Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress

Plant Science

121 (1996)

151

159

Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress Susana

M. Gallego,

Maria

Revised

P. Benavides,

I6 September

Maria

L. Tomaro”

1996

Abstract The relationship between heavy metal ion toxicity and oxidative stress in plant cells was studied. Leaf segments from 14 day old sunflower seedlings were incubated in solutions containing 0.5 mM Fe(II), Cu(II) or Cd(I1) ions for 12 h in the light. Treatment with metal ions studied produced a decrease in chlorophyll and GSH contents as well as increases in lipid peroxidation and lipoxygenase activity. Free radical scavengers, such as sodium benzoate and mannitol, prevented the decrease in chlorophyll and GSH content and the lipid peroxidation and lipoxygenase increases. While Fe(I1) and Cd(I1) ions caused a decrease in superoxide dismutase activity, Cu(I1) ions raised its level. However. all three metal ions caused decreases in other antioxidant enzymes (catalase. ascorbate peroxidase. glutathione reductase and dehydroascorbate reductase). Free radical scavengers protected these enzymes against inactivation. No effect of these scavengers was observed on superoxide dismutase activity. These results indicate that excess Fe(H), Cu(I1) or Cd(H) ions produce oxidative damage in plant leaves. Copyright (? 1996 Elsevier Science Ireland Ltd K~-IvoT&: .4ntioxidants;

Heavy metals;

Hrlianthus

umms;

Oxidative

stress

1. Introduction .4hhruriation.s: APOX, ascorbate peroxidase; BHT. butylated hydroxytoluene; CAT, catalase; DHAR, dehydroascorbate reductase: DTNB, 5.5’ dithio-bis-(2-nitrobenzoic acid); CR. glutathione reductase: LOX, lipoxygenase; MDA, malondialdehyde: SOD. superoxide dismutase; TBA. thiobarbituric acid. * Corresponding author. Tel.: + 54 I 9623276: fax: + 54 I 9625341/9617370: e-mail: [email protected]

Ol67-9452/96!$15.00

Copyright

PfISOl68-9452(96)04528-l

@Z1996 Elsevier

Science

Ireland

In many areas of biology, highly reactive free radicals have been implicated directly in the molecular damage associated with exposure to a wide range of pollutants, heavy metals and other toxins. Toxic levels of metals in soils may be caused by natural soil properties or by agricultural, manufacturing, mining and waste disposal Ltd. All rights

reserved

152

S.M. Guilqp

et al. ; Plant Scirtwe i-71 (1996) 151-159

practices [1,2]. Although some heavy metals are essential as micronutrients. uptake of toxic quantities can be harmful to most plants. Iron and copper are essential elements for many cellular processes [3,4]; however, at toxic concentrations their ions act as efficient generators of reactive oxygen species [3,5-91. It has been reported that excess of iron produce oxidative stress in Nicotiana plumbaginlyolia plants [3] and induce oxygen free radicals in waterlogged plants [6]. Coppermediated free radical formation has been demonstrated in isolated chloroplasts [IO], in intact roots of Silene cucubalus [l 11,in leaf segments [9] and in intact leaves of Phaseofus vulgaris [12]. On the other hand, it has been reported that Cu(I1) ions increase the activities of antioxidant enzymes such as Cu,Zn-superoxide dismutase [ 131, peroxidase [14] and glutathione peroxidase [15]. Cadmium is a major environmental pollutant present in areas with heavy road traffic as well as near smelters and sewage sludge areas. Although not essential for plant growth, this metal is readily taken up by roots and translocated to aerial organs in many species [ 16,171. The oxidative damage caused by metals such as Cu and Fe can be explained by the involvement of changes in redox state (Fenton reaction) [18], but the molecular mechanisms of Cd toxicity are poorly understood. It has been shown that treatment with Cd(I1) can inhibit net photosynthesis [ 191, concomitantly causing structural changes in chloroplasts and a decrease in chlorophyll content [20-221. Recently, evidence has been reported that Cd uptake leads to an oxidative stress acquired in tolerant and sensitive clones of Holcus lanatus [17], and in germinating seedlings of Phaseolus vulgaris [22]. Metal toxicity in plants may result from complex interactions of the major toxic metal ions with other soil components or environmental factors. Sunflower and other important crops are often cultivated in agricultural environments showing low levels of metals. However, low metal concentrations can result in significant accumulation in plant tissues. Different species show different responses to metal toxicity. Moreover, information focused on the relationship of heavy metals and oxidative stress in plants is rather

scarce, SO that it is difficult to draw a general conclusion about critical toxic metal concentrations in soils. All aerobic organisms possess the means to protect themselves from the toxic effects of reduced oxygen species. Plants possess a number of antioxidant molecules (GSH, ascorbic acid, a-tocopherol, carotenoids) and enzymes that protect against oxidative damage. SOD, the first enzyme in the detoxifying process, converts O,- radicals to H?O,. CAT, APOX [23] and a variety of general peroxidases catalyze the breakdown of HzO,. In the ascorbate-glutathione cycle, the enzymatic action of APOX reduces H202 using ascorbate as an electron donor. Oxidized ascorbate is then reduced by reactions catalyzed by monodehydroascorbate reductase. dehydroascorbate reductase and glutathione reductase. GR also plays an essential role in the protection of chloroplasts against oxidative damage by maintaining a high GSH/GSSG ratio [24,25]. Lipoxygenase may be involved in plant growth and development, the biosynthesis of regulatory molecules, wounding and pathogen infection, and plant senescence. The most prevalent source of potential lipoxygenase substrate is found among the polyunsaturated fatty acid chains present in membrane lipids, leading to the loss of membrane integrity [26]. Moreover. MDA level is considered as an essential parameter in order to determine membrane damage. In view of these considerations, we have carried out a study of the effect of Fe(II), Cu(II) or Cd(I1) ion addition on sunflower (Heliunthus unnuus L.) leaf oxidative stress, as well as its prevention by antioxidants, such as sodium benzoate and mannitol.

2. Materials

and methods

2.1. Chemiculs NADPH, GSH, GSSG, DTNB, thiobarbituric acid, glutathione reductase, nitroblue tetrazolium, butylated hydroxytoluene (BHT), and 2vinylpyridine were from Sigma Chemical (St. Louis, MO). All other chemicals were of analytical grade.

S.M. Gallego el al. ! Plant Science 111 (1996) 151.-159

2.2. Plant material and growing conditions Seeds of sunflower (Helianthus annuus L., cv. Mycosol 2, supplied by Agrigenetics) were germinated and grown in perlite-vermiculite in a controlled climate room at 24 + 2°C and 50% RH, with a photoperiod of 16 h and a light intensity of 175 pmol/m’ per s. The plants were irrigated with Hoagland nutrient solution [27]. The second pair of fully expanded leaves were used for all experiments. Leaf discs (12 mm diameter, 0.3 g) were floated abaxial side down under light for 12 h in Petri dishes containing 20 ml of 0.5 mM solution of the corresponding ion (FeSO,, CuClz or CdCl,). When the effect of free radical scavengers was investigated, segments were floated on a 0.5 mM solution of the metal ion containing 10 mM sodium benzoate or 10 mM mannitol. The pH of these solutions was adjusted to 6 with NaOH. Experiments were repeated twice and five replicates were made for each treatment. Controls were incubated in demineralized water. After 12 h treatment, leaf discs were washed with distilled water and extracted for the different determinations. -7.3. Chlorophyll content determination Chlorophyll was extracted by homogenizing and boiling 0.3 g of fresh weight of leaves in 35 ml of 80% ethanol. After centrifugation for 10 min at 5000 rpm, chlorophyll content was analyzed spectrophotometrically on the ethanolic supernatant at 654 nm, as described by Wintermans and De Mots [28]. 2.4. Mulondialdehyde determination Lipid peroxidation was measured as the amount of MDA determined by the thiobarbituric acid (TBA) reaction as described by Heath and Packer [29]. Leaf discs (0.3 g) were homogenized in 2 ml of 0.1% (w/v) trichloroacetic acid (TCA). The homogenate was centrifuged at 10 000 x g for 20 min. To 1 ml of the resulting supernatant, 1 ml of TCA (20%) containing 0.5% (w/v) of TBA, and 100 ~1 BHT (4% in ethanol) were added. The mixture was heated at 95°C for 30 min and then

153

quickly cooled on ice. The contents were centrifuged at 10 000 x g for 15 min and the absorbance was measured at 532 nm. The concentration of MDA was calculated using an extinction coefficient of 155/mM per cm. 2.5. Assay of’ GSH Total glutathione (GSH plus GSSG) was determined in leaf homogenates by spectrophotometry at 340 nm, after precipitation with 0.1 N HCl, using yeast-glutathione reductase, DTNB and NADPH. GSSG was determined by the same method in the presence of 2-vinylpyridine and GSH content was calculated from the difference between total glutathione and GSSG [30]. 2.6. Enzyme preparations and a.s.sa~.v Extracts for determination of CAT, SOD and APOX activities were prepared from 0.3 g of leaf discs homogenized under ice-cold conditions in 3 ml of extraction buffer. containing 50 mM phosphate buffer (pH 7.4). 1 mM EDTA. 1 g PVP, and 0.5% (v/v) Triton X-100 at 4°C. Homogenates were centrifuged at 10 000 x g for 20 min and the supernatant fraction was used for assays. CAT (EC 1.11.1.6) activity was determined in homogenates by measuring the decrease in absorption at 240 nm in a reaction medium containing 50 mM potassium phosphate buffer (pH 7.2) and 2 mM H103. The pseudo-first order reaction constant (k’ = k x [CAT]) of the decrease in H,O, absorption was determined and the catalase content in pmolimg protein was calculated using k=4.7 x lO’/M per s [31]. SOD (EC 1.15. 1.1) activity was measured spectrophotometrically as described by Beyer and Fridovich [32]. In this assay, 1 unit of SOD is defined as the amount required to inhibit the photoreduction of nitroblue tetrazolium by 50%. The specific activity of SOD was expressed as unitsjmg protein. As APOX is labile in the absence of ascorbate [23], 5 mM ascorbate was included for the extraction of this enzyme. APOX (EC 1.11.1 .ll) activity was measured immediately in fresh extracts as described by Nakano and Asada [33], using a reaction mixture (I ml) containing 50 mM potassium

154

S.M. Gallego et al. 1 Plant Science 121 (1996) 1.51-159

phosphate buffer (pH 7.0), 0.1 mM hydrogen peroxide, 0.5 mM ascorbate and 0.1 mM EDTA. Hydrogen peroxide-dependent oxidation of ascorbate was followed by a decrease in the absorbance at 290 nm (C = 2.8/mM per cm). Extracts for determination of GR and DHAR activities were prepared from 0.3 g of leaf discs homogenized under ice-cold conditions in 3 ml of extraction buffer containing 50 mM Tris-HCl buffer (pH 7.6), and 1 mM EDTA. GR (EC 1.6.4.2) activity was measured by following the decrease in absorbance at 340 nm due to NADPH oxidation. The reaction mixture contained leaf extract, 1 mM EDTA, 0.5 mM GSSG, 0.15 mM NADPH, 50 mM Tris-HCl buffer (pH 7.6) and 3 mM MgCl, [34]. B-mercaptoethanol (2 mM) was included for the extraction of DHAR (EC 1.8.5.1). DHAR activity was measured by the formation of ascorbate at 265 nm (Z = 14/mM per cm) in a reaction mixture containing 2.5 mM GSH, 0.1 mM EDTA, 0.2 mM dehydroascorbate and 50 mM sodium phosphate buffer (pH 7.0), as described by Nakano and Asada [33]. Lipoxygenase (EC 1.13.11.12) was extracted according to the method of Lupu et al. [35]. Leaf tissue was homogenized in ice-cold 0.2 M sodium-phosphate buffer (pH 6.5, 1% Triton X-100) and the homogenate was centrifuged at 10 000 x g during 20 min. Lipoxygenase activity was measured spectrophotometrically at 234 nm using linoleic acid (cis-9, cis-12 octadecadienoic acid) as the substrate, according to Ben-Aziz et al. [36]. One ml of substrate solution containing 2.28 x 10 p4 M linoleic acid and 0.25% Tween 20 in 0.2 M citrate-phosphate buffer (pH 6.5) was added to quartz cuvettes in a Beckman DU-65 spectrophotometer. The spectrophotometer was zeroed and 5 to 15 ~1 leaf extract was added to the sample cuvette with thorough mixing. lncreases in absorbance at 234 nm (25°C) were followed for 15 min and rates of increase were calculated from the initial linear portion of at least two separate curves. Activity was expressed as change in absorbance at 234 nm/min per mg protein.

2.7. Protein Determinution Protein concentration was determined according to Bradford [37] using bovine serum albumin as standard. 2.8. Statistics Figures in the text and tables indicate mean values + S.E.M. Differences between control and treated plants were analyzed using Student’s ttest, taking P < 0.05 as significant.

3. Results 3.1. Ejjkct of heavy metal ions on chlorophyll, MDA and GSH content

Leaf treatments with Fe(ll), Cu(ll) or Cd(H), for 12 h in the light, significantly diminished leaf chlorophyll content (30%, 40% and 15%, respectively) with respect to control values (Fig. 1A). Lipid peroxidation measured as MDA content was markedly raised over control values with the three metal ions. A 2.5-fold increase in Fe( II)-treated leaves was detected, while Cd(l1) addition produced a 50% increase with respect to controls. A large increase (3.5-fold) in MDA levels was observed in Cu(ll)-treated leaves (Fig. 1B). Reduced glutathione is one of the main soluble antioxidant compounds in plants. It could, therefore, be expected that if heavy metal ions used in this study induce the formation of oxidants (measured as MDA content), they would also affect GSH leaf levels. Compared to the corresponding controls, GSH content was significantly decreased by treatments with Fe(ll), Cu(l1) or Cd(ll) (400/o, 50% and 20% of the control values respectively) (Fig. 1C). However, no significant changes in GSSG content were observed, so that GSHjGSSG ratio was strongly reduced with respect to control leaves (data not shown). These results clearly indicate that heavy metal ion excess produces oxidative damage.

S.M. Gallego et al.

120 , 100

! Plant Science l-71 (1996) 151-1.59

1

A

-

400

z 6

s ?? =

80

w

-

6 5! 60

-

40

-

20

-

! d

c” % ‘d

155

300

200

I

8

100

0

O-

Copper

CCJlltr01

u

Con

=

Met =

SBe

m

Man

Copper

Control

Cadmium

0

Copper

Con

Met

m

SBe

Cadmium

m

Man

Cadmium

Fig. I. Effect of metal ions and free radical scavengers on chlorophyll (A), MDA (B) and GSH (C) content. Con (control); Met (0.5 mM metal ions); SBe (0.5 mM metal ions + 10 mM sodium benzoate): Man (0.5 mM metal ions + IO mM mannitol). Values are the means of two different experiments with five replicated measurements, and bars indicate S.E.M. *Significant differences (P < 0.05) as assessed by Student’s t-test. Data are expressed as percentages of the control values. Control values: chlorophyll (I .43 + 0.5 mgig FW). MDA (I2 I I nmoljg FW), GSH (85.8 k 2.6 nmol!g FW).

3.2 Effect

of free radical scavengers

When antioxidants such as sodium benzoate or mannitol were administered together with iron or copper ions, it was found that they partially prevented the decrease in chlorophyll and GSH content as well as the increase in lipid peroxidation (Fig. 1A, B, C). However, when free radical scavengers were used simultaneously with cadmium ions, they completely prevented the decrease in chlorophyll and GSH content and the increase in lipid peroxidation levels (Fig. IA, B, C).

3.3. Effect of heavy wetal on lipoxygenase uctivitl‘

ions and antioxidants

The addition of high concentrations of Fe(II), Cu(I1) or Cd(I1) to the incubation medium caused a significant increase in lipoxygenase activity ( 130%. 200% and 52% of controls, respectively) (Fig. 2). LOX oxidized polyunsaturated fatty acids producing hydroperoxides and oxy-free rddicals. Therefore, the increase in LOX may be due to its capacity to form oxidation products. Addition of sodium benzoate or mannitol together with iron or copper ions partially restored

S.M. Gallego et al. /Plant Science 121 (1996) 151.-159

3.4. EfJtict antioxidative

copper

Cadlnium

enzyme activity (Fig. 2). However with cadmium ion treatment, the two antioxidants completely restored lipoxygenase activity.

ions on antioxidant

enzyme

ions and antioxidants

on

enzymes

Fe(II)- or Cd(II)-treated leaves diminished total SOD activity (51% and 27% respectively) compared to corresponding controls. However, Cu(I1) addition resulted in increased SOD activity (18%) (Table 1). In contrast to its effect on SOD, Cu(I1) decreased CAT, APOX, DHAR and GR activities (77%, 60%, 50% and 46%, respectively, of control values) (Table 1). These enzymes showed a similar behavior when leaves were treated with Fe(I1) and Cd(I1) ions. Treatment with iron ions decreased CAT, APOX, DHAR and GR activities (.58%, 50%, 44% and 36% respectively), as also did cadmium ions (30%, 25%. 25% and 18%, respectively) (Table 1). Such enzyme activities were partially restored by adding antioxidants sodium benzoate or mannitol, except when sodium benzoate was administered together with Cd(II), in which case all antioxidative enzymes reached values similar to control leaves (Table 1).

Fig. 2. Effect of metal ions and free radical scavengers on lipoxygenase activity. Con (control): Met (0.5 mM metal ions); SBe (0.5 mM metal ions + 10 mM sodium benzoate); Man (0.5 mM metal ions + IO mM mannitol). Values are the means of two different experiments with five replicated measurements, and bars indicate SEM. *Significant differences (P < 0.05) as assessed by Student’s r-test.

Effect of metal

of metal

activities

Treatment

SOD (U/mg protein)

CAT (pmol/mg protein)

APOX” protein)

Control Fe(II) Fe(II)+SBe Fe(II)+Man Cu(II) Cu(II)+ SBe Cu(I1) + Man Cd(H) Cd(II)+SBe Cd(II)+ Man

59 + 2. I 29 + I.2 50* I.5 44k2.1 70+ 3.1 68 + 3.2 67 k 3.5 43 * I.5 56 k 2.3% 51 + I.6

5.02 2.1 4.2 3.5

8.1 4.1 6.9 5.8 3.2 6.0 5. I 6.1 7.9 7.1

) 0.4 + 0.1 k 0.3 & 0.2

1.2* 0.08 3.8 f 0.2 2.9kO.l 3.5 +0.1 4.8 + 0.3* 4.1 * 0.2

(U/mg

kO.5 * 0.2 _+0.4 + 0.4 * 0.2 k 0.3 * 0.3 k 0.4 + 0.5* kO.3

DHARb protein) 3.2 * 0.2 I.8 kO.1 2.6 & 0. I 2.1 +0.1 I .6 + 0.07 2.3 * 0. I 1.9iO.l 2.4kO.l 3.0 * 0.2* 2.6 + 0.1

(Ujmg

CR’ (Ujmg protein) 0.22 + 0.01 0.14 f 0.01 0.18 + 0.01 0.16~0.01 0.12 + 0.01 0.16 k 0.01 0.14~0.01 0.18 +0.01 0.21 & 0.02* 0.20 & 0.01*

Leaf segments were incubated for 12 h with Fe(II), Cu(I1) or Cd(I1) alone at dose 0.5 mM, or simultaneously with IO mM sodium benzoate (SBe) or IO mM mannitol (Man). Enzymatic activities were assayed as described in Section 2. Data are the means + S.E.M. of two different experiments with five replicated measurements. Treated leaves showed statistically significant differences (PcO.05) as compared with the control group, except where indicated by an asterisk (*). ” One unit of APOX forms I pmol of ascorbate oxidized per min under the assay conditions, ’ One unit of DHAR forms I pmol of ascorbate per min under the assay conditions. ’ One unit of GR oxidizes 1 pmol of NADPH per min under the assay conditions.

S.M.

Gallego

et al. / Plan1 Scirnce

4. Discussion A growing body of evidence indicates that transition metals act as catalysts in the oxidative deterioration of biological macromolecules, and therefore, toxicity associated with these metals may be due, at least in part, to oxidative tissue damage [38]. The addition of high concentrations of Fe(II), Cu(I1) or Cd(I1) ions to the incubation medium, under light, caused a decrease in both chlorophyll and GSH contents, and increased the lipid peroxidation rate (Fig. 1 A, B, C) and lipoxygenase activity (Fig. 2) in sunflower leaf cells. The two transition metals most commonly studied are iron and copper cations. Both are widely distributed in nature and are essential elements. Similar to iron. copper acts as a catalyst in the formation of reactive oxygen species and is known to be at least as effective as iron, in causing oxidative damage [39]. Accordingly, we found that the most pronounced effects were obtained with copper ions, followed by iron ions. Smaller changes in all the parameters assayed were achieved with cadmium ions (Fig. lA, B, C; Fig. 2) suggesting that Cu(I1) is better than Fe(I1) and much more active than Cd(B) as a producer of oxidative stress. Even though sodium benzoate and mannitol are mainly scavengers of hydroxyl radicals, mannitol showed a more efficient antioxidant activity than sodium benzoate in preventing the decrease in chlorophyll and GSH content and the increase in MDA content (Fig. lA, B, C) and lipoxygenase activity (Fig. 2). However, when either antioxidant. sodium benzoate or mannitol, were used simultaneously with Cd ions, they completely prevented the decrease in chlorophyll and GSH content, as well as the increase in lipid peroxidation levels (Fig. lA, B, C) and in lipoxygenase activity (Fig. 2). Superoxide radical, hydrogen peroxide, hydroxyl radical, and singlet oxygen are the four major active oxygen species generated in plant tissues [40]. To mitigate and repair the damage initiated by activated oxygen, plants have developed a complex antioxidant system [40,41]. The pritnary components of this system include free

I21

(1996)

ICI - 159

157

radical scavengers such as carotenoids, ascot-bate, glutdthione and tocopherols, as well as enzymes such as SOD, CAT, APOX, DHAR and GR. When the effect of Fe(I1). or Cd(I1) on antioxidant enzymes was studied, it was found that all of them were decreased. CAT, APOX, DHAR and GR were also decreased when they were assayed in the Cu(I1) treated leaves (Table 1). Cu(I1) was the most effective in causing antioxidant enzyme activity decrease (Table 1). The decrease in enzyme activities by heavy metal ions could result from the attack caused by metal ion-induced oxygen species [ 17,22,42]. It is known that while iron and copper act as catalysts in the formation of reactive oxygen species (Fenton reaction) [ 181, and cause GSH depletion, cadmium does not appear to generate free radicals, but it does elevate lipid peroxidation and decrease GSH content, which results indirectly in active oxygen species production [IS]. A previous report [42] has shown that these oxygen species cause oxidative damage to antioxidant enzymes and, hence. their activities are diminished. However, superoxide dismutase, the other very important antioxidant defense enzyme, showed the opposite behavior in Cu(II)-treated leaves. As shown in Table I, SOD increased during copper treatment. It has been reported that copper ion causes an increase in total SOD activity in oat leaves [9] and excess of copper induces a cytosolic CuiZn-SOD in soybean root [13]. In addition, Cu(I1) ion induced SOD 1 gene in Sacchurotnyces crrrtGciur [43] and its activity was reduced in leaves of Cu-deficient chloronrrt:u mutant [44]. Therefore, the Cu( II)-mediated increase in SOD activity found in this study may be due either to a direct effect of this ion on the SOD gene, or to an indirect effect mediated via an increase in the level of 0; radicals [13]. in agreement with the hypothesis that Cu(I1) plays an important role as a cofactor in SOD protein synthesis and/or protein stability [44]. The decreases in antioxidant enzyme activities produced by Fe(I1) and Cu( II) were partially restored by addition of the antioxidants sodium benzoate or mannitol (Table 1). Unexpectedly. sodium benzoate proved more efficient than mannitol. In the case of Cd(II)-treated leaves, all

S.M. Gallego et al. / Plant Science 121 (1996) 151-159

158

antioxidant enzyme values regained control levels with sodium benzoate addition. However, so far there is no ready explanation for this difference, since both antioxidants are scavengers of hydroxyl radicals. When leaf chlorophyll content and GSH level were decreased by heavy metal treatments, the antioxidant enzyme activities declined, except SOD activity after Cu(I1) treatment. Besides, when free radical scavengers were present, they prevented chlorophyll loss and GSH decrease, as well as a drop in protective oxidative stress enzymes. However, over the 12 h experimental period, antioxidant enzymes were more affected by metal ion treatment than chlorophyll and GSH content (Fig. lA, C; Table 1). The present results, in sunflower leaves, strongly support the likelihood that heavy metal ion excess produces reactive oxygen species which cause oxidative stress. Acknowledgements This work was supported by grants from the Universidad de Buenos Aires (Argentina) and from Consejo National de Investigaciones Cientificas y Tecnicas (CONICET) (Argentina). M.L.T. is a career investigator from CONICET. References [I] R.L.

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25 (1968)

189-

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