Studies on myeloperoxidase activity in the common mussel, Mytilus edulis L.

Camp.Biochem.Physiol.Vol. 99C, No. l/2, pp. 63-68, 1991

0306~4492/91 $3.00 + 0.00 0 1991 Pergamon Press plc

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STUDIES ON MYELOPEROXIDASE ACTIVITY IN THE COMMON MUSSEL, MYTKUS EDULIS L. DANIEL

SCHLENK,*

PAZ GARCIA

MARTINEZ?

and

DAVID

R. LIVINGSTONES

*Duke University Marine Laboratory, Beaufort, NC, 28516-9721. U.S.A. [Telephone: (919) 728-21111; TDepartment of Biochemistry, Faculty of Pharmacy, University of Santiago de Compostela, Santiago de Compostela, Spain; fNERC Plymouth Marine Laboratory, Citadel Hill, Plymouth, Devon PLl 2PB. U.K. (Received 13 June 1990) Abstract-l.

Myeloperoxidase (MPO) activity was indicated in digestive gland tissue and blood cells of Mytilus edulis L. 2. Digestive gland halide-independent putative MPO activity (tetramethylbenzidine (TMB) oxidation) was differentiated from possible catalase-dependent TMB oxidation activity by subcellular distribution and pH profile studies. The apparent K,,, for TMB and HzOz of digestive gland 9000g supernatant were, respectively, 243 and 0.13 PM. 3. Digestive gland halide-independent putative MPO activity (chloramine production from diethanolamine) was observed in the absence of any added NaCl or H, 0,. The process was indicated to be enzymic and largely not dependent on the presence of endogenous metabolites.

(Narain, 1973). Different types of hemocytes are found in M. edulis (see Discussion), one of which, the macrophages, is involved in the clearance of foreign substances from the hemolymph and body tissues (Moore and Lowe, 1977). The phagocytic hemocytes of a number of molluscan species have been shown to produce reactive oxygen metabolites (Ot and/or H,O,), which could serve as substrate for MPO, oiz. in the bivalve Patinopecten yessoensis (Nakamura et al., 1985), and the gastropods Biomphalaria glabrata, Lymnaea stagnalis, Helix aspersa and Planorbarius corneus (Shozawa, 1986; Dikkeboom et al., 1987, 1988). However, it is unknown whether molluscs possess MPO. In adddition, since the normal diet of a filter-feeding bivalve consists of unicellular algae and bacteria (Morton, 1983), digestive diverticula cells may also possess bactericidal activity mediated through MPO. Consequently, the purpose of this study was to examine the digestive gland and blood of M. edulis for the presence of MPO to better understand the role that this enzyme might play in the biotransformation and toxicity of natural and pollutant xenobiotics.

INTRODUCTION The

biotransformation of organic xenobiotics in animals, including marine molluscs, can lead to the activation or detoxication of the parent compound, depending upon the nature and reactivity of the metabolites produced (Miller and Miller, 1985; Livingstone, 1990). The partial reduction of molecular oxygen to various reactive oxygen species (socalled oxyradicals), such as the superoxide anion radical, 0; and its dismutation product hydrogen peroxide, H,O,, can also play a major role in the process of chemical bioactivation and subsequent cellular pathology (Borg and Schaich, 1984; Halliwell and Gutteridge, 1986). Recent studies on the common mussel, Mytilus edulis L. and other bivalve molluscs have indicated a potential for xenobioticstimulated oxyradical production by digestive gland enzymes such as microsomal NADPH-cytochrome c (P-450) reductase and other flavoprotein reductases, so providing one mechanism for linking pollution exposure to biological damage in these organisms (Livingstone et a/., 1990). Oxyradical production and oxyradical/organic xenobiotic interactions can also arise via other enzymes (Morehouse and Aust, 1988). Present in (donor: mammalian leukocytes, myeloperoxidase H,O, oxidoreductase, EC 1.11.1.7; MPO), is a bactericidal enzyme which is able to produce hypochlorous acid from H,Oz and chloride ion (Harrison and Schultz, 1976). MPO is also capable of transforming a host of xenobiotics to free radicals via single electron abstractions (Ritter and MalejkaGiganti, 1989). The oxidized substrates have the potential to either polymerize or stimulate oxyradical production through redox-cycling events (Morehouse and Aust, 1988). Mussels have been shown to possess hemocytes (Amoebocytes) which are thought to serve a function similar to that of mammalian leukocytes

MATERIALS

AND METHODS

Chemicals All biochemicals including 3,3’,5,5’-tetramethylbenzidine, diethanolamine, bovine liver catalase (EC 1.11.1.6) and horseradish peroxidase (1.11.1.7) were obtained from Sigma Chemical, U.K. All other chemicals were of analytical grade and from BDH, U.K. PD-10 Sephadex G-25 columns were from Pharmacia-LKB U.K. Sample preparation Mussels (4-5 cm length) were collected from Whitesand Bay, Cornwall, near Plymouth, and kept for 2-3 days in a system of recirculating seawater to clear gut contents. Digestive glands were dissected out, damp-dried, the crystalline styles removed and the tissues frozen in liquid 63

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DANIEL~CHLENK

nitrogen before storage at -70°C. All subsequent preparation procedures were carried out at 4°C. Tissues were homogenized in a I:4 tissue weight: buffer volume ratio in 10 mM Tris-HCI pH 7.6, containing 0.8 M sucrose, using a Potter-Elvejem electrically-driven homogenizer. The homogenate was centrifuged at 500 g for 15 min, and the resulting supematant at 9000g for 45min. MPO characterization studies were carried out immediately on the 9000g supernatant, or on the latter after it had been passed down a Sephadex G-25 column to remove the low molecular weight fraction (5000 daltons and below) (PD-10 column; bed volume 9m1, equilibration and elution buffer were the same as the homogenization buffer). Subcellular distribution studies of MPO and catalase activities were carried out by centrifuging the homogenate at 500g for 15 min, and the resulting supernatant at 12,000g for 45 min. The 500-9000g pellet was resuspended in a reduced volume of homogenization buffer and the enzyme activities compared between this and the 12,000g supematant. Blood cell samples were obtained as described in Livingstone and Farrar (1984). Blood was removed from the posterior adductor muscle sinus of 10 animals by syringe, pooled and centrifuged at about 1OOOgfor 10min. The resulting pellet containing blood cells was resuspended in a small volume of homogenization buffer (see above), kept on ice and sonicated for 4 x 5 set (MSE probe sonicator, minimum setting) to break open the cells, Both whole blood and the sonicated blood cells were assayed for MPO. MPO and catalase activities

Enzyme assays were carried out at room temperature, or 25°C using a Pye Unicam SP8-200 dual-beam spectrophotometer. All assays were carried out in duplicate and on more than one biological sample (see text for details of sample numbers). MPO has halide-dependent and halideindependent catalytic activities which were assayed as described in Andrews and Krinsky (1982). Halide-independent activity was assayed by the Voxidation of 3,3’,5,5’-tetramethylbenzidine (TMB). The reaction mixture routinely used for assaying mussel putative MPO activity contained in a final volume of 3 ml: 50 mM sodium acetate buffer, pH 5.0 (optimal pH for activity in digestive gland), 0.88 mM TMB and 5 mM H,O,. Conditions of pH and substrate concentration were Garied and the details are given in the text. The

reaction was started by the addition of sample and monitored continuously at 655 nm. Under these conditions, one milliunit of MPO activity is defined as equivalent to a 655 nm absorbance change of O.O114/min (cell path length of 1 cm). Halide-dependent MPO activity was assayed by the production of chloramine from diethanolamine. Incubations contained in a final volume of 1.5 ml: 10 mM diethanolamine in 0.1 M sodium acetate/acetic acid pH 4.3, 1 mM H,O, and 100 mM NaCl. The reaction was &ted by the addition of sample and monitored continuously at 280 nm. Under these conditions, an absorbance change of 0.400/min is observed for 1 fig of MPO activity. Catalase activity was assayed as described in Livingstone et al. (1990). Reaction mixtures contained, in a final volume of 3 ml, 50 mM potassium phosphate buffer pH 7.0 and 19 mM H,O,. The reaction was initiated by the addition of sample and the decrease in H,O, concentration monitored continuously at 240 nm. Activity was quantitated using a molar extinction coefficient of 43.6 M-’ cm-‘. Apparent Michaelis-Menten (K,) constants were determined from weighted Lineweaver-Burk plots. Groups of values for the effects of Sephadex G-25 treatment were compared by Student’s l-test (P < 0.01). Values are given as means k SD. RESULTS

Assays for halide-dependent and halide-independent activities were employed to look for the existence

et al.

of MPO in digestive gland. Unusual results were obtained with the halide-dependent assay. An activity was detected in the absence of any added NaCl or H,O, , i.e. the 9000 g supernatant and diethanolamine alone were sufficient to apparently produce chloramine. The activity was not stimulated by the subsequent addition of NaCl and H,O,. The activity in the 9000g supernatant was eliminated by boiling (1OOC for 15 min), reduced by removal of the low molecular weight fraction (metabolites) and showed linearity with protein (sample) concentration (Fig. la and b), indicating that the process was probably protein-based and enzymic. Halide-independent activity was present in the digestive gland 9000g supernatant (Fig. la) and showed linearity with protein (sample) concentration (Fig. lb). The activity was heat-killable and markedly reduced by passage down a Sephadex G-25 column (Fig. la). No activity was observed in the absence of added H202 or TMB. The activity showed MichaelisMenten kinetics with respect to dependency on the concentration of both substrates, with apparent K,,, for H, O2 and TMB of, respectively, 0.13 and 243 PM (Fig. 2). The pH optimum of the activity was 5.0 (see Fig. 4). The halide-independent assay is not as specific for MPO as the halide-dependent assay because the former reaction can also be catalyzed by other peroxidases. This was observed in preliminary studies with commercial horseradish peroxidase and catalase, albeit the TMB oxidation activities were much lower than the enzymes’ main catalytic activities (data not shown). Since catalase is found in considerable activity in all subcellular fractions of digestive gland of M. edulis, either through appropriate localization in the cell or as a result of contamination (Livingstone et al., 1990; Winston et al., 1990) the

possibility was examined that the digestive gland halide-independent activity might be due to catalase rather than to MPO. The distributions of catalase and TMB oxidation activities between the 50012,000 g pellet (mitochondrial fraction) and 12,000 g supernatant (post-mitochondrial fraction) were compared. The distributions of the two enzyme activities were different, the mitochondrial fraction possessing a larger percentage of TMB oxidation activity, whereas the reverse was observed for catalase activity (Fig. 3). In addition, in the absence of the availability of purified catalase from M. edulis or any other mollusc, the pH profiles for TMB oxidation were compared between the digestive gland 9000g supernatant and a commercial pure catalase (bovine liver). The pH profiles were very different, the mussel apparent MPO having a pH optimum of 5.0 compared to 3.5 for catalase-dependent TMB oxidation (Fig. 4) The results indicate that the digestive gland TMB oxidation activity is, at least in part, not due to the catalytic action of catalase. Halide-independent putative MPO activity was also observed in blood from mussels, but halidedependent activity, either in the presence or absence of added NaCl and Hz02 could not be detected. Whole blood and the sonicated cells from the blood contained, respectively, 79.4 f 24.0 (N = 3) and 29.5 f 8.3 (N = 3) milliunits halide-independent activity per ml original blood, i.e. 37% of the activity

Myrilus myeloperoxidase

65

(4

z

e O.WO

: 9000 x g

G-25

Boiled

Sample

0.5 -

.

-

Independent Dependent

-

-10

0.4 -6 0.3 -6 0.2 -

Fig. I(a) Effect of boiling and Sephadex G-25 chromatography on “halide-dependent” and “halide-independent” putative myeloperoxidase (MPO) activities in mussel digestive gland 9000g supernatant. Halide-dependent activities were observed in the absence of any added NaCl or H,O, (see Results). Values are means + SD (N = 3). Halide-dependent and halide-independent activities are expressed, respectively, as pg and milliunits of MPO (see Materials and Methods for definition of units). (b) Dependence of “halide-dependent” and “halide-independent” putative myeloperoxidase activities in mussel digestive gland 9000g supernatant on protein (sample) concentration. Values are the means of two determinations. Other details as for Fig. l(a).

0.7

2 z 2 z a = m E :

0.4 0.3 0.2 -

-100

0

100

200 l/r

300

400

6

(mm)

Fig. 2. Dependence of halide-independent putative myeloperoxidase activity of digestive gland 906Og supematant on tetramethylbenzidine and hydrogen peroxide concentration, displayed on a LineweaverBurke reciprocal plot. Values are means k range (N = 2) or SD (N = 3). Other details as for Fig. l(a). CBPC W,I,I--E

66

DANIELSCHLENKef al.

40

f s s r

20

t 0 rv+Q Enzyme

Acthtty

Catah

Fig. 3. Subcellular distribution of catalase and halide-independent putative myeloperoxidase activities in digestive gland. Values are means + SD (N = 3). Percentage distributions of activities between the two fractions are shown on the Figure. Catalase activity is in nmol/min-‘. Other details as for Fig. I(a).

was in the cells (assuming sonication)

and Krinsky, 1985). In the mussel, diethanolamine reacted with 9000g supernatant from the digestive gland, forming chloramine or another compound that absorbed light at 280nm, without the need for the addition of either halide or H202. The reaction was indicated to be enzymic-mediated, and was significantly reduced by removal of low molecular weight metabolites from the sample, indicating that endogenous halide ions and Hz02 could have been responsible for part, but not all, of the apparent chloramine formation. Because of the unusual results for the halide-dependent activity, attention was focused on the halideindependent activity (TMB oxidation) for the further investigation of the existence of MPO. TMB oxidation activity was detected in blood cells and digestive gland of the mussel. The blood of various bivalve molluscs contains a distinct phagocytic group of cells known as hemocytes which seems to be analogous to white blood cells in vertebrates (Narain, 1973). In M. edulis, macrophages are involved in the clearance of injected carbon particles (Moore and Lowe, 1977), and increase in numbers in response to shell damage (Bubel et al., 1977). The classification or identification of different types of hemocytes in M. edulis

no loss of activity through and 63% in the serum. DISCUSSION

MPO has been purified in several vertebrate species (Bos et al., 1978; Bakkenist et al., 1978). The molecular weight of the protein has been shown to vary among species; 65,000 for guinea-pig and 144,000 for human (Bakkenist et al., 1978). The formation of hypochlorous acid (HOCI) by the enzyme serves a bactericidal function in phagocytic white blood cells (leukocytes) and MPO occurs primarily within the lysosomal compartment of the cell (Bos et al., 1978). The detection of MPO in the mussel studies was based upon the measurement of the enzyme’s catalytic activities. The most specific assay for MPO is the quantification of hypohalous acid formation (usually HOC]) which is dependent upon the participation of halide in the reaction, i.e. the halide-dependent MPO activity. Upon the addition of H,O,, MPO is able to remove two electrons from chloride, which can then react with an added substrate (diethanolamine) leading to the formation of a product (chloramine) which is spectrophotometrically detectable (Andrews

0

3

4

5

6

7

8

PH

Fig. 4. The dependence of TMB oxidation catalyzed by commercial pure catalase (bovine liver) and digestive gland YOOOgsupernatant (halide-independent putative myeloperoxidase activity) on pH. Values are the means of two determinations. For details of enzyme activity units see Fig. l(a).

Myfilus

myeloperoxidase

and other bivalves has been a matter of discussion and some disagreement. Three morphologically distinct types of hemocyte were identified in M. edulis-lymphocytes, macrophages and eosinophilic granulocytes (Moore and Lowe, 1977). This was disputed and the results reinterpreted as showing two types of hemocyte-hyalinocytes and granulocytes-with the macrophage redefined as a small granulocyte (Cheng, 1981). More recently, however, support for the original classification has come from differential lectin binding studies which revealed three types of cell with distinct carbohydrate compositions, namely hyalinocytes and two forms of granulocyte (Pipe, 1990). The occurrence of TMB oxidation activity in the cellular component of the blood is indicative that hemocytes possess MPO, but in which of the cell types it is present will require histochemical localization studies to establish. Also of interest is the occurrence of a proliferative atypical hemocytic condition in M. edulis from polluted environments (Lowe and Moore, 1978), possibly indicating an interaction between organic xenobiotic metabolism and MPO presence. TMB oxidation activity was observed in both the mitochondrial and post-mitochondrial fractions of the digestive gland. Since only approximately 2% of digestive gland tissue consists of hemocytes (R. K. Pipe, personal communication), the likelihood is that TMB oxidation activity is also present in other cell types such as digestive cells (Moore et al., 1978). Bivalves are known to regularly feed on bacteria and unicellular algae (Morton, 1983). Consequently, bactericidal enzymes such as MPO would probably be necessary in digestive gland cells to digest this material obtained from the diet. Although TMB oxidation activity was present in the post-mitochondrial fraction, this may have been due to the relative ease of lysosome fragmentation during tissue homogenization releasing enzyme into the supernatant (Livingstone and Farrar, 1984). A cytosolic localization is also a possibility. The marked reduction in the TMB oxidation activity, following removal of the low molecular weight fraction, is possibly indicative of a need for certain stabilizing agents or cofactors for maximal activity. Since HOC1 is an extremly strong oxidant, certain substrates such as TMB are prone to one-electron oxidations which leads to the formation of a dimerized end-product, which absorbs light at 655 nm (Andrews and Krinsky, 1982). However, since other oxidants may cause the same reaction, the measurement of TMB oxidation lacks the specificity for MPO of the halide-dependent activity. For example, commercial catalase catalyzed the oxidation of TMB, presumably through the formation of a compound I (oxidation state equivalent to Fe V) and its abstration of electrons from TMB (Andrews and Krinsky, 1985). Consequently, due to the ubiquitous nature of catalase in mussel digestive gland subcellular fractions (see before), the possibility was explored that the observed halide-independent activity might be due to catalase. This was indicated not to be so from the different distributions of TMB oxidation and catalase activities between the mitochondrial and post-mitochondrial (cytosol plus microsomes) fractions. Catalase is virtually totally localized in the peroxisomes in M. edufis digestive gland, and its distribution between the two

61

fractions is due to part sedimentation of intact peroxisomes at 12,000 g and part disruption of peroxisomes releasing catalase into the medium (D. R. Livingstone, F. Lips and R. K. Pipe, unpublished data). The low pH 5 optimum of TMB oxidation by digestive gland 9000g supernatant is consistent with a lysosomal location, the enzymes of which generally have acidic pH optima (Moore, 1985), and indicative that cytosolit peroxidases, if they exist, are unlikely to be contributing to the activity. The pH optimum was also different from that of the catalase-catalyzed TMB oxidation, consistent with the mussel TMB oxidation activity not being catalase-derived (this experiment can only be regarded as an indication, of course, as the mussel catalase may have different properties from the bovine liver one used in the study). The high percentage of TMB oxidation activity in the mitochondrial fraction is surprising, given the fragility of the lysosomes (in subcellular characterization studies of digestive gland, the lysosomal marker enzyme was over 90% present in the 100,OOOg supernatantLivingstone and Farrar, 1984). Possible explanations for this include sedimentation of intact blood cells present in the digestive gland, a tight association of activity with lysosomal membranes (either sticking to, and sedimenting with, mitochondria, or in aggregates sufficiently heavy enough to be sedimented themselves), or a mitochondrial presence for the enzyme activity. Significant differences in apparent K,,, values were observed for H,O, and TMB for the halide-independent activity, indicative of differences in substrate affinities. Since H2 0, is a natural substrate for MPO, its higher affinity (lower apparent K,,,) is perhaps not surprising. Similarly, the lower affinity for TMB is possibly a consequence of it not being a typical natural substrate for the enzyme. The demonstration of TMB oxidation activities in the blood cell particulate fraction and the digestive gland, and the subcellular distribution and pH studies, are indicative of the existence of MPO in M. edulis. However, the non-detection in blood and unusual results in digestive gland for the MPO halide-dependent activity mean that more studies are required before a definitive conclusion can be made. The existence of MPO in bivalves and other molluscs would be of significance from a toxicological standpoint. The oxidation of TMB involves two separate one-electron oxidations leading to the formation of free radicals (Andrews and Krinsky, 1982). Many pollutants are likewise vulnerable to one-electron oxidations, which may initiate redox-cycling events causing the production of oxyradicals and possible initiation of lipid peroxidation and DNA damage (Kasai and Nishimura, 1986; Morehouse and Aust, 1988). MPO has been shown to bioactivate several xenobiotics in mammals (Tsuruta et al., 1985; Uetrecht and Zahid, 1988; Thompson et al., 1989), and therefore the same could occur in M. edulis and other molluscs. REFERENCES

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