Glutathione enzymes, glutathione content and t-butyl hydroperoxide induced lipid peroxidation in the gill and digestive gland of the estuarine clam, Rangia cuneata

Camp. Eiochem. Physiol. Vol. 106C, No. 3, pp. 809-814, Printed in Great Britain

1993 0

0742-8413/93 $6.00 + 0.00 1993 PergamonPressLtd

GLUTATHIONE ENZYMES, GLUTATHIONE CONTENT AND t-BUTYL HYDROPEROXIDE INDUCED LIPID PEROXIDATION IN THE GILL AND DIGESTIVE GLAND OF THE ESTUARINE CLAM, RANGIA CUNEATA PHILIPC. DARBY,* EVAN P. GALLAGHER?and RICHARDT. DI GIULIO*$ *Ecotoxicology Laboratory, School of the Environment, Duke University, and TDepartment

of Environmental

Health,

University

of Washington,

(Received 25 May 1993; accepted for publication

Durham, NC 27706 U.S.A.; Seattle, WA 98195, U.S.A.

14 June 1993)

Abstract-l.

Reduced glutathione (GSH), glutathione reductase (GSSG-reductase) and glutathione peroxidase (GSH-peroxidase) activities were measured in the gill and digestive gland of Rungia cuneata. 2. Substantial GSH concentrations were found in both gill (820 & 80 nmole/g tissue) and digestive gland (930 k 130 nmole/g tissue). The digestive gland exhibited 2.5-fold greater GSSG-reductase activities and 0.5-fold lower GSH-peroxidase activities relative to the gill. 3. In uiuo exposure to t-butyl hydroperoxide (BHP) elicited a dose-dependent increase (P -K 0.05) in lipid peroxidation in both tissues. Lipid peroxidation occurred earlier and to a greater extent in the digestive gland versus the gill. GSH concentrations in both tissues were unaffected by BHP exposure. 4. The study results indicate that gill and digestive gland differ in susceptibility to BHP induced oxidative damage, and the difference is accounted for by differences in tissue GSH metabolism.

INTRODUCTION The growing bank of evidence linking the presence of

oxidants to a variety of toxicological responses, and the apparent ubiquity of antioxidant defenses among organisms, continues to motivate numerous studies of oxidative stress and antioxidant defense across a wide range of phyla (Smith and Shrift, 1979; Morrill et al., 1988; Sundquist and Fahey, 1989; Ahmad and Pardini, 1990; Vanoni et al., 1990; Chang et al., 1991; Winston and Di Giulio, 1991). There exists considerable variation among organisms as to the role of specific antioxidant defenses (i.e. glutathione, tocopherol, superoxide dismutase, etc.) in protecting against oxidant mediated damage. The peroxidation of membrane polyunsaturated fatty acids (lipid peroxidation) has been examined extensively as an index of oxidative damage, although the involvement of lipid peroxidation in toxicity has not been fully elucidated (Singh et al., 1992). Organic peroxides, such as t-butyl hydroperoxide (BHP), have been shown to elicit lipid peroxidation through the formation of organic peroxy radicals, e.g. t-butyloxy radical in the case of BHP exposure (Thornalley et al., 1983; Rush et al., 1985). Glutathione provides protection against organic peroxide mediated toxicity both as an enzyme cofactor for glutathione peroxidase and as a free radical scavenger (Rush et al., 1985; Bruggeman et al., 1988; Ochi, 1988; Ochi and Miayaura, 1989). Elevated levels of malondialdehyde, a product of lipid peroxidation, have been shown to SAuthor to whom correspondence

should

be addressed.

correspond to a decrease in GSH upon exposure to BHP in mouse and rat isolated hepatocytes (Rush et al., 1985, 1986). Previous studies have revealed a critical balance between glutathione peroxidase-mediated and non-enzymatic glutathione oxidation, glutathione reductase activity, and cellular redox status in an organism’s ability to protect against oxidant stress (Nishiki et al., 1976; Rush et al., 1985, 1986). In this regard, alterations in the “prooxidantantioxidant balance” may lead to unsequestered radical formation and damage to cellular macromolecules (Sies, 1985). BHP has been used extensively in oxidative stress studies, especially those investigating antioxidant properties of glutathione and its associated enzymes (Srivastava et al., 1974; Nishiki et al., 1976; Oshino and Chance, 1977; Rush et al., 1985, 1986; Sies, 1985; Ochi, 1988; Ochi and Miyaura, 1989). The steric hindrance caused by the bulky tert-butyl component of the peroxide prevents reduction by catalase, therefore limiting the enzymatic breakdown of BHP to glutathione peroxidase (Sies, 1985). In mammals, inorganic and organic peroxide detoxification by glutathione involves two distinct forms of glutathione peroxidase; selenium (Se)-dependent glutathione peroxidase activity catabolizes both hydrogen peroxide and organic peroxides such as cumene hydroperoxide and BHP, whereas non associated with certain Se-dependent activity, isozymes of glutathione transferase, can reduce organic peroxides but not hydrogen peroxide (Lawrence and Burk, 1976, 1978; Proshaka and Ganther, 1977). Few studies have investigated oxidant damage and

809

P. C. DARBY et al.

810

oxidant defense in aquatic invertebrates, especially in regard to the protective role of glutathione. Blum and Fridovich (1984) reported glutathione peroxidase activity in the tube worm (Rzftiapachyptilu), and in two bivalves, Calyptogena magnz$ca and Mercenaria mercenaria. The crayfish (Orconcetes limosus) and a snail (sp.) were also reported to have glutathione peroxidase activity (Smith and Shrift, 1979). Glutathione peroxidase activities towards hydrogen peroxide and an organic peroxide were measured as part of a multi-drug resistance study using the mussel Mytilus galloprovincialis (Kurelec and Pivcevic, 1991). Winston et al. (1990) also measured glutathione peroxidase using hydrogen peroxide and an organic peroxide, as well as glutathione reductase in the common marine mussel, Mytilus edufis. Glutathione reductase from M. edulis has been purified and characterized by Ramos-Martinez et al. (1983). Glutathione concentrations have been reported in Geukensiu denzissa (Wenning and Di Giulio, 1988), M. edulis (Suteau et al., 1988; Viarengo et al., 1989), and M. provincialis (Ribera et al., 1989). Wenning et al. (1988) reported a transient increase in GSH in G. demissa following exposure to paraquat. The main objectives of this study were: (1) to report and compare reduced glutathione concentrations, glutathione peroxidase activities, and glutathione reductase activities in the gill and digestive gland of the wedge clam, R. cuneata; (2) to investigate lipid peroxidation in the gill and digestive gland following an in vivo exposure to BHP; and (3) to examine the relationship between GSH metabolism and susceptibility to BHP-induced oxidative damage in the gill and digestive gland. The data are compared to previous studies of oxidant defense and toxicity in bivalves, and the relationship between antioxidant levels and susceptibility to lipid peroxidation between the two issues is discussed.

MATERIALS AND METHODS

Chemicals

All buffer materials, GSH, GSSG, GSSG reductase, N-ethylmaleimide (NEM), O-phthaladehyde (OPT), BHP, thiobarbituric acid (TBA), sodium azide and reduced B-nicotinamide adenine dinucleotide phosphate (NADPH) were purchased from Sigma Chemical Company, (St. Louis, MO, U.S.A.). Hydrogen peroxide and phosphoric acid were obtained from Fisher Scientific (Springfield, NJ, U.S.A.). Animals

Wedge clams (Rungia cuneata) were collected from the Neuse River, 19 km north of Beaufort, North Carolina. Individuals ranged in size from 5 to 8 cm in shell length. Barnacles and other material were cleaned from the shell surfaces. Animals were transferred to Duke University, Durham, NC, and main-

tained in aerated, 11 ppt artificial salt water (Instant Ocean, Aquarium Systems, OH, U.S.A.) at ambient temperature (21°C) for 6 days. No food was provided throughout the studies. In vitro analysis of GSH, GSH-peroxidase and GSSG reductase in gill and digestive gland Clams were removed from their holding tanks as described above, and dissected for gill and digestive gland. Gills and digestive glands from three to four clams were pooled to yield sufficient tissue for biochemical analysis. Excised tissue was placed in ice cold 70 mM phosphate buffer (pH 7.4) and homogenized at 4°C with a Brinkman Polytron (Westbury, MO, U.S.A.) to yield a 40% (w/v) homogenate. The homogenate was separated for protein determination, enzyme analyses, and glutathione concentrations. Reduced glutathione concentrations (GSH) were determined by the method of Hissin and Hilf (1976) with fluorescence products of the reaction of OPT with GSH at pH 8.0 measured with excitation and emission wavelengths of 420 nm and 350 nm, respectively. Enzyme analyses were performed using cytosolic preparations of the homogenates, prepared by centrifuging the homogenates at 10,OOOg for 20 min, followed by 100,OOOg for 40 min. The method of Massey and Williams (1965) was used to determine glutathione reductase activity. Glutathione peroxidase was measured according to Lawrence and Burk (1976) using both BHP, and in a some samples, H,O,, as substrates. To eliminate the possibility of catalase degradation of H,O,, sodium azide was included in the peroxidase assay. Protein determinations for all samples were performed according to Lowry et al. (1951) with bovine serum albumin as the standard. Tissue GSH concentrations were determined on freshly prepared homogenate; the remainder of the tissue was frozen at -70°C for I or 2 days prior to analysis. Effect of BHP exposure on in vivo MDA production and GSH concentrations Range jinding tests. In order to determine the appropriate dose of BHP for the lipid peroxidation experiment, a range finding test was conducted. BHP was tested at 0.0, 0.5, 1.0, 2.0,4.0 and 10 mM. Clams were dosed in 11 ppt artificial sea water under static conditions, with three clams for each dose tested. After 36 hr, no deaths occurred at 0.0, 0.5, or 1.0 mM, and two deaths occurred at each of the higher doses. Death was assessed via failure to close the shell when prodded. The lack of 100% mortality at any level and the tendency for bivalves to close their valves during chemical exposure precluded ob. . taming a LC~~. Based on rangefinder information, 0.2 and 0.8 mM BHP were chosen as the non-lethal doses for the biochemical response study. BHP exposure protocol and tissue sampling preparation. Clams were placed in 38-l aquaria containing

aerated

artificial

salt water as described

above,

Glutathione metabolism in R. cuneata Table I. Reduced glutathione

811

(GSH) concentrations and glutathione enzyme activities in the gill and digestive Rangia cunca~a. Mean f SD; n = 12 except where noted

gland of

Tissue Parameter *Reduced glutathione tGlutathione peroxidase fGlutathione

reductase

Substance

Gill

H,G, BHP GSSG

820 f 80 1.1 +0.6 (n =4) 1.6kO.7 53.6 +_ 18.0

Digestive 930 2.0 3.3 20.8

* f * f

r-test

gland

130 0.8 (n = 4) 1.3 7.4

P P P P

< < < <

0.05 0.05 0.05 0.05

*Reduced glutathione determined by the method of Hissin and Hill (1976). Units are nmoles GSH/g tissue. tGlutathione peroxidase activity determined by the method of Lawrence and Burk (1976). Units are nmoles NADPH/mg protein/min. SGlutathione reductase activity determined by the method of Massey and Williams (1965). Units are nmoles NADPHjmg protein/min.

with 40 clams per tank. Appropriate amounts of BHP were diluted in 1 1of sea water and added to the tanks to give nominal concentrations of OmM (control), 0.2mM and 0.8mM BHP. All tanks were drained and redosed at 15 hr from the initial BHP administration. Clams from each dose and the control were sampled at 6, 12 and 30 hr after the initial BHP administration (n = 4). Gill and digestive glands from three to four clams were pooled to yield sufficient tissue for biochemical analysis. Excised tissue was prepared as described above prior to protein analysis. MDA production was determined on freshly prepared 10% (w/v) tissue homogenates by the fluorometric method of Tanizawa et al. (1981). Gill and digestive gland reduced glutathione concentrations were measured in 40% (w/v) tissue homogenates using the method of Hissin and Hilf (1976). Statistical

analyses

Significant differences in GSH and enzyme activities between the gill and digestive gland were assessed using t-tests. Comparisons between treatments and control within a time block were analyzed with the two way analysis of variance (ANOVA). Multiple comparisons among the means were made using Tukey’s significant difference test (P < 0.05). All statistical analyses were performed using SAS (SAS Institute Inc., Cary, NC). The criterion for determining statistical significance for t-tests and multiple comparisons was P < 0.05.

with BHP as the substrate. A similar relationship between gill GSH-peroxidase was observed (1.1 &-0.6 nmoles/mg/min) and digestive gland GSHperoxidase (2.0 f 0.8 nmoles/mg/min), when H202 was used as a substrate. Mean GSH concentrations in the gill (820 k 80 nmoles/g) were slightly lower than observed in the digestive gland (930 f 130 nmoles/g). Lipidperoxidation and GSH concentrations digestive gland from BHP exposed clams

in gill and

No mortality was observed in any of the controls or treatments during the course of the BHP exposure study. A dose dependent increase in lipid peroxidation was observed in both gill and digestive gland (Table 2). The gill showed no sign of lipid peroxidation until 30 hr in the 0.8 mM BHP treated clams (P < 0.05) (Table 2). In the digestive gland preparations, accumulation of TBA reactive products appeared at all time points for BHP exposed animals, with statistically significant levels (P < 0.05) detected in the 30 hr, 0.8 mM exposed clams. At 30 hr, MDA production in the digestive gland was approximately 27-fold and 7-fold higher than observed in the gill of 0.2 mM and 0.8 mM BHP treated clams, respectively. MDA accumulation was also observed in the digestive gland of control clams at 30 hr. The GSH concentrations in both the gill and digestive gland did not appear to be affected by the BHP treatments; variability among the clams was great (Table 3).

RESULTS

Glutathione enzyme activities and glutathione tration in gill and digestive gland

concen-

The glutathione enzyme activities and GSH concentrations in Rangiu cuneata gill and digestive gland preparations are shown in Table 1. The means for each of the enzyme activities and the GSH concentrations in the two tissues were significantly different (P < 0.05). These data reveal marked differences in enzyme activities between the two tissues. Mean gill glutathione reductase activity (53.6 f 18 nmoles/ mg/min) is 2.5 times that found in the digestive gland (20.8 f 7.4 nmoles/mg/min), but mean gill GSH-peroxidase activity (1.6 f 0.7 nmoles/mg/min) was 50% that of the digestive gland (3.3 f 1.3 nmoles/mg/min),

Table 2. The effect of r-butyl hydroperoxide concentration on lipid peroxidation (nanomoles malondialdehyde/mg tissue) in the gill and digestive gland of the clam, Rangia cuneom. Means + SD. n = 4 except where noted Hours after initial exposure 6 12

1uW 0 0.2 mM 0.8 mM 0 0.2 mM 0.8 mM

Gill BDL* 1.8 f 3.5 BDL BDL (n = 3) BDL BDL Digestive gland BDL (n = 3) 3.3 f 0.95 BDL I.5 + 0.75 12 + 3.25 I I.8 f 2.03t

30 BDL I.8 i 3.5 Il.3 * 3.9t 17.3 k 1.65 49.5 f 6.63 78.8 f 10.6Ot

Lipid peroxidation was determined by Tanizawa ef a/. (1981). ‘Below detection limit. Practical limit of assay sensitivity is 0.5 nmole MDA/mg tissue. tsignificantly elevated when compared to controls within exposure period (P < 0.05).

812

P. C. DARBY

Table 3. Reduced glutathione (GSH) concentrations (nmole GSH/g tissue) in the gill and digestive gland of the clam, Rartgia cuneufa, foIlowing exposure to t-butyl hydro~roxide @HP) Means & SD. Hours after initial exposure 12 6 [RHPI 0 0.2 mM 0.8 mM 0 0.2 mM 0.8 mM

Gill 780 + 390 760 + 320 750 * 520 670 + 240 7705600 750 ?r: 360 Digestive gland 1080 + 760 830 f 320 1050*400 800 f 360 1170*840 780 + 120

GSH was determined

30 910 rt 320 830 rt 480 870 + 480 890 + 480 920 & 200 940 f 360

by Hissin and Hilf (1976).

DISCUSSION

The study results presented here provide additional evidence that bivalves, specifically fz. cuneata, possess antioxid~t defenses associated with glutathione. Previous work done in this lab has quantified activities of SOD and catalase in R. cuneata (Wenning and Di Giulio, 1988) which, coupled with the information in this study, demonstrates that R. cuneafa possess a full suite of antioxidant defenses. R. curzeafa maintain glutathione peroxidase activity towards both hydrogen peroxide and organic peroxides. Glutathione peroxidase activity towards BHP in the gill (Table 1), though quite low relative to vertebrate species, was similar to the levels recorded by Blum and Fridovich (1984) for M. mercenaria and C. magnifzca. Glutathione peroxidase assayed with BHP in the R. cuneatu digestive gland exhibited 50% lower activity than observed in M. edulis (Winston ef al., 1990). GSHperoxidase activity towards H202 was also measured, and appeared somewhat lower than the recorded activity for the digestive gland of M. edulis (Winston et al., 1990). GSH content based on the wet weight of gill and digestive gland samples from R. cuneata appears to be quite similar to those measured in M. edulis (Viarengo et al., 1989), but were 2-3 times higher than the glutathione levels found in M. galioprovincialis (Ribera et al., 1989). In the in uiuo experiment of the present study, the elevated lipid peroxidation, as indicated by the levels of MDA, correlated with an increase in BHP nominal concentration (Table 2) and indicates that the antioxidant defenses were overwhelmed. This is consistent with previous reports involving the mechanisms of organic peroxide toxicity (Rush et al., 1985; Sies, 1985). Comparisons between the gill and digestive gland biochemical responses to BHP reveal some notable differences in the two tissues. Production of MDA in digestive gland homogenate exceeded that of the gill at all time points, and by 7-fold after 30 hr exposure of 0.8 mM of the hydroperoxide. Ribera et al. (1989) also demonstrated tissue differences in MDA levels in the bivalve, Myths galloprovincialis. The baseline concentrations of MDA were approximately two times higher in M. prov~nc~~~~digestive gland versus the gill. In the present study, lipid peroxidation was observed in the digestive glands of

et al.

control clams more frequently and at higher levels than in the gills (Table 2). The apparent difference in MDA production between the gill and digestive gland may be due to differences in antioxidant levels or possibly in the susceptibility of the lipid membrane components to oxidation (Rush et al., 1985; Singh et al., 1992). The relationships between concentrations of GSH, activities of GSSG reductase and GSH peroxidase, and oxidant mediated toxicity in previous studies demonstrate a critical role for GSH in antioxidant defense. Studies using cultured hamster cells exposed to BHP indicated that protection against hydroperoxide damage relies more heavily on GSH as a radical scavenger than as a substrate for GSH peroxidase, at least at the concentrations up to 0.5 mM BHP applied to these cells (Ckhi and Miyaura, 1989). In R. ~~euf~ gill and digestive gland, differences in reduced glutathione concentrations, glutathione enzymes, and the extent of lipid peroxidation upon exposure to BHP were observed. However, because GSH concentrations did not appear to change in either tissue during the course of this experiment, the role of GSH in protecting against BHP-induced lipid peroxidation could not be determined. The maintenance of membrane lipid integrity in the presence of oxidizing agents also depends on the availability of radical scavengers other than GSH (e.g. ascorbic acid, B-carotene, tocopherol). Analysis of ascorbic acid and tocopherol in gill, digestive gland, and other tissues of the mussel M. provincialis indicated that these two antioxidant vitamins may account for the variation in the selective tissue MDA production levels (Ribera et al., 1989). In a comparative study on the relationship of age and oxidative stress, depressed vitamin E and GSH contents in older mussels (M. e&Es, L.) were found to correlate with elevated MDA production during an oxygen mperfusion experiment (Viarengo et al., 1989). The authors demonstrated that sustained levels of vitamin E and GSH were linked to the protection against peroxidation of membrane lipids. Differences in species susceptibility to lipid peroxidation may also reflect inherent differences in membrane structure as observed between mice and rat isolated membrane preparations (Rush er al., 1985), and between rat, rabbit and rainbow trout microsomes (Singh et al., 1992). The ~roxidation process depends on the presence of polyunsaturated fatty acid (PUFA) components, and the degree of peroxidation depends on their percent composition in the membrane (Kappus, 1985; Singh et al., 1992). Ribera et ai. (1989) reported that the M. provjne~ulis digestive gland contains a higher percentage of unsaturated fatty acids relative to the gill, but the relative contribution to the higher MDA in digestive gland is uncertain. Singh et al. (1992) found that the composition of fatty acids in trout rendered the membrane lipids more susceptible to lipid peroxidation compared to representative fatty acid mixtures based on

Glutathione

metabc slism in R. cuneata

rat and rabbit membrane lipid composition. However, trout microsomes incubated with a peroxidizing system were refractory to lipid peroxidation relative to rabbit and rat microsomes. The role of tocopherol (vitamin E), as discussed above, appeared to explain the difference as Singh et al. (1992) reported that trout vitamin E concentrations were 25 and 60 times greater than vitamin E concentrations in rat and rabbit microsomes, respectively. The level of lipid peroxidation rose dramatically in trout microsomes after 1 hr of incubation when FeCl,/H,O, was used as the peroxidizing system. The concurrent drop in vitamin E levels supported a critical role for vitamin E in protection against lipid peroxidation (Singh et al., 1992). In summary, R. cuneatn possess reduced glutathione, GSH-peroxidase activity against both hydrogen peroxide and organic peroxides, and GSSG-reductase activity in both the gill and digestive gland. The in vivo application of BHP to R. cuneutu resulted in oxidative damage in the gill and digestive gland as indicated by the accumulation of lipid peroxidation products. The higher levels of MDA in the digestive gland relative to the gill may reflect inherent differences in these tissues’ ability to protect against oxidant stress. There exist quantitative differences in the antioxidant enzymes between the two tissues, but the maintenance of GSH concentrations in each tissue during the course of BHP exposure does not indicate a relationship between GSH metabolism and protection against BHP-induced lipid peroxidation. However, other factors (e.g. percent lipid composition, tocopherol) inherent to the structure or the biochemical profile of each of the tissues may contribute to tissue differences in susceptibility to lipid peroxidation. REFERENCES

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