Microsomal enzyme activities, superoxide production, and antioxidant defenses in ribbed mussels (Geukensia demissa) and wedge clams (Rangia cuneata)

Camp. B&hem. Physiol. Vol. WC, No. I,

pp. 21-28,

1988 0

Printedin Great Britain

0306-4492/88 $3.00 + 0.00 1988 Pergamon Press plc

MICROSOMAL ENZYME ACTIVITIES, SUPEROXIDE PRODUCTION, AND ANTIOXIDANT DEFENSES IN RIBBED MUSSELS (GEUKENSIA DEMISSA) AND WEDGE CLAMS (RANGIA CUNEAZ-2) RICHARD J. WENNING*

and

RICHARD

T. DI GIULIO~

Ecotoxicology Laboratory, School of Forestry and Environmental Studies, Duke University, Durham, NC 27706, USA. Telephone: (919) 684-2802 (Received 27 April 1987)

Abstract-l. Microsomes of hepatopancreae from the ribbed mussel, Geukensia den&a, and the wedge clam, Rang& cuneuta, were examined for their ability to catalyze the reduction/oxidation cycling of xenobiotics, particularly paraquat, and subsequently stimulate superoxide anion (0;) production. 2. Levels of the microsomal electron transport components cytochrome P-450, cytochrome b,, NADH-cytochrome c reductase, and NADPH-cytochrome c reductase were similar in both bivalves and like those seen in other species of molluscs. 3. In vitro studies indicated a dose-dependent increase in the rate of 0; generated in microsomal fractions incubated with paraquat; the highest concentration of paraquat employed (4mM) elicited an 81% increase in cytochrome c reduction in mussels and a 135% increase in clams. In both species, cytochrome c reduction was inhibited by the addition of exogenous superoxide dismutase (SOD). 4. Activities of SOD and catalase and the concentration of reduced glutathione were determined in the hepatopancreas of these bivalves. Similar values for these antioxidants were observed in both species.

INTRODUCTION

In earlier studies by Vandermeulen and Penrose (1978) and Lee et al. (1972), the inability of Mya urenuria, Mytilus edulis, and Ostreu edulis to metabolize petroleum aromatic hydrocarbons prompted some researchers to conclude that bivalve molluscs did not possess a monooxygenase capacity. However, the presence of nitroreductase activity (Carlson, 1972) and azoreductase activity (Hanzel and Carlson, 1974) in Mercenuriu mercenuria, low rates of aldrin epoxidation in Anodontu cygneu (Khan et al., 1972) and Mytilus culiforiunus (Kreiger et al., 1979), and benzo[a]pyrene hydroxylation in the mussels M. culiforiunus (Trautman et al., 1979), Mytilus edulis and Modiolus modiolus (Stegeman, 1980), Mytilus galloprooinciulis (Viarengo et al., 1986) and the oyster Crussostrea uirginicu (Anderson, 1978) have been reported in microsomal preparations from digestive gland tissue. Additional evidence for the presence of a bivalve MFO system has been provided by the activation of mammalian carcinogens to frameshift mutagens in M. mercenariu (Anderson and Doos, 1983) and the inducibility of NADPH-neotetrazolium reductase by aromatic hydrocarbons in M. edulis (Moore et al., 1980). Most of these reactions are known to be catalyzed by the cytochrome P-450 system in marine fish and terrestrial mammals. The presence of such a microsomal electron transport chain in bivalve molluscs has been established in recent years by the works of Livingstone and Farrar (1984) on M. edulis, and Stegeman (1985) on M. edulis, Arca zebra, and Macrocullista maculuta. Given the potential significance of free radical intermediates in the metabolism of certain xenobiotics and their enhancement of oxygen toxicity through the generation of active oxygen species, it is important to understand the mechanisms underlying

Investigations reported to date have demonstrated that marine fish are capable of essentially the same cytochrome P-450-dependent monooxygenase reactions for the biotransformation of xenobiotics that are present in mammals, although, in general, enzyme activities are considerably lower (Pohl et al., 1974; Chambers and Yarbrough, 1976; Bend and James, 1978; James et al., 1979). However, the nature and characteristics of these activities in molluscs have not been fully established, partly due to reaction rates even slower than those observed in marine fish. Although these reactions are most often detoxifications, there are implications that some reactions, including those involving reduction/oxidation (redox) cycling compounds, can be activations to more toxic compounds. Probably the best characterized redox compounds are the bipyridylium cations (such as diquat and paraquat), which accept electrons from an electron carrier (such as NADPH) to give rise to free radical intermediates which are in turn capable of diverting a portion of the electron flow to the intracellular production of superoxide (0;) and hydrogen peroxide (H202) (Hassan and Fridovich, 1979; Rotilio et al., 1985). The interaction of these compounds with mitochondrial or microsomal electron transport components, namely cytochrome P-450, cytochrome bS, NADH- and NADPH-dependent cytochrome c reductases, requires molecular oxygen and NADPH for activity (Hassan and Fridovich, 1978).

*Present address: EnviroLogic Data, Inc., 4 Milk Street, Portland, ME 04101, USA. tAuthor to whom correspondence should be addressed. 21

RICHARD

22

J. WENNINGand

this phenomenon in different species that are likely to be exposed to redox cycling compounds. These compounds include phenolics, quinones, nitroaromatics, azo dyes and bipyridiliums (Mason, 1982) and are common aquatic contaminants. The major objectives of this study were (1) to describe the key components of the microsomal electron transport chain (cytochrome P-450, cytochrome b,, NADHand NADPH-dependent cytochrome c reductases), which are known to be involved in reactions with redox-active compounds, in the marine bivalve Geukensia demissa and the estuarine bivalve Rangia cuneata, and (2) to determine the in vitro ability of paraquat, a classic redox compound, to stimulate 0; production in bivalve hepatopancreas microsomes and compare this stimulation with that generated in channel catfish (Zctalurus punctatus) and rat microsomes. Furthermore, we report baseline information on antioxidant enzyme activities (superoxide dismutase and catalase) and glutathione con~ntrations in these two bivalve species. Relatively few studies have examined the antioxidant system in bivalves. Quantification of superoxide dismutase (SOD), catalase, and glutathione peroxidase activities in Cuiyptogena magn&a and M. mercenaria (Bfum and

Fridovich, 1984), glutathione reductase activity in M. edulis (Ramoz-Martinez et al., 1983), and catalase activity in M. califoriartus (Marks and Fox, 1937)

represent the few studies of baseline activities we are aware of in the literature. Recently, our laboratory investigated in viuo responses of the antioxidant system in G. demissu to paraquat (Wenning et al., 1987).

RICHARD T. Dr GIULIO

and placed in ice-cold bomogeni~tion buffer. The crystalline style was removed from the hepatopancreas immediately upon dissection. Organs from 8 individuals were pooled without regard to sex. The tissues were weighed and homogenized in buffer at a I:4 w/v ratio (tissue weight: buffer volume) with a Tellon@ pestIe-type motorized tissue grinder (A. C. Thomas Co., Philadelphia, PA, USA) as described by Livingstone and Farrar (i984). The homagenization buffer consisted of ice-cold 20 mM Tris-HCl @H 7.6) with 0.5 M sucrose, 0.15 M KCI, 1mM DTT, 1mM EDTA, and 100 PM PMSF (added to the buffer from a stock solution in isopropanol just before homogenization to prevent its aqueous inactivation; James, 1978). The microsomal fraction was obtained by differential centrifugation of the homogenate at 500g for 10min (nuclear), 12,OOOg for 30 min (mitochondrial), and l~,O~g for 90 min (microsomal). The microsomal pellet was washed in homogenization buffer, spun at 100,000 g for 90 min, and resuspended in microsomal buffer consisting of 20mM Tris-HCl @H 7.4) with 1mM EDTA, 1mM DTT, and 20% w/v glycerol to yield a concentration of between 4 and 12 mg protein/ml. Microsomes from channel catfish and rats were prepared by a modification of Fingennan er al. (1983). Tissue was excised and homogenized in a 1: 5 w/v ratio in ice-cold Tris-HCl (PH 7.4) buffer with 0.25 M sucrose using the same tissue grinder described above. Microsomes were isolated by differential centrifugation of the homogenate at 10,OOOgfor 20 min, followed by filtration of the supernatant through a Buchner funnel using twice-folded Kimwipes to remove any lipid material. The filtered supematant was then spun at 105,~g for 60min. The microsomal pellet was washed in homogenization buffer, spun at 105,OOOgfor 60 min, and resuspended in microsomal buffer consisting of 0.05 M Tris-HCl (pH 7.4) with 20% w/v glycerol. 3jochemica~assays

MATERIALS

AND METHODS

Chemicals Paraquat, phenylmethyIsulpbony1 fluoride (PMSF), dithiothreitol (DTT), horse heart cytochrome c, bovine-type superoxide dismutase, nitrofurantoin, and other chemicals were purchased from Sigma Chemical Co. (St Louis, MO, USA). B-nicotinamide adenine dinucleotide, reduced form (NADH), ~-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and xanthine oxidase were purchased from U.S. Biochemical Co. (Cleveland, OH, USA), Animals Ribbed mussels (leukemia demissa) were obtained from several localities in Carteret County along the central North Carolina coast during the summer and fall of 1986. Individuals collected ranged in size from 6 to 8 cm in shell length and were held in 7 1,aerated, static aquaria with 30 parts per thousand (ppt) artificia1 seawater (Instant Ocean) at 25°C for a period of 24 hr prior to dissection. Wedge clams (Rangia cuneata) were collected from the Neuse River in Croatan National Forrest, New Bern County, North Carolina during the summer and fall of 1986. Individuals collected ranged in size from 5 to 8 cm in shell length and were held in 7 I, aerated, static aquaria with 15 ppt artificial seawater at 25°C for a period of 24 hr prior to dissection. Yearling channel catfish (Ictafurwpuncrarus)approximately 15 cm in length were obtained from Cape Fear Fish Farm (Raleigh, NC, USA) and maintained in a 12001, aerated, sand-filtered staintess steel aquarium at 20°C. Male, Sprague-Dawley rats weighing 300-400 g were obtained from the Duke University Vivarium. Microsomalpreparation Bivalves were opened and the hepatopancr~s

was excised

All enzyme assays were carried out at 25°C using freshly prepared microsomes. Spectrophotometric measurements were performed on a Shimadzu model UV-260 scanning spectrophotometer (Kyoto, Japan). Protein concentrations were determined according to Lowry et al. (1951). Cytochrome P-450 concentrations in hepatopancreas microsomes were analyzed from the carbon monoxide difference spectra of sodium dithionite treated samples according to the method of Omura and Sato (1964). Resuspended samples containing between 1.0 and 4.0mg microsomal protein/ml were placed in 50mM Tris-HCI (PH 7.6) buffer and the reduction was followed over 20 min between 400 and 5lOnm. The cytochrome P-450 content was determined assuming an extinction coefficient of 91 mM_’ cm-’ (450-480). Cytochrome b, was estimated in the same resuspension using sodium dithionite as the reductant as described by Stegeman and Binder (1979). Cytochrome b, concentrations were measured using an extinction coefficient of 171 mM_’ cm-’ (423-500). NADPH~ytochrome c reductase activity in hepatopancreas microsomes was determined by a modification of the method of Stegeman (1985) with a reaction mixture containing 0.175 mM NADPH, 0.5 mM KCN, and 0.08 mM horse heart cytochrome c in 0.2M potassium phosphate buffer (pH 7.7). For comparative purposes, NADPH-cytochrome c reductase activity was also measured by the methods of Livingstone and Farrar (1984) and by Omura and Takesue (1970). NADH-Cytochrome c reductase activity in hepatopancreas microsomes was determined using the conditions for NADPH-cytochrome c reductase, with 0.4 mM NADH replacing the NADPH. The reduction of cytochrome c was followed at 550 nm. The effects of the redox cycling compounds paraquat and nitrofurantoin on the rate of cytochrome c reduction in the hepatic microsomal fractions of rat, channel catfish, and mussel were analyzed by a m~ifi~tion of the method

Microsomal-mediated

0;

described by Stegeman (1985). Either 1 mM paraquat (added from a stock solution in distilled deionized water) or 1 mM nitrofurantoin (added from a stock solution in DMSO) was added to a 1 ml reaction mixture of 5 PM horse heart cvtochrome c. 50uM NADPH. 20uM KCN. and 7.5,ug microsomal protein/ml in 50 mM potassium phosphate buffer (pH 7.5) with 0.1 mM EDTA. The effect of paraquat on cytochrome c reduction in hepatopancreas microsomes from the mussel and clam was analyzed by the addition of either 0.5, 1.0, 2.0, or 4.0mM paraquat to a reaction mixture of 0.175 mM NADPH, 0.05 mM KCN, 0.125 mM horse heart cytochrome c, and 27 pg microsomal protein/ml in 0.2 M potassium phosphate buffer (PH 7.4) with 0.1 mM EDTA. The inhibitory effect of SOD on paraquat-stimulated cytochrome c reduction was determined by the addition of bovine-type SOD to the reaction mixture from a stock solution prepared in distilled deionized water. For the analysis of catalase and GSH, the hepatopancreae of two mussels or clams were pooled for each sample and homogenized in a I:2 w/v ratio in 0.15 M potassium phosphate buffer @H 7.4). The activity of catalase was determined in the cytosolic fraction at 240 nm according to the method of Luck (1963). GSH concentrations were determined in whole homogenates treated with 4% sulfosalicyclic acid by the method of Jollow ef al. (1974). For the analysis of SOD, the hepatopancreae of two mussels or clams were pooled for each sample and homogenized in 50 mM potassium phosphate buffer (PH 7.8) with 0.1 mM EDTA, spun at 12,000 g for 30 min, and dialyzed at 4°C against changes of this buffer overnight as described by Blum and Fridovich (1984). SOD was determined with the xanthine-xanthine oxidase 0; generating system described by McCord and Fridovich (1969) based upon a definition of one unit of SOD as the amount of enzyme that inhibits the reduction of cytochrome c by 50%. To determine the relative activites of CuZnSOD and MnSOD present in the tissue homogenate, 5 mM potassium cyanide was added to the reaction mixture to instantaneously and completely inhibit CuZnSOD (Blum and Fridovich, 1984). Statistics

23

production in bivalves

Table I. Microsomal protein and cytochrome concentrations, and enzyme activities in the hepatopancreas of the ribbed mussel (Geukensia demissa) and the wedge clam (Rnngia cuneara)*

Microsomal protein (mgiml) Cytochrome P-450 (nmol/mg protein) Cytochrome b,

(nmol/mg protein) NADH Cyt c (b,) reductase (nmol/min/mg protein) NADPH Cyt c (P-450) reductase (nmol/min/ma protein)

G. denim

R. cuneata

IO.44+ 0.49

4.14 f 0.43

ND

ND

0.075 * 0.001

0.0637

--t

43.33f I .24 7.62+ 0.291

8.81f 0.33

‘Values are means + SEM for 4 pooled samples consisting individuals each, except where noted. tMean of a replicate assay on a single pooled sample. fNot determined. ND, not detected.

of 8

G. demissa than from R. cuneata at microsomal protein concentrations ranging from 25 to 100 pg/ml (Table 2). In each species, there were significant differences (P < 0.05) in the rates of cytochrome c reduction observed at every concentration of microsomal protein, with rates decreasing as the concentration of protein increased. However, the overall ranges of reduction rates were relatively small for both species across the range of microsomal proteins employed. The assay system used to characterize the enzyme appears to affect its activity. A comparison of three methods, using R. cuneata preparations, which have been used by various investigators to examine NADPH-cytochrome c reductase activities in bivalve hepatopancreas microsomes, is shown in Fig. 2. At any given microsomal protein concentration, NADPH-cytochrome c reductase activity determined by the method of Livingstone and Farrar (1984) was consistently higher than Stegeman’s (1985) method, which in turn was consistently higher

For the in vitro analysis of microsomal oxygen reduction, differences between treatments were assessed by multifactor analysis using least significant difference (LSD) testing. Results are expressed as the mean k SEM. The Statgraphic statistical software system for the IBM computer was employed for these analyses (STSC Inc., Rockville, MD, USA).

._P-450

RESULTS

Microsomal

heme content and enzyme activity

microsomal concentrations of cytochromes P-450 and b, and activities of NADH- and NADPHdependent cytochrome c reductases from hepatopancreae of G. demissa and R. cuneata are shown in Table 1. Cytochrome P-450 was not detectable by difference spectroscopy in either the mussel or the clam, despite repeated scans over a 20 min period. Using microsomal suspensions containing l&4.0 mg microsomal protein/ml, only cytochrome b, could be detected in either species. Representative spectra for cytochromes P-450 and b, using a suspension of 3.15 mg microsomal protein/ml from the hepatopancreas of G. demissa are shown in Fig. 1. NADPH-cytochrome c reductase activities were present in mussel and clam microsomes (Table 1). NADPH-cytochrome c reductase activities were slightly higher in hepatopancreas microsomes from The

I 400

I 450

I 500

Fig. 1. Difference spectra of hepatopancreas microsomal cytochromes P-450 and b, from Geukensiu demissa. Spectra were obtained as outlined in Material and Methods. Absorption maxima were at 453 and 415 nm on P-450 spectra and at 427nm on b, spectra in a sample that contained 3.15 mg microsomal protein/ml.

RICHARD

24

J.

and

WENNING

Table 2. In vitro cytochromc c reduction (nmol/min/mg protein) at various concentrations of hcpatopancreas microsomal protein from ribbed mussels (Geukensia demissa) and wedge clams (Rangia cuneata)* Microsomal

urotein

(u P)

G. demissat

R. cuneata t

II.79 f ND 9.58 k 8.81 k 6.62 f 7.68 + 6.99 f

ND 10.54 * ND 7.62 f 6.57 + 5.29 k 4.92 f

10.0 12.5 20.0 25.07 50.0 75.01 lOO.O$

1.55 0.42 0.33 0.52 0.38 0.56

RICHARD

T. DI GIULIO

Table 4. The effect of superoxide dismutase (SOD) on cytochrome c reduction in the presence of 4mM paraquat in the microsomal fraction of the hepatopancreas of the ribbed mussel (Geuknsin demissa) and the wedge clam (Raneia cuneatoV nmol cyt c reduced/min/mg protein G. akmissa R. cuneata

Treatment 0 mM paraquat 4 mM paraquat + 0.4 pg SOD + I pg SOD + IOpg SOD + IO pg boiled SOD

0.71 0.29 0.10 0.02 0.09

‘Values are means + SEM for 4 pooled samples, consisting of 8 individuals each. Assayed at 25°C in a I ml reaction mixture of 0. I75 mM NADPH, 0.05 mM KCN, and 0.08 mM cytochrome c in 0.2 M KPO, buffer (pH 7.7). tWithin each species, each mean is significantly different from all other means (P < 0.05). fBetwecn species, means are significantly different from each other (P < 0.05). ND not determined.

2.99 6.11 2.89 2.32 1.70 5.58

f 0.09t kO.l4$ + 0.17t f O.OS# f 0.0911 k 0.251

2.60 f 0.26t 5.16 f 0.401 2.78 f 0.14t 2.42 _t 0.38t 1.79*0.11§ 4.63 f 0.41 I/

‘Values are mean k SEM for 4 pooled samples of 8 individuals each. Assayed at 25°C in a I ml reaction mixture of 0.175mM NADPH, 0.05 mM KCN, 0.125 mM cytochrome c, and 27 fig microsomal protein in 0.2 M KPO, buffer (pH 7.4) with 0.1 mM EDTA. t$#IITIFor each species, means not sharing the same letter arc significantly different from each other (P < 0.05).

with NADPH, was strongly dose-dependent. This effect was generally highly significant (P < 0.001) in both species. The addition of exogenous SOD to the reaction mixture significantly inhibited (P < 0.05) the reduction of cytochrome c in a dose-dependent manner, further indicating the presence of an in vitro paraquat-stimulated 0; generating system in both G. demissa and R. cuneata (Table 4). As expected, boiled SOD, which represents the inactivated enzyme, has only a marginal effect on cytochrome c reduction; thus, inhibition by SOD was an enzymatic effect.

than the method developed by Omura and Takesue (1970). Paraquat-stimulated microsomal oxygen reduction

The relative in vitro rate of cytochrome c reduction in hepatopancreas microsomes from G. demissa and R. cuneata at different concentrations of paraquat are shown in Table 3. These results demonstrate that the rate of cytochrome c reduction in the presence of paraquat, which suggests the generation of superoxide in hepatopancreas microsomes supplemented

Table 3. In vitro reduction of cytochrome c by various concentrations of paraquat in the microsomal fraction of the hepatopancreas of the ribbed mussel (Geukensia demirsa) and wedge clam (Rangia cuneata)* nmol cyt c reduced/min/mg protein R. cuneafa G. demissa % chanae

Concentration of uaraauat 0 0.5 mM I .O mM 2.0 mM 4.0 mM

3.17 + o.ost 4.00*0.llf 4.62 f 0.215 5.23 + 0.1311 5.76 f O.OBll

2.35 4.03 4.20 5.20 5.53

26% 45% 65% 81%

f * k f +

% change

0.08t 0.55$ O.l6$ 0.2% 0.1s

72% 79% 121% 135%

*Values are mean f SEM for 4 pooled samples of 8 individuals each. Assayed at 25°C in a 1 ml reaction mixture of 0.175 mM NADPH, 0.05 mM KCN, 0.125mM cytochrome c, and 27 pg microsomal protein in 0.2 M KPO, buffer (pH 7.4) with 0.1 mM EDTA. t$§liljFor each species, means not sharing the same letter are significantly different from each other (P < 0.001).

ii =

0

I

I

IO

20

I 30

I 40

I 50 Protein

I 60

I 70

I 80

I 90

I 100

(cg)

Fig. 2. Comparison of NADPH cytochrome c reductase activities in hepatopancreas microsomes of Rangiucuneataby the methods of (A) Livingstone and Farrar (1984), (B) Stegeman (1985),and (C) Omura and Takesue (1970). Values are mean f SEM (N = 3).

0;

Microsomal-mediated

production

25

in bivalves

Table 5. Effects of paraquat and nitrofurantoin on cytochrome c reduction (nmol/min/mg protein) in hepatic microsomal preparations from rat, channel catfish (Icralurus punctarus) and ribbed mussel (Geukensia demissa)’ Channel

Rat Baseline Paraquat (I mM) % change Nitrofurantoin (I mM) %change

32.06 k 0.32 91.43 * 1.31 185% 120.43 f 7.67 216%

catfish

13.33 f 0.58 23.81 f 0.34 19% 23.19 + 0.89 14%

Mussel

I .90 +

0.00 4.66 If: 1.16 145% 3.81 + 0.62 100%

*Values are mean f SEM of duplicate measurements. Assayed at 25°C in a I ml reaction mixture of 50pM NADPH, 20 PM KCN, 5pM cytochrome c, and 7.5 pg microsomal protein in 50 mM KPO, buffer (pH 7.5) with 0.1 mM EDTA.

Cytochrome c reductions in NADPH-supplemented hepatic microsomes from rat, channel catfish, and G. demissa in the presence of paraquat and another aromatic redox compound, nitrofurantoin, are shown in Table 5. Both baseline and xenobioticstimulated reduction rates were greatest in rat microsomes, followed by catfish microsomes, and finally mussel microsomes. However, paraquat-stimulated rates relative to baseline rates were similar for rat and mussel microsomes; both were well above relative Table 6. Activities of copper/zinc (CuZn) and manganese (Mn) superoxide dismutases (SOD), catalase and glutathione (GSH) concentrations in the hepatopancreas of the ribbed mussel (Geukensia demissa) and the wedge clam (Rongia cuneafa)’ G. demissa Total SOD7 CuZnSOD MnSOD CATf GSH§

15.69 13.88 1.65 15.30 2345

f f f f +

0.67 0.63 0.13 0.70 76

(6) (6) (6) (8) (8)

R. cuneala 12.42 11.62 0.81 15.30 2703

?r.0.98 f 0.94 f 0.04 f 1.00 f 68

(8) (8) (8) (7) (8)

*Two individuals were pooled for each sample and prepared as a 50% homogenate. Values are mean k SEM (A’). tUnits/mg protein. One unit of enzymatic activity is defined as the amount of dialyzed 12,000g supernatant which causes a 50% inhibition of the xanthine-xanthine oxidase mediated reduction of cytochrome c in the presence or absence of 5mM KCN at 25°C (McCord and Fridovich, 1969). fUnits/mg protein. One unit of catalase is defined as the amount of enzyme, in the presence of H,O,, required for a decrease in the optical density from 0.450 to 0.400 at 240 nm (Luck, 1963). §nmol GSH/mg protein. Table 7. Comparison

of cytochrome

Species Acra zebra Crassostrea uirginica Geukensia demissa Lirlorina litmrea L. Macrocallisra macula& Mytilus galloprouincialis Mytilus edulis L.

Myrilus cali/orianus Rangia cunenta Thais hoemasroma

rates for catfish. Nitrofurantoin-stimulated rates relative to baseline rates were similar in catfish and mussel microsomes; both were considerably below relative rates for rat microsomes. Antioxidant enzymes hepatopancreae

and

glutathione

orotein*

2.9 10.4 f 0.55 6.6 + 0.3 4.9 121.7 f 1.0 F: 3.1 +O.l M: 3.9 f 0.1 5.0 f 2.5 1.0 - 3.01 4.7 + 0.4$ 7.1 f 0.6

bivalve

Given the apparent ability of the redox compounds paraquat and nitrofurantoin to generate active oxygen species in hepatopancreas microsomes from G. demissa and R. cuneata, the activities of SOD and catalase and the concentrations of GSH in these bivalves were of interest. The activities of catalase, CuZnSOD, and MnSOD and concentrations of GSH in hepatopancreas tissue from G. demissa and R. cuneata are shown in Table 6. In both species, considerably more CuZnSOD activity than MnSOD activity was observed. G. demissa hepatopancreae appeared to display more activity of both types of SOD than R. cuneata hepatopancreae. Catalase activities and glutathione concentrations were very similar in the hepatopancreae of both species. DISCUSSION

A current explanation for the mechanism of toxicity of redox cycling compounds, such as paraquat, involves the oxidations of lipid membranes and cellular reductants by activated oxygen species (H,O,, O;, -OH) which are formed during the NADPHdependent microsomal electron transfer process and involve the autoxidation of the flavoprotein reductase and the oxy-cytochrome P-450 complex (Hassan and

P-450 levels and NADPH cytochrome c reductase activity in hepatopancreas selected marine molluscs

Microsomal

in

P-450t 0.106 BDL trace 0.079 BDL trace 0.13+0.06 O.lO~O.02 BDL BDL

NADPH

cyt c red.# 8.3 2.9 k 8.8 f 7.5 * 4.0 11.8?

F: 18.4 f M: 12.4 5 8.1 f 7.6 f 13.8 f

Note: values are mean + SEM *Concentrations expressed as mg microsomal protein/g wet wt, unless otherwise indicated. tconcentrations were assayed by Omura and Sato (1964) and expressed as nmol/mg protein. SActivities were assayed by various methods and expressed as nmol/min/mg protein. §mg microsomal protein/ml. BDL below detection limits.

0.2 0.3 0.4 3.0 4.6 1.3 0.5 0.3 1.1

microsomes

from

Reference Stegeman (1985) Chambers er al. (1975) This study Livingstone et al. (1985) Stegeman (1985) Ade et al. (1984) Gilewicz ef al. (1984) Livingstone et al. (1984) Stegeman (1985) Krieger et al. (1979) This study Livinastone el al. (19861

26

RICHARD J. WENNING and RICHARD T. DI GKJLIO

Fridovich, 1979; Bus, 1982; Freeman and Crapo, 1982). Microsomal NADPH-cytochrome c reductase and NADPH have been shown in vitro to catalyze paraquat reduction (Gage, 1968; Bus et al., 1974). The inhibition of microsomal paraquat reduction by antibody to rat liver NADPH-cytochrome c reductase has been cited as evidence that NADPHcytochrome c reductase was the microsomal enzyme which catalyzed the univalent reduction of paraquat (Bus et al., 1974). The stimulation of NADPHcytochrome c reductase activities in rats (Vuksa et al., 1983; Bus et al., 1974) and rabbits (Ilett et al., 1974) indicated that NADPH-cytochrome c reductase was involved in the enzymatic formation of paraquat radicals. The results of this study suggest that G. demissa and R. cuneata possess the microsomal electron transport system required for the activation of redox cycling compounds. Activities of cytochrome c reductase in these species appears similar to activities reported for other bivalves (Table 7) although caution must be taken in making such comparisons due to the use of different assay techniques by investigators. Despite the presence of NADH- and NADPHdependent cytochrome c reductase activities, neither G. demissa nor R. cuneata microsomes exhibited an absorption peak in the region of 450 mm. However, this apparent absence of cytochrome P-450 could be misleading, as an absorption maximum representing or including cytochrome P-420 was detected in each analysis of G. demissa and R. cuneata. Similar findings have been reported in hepatopancreas microsomes by other investigators (Gilewicz et al., 1984; Livingstone and Farrar, 1984; Stegeman, 1985). Numerous environmental and biological parameters associated with specimen handling and sample preparation can affect the stability of cytochrome P-450 (Stegeman, 1980). The protease inhibitor PMSF in the homogenization buffer may not have totally neutralized the considerable amount of digestive enzymes present in the bivalve hepatopancreae. These highly reactive enzymes are very inhibitory to chemical and drug-metabolizing activity in various vertebrate and invertebrate tissues (Pohl et al., 1974) and, in conjunction with the potential problems associated with sample handling, could account for the apparent lack of cytochrome P-450 and the presence of an absorption maximum at 420 nm. However, it has been shown with redox cycling compounds, such as paraquat and nitroaromatics, that NADPHcytochrome c reductase and not cytochrome P-450 is the key microsomal component in 0; production in mammalian systems (Bus et al., 1974; Harada and Omura, 1980). In addition, Washburn (1987) demonstrated in channel catfish (Zctalurus punctatus) that cytochrome P-450 does not play an in vitro role in 0; production in hepatic microsomes incubated with nitrofurantoin and suggested that NADPHcytochrome c reductase was the key microsomal enzyme responsible for univalent nitroreduction in this aquatic species. Ionic strength, pH, and the NADPH concentration of the experimental buffer solution have been shown to influence the activity of NADPH-cytochrome c reductase in rat liver microsomes (Phillips and Langdon, 1962). These factors, particularly the ionic

strength of the buffer solution and the concentration of NADPH added to the reaction mixture, appear to be the critical factors accounting for the differences in reductase activities noted in comparisons we conducted of three methods for determining NADPHcytochrome c reductase: (1) 50 mM Tris, 0.26 mM NADPH (Livingstone and Farrar, 1984); (2) 200 mM potassium phosphate, 0.175 mM NADPH (Stegeman, 1985); and (3) 100mM potassium phosphate, 0.10 mM NADPH (Omura and Takesue, 1970). Variations between these biochemical methods, and others not considered in this study, may also account for the apparent differences in baseline NADPHcytchrome c reductase activities reported by various researchers in the literature (Table 7). McCord and Fridovich (1969) first employed the reduction of cytochrome c as a measure of 0, production, which in their study was produced as a by-product of xanthine oxidase activity. Although xanthine oxidase does not produce free radical intermediates in viuo, other intracellular enzymatic sources for 0; generation exist, deduced either from the observation that SOD will inhibit their activity or from electron spin resonance measurement of free radical intermediates during enzyme catalysis (Freeman and Crapo, 1982). Specificity for 0; generation is achieved not by measuring total cytochrome c reduction, but rather the SOD-sensitive component of that reduction. In this study, in vitro experiments with mussel and clam hepatopancreas microsomes incubated with paraquat displayed dose-dependent increases in the rates of the reduction of cytochrome c. The inhibition of paraquat-stimulated cytochrome c reduction by exogenous SOD indicates that 0; production is indeed stimulated by paraquat. It is unclear why the addition of SOD reduced the rate of cytochrome c reduction to below baseline rates; however, it is possible that this represents a nonenzymatic protein effect. In the absence of paraquat, the baseline rate of NADPH-dependent cytochrome c reduction was not affected by the addition of exogenous SOD (data not shown). This insensitivity to SOD indicates that in the absence of paraquat, cytochrome c is directly reduced by the flavoprotein reductase and does not involve 0;. These results indicate that molluscs can univalently reduce paraquat to its anionic form, thereby catalyzing a redox cycle capable of generating oxyradicals. However, studies with xanthine oxidase and other enzymatic sources of free radicals have shown that modulation of enzyme activities, cofactor availability, substrate concentration, and oxygen tension can combine to affect the rates of intracellular 0; production (Fridovich, 1970; Freeman and Crapo, 1982). The capabilities of different species to respond to the presence of redox cycling compounds are illustrated in the comparisons of fish, mammal, and invertebrate microsomal cytochrome c reduction in the presence and absence of either paraquat or nitrofurantoin (Table 5). In accordance with observations by Ade et al. (1984), Bend and James (1978), and Livingstone and Farrar (1984), baseline rates of NADPH-cytochrome c reductase activities were highest in hepatic microsomes from a mammalian species, the rat, and lowest in an invertebrate species, the mussel; activities in the teleost fish were inter-

Microsomal-mediated 0; production in bivalves mediate. However, xenobiotic-stimulated 0; production rates relative to baseline rates were much more similar among these species than absolute baseline or stimulated rates. While these observations may suggest similar sensitivities among these species to redox cycling compounds, other factors such as various pharmacokinetic variables, target tissues, and biochemical defense mechanisms must be considered before reasonable hypotheses in this area can be formulated. If, indeed, 0; free radical stimulation by paraquat is a valid mechanism for its toxicity, then the endogenous antioxidant defense system of the hepatopancreas can be expected to play an important role in controlling the extent of paraquat-stimulated 0; cytotoxicity. SOD and catalase are inducible enzymes (Fridovich, 1974) which react with activated oxygen species (0; and H,Oz, respectively), both in the cytosol and in subcellular organelles, to reduce the cytotoxic effects of these highly reactive molecules (Halliwell and Gutteridge, 1985). In addition to these enzymatic components, non-enzymatic radical scavengers such as GSH, ascorbate, and a-tocopherol act as reducing agents by donating electrons to 0; or the hydroxyl radical (-OH) (Halliwell and Gutteridge, 1985). Antioxidant enzyme activities have been reported in various mammalian and nonmammalian species (Matkovics et al., 1977), and in several marine and freshwater fishes (Wdzieczak et al., 1982; Mather-Mihiach and Di Giulio, 1986). Determinations of SOD and catalase in G. demissa and R. cuneata in our laboratory are very similar to activities found in Calyptogena magniJica and Merecenaria mercenaria by Blum and Fridovich (1984). The predominance of cytosolic CuZnSOD over mitochondrial MnSOD in both species appears typical for all eukaryotes examined (Fridovich, 1983). In conclusion, the results of this study support the hypothesis that both G. demissa and R. cuneata possess a microsomal electron transfer system which can stimulate 0; production by redox cycling compounds. The components of the cytochrome P-450 system found in these bivalves are similar to observations made by researchers in other bivalve molluscs. The inhibition of paraquat-stimulated cytochrome c reduction by exogenous SOD in hepatopancreas microsomes demonstrates the ability of paraquat to generate 0; in uitro. In addition, each species has at least some of the elements of an antioxidant defense system (SOD, catalase, and GSH) that may provide some protection against oxidative stress, including that imposed by redox cycling compounds. We have recently observed apparently adaptive responses by this system in G. demissa exposed in vivo to paraquat (Wenning et al., 1987). Acknowledgements-This study was supported in part by grants from the U.S. Environmental Protection Agency, the University of North Carolina Water Resources Research Institute. and the North Carolina Board of Science and

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