatments were assessed by multifactor analysis using least significant difference (LSD) testing. For the in vivo analysis, the differences among treatments within an exposure period and among exposure periods within a treatment group were assessed with a two-way ANOVA and by multifactor analysis using LSD testing for multiple comparisons. Results are expressed as the mean _ S.E.M. The Statgraphic statistical software system for the IBM computer was employed for these analyses (STSC Inc., Rockville, MD, U.S.A.).

RESULTS

In vitro study

Paraquat-stimulated cytochrome c reduction The relative in vitro rate of cytochrome c reduction in the microsomal fraction of the mussel hepatopancreas at different doses of paraquat is shown in Table I. These results demonstrate that the rate of cytochrome c reduction, which suggests the generation of superoxide in hepatopancreas microsomes supplemented with NADPH, is significantly enhanced (/7<0.001) by paraquat in a dose-dependent manner. The addition of SOD to the

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TABLE 1

In vitro reduction of cytochrome c by various concentrations of

the hepatopancreas of the mussel, Modiolus demissus ~. paraquat in the microsomal fraction of

Concentrat ion of paraquat mm cyt. c r educed /min . /mg protein b

0 3.175 + 0.051

0.5 mM 3.998 _+ 0.106

1.0 mM 4.615 + 0.206

2.0 mM 5.232 + 0.128

4.0 mM 5.761 _+ 0.078

Assayed at 25°C in a 1 ml reaction mixture of 0.175 mM NADPH, 0.05 mM KCN, 0.125 mM

cytochrome c, and 27 #g microsomal protein in 0.2 M KPO4 buffer (pH 7.4). Values are mean _+ SEM

(n=4) .

b Each mean is significantly different from all other means (p<0.001) .

TABLE II

The effect of superoxide dismutase on cytochrome c reduction in the presence of 4 mM paraquat in the

microsomal fraction of the hepatopancreas of the mussel, Modiolus demissus ~.

Treatment mm cyt. c r educed /min /mg protein

0 mM paraquat 2.994 +_ 0.091 b

4 mM paraquat 6.107 + 0.141 c

+ 10 #g SOD 1.698 + 0.091 a

+ 1 /zg SOD 2.315 + 0.075 e

+ 0.4 #g SOD 2.888 + 0.166 b

+ 10 #g boiled SOD 5.578 + 0.253 f

a Assayed at 25°C in a 1 ml reaction mixture of 0.175 mM NADPH, 0.05 mM KCN, 0.125 mM

cytochrome c, and 27 #g microsomal protein in 0.2 M KPO4 buffer (pH 7.4). Values are mean + SEM

(n = 4).

b,c,d.e,f Means not sharing the same letter are significantly different from each other (p<0 .05) .

reaction mixture significantly inhibited (t7 < 0.05) the reduction of cytochrome c in a dose-dependent manner, confirming the presence of an in vitro paraquat- stimulated 0 2 - generating system in the mussel (Table II). As expected, boiled SOD, which represents the inactivated enzyme, had only a marginal effect on cytochrome c reduction that was probably the result of the incomplete deactivation of the enzyme by heat.

In vivo study

Residualparaquat The concentration of paraquat measured in each treatment aquarium at each sampling period dropped by an average of 10°70 at 12 h, 25°7o at 24 h, and 56°70 at 36 h from the initial concentration applied to each aquarium (Table III).

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Superoxide dismutase activity At 12 h, SOD activities were significantly elevated (p < 0.05) above controls in all treatment groups (Table IV). However, SOD activities at this time were not induced in a dose-dependent manner; 0.5 mM para- quat induced the greatest enzyme activity followed distantly, in order, by 1.0 and 2.0 mM. Although minor changes were observed at times other than 12 h, SOD ac- tivities in the treatment groups generally followed controls. Except at 24 h, SOD ac- tivity in the 2.0 mM group was never higher than either the 0.5 mM or 1.0 mM

TABLE III

Concentrations (mM) of paraquat measured in treatment aquaria at each sampling period a.

Initial (paraquat) Hours after initial dose

6 12 24 36

0.5 mM 0.46 ± 0.04 0.44 +_ 0.03 0.34 +_ 0.03 0.20 + 0.03

1.0 mM 0.90 + 0.04 0.81 + 0.02 0.72 ± 0.10 0.40 + 0.04

2.0 mM 1.84 + 0.05 1.76 + 0.03 1.35 + 0.04 0.80 +_ 0.03

a The concentration of reduced paraquat was determined spectrophotometrically from a standard curve

in 1 ml samples bubbled with nitrogen and reduced with sodium dithionite at 605 nm. Values are means

+_ S E M ( n = 3 ) .

TABLE IV

Effects of various initial concentrations of paraquat and durations of exposure on the activities of

superoxide dismutase and catalase in vivo in the hepatopancreas of the mussel, Geukensia demissa. Mean

± SEM (n).

Initial (paraquat) Hours after exposure

in aquaria 6 12 24 36

Catalase (U/mg protein) b

control 24.10 ± 2.31(3)* 14.60 +_ 0.24(3) 12.34 + 0.95(3) 14.30 +_ 1.33(2)

0.5 mM 29.28 + 1.48(4) a* 24.30 + 1.81(3) a* 13.14 ± 0.37(2)* 19.27 _+ 0.34(2) a*

1.0 mM 40.24 _+ 4.04(4) a* 23.20 + 1.53(4) a* 11.22 ± 1.43(3)* 16.71 + 2.90(3)*

2.0 mM 42.23 ± 12.45(4) a'* 14.38 ± 1.50(4) 10.03 _ 1.94(3)* 14.22 _+ 2.19(3)

Superoxide dismutase (U/mg protein) c

control 16.73 + 0.90(4) 12.60 ± 3.63(2)* 15.88 +_ 1.53 (3) 18.43 ±

1.042(2)*

0.5 mM 16.59 + 1.26(4) 39.49 + 4.16(2) a* 14.18 + 0.07(2) a* 17.65 _+ 1.09(3)

1.0 mM 18.07 _+ 1.21(4) a* 24.55 ___ 7.34(2) a* 12.52 + 0.54(3) a* 16.21 + 0.79(2) a*

2.0 mM 13.54 ± 0.72(4) a 21.45 _+ 2.73(3) a* 14.13 + 0.26(32) a 14.91 ± 0.82(3) a

a Statistically significant from control within exposure period (,o<0.05).

* Statistically significant from other exposure periods within treatment group Lo<0.05).

b Catalase activity was determined by Luck (1963); 1 unit is defined as the amount of enzyme, in the

presence of HzO2, required for a decrease in the optical density from 0.450 to 0.400 at 240 nm.

c SOD activity was determined by McCord and Fridovich (1969); 1 unit is defined as the amount of en-

zyme that inhibits the reduction of cytochrome c by 50°70.

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g r o u p s . A l t h o u g h s ign i f i c an t d i f f e r e n c e s due to t i m e were o b s e r v e d , these d i f -

f e r ences , excep t at 12 h, were r e l a t ive ly m i n o r .

Catalase activity C a t a l a s e ac t i v i t y d e m o n s t r a t e d s i gn i f i c an t d i f f e r e n c e s

(t7 < 0 .05) due to b o t h t r e a t m e n t a n d t i m e o f e x p o s u r e at 6 h ( T a b l e IV) . C a t a l a s e

ac t iv i t i e s in t he 0.5 m M t r e a t m e n t a t 12 h a n d 36 h a n d in t he 1.0 m M t r e a t m e n t

a t 12 h w e r e a lso s i g n i f i c a n t l y h i g h e r t h a n the ac t i v i t y in t he c o n t r o l g r o u p . E x c e p t

a t 6 h, ca t a l a se ac t iv i ty in t he h ighes t t r e a t m e n t g r o u p (2.0 m M ) was neve r

s i g n i f i c a n t l y d i f f e r e n t f r o m the c o n t r o l g r o u p o r h i g h e r t h a n the l o w e r t r e a t m e n t

g r o u p s .

Reduced glutathione levels A l t h o u g h all t r e a t m e n t m e a n s were g e n e r a l l y

g r e a t e r t h a n c o n t r o l m e a n s at all t i m e po in t s , n o c ons i s t en t d o s e - r e s p o n s e r e l a t i o n -

sh ip was o b s e r v e d a m o n g t r e a t m e n t g r o u p s ( T a b l e V). A m o n g all t r e a t m e n t g r o u p s ,

t he l o w e s t m e a n G S H c o n c e n t r a t i o n s were o b s e r v e d at 24 h.

Lipid peroxidation L i p i d p e r o x i d a t i o n , as m e a s u r e d by the c o n c e n t r a t i o n o f

m a l o n d i a l d e h y d e , was s i gn i f i c an t l y h i g h e r (p < 0 .05) in t i ssue o f musse l s e x p o s e d to

1.0 a n d 2 .0 m M p a r a q u a t at 6, 12, a n d 24 h a f t e r in i t ia l e x p o s u r e t h a n in t he c o n t r o l

g r o u p s (Tab le V). L i p i d p e r o x i d a t i o n in t r e a t m e n t g r o u p s t e n d e d to be h i g h e r t h a n

c o n t r o l s at all t i m e po in t s , bu t , e x c e p t i n g the 6 h e x p o s u r e , d i f f e r e n c e s a m o n g t r ea t -

m e n t g r o u p s were r e l a t ive ly m i n o r .

TABLE V

Effects of various initial concentrations of paraquat and durations of exposure on reduced glutathione concentrations and lipid peroxidation in vivo in the hepatopancreas of the mussel, Modiolus demissus. Mean + SEM.

Initial (paraquat) Hours after exposure

in aquarium 6 12 24 36

Glutathione (nmol reduced glutathione/mg tissue) b

control 1882 + 191(4) 1796 + 291(4) 855 +_ 171(3)* 1792 + 168(3) 0.5 mM 1961 -!-_ 293(4) 2264 _+ 103(4) a* 1890 ___ 168(3) a 2055 +_ 98(3) a 1.0 mM 1959 _+ 155(4) 2099+ 244(3) a* 1683 + 153(3) a* 1887 + 270(3) 2.0 mM 2384 _+ 162(4) a* 1812 + 72(4)* 1275 ___ 104(3) a* 2032 + 221(2) a*

Lipid Peroxidation (nmol malondialdehyde/mg tissue) c

control 0.121 + 0.014(4)* 0.087 ___ 0.011(3) 0.100 + 0.005(3) 0.080+0.007(2) 0.5 mM 0.128 + 0.023(4)* 0.085 _+ 0.013(3)* 0.190 _+ 0.001(2) ~* 0.108+0.018(3) a* 1.0 mM 0.148 _+ 0.008(3) ~ 0.141 _+ 0.003(3) a 0.183 _+ 0.010(3) a* 0.085+0.007(3)* 2.0 mM 0.704 _+ 0.046(4) ~* 0.134 _+ 0.013(3) a* 0.150 + 0.005(3) a* 0.094_+0.018(3)*

a Statistically significant from control within exposure period at p<0.05. * Statistically significant from other exposure periods within treatment group at p<0.05. b GSH determined by Jollow et al. (1974). c Lipid peroxidation determined by Tanizawa et al. (1981).

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DISCUSSION

The first step in the proposed mechanism for the in vivo toxicity of paraquat in the mussel hepatopancreas requires that paraquat undergo a single electron reduc- tion and subsequent oxidation at the expense of 02. Microsomal NADPH- cytochrome c reductase and NADPH have been shown in vitro to catalyze paraquat reduction (Bus et al., 1974; Gage, 1968). The reduction of cytochrome c was shown by McCord and Fridovich (1968, 1969) to be mediated by O2- produced as a by- product of xanthine oxidase and inhibited by SOD. In vitro experiments with mussel hepatopancreas microsomes indicate a dose-dependent increase in the rate of the reduction of cytochrome c. Specificity for O2- generation is achieved not by measuring total cytochrome c reduction, but rather the SOD-inhibitable component of that reduction. The inhibition of paraquat-induced cytochrome c reduction by exogenous SOD indicates that 02 - production is stimulated in the presence of para- quat. While absolute paraquat-stimulated Oz-production in this bivalve is con- siderably below that observed in rats or channel catfish (Ictalurus punctatus), the relative increase above baseline is similar (Wenning and Di Giulio, 1987).

In interpreting the results of the in vivo study, it is important to consider the declining paraquat concentrations that Occurred in this static test. The concentration of paraquat at 6 and 12 h dropped by less than 10%0 in each of the treatment aquaria. Measurements of SOD, catalase, GSH, and lipid peroxidation at these ex- posure periods probably reflect the influence of the initial concentrations of para- quat that were applied. However, paraquat concentrations at 24 h dropped by an average of 25% from the initial dose and by an average of 56% of 36 h. Therefore, the analysis of SOD, catalase, GSH, and malondialdehyde at these exposure periods must take into consideration the reduced concentrations of paraquat measured in the aquaria.

In accord with in vivo experiments with E. coli (Hassan and Fridovich, 1979), microplasmodia (Allen et al., 1985), carp (Matkovics et al., 1984), and mice (Matkovics et al., 1980) SOD activity in the mussel hepatopancreas was stimulated and probably induced by exposure to paraquat for 12 h. Given that paraquat con- centrations were relatively unchanged in the first 12 h of exposure, de novo synthesis may have been reponsible for the elevation in SOD activities measured between 6 and 12 h from initial exposure. If the concentration of paraquat in the subsequent exposure periods had been maintained, it is likely that the elevated enzyme activity measured at 12 h would have been maintained or elevated further in the hepatopan- creas at 24 and 36 h. However, the SOD response to paraquat observed in these ex- periments is not dose-dependent; greatest activity occurred in the lowest treatment group (0.5 mM). At higher concentrations of the herbicide, the damaging effects of paraquat-stimulated 02-may have affected cellular protein synthesis processes in- volved directly or indirectly with the generation of the SOD enzyme. Research into the effect of paraquat on the expression of the Sod gene in E. coli by Hassan and

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Fridovich (1977, 1979) and in maize by Matters and Scandalios (1986) support this hypothesis.

Given that SOD dismutates the superoxide radical to hydrogen peroxide, one would expect that an increase in the cellular levels of this strong oxidant would stimulate the activity of catalase. However, despite elevated catalase activities in treated mussels relative to controls, the activity of the enzyme decreases at the same time as the activity of SOD increases at 12 h. Matters and Scandalios (1986) measured a 15% increase in catalase activity and a 40% increase in SOD activity in maize leaves exposed to 0.01 mM paraquat for 12 h. In their opinion, the concen- tration of hydrogen peroxide generated by the elevated SOD activity would be ade- quately detoxified by the smaller increase in catalase activity and protect the cell from injury. Our observations of elevated catalase activity and GSH concentrations in the mussel hepatopancreas agree with the observations of Matters and Scandalios (1986) regarding the disproportionate increases in SOD and catalase activities. However, in animal models, paraquat does not always stimulate both SOD and catalase enzyme activities. Matkovics et al. (1984) observed a reduction in catalase activity and an increase in SOD activity in carp (Hypophthalmichthys molitrix) hepatic tissue exposed to 0.1 ppm, 1.0 ppm, and 10.0 ppm paraquat for 24 h. And, in mice, Matkovics et al. (1980) found that while an LDs0 concentration of paraquat stimulated catalase activity in the liver, an LD100 dose depressed the enzyme's activi- ty; SOD activity exhibited the opposite effect at both doses in the same tissue. Similar observations have been made in rats by Frank (1981) and in houseflies by Allen et al. (1984).

Contrary to much of the literature concerning the association of GSH depletion with lipid peroxidation (Bus et al., 1976; Brigelius et al., 1981; Bus, 1982; Sandy et al., 1986; Beloqui and Cederbaum, 1986) GSH concentrations in the mussel hepatopancreas were elevated in response to paraquat at all exposure periods. From kinetic considerations, H202 would be expected to be preferentially metabolized by GSH peroxidase because of its lower Km for H202 than catalase as shown by Cohen and Hochstein (1963) in mammalian erythrocytes. However, catalase has a greater affinity for H202 at higher concentrations of the oxidant (Chance et al., 1979). Thus, cells may avoid GSH depletion and GSSG accumulation by depending more on catalase during increased rates of cellular H202 production. Among the most significant effects of 1 mM paraquat administration in the housefly (Allen et al., 1984) is an increase in catalase activity and GSH concentration but not in GSSG levels, which suggests that the synthesis of GSH was independent of the rate of GSH oxidation. In their opinion, the elevation of GSH without a corresponding increase in its oxidation rate may be a compensatory response induced by an imbalance in the cellular redox state. Since both NADPH and GSH play an important role in maintaining an optimal cellular redox state (Chance et al., 1979) and the formation of O2- by paraquat requires NADPH, the increase in GSH levels may compensate for the loss of NADPH. Bus (1982) has noted that, in addition to scavenging

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radicals by allowing itself to lose a hydrogen ion, GSH provides reducing equivalents for glutathione peroxidase to convert lipid hydroperoxides to stable lipid alcohols. Pretreatment of rat hepatocytes with BCNU (a specific inhibitor of glutathione reductase) by Sandy et al. (1986) demonstrated the importance of the glutathione redox state in protecting against bipyridyl and active oxygen cytotoxici- ty. Pretreated cells exposed to 1.5 mM diquat or paraquat lost most of their GSH in 15 min and were nearly all dead after 90 min, suggesting the critical role of GSH in thiol homeostasis and cell viability following toxic insult. In microplasmodia (Allen et al., 1985) and in rat alveolar type II cells (Hagen et al., 1986) increases in GSH concentrations have been noted after in vivo exposure to paraquat, suggesting either an increase in the rate of synthesis of GSH or an increase in the activity of glutathione reductase to reduce GSSG.

Peroxidation of fatty acids containing 3 or more double bonds will produce malondialdehyde. The presence of this oxidation by-product can be measured with thiobarbituric acid which, although not a specific or quantitative indicator of fatty acid oxidation, correlates with the extent of lipid peroxidation stimulated by para- quat (Tanizawa et al., 1981). If redox cycling is important for the toxicity of para- quat, one would also expect that the active oxygen species produced would stimulate lipid peroxidation. While this has been observed in microsomal fractions in vitro by Bus et al. (1974, 1976a) and by Trush et al. (1982), in vivo studies with rodents (Stef- fen and Netter, 1979; Steffen et al., 1980; Matkovics et al., 1980; Shu et al., 1979) and in houseflies (Allen et al., 1985) indicate that lipid peroxidation may not be a critical factor in paraquat toxicity. From our investigation, if we consider only the 6 and 12 h exposure periods, it appears that lipid peroxidation is a significant ele- ment of the toxicity of paraquat at higher concentrations in the mussel hepatopan- creas. And, at 24 and 36 h, when the concentrations of paraquat in the treatment aquaria dropped by as much as 56°70, significant concentrations of malondialdehyde were detected above controls. These observations are supported by the work of Gabryelak and Klekot (1985) in which lipid peroxidation in two species of carp, C. carpio and C. carrasius, was significantly elevated by 10 ppm paraquat from 4 to 96 h. Furthermore, Matkovics et al. (1984) observed lipid peroxidation in carp (H. molitrix) exposed to as little as 0.1 ppm paraquat for 24 h.

In conclusion, the results of this study support the hypotheses that these bivalves can activate redox cycling compounds and demonstrate both in vivo and in vitro responses typical of oxidative stress, as has been observed in other organisms. The SOD-inhibitable reduction of cytochrome c by paraquat in mussel hepatopancreas microsomes demonstrates the ability of the compound to generate O2- in vitro. And observed in vivo responses, such as increased activities of antioxidant enzymes, enhanced lipid peroxidation, and perhaps increased GSH concentrations, suggest that paraquat-mediated 02-generation is biologically significant in these animals. Mussels are thought to be an excellent indicator species in coastal pollution monitor- ing programs. Further research is needed to determine the utility of oxidative stress- mediated mechanisms for assessing the impacts of aquatic pollutants.

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ACKNOWLEDGEMENTS

T h i s s t u d y w a s s u p p o r t e d in p a r t b y g r a n t s f r o m t h e U . S . E n v i r o n m e n t a l P r o t e c -

t i o n A g e n c y , t h e U n i v e r s i t y o f N o r t h C a r o l i n a W a t e r R e s o u r c e s R e s e a r c h I n s t i t u t e ,

a n d t h e N o r t h C a r o l i n a B o a r d o f S c i e n c e a n d T e c h n o l o g y .

REFERENCES

Allen, R.G., K.J. Farmer, R.K. Newton and R.S. Sohal, 1984. Effects of paraquat administration on longevity, oxygen consumption, lipid peroxidation, superoxide dismutase, catalase, glutathione reduc- tase, inorganic peroxides, and glutathione in the adult housefly. Comp. Biochem. Physiol. 78C, 283-288.

Allen, R.G., R.K. Newton, K.J. Farmer and C. Nations, 1985. Effects of the free radical generator para- quat on differentation, superoxide dismutase, glutathione, and inorganic peroxides in microplasmodia of Physarum polyeephalum. Cell Tissue Kinet. 18, 623-630.

Autor, A.P., 1974. Reduction of paraquat toxicity by superoxide dismutase. Life Sci. 14, 1309-1319. Beloqui, O. and A.I. Cederbaum, 1986. Prevention of microsomal production of hydroxyl radicals, but

not lipid peroxidation, by the glutathione-glutathione peroxidase system. Biochem. Pharmacol. 35, 2663-2669.

Blum, J. and I. Fridovich, 1984. Enzymatic defenses against oxygen toxicity in the hydrothermal vent animals Rifta pachyptila and Calyptogena magnifiea. Arch. Biochem. Biophys. 228, 617-620.

Brigelius, R., A. Hashem and E. Lengfelder, 1981. Paraquat-induced alterations of phospholipids and GSSG-release in the isolated perfused rat liver, and the effects of SOD-activated copper complexes. Biochem. Pharmacol. 30, 349-354.

Bus, J.S., 1982. Oxygen activation and lipoperoxidative mechanisms of toxicity of pesticides and other xenobiotics. In: Pesticide chemistry: human welfare and the environment, Vol. 3, edited by J. Meyamoto and P.C. Kearney, Pergamon Press, Oxford, pp. 457-462.

Bus, J.S., S.D. Aust and J.E. Gibson, 1974. Superoxide and singlet-oxygen catalyzed lipid peroxidation as a possible mechanism for paraquat (methyl viologen) toxicity. Biochem. Biophys. Res. Commun. 58, 749-755.

Bus, J.S., S.D. Aust and J.E. Gibson, 1976a. Paraquat toxicity: proposed mechanism of action involving lipid peroxidation. Environ. Health Perspect. 16, 139-146.

Bus, J.S., S.Z. Cagen, M. Olgaard and J.E. Gibson, 1976b. A mechanism of paraquat toxicity in mice and rats. Toxicol. Appl. Pharmacol. 35, 501-513.

Chance, B., H. Sies and A. Boveris, 1979. Hydroperoxide metabolism in mammalian organs. Physiol. Rev. 59, 527-603.

Cohen, G. and P. Hochstein, 1963. Glutathione peroxidase: the primary agent for the elimination of H202 in erythrocytes. Biochemistry 2, 1420-1426.

Fisher, H.K., J.A. Clements and R.R. Wright, 1973. Enhancement of oxygen toxicity by the herbicide paraquat. Am. Rev. Res. Dis. 107, 246-252.

Frank, L., 1981. Prolonged survival after paraquat" role of the lung antioxidant enzyme system. Biochem. Pharmacol. 30, 2319-2324.

Gabryelak, T. and J. Klekot, 1985. The effect of paraquat on the peroxide metabolism enzymes in erythrocytes of freshwater fish species. Comp. Biochem. Physiol. 81C, 415-418.

Gage, J.C., 1968. Actions of paraquat and diquat on liver cell fractions. Biochem. J. 109, 757-761. Goldberg, E.D., V.T. Bowen, J.W. Farrington, G. Harvey, J.H. Martin, P.L. Parker, R.W.

Risebrough, W. Robertson, E. Schneider and E. Gamble, 1978. The mussel watch. Environ. Conserv. 5, 101-125.

169

Hagen, T.M., L.A. Brown and D.P. Jones, 1986. Protection against paraquat-induced injury by ex- ogenous GSH in pulmonary aveolar type II cells. Biochem. Pharmacol. 35, 4537-4542.

Halliwell, B. and J.M.C. Gutteridge, 1985. Free radicals and toxicology. In: Free radicals in biology and medicine, edited by B. Halliwell and J.M.C. Gutteridge, Clarendon Press, Oxford, pp. 206-246.

Hassan, H.M. and I. Fridovich, 1977. Regulation of the synthesis of superoxide dismutase in Escherichia coli. J. Biol. Chem. 252, 7667-7672.

Hassan, H.M. and I. Fridovich, 1978. Superoxide radical and the oxygen enhancement of the toxicity of paraquat in Escherichia coli. J. Biol. Chem. 253, 8143-8148.

Hassan, H.M. and I. Fridovich, 1979. Intracellular production of superoxide radical and of hydrogen peroxide by redox active compounds. Arch. Biochem. Biophys. 196, 385-395.

Hassan, H.M., 1984. Exacerbation of superoxide radical formation by paraquat. In: Methods in en- zymology, Vol. 105, edited by L. Packer, Academic Press, NY, pp. 523-532.

James, G.T., 1978. Inactivation of the protease inhibitor phenylmethylsulphonyl fluoride in buffers. Anal. Biochem. 86, 574-579.

Jollow, D.J., J.R. Mitchell, N. Zampaglione and J.R. Gillette, 1974. Bromobenzene-induced liver necrosis: protective role of glutathione and evidence for 3,4-bromobenzene oxides as the hepatotoxic metabolite. Pharmacology 1 l, 151-169.

Keeling, P.L. and L.L. Smith, 1982. Relevance of NADPH depletion and mixed disulphide formation in rat lung to the mechanism of cell damage following paraquat administration. Biochem. Pharmacol. 31, 3243-3249.

Livingstone, D.R. and S. Farrar, 1984. Tissue and subcellular distribution of enzyme activities of mixed- function oxygenase and benzo[a]pyrene metabolism in the common mussel Mytilus edulis. Sci. Total Environ. 39, 209-235.

Lowry, O.H., N.J. Rosebrough, A.C. Farr and R.J. Randall, 1951. Protein measurements with the folin phenol reagent. J. Biol. Chem. 193, 265-275.

Luck, H., 1963. Catalase. In: Methods in enzymatic analysis, Academic Press, NY, pp. 885-894. Marks, G.W. and D.L. Fox, 1937. Studies on catalase from the California mussel. In: Scripps Institution

of Oceanography Technical Series Bull. No. 3 1931-1935, edited by G.F. McEwen, E.G. Moberg, F.B. Summer and T.W. Vaughan, U.C. Berkley Press, Berkeley, CA, pp. 297-310.

Mason, R.P. and C.F. Chignell, 1982. Free radicals in pharmacology and toxicology. Pharmacol. Rev. 33, 189-211.

Matkovics, B., L. Szabo, Sz.I. Varga, K. Barabas and G. Berensci, 1980. In vivo effects of paraquat on some oxidative enzymes in mice and experiments to support the defence against the poisoning. In: Biological and clinical aspects of superoxide and superoxide dismutase, edited by W.H. Bannister and J.V. Bannister, Elsevier/North Holland Press, NY, pp. 367-380.

Matkovics, B., L. Szabo, Sz.I. Varga, K. Barabas, G. Berensci and J. Nemcsok, 1984. Effects of a her- bicide on the peroxide metabolism enzymes and lipid peroxidation in carp fish (Hypophthalmichthys molitrix). Acta Biol. Hung. 35, 91-96.

Matters, G.L. and J.G. Scandalios, 1986. Effect of the free radical generating herbicide paraquat on the expression of the superoxide dismutase (Sod) genes in maize. Biochem. Biophys. Acta 882, 29-38.

McCord, J.M. and I. Fridovich, 1968. The reduction of cytochrome c by milk xanthine oxidase. J. Biol. Chem. 243, 5753-5760.

McCord, J.M. and I. Fridovich, 1969. Superoxide dismutase: an enzymatic function for erythrocuprein (hemocuprein). J. Biol. Chem. 244, 6049-6055.

Popham, J.D. and J.M. D'Auria, 1983. Combined effect of body size, season, and location on trace ele- ment levels in mussels (Mytilus edulis). Arch. Environ. Contam. Toxicol. 12, 1-14.

Ramos-Martinez, J.I., T.R. Bartolome and R.V. Pernas, 1983. Purification and properties of glutathione reductase from hepatopancreas of Mytilus edulis L. Biochem. Physiol. 75B, 689-692.

Rosas, I., A. Baez and R. Belmont, 1983. Oyster (Crassostrea virginica3 as indicator of heavy metal pollution in some lagoons of the Gulf of Mexico. Water Air Soil Poll. 20, 127-135.

170

Sandy, M.S., P. Moldeus, D. Ross and M.T. Smith, 1986. Role of redox cycling and lipid peroxidation in bipyridyl herbicide cytotoxicity. Biochem. Pharmacol. 35, 3095-3101.

Shu, H., R.E. Talcott, S.A. Rice and E.T. Wei, 1979. Lipid peroxidation and paraquat toxicity. Biochem. Pharmacol. 28, 327-331.

Steffen, C. and K.J. Netter, 1979. On the mechanism of paraquat action on microsomal oxygen reduc- tion and it's relation to lipid peroxidation. Toxicol. Appl. Pharmacol. 47, 593-602.

Steffen, C., H. Muliawan and H. Kappus, 1980. Lack of in vivo lipid peroxidation in experimental para- quat poisoning. Naunyn-Schmiedeberg's Arch. Pharmacol. 310, 241-243.

Stegeman, J.J., 1985. Benzo(a)pyrene oxidation and microsomal enzyme activity in the mussel (Mytilus edulis) and other bivalve mollusc species from the Western North Atlantic. Mar. Biol. 89, 21-30.

Talcott, R.E., H. Shu and E.T. Wei, 1979. Dissociation of microsomal oxygen reduction and lipid perox- idation with the electron acceptors paraquat and menadione. Biochem. Pharmacol. 28, 665-671.

Tanizawa, H., Y. Sazuka and Y. Takino, 1981. Micro-determination of lipoperoxides in the mouse myocardium by thiobarbituric acid fluorometry. Chem. Pharmacol. Bull. 29, 2910-2914.

Trush, M.A., E.G. Mimnaugh, E. Ginsburg and T.E. Gram, 1982. Studies on the in vitro interaction of mitomycin c, nitrofurantoin, and paraquat with pulmonary microsomes. Biochem. Pharmacol. 31, 805-814.

Wenning, R.J. and R.T. Di Giulio, 1987. Microsomal enzyme activities, superoxide production, and an- tioxidant defenses in ribbed mussels (Geukensia demissa) and wedge clams (Rangia cuneata). Comp. Biochem. Physiol. Pt. C. (in press).