Protection of glycolytic enzymes by metallothioneins from oxidative damage in the digestive gland of green lipped mussel Perna viridis.

ABSTRACT We evaluated the capacity of metallothioneins (MT) in maintaining anaerobic metabolism in the green lipped mussel Perna viridis during experimentally induced oxidative stress. Mussels were exposed to 50 [micro]g/L of cadmium during seven days, followed by recuperation in the sea during 35 days. Other groups of cadmium pre-exposed organisms and their respective controls were exposed to 200 and 500 mM of ferric iron/100 mM ascorbate to promote the formation of reactive oxygen species (ROS) by the Fenton reaction. Oxidative damage was evidenced by the malondialdehyde (MDA) production through thiobarbituric acid reactive substances (TBARS). The activities of the enzymes, hexokinase (HK), piruvate kinase (PK), and glucose-6-P dehydrogenase (G6PDH) in the digestive gland and the survival during anoxia were evaluated. Exposure of organisms to cadmium led to a significant bioaccumulation of this metal with concomitant high MT levels. Survival during prolonged periods of anoxia was reduced only in the iron/ascorbate exposed organisms, which exhibited an increase in MDA concentration; however, the cadmium-preexposed and control organisms showed similar resistance to anoxia. The iron/ ascorbate-exposed organisms showed a decrease in the activities of HK in the cytosol and HK, PK, and G6PDH of the particulate fraction. In comparison with the control group, the cadmium pre-exposed/iron-exposed organisms did not show significant differences in lipid peroxidation, associated with elevated MT levels. In conclusion, the data obtained suggest that of the glycolytic enzymes, HK and PK and G6PDH are targets to the attack by ROS and that the MT can protect molecules against these effects. MT appears to protect against ROS induced injury, allowing maintenance of metabolic control by glycolytic enzymes that are important during anaerobic metabolism.

KEY WORDS: enzymatic activities, glucose-6-phosphate dehydrogenase, hexokinase, metallothioneins, oxidative stress, Perna viridis, pyruvate kinase

INTRODUCTION

Oxidative stress of marine molluscs affect many levels organization through generation of reactive oxygen species (ROS) and resulting oxidative damage of membranes, proteins, and DNA (Winston & Di Guilio 1991, Storey 1996, Livingstone 2001). Evidence has been reported showing that oxidative stress can reduce the capability of intertidal organisms to cope with oxygen decreased availability and that metallothioneins (MT) play an important role in the antioxidant defenses against such oxidative insults. Accordingly, Viarengo et al. (1999) observed that the treatment of the mussel Mytilus galloprovicialis with nontoxic doses of Cd, which increased the tissue levels of MT, provided protection against oxidative effects of a subsequent exposure to prooxidant chemical, such as iron and hydrogen peroxide. The oxidative toxicity reflected by membrane lipid peroxidation and decreased tolerance to anoxia, was less pronounced in Cd pre-exposed as compared with nonexposed mussels. Several lines of research have demonstrated the role of MT in the homeostasis and detoxification pathways of transition metals (Hamer 1986, Kagi & Shaffer 1988, Roesijadi 1992, Davis & Cousins 2002) and the antioxidant properties in diverse biological systems (Thornalley & Vasak 1985, Thomas et al. 1986, Kang 1999, Viarengo et al. 1999, Davies 2000, Kondoh et al. 2003); however, limited attention has been given to the MT antioxidant influences on the control of anaerobic metabolism in anoxic tolerant molluses.

The biochemical mechanisms conferring hypoxia or anoxia tolerance have been analyzed in bivalves and gastropods, with most of the studies conducted on Mytilus galloprovincilis (Wang & Widdows 1993, Isani et al. 1995), M. edulis (De Zwaan et al. 1991, De Zwaan et al. 1995), Littorina littorea (Greenway & Storey 2000, Greenway & Storey 2001, Ladare & Storey 2002). In hypoxia, metabolic energy demands are chiefly met by reliance on glycolysis. When oxygenation reaches low critical levels, conservative strategies favor reversible inhibition of the glycolytic pathway, coupled with additional substrate-level phosphorylations (Hochachka & Somero 2002). The ATP yield per hexose is maximized by the accumulation of end products such as succinate or propionate. At this stage, the ATP turnover is strongly inhibited and the metabolic rate falls to <10% of the aerobic rate at the same temperature (Hochachka & Somero 2002). Thus, anoxic survival can be extended despite a fixed amount of fermentable fuel. Anaerobic energy metabolism is controlled by the interplay of biochemical mechanisms, which include allosteric control by key metabolites, reversible enzyme binding with subcellular and structural elements, and reversible protein phosphorylation (Clarke et al. 1984, Lazou & Beis 1989, Brooks & Storey 1993, Gutowicz & Terlecki 2003).

Among anoxic tolerant organisms, a decreased binding of glycolytic enzymes to subcellular structure is noted when glycolytic flux decrease and an increased binding when the flux is raised (Lushchak et al. 1997, Lushchak et al. 1998). Such a binding can modify the conformation of the glycolytic enzymes bringing about alteration of regulatory catalytic properties. The binding can be influenced by the phosphorylation states of the protein transition (Brooks & Storey 1993, Greenway & Storey 2001). The harmful effects of oxidative stress on mollusc anoxic tolerance suggest that oxidative injuries may occur at molecular and cell structures impinging the metabolic regulation that allows survival of hypoxia and anoxia.

This research was aimed at examining whether experimentally induced oxidative stress perturbs the maximal activities of both cell particulate bound and soluble glycolytic regulatory enzymes (namely pyruvate kinase, EC 2.7.1.40: hexokinase, EC 2.7.1.1), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) in the digestive gland of the green lipped-mussel Perna viridis, in line with depressed anoxic tolerance. In addition, the possible protection of MT against the oxidative iron toxicity was assessed after recovery from short-term exposure to a low Cd concentration, without noticeable Cd harms to the organism. The digestive gland displays marked antioxidant defense and can synthesize MT, it seemed to be the best to conduct this study.

Perna viridis is broadly distributed in tropical marine ecosystems, and has recently colonized Caribbean habitats (Lodeiros et al. 1999). The green-lipped mussels are commonly found in muddy-benthos along the northeastern Venezuelan coast, which are at the risk of contamination by oxidant xenobiotic associated with anthropogenic activities.

MATERIALS AND METHODS

Organisms and Experimental Treatments

Green-lipped mussel Perna viridis (60-80 mm) was collected from "La Esmeralda" (10[degrees]26'N, 64[degrees]01'O) a locality near Carupano in eastern Venezuela. The animals were kept 40 L-aquaria (1 animal/liter) for 2 wk prior to the experiments in well-aerated seawater (26 [+ or -] 2[degrees]C, salinity 36 [per thousand], pH 7.8 and oxygen saturation >90%). The water was changed daily and organisms were fed with 100 mL of microalgae Tetrasehnis chuii (density 20,000 cells/mL). To induce an increase of metallothionein concentrations in the digestive gland of mussels, the organisms were exposed for 7 days to 50/[micro]g/L cadmium and transferred to the Gulf of Cariaco (10[degrees]41'N 63[degrees]24'W) for a 35-day detoxification period. Then, control and Cd-preexposed animals were placed in aquaria containing a mixture of 200 and 500 mM Fe[Cl.sub.3], each with 100 mM ascorbate for 7 days, to promote acute oxidative stress. In addition, a 100 mM ascorbate control group was assayed.

Cadmium and Iron Concentration

The dry mass of the digestive gland was estimated after drying at 60[degrees]C for 48 h. The dry samples were digested with nitric acid (HN[O.sub.3], 69%) for 24 h, and then placed in a water bath at 60[degrees]C for 6 h. The digested tissues were filtered through No.42 Whatman paper and the solution volume was adjusted to 10 mL with deionized water. The concentration of cadmium and iron was then determined using atomic absorption spectrophotometry with air-acetylene flame and a deuterium background corrector (Perkin Elmer, model 3110), taking the mean of three readings. The method was verified using NIST SRM 1566a Oyster Tissue, TORT-2 lobster hepatopancreas y DORM-2 dogfish muscle of National Research Council of Canada (Nova Scotia, Canada). The average values of metals were within 90% to 98% of the certified values.

MT Content

Metallothionein content in digestive glands was evaluated according to Viarengo et al. 1997. The tissue was rapidly dissected; damp dried and stored at -40[degrees]C during 3-5 days. One gram of tissue sample was homogenized in three volume of 500 mM sucrose, containing 20 mM Tris-HCl buffer pH 8.6 with 0.6 [micro]M of leupectine, 500 [micro]M phenylmethyl-sulphonyl fluoride (PMSF) as antiproteolytic agents and 0.01% of dithiotritol (DTT) as a reducing agent. The homogenate was centrifuged at 30,000 g for 20 rain (0-4[degrees]C) to obtain the supernatant containing metallothioneins. To the supernatant (1 mL) was added 1.05 mL of cold (-20[degrees]C) ethanol and 80 [micro]L of chloroform, followed by a centrifugation at 6,000 g for 10 min at 0-4[degrees]C. The supernatant was combined with 1 mg RNA, 40 [micro]L of 37% HCl and with three volume of cold ethanol (to a final concentration of 87%). The sample was maintained at -20[degrees]C for 1 h, and then centrifuged at 6,000 g for 10 min. The metallothionein-containing pellet was washed with 87% ethanol: 1% chloroform in homogenizing buffer, centrifuged at 6,000 g for I 0 min and dried under a nitrogen gas stream. The pellet was resuspended in 150 [micro]L of 250 mM NaCl and 150 [micro]L of 1 M HCl containing 4 mM ethylene diaminetetraacetic acid (EDTA). A volume of 4.2 mL of 2 M NaCl containing 0.43 mM 5,5'-dithibis-2-nitrobenzoic acid (DTNB) buffered with 0.2 M Na-phosphate pH 8, was added to the sample at room temperature (Ellman 1958). The sample was finally centrifuged at 3,000 g for 5 min; the absorbance of the supernatant was evaluated at 412 nm and metallothionein concentration was estimated using reduced glutathione (GSH) as a standard. The amount of MT was defined assuming a cysteine content in mussel MT of 29% (Khoo & Patel 1999).

Lipid Peroxidation

Eight individuals were sampled to estimate lipid peroxidation by formation of thiobarbituric acid reactive substances (TBARS). The digestive glands were homogenized at 5% (w/v) in 50 mM Imidazole-HCl buffer at 4[degrees]C. The extract was centrifuged at 12,000 g for 20 min. Subsequently, 250 [micro]L of supernatant were placed in Eppendorf vials and incubated in water at 37[degrees]C during 15 min. Then, the vials were placed in ice, and 250 [micro]L of trichloroacetic acid (12.5%) and 0.8 M HCl, and 500 [micro]L of 1% thiobarbituric acid (TBA) were added. The samples were placed in water at 90[degrees]C with constant agitation for 10 rain and then centrifuged at 1500 g for 10 min. at 4[degrees]C. The absorbance was measured at 535 nm and malondialdehyde (MDA) concentration was estimated using an extinction coefficient 1.56 x [10.sup.5] [m.sup.-1]. [cm.sup.-1]. Lipid peroxidation was expressed in nmol MDA.[g.sup.-1] wet mass.

GSH Determination

Reduced glutathione (GSH) was quantified using a commercial assay kit (Bioxytech GSH-400), and was expressed as nmol GSH.[g.sup.-1] wet mass.

Preparation of Enzyme Extracts

The digestive glands were homogenized at 0-4[degrees]C (1:4 w/v basis) in 500 mM sucrose containing 10 mM DTT and 500 [micro]M PMSF. The homogenate was centrifuged for 5 min at 4[degrees]C at 23,000 g: after centrifugation the supernatant was removed and diluted with 4 volume of a stabilization buffer, which contained 100 mM potassium phosphate (pH 7.5), 1 mM EDTA, 1 mM ethylene glycol-bis-(2-aminoethyl) tetraacetic acid (EGTA), 25 mM NaF, 0.1 mM fructose-l,6-bisphosphate, 0.1 mM ATP and 10 mM DTT (Plaxtron & Storey 1986). This diluted fraction was then stored on ice for later measurement of soluble enzyme activity. After centrifugation, the pellet was resuspended in five volumes of stabilization buffer, vortexed, and centrifuged at low velocity (1,500 g) for 2 min. The supernatant was used for measuring the particulate enzyme activity.

Measurement of Enzyme Activities

All enzymes were measured at 25[degrees]C by monitoring at 340 nm after the absorbance changes of NADPH or NADH (ext. coeff. 6.2 m[M.sup.-1]. [cm.sup.-1]) using a Perkin-Elmer spectrophotometer. The following incubation mixtures provided optimal conditions for measuring reaction rates proportional to the quantity of enzyme extracts. Hexokinase (HK, EC 2.7.1.1): 100 mM Tris-HCl buffer, pH 8, 10 mM D-glucose, 5 mM ATP, 100 mM Mg[Cl.sub.2], 1 mM NAD[P.sup.+], and 1 unit (U) NAD[P.sup.+]-dependent glucose-6-phosphate dehydrogenase (G6PDH), 200 [micro]L of enzymatic extract; Pyruvate Kinase (PK, EC 2.7.1.40): 50 mM Imidazole-HCl, 13 mM MgS[O.sub.4], 100 mM KC1, 5 mM phosphoenolpyruvate, 5 mM AD[P.sup.+], 0.2 mM NADH and 1 U lactate dehydrogenase and 250 [micro]L of enzymatic extract; Glucose-6-Phosphate Dehydrogenase (G6PDH, EC 1.1.1.49): 50 mM Imidazole-HCl, pH 7.5, 5 mM MgS[O.sub.4], 0.05 mM NAD[P.sup.+], 2 mM Glucosa-6-phosphate and 200 [micro]L of enzymatic extract. Enzyme activities were expressed as in international enzyme units ([micro]mol substrate converted to product per minute) per gram wet-mass.

Anoxia Survival

Twenty organisms from each experimental treatment were placed in closed 6L-vessels containing seawater (26[degrees]C [+ or -] 2[degrees]C, salinity 36 [per thousand]) that had been equilibrated and maintained with [N.sub.2] gas atmosphere. Mussel were renewed and placed in freshly prepare anoxic water every day at which time survivors were counted. Open valves or absence of muscular activity was considered as an indication of death.

[FIGURE 1 OMITTED]

Statistical Analysis

Non-transformed data were subjected to tests of normality and homogeneity of variance. One-way analysis of variance (ANOVA) was used to examine the effects of cadmium and iron exposure. Multiple comparisons among the mean values used the Bonferroni method with a [alpha] < 0.05 (Sokal & Rohlf 1981).

RESULTS

Heavy Metal Concentration and MT Content

Green-mussels exposed to 50 [micro]g/L cadmium showed a significant increase (~21.42 fold) in Cd (Fs = 157.48, P < 0.001) and MT levels (~3.23 fold; Fs = 25.01, P < 0.001) in the digestive gland. The detoxification (35 d) of Cd exposed organisms significantly decreased the tissue Cd concentration to ~65% of that found for Cd-exposed individuals, but MT levels remained essentially unchanged (Fig. 1). The iron/ascorbate-exposed mussels exhibited significant Fe accumulation in the digestive gland, both in the presence and absence of Cd preexposure (Fs = 79.39, P < 0.001; Fig. 2). No significant difference was recorded in MT concentrations between mussels exposed only to iron and their respective controls. The MT values were similar between the Cd and Cd/Fe exposed bivalves (Fig. 3).

Lipids peroxidation and GSH

The amount of MDA in the digestive gland did not differ significantly among the control, Cd exposed and Cd-depurated mussels (Fs = 0.182, P > 0.05), with mean values ranging from 163.62-177.43 nmol/g wet tissue. In contrast, the tissue MDA levels increased 1.52- and 1.91-fold in 200 and 500 mM Feexposed mussels, respectively, compared with controls. On the hand, the preexposure to Cd significantly inhibited the MDA formation in the Fe[Cl.sub.3]/ascorbate treated individuals; the corresponding mean values were essentially similar to that of the controls. The content of reduced glutathione in the digestive gland was not altered by the Cd-exposure and depuration (Fs = 0.17, P > 0.05). Likewise, Cd/Fe treatments did not affect the tissue GSH concentration. However, the exposure to the higher Fe doses resulted in an increased level of GSH (Fs = 4.18, P < 0.01) (Fig. 4).

[FIGURE 2 OMITTED]

Enzymatic Determination

The Fe-exposed organisms showed a marked decrease in the activities of HK in the cytosol fraction (Fig. 5). In the particulate fraction, HK and G6PDH activities decreased in organisms under the different Fe exposures; PK activity only dropped at the higher Fe concentration (Fig. 6). Cadmium exposure did not exert effect on the glycolitic enzyme activities, but Cd-preexposure prevented the iron induced enzymatic inhibition.

[FIGURE 3 OMITTED]

Anoxia Survival

The resistance to prolonged periods of anoxia was reduced only in the Fe-exposed organisms, associated to an increase in TBARS concentrations; however, the Cd-preexposed and control organisms showed similar resistance to anoxia (1-2 difference days) (Fig. 7). Hence, cadmium preexposure prevented iron induced decrease in survival during anoxia.

DISCUSSION

This study shows significant alterations in the activity of cytosolic HK, particulate-bound HK, PK and G6PDH in the digestive gland of the green-mussel Perna viridis after 7-d exposure to sublethal concentrations of iron/ascorbate. The development of oxidative stress ensued the chemical exposure, associated with changes in the activities of the enzymes and lowered tolerance to anoxia. Conversely, Cd pre-exposed mussels followed by a depuration period (35 d) prior to iron/ ascorbate treatments did not display the foregoing biological changes; neither did the 7d-Cd exposed and metal nonexposed mussels. The Cd-experiments rose the tissue levels of metallothioneins (MT), which are known to possess antioxidant properties through reactive oxygen species (ROS) scavenging, protecting cells and organisms from oxidative stress (Hidalgo et al. 1988, Anderson et al. 1999, Viarengo et al. 1999, Kondoh et al. 2003). The proceeding discussion is focused on the implications of oxidative stress on the limited activity of the tested enzymes, constraining the metabolic capacity of the mussels to survive hypoxia or anoxia. Furthermore, it is suggested that MT may have important role in the antioxidant defenses for normal functioning of the glycolityc pathways to endure anoxic tolerance, when survival is threatened by internal and external prooxidant stimuli.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

The digestive gland in the organisms exposed only to iron/ ascorbate dropped the soluble and particulate HK activity to ~50% to 70% levels of corresponding activity recorded for both control and Cd-pre-exposed mussels. The inhibition of HK was associated with increased iron uptake by the tissue, displaying a marked rise of MDA. The elevated tissue burden of iron probably increased the levels of the redox forms of the metal, which further promote the generation of ROS by the Haber-Wiess reaction (Fridovich 1998, Hermes-Lima 2004), and hence the oxidative damage ensued. Likely, iron directly and/or the iron mediated oxidative stress targeted the particulate-bound and soluble HK catalytic function. The HK activity is dithiol dependent (Chou & Wilson 1974, Subbarao & Kenkare 1977, Magnini et al. 1980); two essential cysteine residues within the catalytic center may interact with Fe to form an inactive catalytic-iron complex. Also, these thiols may readily be oxidized by ROS to disulphyde bonds, destabilizing the enzyme. Similar, observations have been previously reported for HK in the digestive gland from mussels, and rabbit erythrocytes exposed to iron/ascorbate or heavy metals (Lai & Blass 1984a, Lai & Blass 1984b, Stocchi et al. 1994, Canesi et al. 1998, Canesi et al. 1999, Jannaschk et al. 1999).

[FIGURE 6 OMITTED]

The particulate bound PK and G6PDH also lost activity in the digestive gland, as its iron and MDA contents heightened. However, soluble PK and G6PDH remained unaltered. These enzymatic changes may also be explained as the result of ROS overload induced by iron, causing molecular or cell structural damage, exerting inhibitory influence on the particulate-bound enzymes. Evidence of the binding of some glycolytic enzymes to insoluble proteins and membrane structures has been given for vertebrates and nonveretebrates (Clarke et al. 1984, Plaxtron & Storey 1986, Brooks & Storey 1993, Lushchak et al. 1997, Lushchak et al. 1998, Lushchak et al. 2001), demonstrating that the ability of the binding of glycolytic enzymes to subcellular specific structures plays a marked role in the regulation of glycolysis as determined by the ordering of the energy consuming pathways. Such a binding can modify the conformation state of the enzymes changing their kinetic properties, and thus their regulatory the catalytic responses (Gutowicz & Terlecki 2003).

[FIGURE 7 OMITTED]

The role of glycolytic enzyme partition between soluble and particulate cell fractions in the control of anaerobic metabolism has been assessed for some anoxic tolerant marine molluscs (Plaxtron & Storey 1986, Hochachka & Somero 2002,); the results demonstrate a decreased binding of several glycolytic enzymes in line with a suppressed metabolic rate during environmental anoxia, as opposed to an increased binding of enzymes concurrently with an increases glycolytic flux as oxygenation is favored (Plaxtron & Storey 1986, Lushchak et al. 1997, Lushchak et al. 1998). The biochemical processes underlying the cellular partition of enzymes for the control of the glycolytic rate could be potential targets for the damaging action of ROS under oxidative stress. Some of the well known consequences of the oxidative damage include protein oxidation, lipid peroxidation, protein cross-linking, impaired mechanisms ofgene expression, ultimately resulting in cell metabolic dysfunction (Halliwell & Gutteridge 1984, Di Guilio et al. 1995, Fridovich 1998, Livingstone 2001). Accordingly, the development of oxidative stress in P. viridis could modify the cells regulation on particulate bound PK, HK, G6PDH, and possibly other glycolytic enzymes in the digestive gland, and thus pose a threat of perturbations on the metabolic capacity to withstand period of oxygen lack. The enzyme PK is key element in the metabolic control of glycolytic flux rate through the hypoxic and anoxic energy producing pathways; the couple HK and G6PDH is important in the glycolytic generation of reducing equivalents (NADPH), supporting the cellular antioxidant power. Hence, the reduced tolerance to anoxia showed by iron/ascorbate exposed mussels may be linked, at least partially, to the oxidative alteration on the functional properties of the particulate bound enzymes, limiting the use of fermentable fuel and the supply of NADPH to sustain hypoxic or anoxic survival. Interestingly, soluble PK and G6PDH apparently are not readily targets for oxidative stress, though they may be affected if a strengthened tissue ROS generation emerges.

A noteworthy finding is the protection afforded by the Cd-exposure against the noxious oxidative effects on iron/ ascorbate exposure, associated with increased levels of MT in digestive gland of P. viridis, implying the possible involvement of the protein in the antioxidant pathways to prevent, or aminorate, oxidative injures at molecular, cellular and tissue levels. MT are sulphydril rich proteins having high affinity for group IB and IIB metal ions, thereby playing an important role in the metal homeostasis and detoxification (Roesijadi 1992, Davis & Cousins 2002). In addition, the MT are known to display antioxidant properties, mainly derived from sulphydryl nucleophylicity and metal complexity (Sato & Bremmer 1993, Kang 1999, Viarengo et al. 2000). MT biosynthesis is inducible by heavy metals (Freedman et al. 1989, Palmitier 1998); therefore, the persistent overproduction of MT in the digestive gland of P. viridis through Cd-exposure could lead to an increased antioxidant capacity, and improved metal complexing, including iron ion chelation, to control the free radical generation by the Fenton reaction, facilitating the normal physiological and biochemistry adjustments to hypoxia or anoxia.

The augmented MT-production in the Cd-treated mussels should expand the intracellular pool of the thiol groups involved in the antioxidant defense systems, in which glutathione (GSH) is an essential component. GSH is capable of complexing heavy metals and acting as scavenger of oxyradicals, especially [O.sub.2.sup.-] and HO-, and other reactive species (Winston 1991, Seis 1999). The GSH indirectly support the detoxification of free radical components by its function as cosubstrate of the antioxidant enzymes such as glutathione peroxidases (GPxs) (Regoli & Principato 1995). It is also cosubstrate of glutathione-S-transferase, which is important in the process of detoxification, including peroxidasic activity. Furthermore, several lines of evidence, derived mainly from in vivo and in vitro studies in vertebrates systems, have indicated that MT can buffer GSH concentrations attenuating its depletion by oxidant effects, by means of redox mechanisms associated to the protein-metal coordination chemistry (Hai'dara et al. 1999, Fabisiak et al. 2002). In virtue of these facts, it may be asserted that even with the stabilized GSH at control levels in the digestive gland of the Cd-Fe treated mussels, the rise of MT amount possibly set up antioxidant mechanisms making possible the organisms to cope up with the potential harmfulness of the oxidative elements, facilitating the normal response to anaerobiosis. The increase in GSH levels in 500 mM Fe-exposed organisms reflects an antioxidant response to exacerbate oxidative stress.

Moreover, current studies have demonstrated that MT can afford cell defense against iron induced oxidative stress by preventing enhanced lysosomal autophagy and concomitantly oxidative injuries (Baird et al. 2006, Terman et al. 2006). Lysosomes contain pool of redox active-iron resulting from engulfed ferrous materials that could make these organelles susceptible to oxidative damage. The degradation of the autophagic iron material leads to intralysosomal release of redox active- iron that upon transportation to the cytosol magnifies its cytosolic pool, endangering the cell viability. Likewise, lysosomes carrying an augmented charge of labile active iron are prone to oxidative attack, with membrane destructive consequences followed by expanding cell deterioration. Apparently, Mt exert protection against iron triggered oxidative lesions no only by sequestering cytosolic but also intralysosomal labile redox iron (Baird et al. 2006). The latter metal chelating activity was presumed feasible because apoMT showed iron binding capability in acidic and reducing lysosomal-like environment; thus, the metalloprotein would stabilized lysosomes in iron enriched cells. The foregoing antioxidant scheme has been adequately explained for the iron-binding protein ferritin. On this concern, the mechanistic action of MT is fare from being cleared.

The resistance to oxidative stress of the Cd-Fe treated mussels could be caused by oxidative defenses other than MT, specially the iron-binding protein ferritin that prevents iron from participating in Fenton type reactions and ensuing peroxidation of membranes (Ladare and Storey 2004). However, in mussels exposed to Fe without previous Cd-treatment, the MT in the digestive gland was held at the basal values, and TBARS rose significantly, implying deficiencies in the antioxidant defenses in the tissue to overcome the oxidative harms, reflected herein by inhibition of certain key enzymes of glucose metabolism. The defective antioxidant protection was possible related to a certain extent to the inhibition of HK and G6PDH reactions, limiting the generation of reductive power, NADPH, by the pentose phosphate pathway to meet the reductive requirements of some glutathione-dependent antioxidant reactions.

In agreement with the above assertions, a Cd-dependent resistance to oxidative stress has previously been reported for the hypoxic-tolerant mussels Mytilus galloprovincialis (De Zwaan et al. 1991), and it has been ascribed to MT over expression (Viarengo et at. 1999, Viarengo et al. 2000). Anoxic-tolerance tests on the whole organisms showed that anoxic survival was lowered in mussels treated with Fe, being such a effects less pronounced in Cd-preexposed mussels compared with the nonexposed ones. Furthermore, biochemical and digital image data concerning the digestive gland provided indication that MT has physiological role in the protection of cells against toxic effects of free radicals and possibly entire organisms from oxidative stress.

Interestingly, English and Storey (2003) provided compelling evidence that upregulation of MT gene expression is a response to both anoxia and freezing stresses in the marine gastropod Littorina littorea, possibly contributing to survival under these stressing conditions. North blot hybridization analysis of MT gene against total RNA from foot muscle and hepatopancreas from control, anoxia and freezing treated snails revealed a 2.5-6 fold increased of MT transcripts, within l h exposure to the stresses, remaining high a levels at 12 or 24 h. Upon 24 h recovery from either stress, transcript levels dropped in some cases but remained elevated in the hepatopancreas. Because freezing creates an ischemic state leading to tissue anoxia, it was conceived that the oxygen lack also promoted the activation of MT gene in frozen snail. On the basis of general documentation on the antioxidant protective effects of MT against oxidative stress in variety biological systems (vertebrates and invertebrates) during environmental stress, it was proposed that upregulation of MT may be of primary impor tance in the antioxidant system that supports freezing and anoxia survival in Littorina littorea. Furthermore, Ladare and Storey (2004) demonstrated that anoxia induced the ferretin-heavy chain gene in the hetopancreas of L. littorea; ferritin transcripts were actively produced and translated during the anoxic period, followed by a decrease to control levels during normoxic recovery. The anoxia-induced response appears to be mediated by cGMP in a yet hitherto manner. These observations suggested an important role of ferritin-heavy chains in the control of free iron levels, diminishing the generation toxic free radicals by fenton- linked reactions during anoxic episode. In addition, the accumulation of ferritin heavy chain transcripts during anoxia would ensure available template for the protein biosynthesis through the aerobic recovery, precluding iron activation of excessive formation of harmful oxyradicals. Consequently, ferritin upregulation represents an important component of the antioxidant defenses that endure the tolerance of L. littorea to natural anoxia. In the above general context, the knowledge for the role of MT and ferrtin upregulation in the control of oxidative stress is limited in anoxic tolerant tropical molluscs. Although there must be interspecific differences in the antioxidant responses of these metalloproteins to hypoxic or anoxic environmental stresses, it is evidenced herein that MT may be an important component of the antioxidant system that endures the anaerobic tolerance of P. viridis subjected to oxidative stress; possibly by participating in the control of cellular redox-active iron concentration.

In conclusion, the data obtained suggest that the regulatory glycolytic enzymes, HK and PK, and G6PDH are sensitive targets to the attack of ROS and that the MT may protect against oxidative stress acting as free-radical scavenger. The MT appears to play an important function against the ROS induced injury, allowing the maintenance of cell-partitioned activity of certain glycolytic enzymes that are important during anaerobic metabolism.

ACKNOWLEDGMENTS

The study was supported by the Science Coordination Office of Oriente University-Venezuela. The authors thank to Dr. Helga Guderley for her academic councils.

LITERATURE CITED

Anderson, S. R., K. M. Patel & G. Roesijadi. 1999. Oyster metallothionein as an oxyradicals scavenger: implication for hemocyte defense responses. Dev. Comp. Immunol. 23:443-449.

Baird, S. K., T. Kurz & U. T. Brunk. 2006. Metallothionein protects against oxidative stress-induced lysosomal destabilization. Biochem. J. 394:275 283.

Brooks, S. P. J. & K. B. Storey. 1993. Control of glycolytic enzymes binding: effect of changing enzyme substrate concentrations on vivo enzyme distributions. Mol. Cell. Biochem. 122:1-7.

Canesi, L., C. Ciacci, G. Piccoli, V. Stocchi, A. Viarengo & G. Gallo. 1998. In vitro and in vivo effects of heavy metals on mussel digestive gland hexokinase activity: the role of glutathione. Comp. Biochem. Physiol. C 120:261-268.

Canesi, L., C. Ciacci, M. Betti, M. Malatesta, G. Gazzanelli & G. Gallo. 1999. Growth factors stimulate the activity of key glycolytic enzymes in isolated digestive gland cells from mussels (Mytilus galloprovincialis Lam.) through tyrosine kinase-mediated signal transduction. Gen. Comp. Endocrinol. 116:241-248.

Chou. A. C. & J. E. Wilson. 1974. A study of sulphydrylic groups of rat brain hexoquinase. Arch. Biochem. Biophys. 163:191-199.

Clarke, F. M., P. Stephan, G. Huxham, D. Hamilton & D. J. Morton. 1984. Metabolic dependence of glycolytic enzyme binding in rat and sheep heart. Eur. J. Biochem. 138:643-649.

Davies. K. J. A. 2000. Oxidative stress, antioxidant defenses, and damage removal, repair, and replacement systems. IUBMB Life 50:279-289.

Davis, S. R. & R. J. Cousins. 2002. Metallothionein expression in animals: a physiological perspective on function. J. Nutr. 130:1085-1088.

De Zwaan, A., P. Cortesi, G. van Thillart. J. Roos & K. B. Storey. 1991. Differential sensitivities to hypoxia by two anoxia-tolerance marine mollusk: a biochemical analysis. Mat'. Biol. 111:343-351.

De Zwaan, A., P. Cortesi & O. Cattani. 1995. Resistance of bivalves to anoxia as a response to pollution-induced environmental stress. Sei. Total Environ. 171:121-125.

Di Guilio, R. T., W. H. Benson, B. M. Sander & P. A. Vanveld. 1995. Biochemical mechanisms: metabolism, adaptation and toxicity. In: G. M. Rand, editor. Fundamentals of aquatic toxicology. 2nd ed. pp. 223-255.

Ellman, G. L. 1958. A colorimetric method for determining low concentrations of mercaptans. Arch. Biochem. Biophys. 74:443-450.

English, T. E. & K. B. Storey. 2003. Freezing and anoxia stress induce expression of metallothionein in the foot muscle and hepatopancreas of the marine gastrop Littorina littorea. J. Exp. Biol. 206:2517-524.

Fabisiak, J. P., G. G. Borisenko, S. X. Liu, V. A. Tyurin, B. R. Pitt & V. E. Kagan. 2002. Redox sensor function of metallothioneins. Methods Enzymol. 353:268-281.

Freedman, J. H., M. R. Cirrolo & J. Persach. 1989. The role of glutathione in copper metabolism and toxicity. J. Biol. Chem. 264:5598-5607.

Fridovich, I. 1998. Oxygen toxicity: a radical explanation. J. Exp. Biol. 201:1203-1209.

Greenway, S. C. & K. B. Storey. 2000. Seasonal changes and prolongs anoxia effect the kinetic properties of phosphofructokinase and piruvate kinase in oysters. J. Comp. Physiol. [B] 170:285-293.

Greenway, S. C. & K. B. Storey. 2001. Effects of seasonal changes and prolonges anoxia on metabolic enzymes of Littorina littorea. Can. J. Zool. 79:907-915.

Gutowicz, J. & G. Terlecki. 2003. The association of glycolytic enzymes with cellular and model membranes. Cell. Mol. Biol. Lett. 8:667-680.

Haidara, K., P. Moffatt & F. Denizeau. 1999. Metallothionein induction attenuates the effects of gluthatione depletory in rat hepatocytes. Toxicol. Sci. 49:297-305.

Halliwell, B. & J. M. Gutteridge. 1984. Oxygen toxicity, oxygen radical, transition metal and disease. Biochem. J. 219:1-14.

Hamer, D. 1986. Metallothioneins. Annu. Rev. Biochem. 55:913-951.

Hermes-Lima, M. 2004. Oxygen in biology and biochemistry: role of free radicals. In: K. B. Storey, editor. Functional metabolism: regulation and adaptation. New York: John Wiley & Sons, Inc. pp. 319-368.

Hidalgo, J., L. Champmany, M. Borras, J. S. Garvey & A. Armario. 1988. Metallothionein response to stress in rats: role in free radical scavenging. Am. J. Physiol. 18:E518-E524.

Hochachka, P. W. & G. N. Somero. 2002. Biochemical adaptations: mechanism and process in physiological evolution. New York. USA: Oxford University Press. pp. 101-123.

Isani, G., O. Cattani, M. Zurzolo, C. Pagnucco & P. Cortesi. 1995. Energy metabolism of the mussel, Mytilus galloprovincialis, during long-term anoxia. Comp. Biochem. Physiol. C 100:103-113.

Jannaschk, D., M. Burgos, J. J. Centerlles, J. Ovadi & M. Cascate. 1999. Application of metabolic control analysis in the study of toxic effects of copper in muscle glycolysis. FEBS Lett. 445:144-148.

Kagi, J. H. R. & A. Shaffer. 1988. Biochemistry of metallothioneins. Perspectives in Bioehemistry. 27:8509-8515.

Kang, Y. J. 1999. The antioxidant function of metallothionein in the heart. Soc. Exp. Biol. Med. 222:263-273.

Khoo, H. W. & K. H. Patel. 1999. Metallothionein cDNA, promoter, and genomic sequences of the tropical green mussel, Perna viridis. J. Exp. Zool. 284:445-453.

Kondoh, M., K. Kamada, M. Kuronaga, M. Higashimoto, M. Takiguchi, Y. Watanabe & M. Sato. 2003. Antioxidant property of metallothionein in fasted mice. Toxicol. Lett. 143:301-306.

Ladare, K. & K. Storey. 2002. A profile of the metabolic responses to anoxia in marine invertebrates. Cell and molecular responses to stress. In: K. B. Storey & J. M. Storey, editors. Sensing, signaling and cell adaptation. Vol. 3. Amsterdam: Elsevier Press. pp. 1-24.

Ladare, K. & K. Storey. 2004. Accumulation and translation of ferritin heavy chain transcripts following anoxia exposure in marine invertebrate. J. Exp. Biol. 207:1353-1360.

Lai, J. C. & J. P. Blass. 1984a. Inhibition of brain glycolysis by aluminum. J. Neurochem. 42:438-446.

Lai, J. C. & J. P. Blass. 1984b. Neurotoxic effects of copper: inhibition of glycolysis and glycolytic enzymes. Neurochem. Res. 9:1699-1710.

Lazou, B. M. & I. Beis. 1989. Evidence for glycolytic enzyme binding during anaerobiosis of the foot muscle of Patella caerulea (L.). J. Comp. Physiol. [B] 158:771-777.

Livingstone, D. R. 2001. Contaminant-stimulated reactive oxygen species production and oxidative damage in aquatic organisms. Mar. Pollut. Bull. 42:656-666.

Lodeiros, C., B. Marin & A. Prieto. 1999. Catalogo de moluscos marinos de las costas nororientales de Venezuela. Clase Bivalvia. APUDONS. Cumana, Venezuela. 110 pp.

Lushchak, V. I., T. V. Bahnjukova & A. V. Spichenkov. 1997. Modification of pyruvate kinase and lactate dehydrogenase in foot muscle of the sea mussel Mytilus galloprovincialis under anaerobiosis and recovery. Braz. J. Med. Biol. Res. 30:381-385.

Lushchak, V. I., T. V. Bahnjukova & K. B. Storey. 1998. Effect on hypoxia on the activity and binding of glycolytic and associated enzymes in sea scorpion. Braz. J. Med. Biol. Res. 31:1059-1067.

Lushchak, V. I., L. P. Lushchak, A. A. Mota & M. Hermes-Lima. 2001. Oxidative stress and antioxidant defenses in goldfish Carassius auratus during anoxia and reoxygenation. Am. J. Physiol. Regul. Integr. Comp. Physiol. 280:R100-R107.

Magnini, M., V. Stocchi, P. Ninfali, M. Dacha & X. Fornaini. 1980. Action of oxidized and reduced glutathione on the rabbit red blood cell hexokinase. Biochem. Byophys. Acta. 615:113-120.

Palmitier, R. D. 1998. The elusive function of metallothionein. Proc. Natl. Acad. Sei. USA 95:8428-8430.

Plaxtron, W. C. & K. B. Storey. 1986. Glycolitic enzyme binding and metabolic control in anaerobiosis. J. Comp. Physiol. [B] 156:635-640.

Regoli, F. & G. Principato. 1995. Glutationa, glutationa-dependent and antioxidants enzymes in mussel, Mylitus gallovincialis, exposed to metal under field and laboratory conditions: implications for the use of biochemical biomarkers. Aquat. Toxicol. 31:143-164.

Roesijadi, G. 1992. Metallothioneins in metal regulation and toxicity in aquatic animals. Aquat. Toxicol. 22:81-113.

Sato, M. & 1. Bremmer. 1993. Oxygen free radicals and metallothionein. Free Rad. Biol. Med. 14:325-337.

Seis, H. 1999. Glutathione and its role in cellular functions. Free. Rad. Biol. Med. 27:916-921.

Sokal, R. R. & J. F. Rohlf. 1981. Biometry: the principles and practice of statistic in biological research, 2nd ed. New York: W.H. Freeman & Co. 859 pp.

Stocchi, V., B. Biagiarelli, M. Fiorani, F. Palma, G. Piccoli, A. Cucchiarini & M. DachS_. 1994. Inactivation of rabbit red blood cell hexokinase activity promoted "in vitro" by an oxygen radical generating system. Arch. Biochem. Biophys. 311:160-167.

Storey, K. B. 1996. Oxidative stress: animal adaptations in nature. Braz. J. Med. Biol. Res. 29:1715-1733.

Subbarao, B. & U. W. Kenkare. 1977. Reaction of brain hexokinase with tetranitromethane: oxidation of essential thiol groups. Arch. Biochem. Biophys. 181:8-18.

Terman, A., B. Gustafsson & U. T. Brunk. 2006. The lysosomal mitochondrial axis theory ofpostmitotic aging and cell death. Chem. Biol. Interact. 163:29-37.

Thomas, J. P., G. J. Bachowski & W. G. Albert. 1986. Inhibition of cell membrane lipid peroxidation by cadmium and zincmetallothioneins. Biochim. Biophy. Act. 884:448-461.

Thornalley, P. J. & M. Vasfik. 1985. Possible role metallothionein in protection against radiation-induced oxidative stress. Kinetics and mechanism of its reaction with superoxide and hydroxyl radicals. Biochim. Biophy. Act. 827:36-44.

Viarengo, A., E. Ponzano, F. Dondero & R. Fabbri. 1997. A simple spectrophotometric method for metaltothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs. Mar. Environ. Res. 44:69-84.

Viarengo, A., B. Burlando, M. Cavaletto, B. Marchi, E. Ponzano & J. Blasco. 1999. Role of metallothionein against oxidative stress in mussel Mytilles galloprovincialis. Am. J. Physiol. 227:1612-1619.

Viarengo, A., B. Burlando, N. Caratto & I. Panfoli. 2000. Antioxidant role of metallothioneins: a comparative overview. Cell. Mol. Biol. 46:407-417.

Wang, W. X. & J. Widdows. 1993. Metabolic responses of the common mussel Mytilus edilis to hypoxia and anoxia. Mar. Ecol. Prog. Ser. 95:205-214.

Winston, G. W. 1991. Oxidants and antioxidants in aquatic animals. Comp. Biochem. Physiol. C 110:173-176.

Winston, G. W. & R. T. Di Giulio. 1991. Prooxidant and anti-oxidant mechanisms in aquatic organisms. Aquat. Toxicol. 19:137-161.

E. ZAPATA-VIVENES * AND O. NUSETTI

Departamento de Biologia, Nucleo de Sucre, Universidad de Oriente. Cumana, Venezuela

* Corresponding author. E-mail: [e-mail]