Chromosomal sensitivity of human lymphocytes to bleomycin

Chromosomal Sensitivity of Human Lymphocytes to Bleomycin Influence of Antioxidant Enzyme Activities in Whole Blood and Different Blood Fractions Alejandro D. BolzAn, N6stor O. Bianchi, Marcelo L. Larramendy, and Martha S. Bianchi

ABSTRACT: The activity of the antioxidant enzymes catalase (CAT), peroxidases (POD), and superoxide dismutases (SOD) in whole blood and different blood fractions was analyzed in 20 normal human beings and correlated with the chromosomal sensitivity of lymphocytes to bleomycin (BLM) (measured as frequency of dicentric chromosomes per BLM dose). Our results demonstrate that both the physiologic activities of the enzymes and the chromosomal sensitivity to BLM exhibit an ample and significant interindividual variability. An inverse and linear correlation between chromosomal sensitivity to BLM and the concentration of 1) CAT and POD in plasma and 2) SOD in whole blood, erythrocytes, and plasma was found. On the other hand, the chromosomal sensitivity to BLM showed a direct correlation with the concentration of SOD and POD in mononuclear leukocytes. It is suggested that a determination of antioxidant e n z y m e (AOE) activities in a given cell population may serve to predict the chromosomal sensitivity to BLM.

INTRODUCTION

MATERIALS AND METHODS

The bleomycins (BLMs) are a structurally related group of antibiotics derived from Streptomyces verticillus [1]. Because of its cytotoxic actions, these compounds are usually used in the treatment of squamous cell carcinoma, Hodgkin disease, reticulosarcoma, and testicular tumors [2]. Cellular damage by BLMs is accepted to occur as a consequence of the degradation of nuclear deoxyribonucleic acid (DNA) [3, 4]. Therefore, information about the factors that modulate the cytotoxicity of BLMs are of therapeutic interest. In a preliminary report, we found evidence suggesting an inverse correlation between the chromosomal sensitivity of human lymphocytes to BLM and the levels of superoxide dismutase (SOD) in whole blood, erythrocytes, and plasma [5]. By analyzing a larger number of normal human donors, we confirm here our preliminary report, and we also demonstrate the existence of correlation between chromosomal sensitivity to BLM and plasma levels of catalase (CAT) and peroxidases (POD).

Blood Samples

From IMBICE (Instituto Multidisciplinario de Biologia Celular) (A. D. B., N. O. B., M. S. B.), La Plata, Argentina, and Laboratorio de Citogen~tica (Facultad de Ciencias Naturales y Museo de La Plata) y Cdtedra de Gen~tica y Mejoramiento Animal y Vegetal (Facultad de Ciencias Agron6micas y Forestales) (M. L. L.), Universidad Nacional de La Plata, La Plata, Argentina. Address reprint requests to: Dr. Alejandro D. Bolzdn, IMBICE, C.C. 403, La Plata, Argentina. Received March 9, 1992; accepted June 12, 1992.

Human blood samples were obtained from 20 healthy male voluntary donors (20-40 years old) selected according to the recommendations reported elsewhere [6].

Lymphocyte Cultures, Bleomycin Treatment, and Cytogenetic Analysis Aliquots of 1.0 ml of heparinized whole blood from each donor were added to 9.0 ml of Ham's F10 medium (GIBCO, Grand Island, NY) and treated with 0 (controls), 10,100 or 200/~g/ml of BLM (Lab. Dr. Gador, Argentina) for 3 hours at room temperature. Thereafter, the cells were washed three times in Hanks' basic salt solution, resuspended in complete culture medium [80% Ham's F10, 17% fetal calf serum, 3% PHAoM (GIBCO), penicillin (100 U/ml), streptomycin (100/~g/ml)] and incubated for 48 hours at 37°C until harvesting. Cells were treated with colchicine (0.1 ~g/ml, Sigma Chemical Co., St. Louis, MO) during the last 3 hours of culture. Chromosome preparations were stained with Giemsa. Cultures were set up in duplicate for each sample. A total of 100 metaphases was analyzed per sample and the frequency of dicentric chromosomes evaluated. The analysis of BLM-induced chromosome aberrations was restricted to dicentric chromosomes as indicator of chromosome damage according to the recommendations of the UNSCEAR (1969, cited by Kucerova and Polivkova [7]). Policentric chromosomes were scored as dicentrics ac133

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134 cording to: number of dicentrics -- number of centromeres - 1.

Antioxidant Enzyme (AOE) Activity Assay CAT, POD, and SOD activities were measured in whole blood, erythrocytes, plasma and mononuclear leukocytes. One ml of whole blood was diluted in 9.0 ml of 0.07 M phosphate buffer solution (pH 7.0) (dilution 1:10) and lysed with 0.1% Triton X-100 (Sigma Chemical Co., St. Louis, MO) plus sonication (W-225 R Sonicator, Heat Systems, Ultrasonic Inc., New York). Enzyme activity determinations were made on supernatants after centrifugation at 20,000 × g for 15 minutes at 4°C. Mononuclear leukocytes were purified from whole blood by the Ficoll-Hypaque (Pharmacia Fine Chemicals, Uppsala, Sweden) gradient separation technique of B6yum [8]. Erythrocytes were obtained from red blood cells pellets of Ficoll-Hypaque gradients. (No attempt to isolate contaminant polymorphonuclear leukocytes was made.) After separation, mononuclear and red cells were resuspended, lysed, and sonicated in the same buffer solution as used for whole blood samples. Afterwards, the samples were centrifuged (20,000 × g for 15 minutes, 4°C), and the analyses of AOE activities were performed in the supernatants. Plasma samples were obtained after centrifugation of whole blood at 20,000 x g for 15 minutes at 4°C. Whole blood and blood fractions were immediately used for enzyme dosages or stored in liquid nitrogen until use. CAT and POD activities were determined according to LUck [9, 10]. SOD activity was determined by measuring the inhibition of epinephrine autoxidation to adrenochrome [11]. One unit of SOD activity was defined as the amount of enzyme able to induce a 50% inhibition in the rate of epinephrine autoxidation. Enzyme activities were expressed as units per milligram of hemoglobin (U/mgHb) for whole-blood and red-cells samples, and as units per milligram of protein (U/mgPt) for plasma and mononuclear leukocyte fractions.

Statistical Analysis Analysis of variance (ANOVA) was used for chromosome aberrations and AOE activity data. The Tukey's test for comparisons was employed to determine the significance of dicentric value differences between control and BLMtreated samples. The correlation analysis between chromosomal sensitivity to BLM (expressed as frequency of dicentrics per 100 cells per BLM dose) and AOE activities was analyzed in an IBM PC by using the Statgraphics software (Statistical Graphics Corporations). Because some dicentric frequencies followed a Poisson distribution, the obtained values as well as the enzyme activities values were transformed into their logarithms for the correlation analysis. In order to determine whether the observed distribution of dicentrics differed significantly from the Poisson law, we used the Papworth test as described by Savage [12]. This test gives a Z index (unit normal deviate); Z < 2 indicates Poisson distribution; conversely, Z > 2 indicates heterogeneous dispersion of data. The level of significance chosen was p -< 0.05.

A.D. Bolz~in et el.

RESULTS Table 1 summarizes the frequency of dicentrics induced by BLM and the values of CAT, POD, and SOD activities in whole blood and blood fractions of each donor. As previously demonstrated [5, 13], BLM induces a clear dose-response increase in the dicentric frequencies over control values (p -< 0.05) in 17 of the 20 donors studied (Fig. 1). In the remaining donors (donors 2, 5, and 10. Table la), though the dose-response curve was not clear, the chromosomal sensitivity to the antibiotic was evident. Interdonor variations in the chromosomal sensitivity to BLM were significant for all of the doses used (p -< 0.05). Moreover, the interindividna] variability of CAT, POD, and SOD activities in the different blood fractions analyzed was also statistically significant (p -< 0.05). The Papworth test gave a Z index above 2 in the majority of individuals for each BLM dose. This indicates overdispersion of data corresponding to a non-Poisson distribution of dicentric frequencies. Similar findings have been reported by other authors [14] and are known to be due to intercellular variation in the sensitivity to BLM resulting from cell to cell differences in the level of DNA methylation [15, 16], the structure of the chromatin [17, 18], or the cell membrane permeability to BLM [19]. The coefficients of correlation between the yield of BLMinduced dicentrics and the CAT, POD, and SOD activities in whole blood and different blood fractions analyzed are shown in Table 2. A regression test showed a linear, negative, and significant correlation between the yield of dicentrics and the activity of 1) CAT and POD in plasma for the BLM dose of 100/xg/ml 2) SOD in whole blood for BLM doses of 10 and 200/.tg/ml 3) SOD in erythrocytes for all the BLM doses used, and 4) SOD in plasma for BLM doses of 100 and 200/xg/ml (p < 0.05) (Table 2). On the other hand, we also observed a linear, positive, and significant

Figure 1 Metaphase figure from donor 5 showing a dicentric chromosome (a) and the resulting acentric fragment (b) induced by bleomycin (100 ~g/ml).

b

0

10

100

200

0

26

24

100

200

16

24

10

0

10

0

14

100

200

11

4

19

0

10

0

100

200

9

5

19

0

10

0

100

200

19

25

0

10

0

38

11

100

200

38

0

l0

0

35

106

200

30

0

10

0

20

Failed

100

200

51

6

25

0

10

0

100

200

9

3

21

O

10

0

100

200

11

21

O

10

10

9

8

7

6

5

4

3

2

1

30.63 ÷ 0.23

17.27 + 0.37

10.85 + 0.34

14.42 ± 0.52

6.53 + 0.70

6.33 + 0.05

10.25 ÷ 0.44

10.67 + 0.48

1 0 . 4 1 -+ 0 . 2 6

7.48 ÷ 0.26

U/mgHb

3

CAT

(/zg/ml)

Donor

BLM

4.9

8.0

8.6

1. 0

2.0

÷

196.8 ÷

128.1

143.8 +

9.8

2.0

1.5

234.8 ± 30.5

394.5 ÷ 21.6

611.9 *

132.0 +

89.9 +

20.5 ±

11.1 +

>U/mgttb

POD

Whole Blood SOD

2.25 + 0.04

1,27 ÷ 0.01

1.73 + 0.01

1.69 ± 0.61

2.62 ÷ 0.02

1.56 + 0.05

1.48 + 0.05

1.72 + 0.06

4.76 ÷ 0.36

2.96 ÷ 0.08

U/mgHb

28.93 ÷ 0.08

17.19 + 0.06

10.63 + 0.12

13.95 + 0.04

6 . 7 6 -+ 0 . 9 3

7.79 * 0.35

18.64 + 0.85

16.22 + 0,85

1 1 . 5 0 -+ 0 . 3 3

9.72 * 0.07

U/mgHb

CAT

3.1

1.5

7.4

2.7

103.9 ÷

114.8 ÷

157,9 +

4.6

3.3

1.9

7 4 2 . 5 + 37.1

828.2 ÷ 10.6

269.8 ÷

82.2 +

94,9 + 13.1

24.3 ±

24.1 +

>U/mgHb

POD

Erythrocytes SOD

+

0.04

1.66 T 0.02

1,13 ÷ 0,06

1.37 * 0.02

1.77 ± 0.03

1.19 ÷ 0.61

0.79 + 0.03

0.73 ÷ 0.03

1.21 + 0.01

2.04 ± 0.01

1.77

U/mgHb

1 0 . 6 8 ± 1.71

2.81 + 0.26

14.89 ± 0.35

5.84 + 0.93

4.25 + 0.08

8.14 + 0.28

9.57 + 0.40

1.15 ± 0.12

0.37 + 0.04

0.34 ÷ 0.06

U/mgPt

CAT

7.44 ± 0.33

7.27 * 0.14

4.36 + 0.40

4.17 + 0.18

10,96 + 0.74

24.87 + 0.61

5 .5 1 * 0 . 1 2

1.27 * 0.06

3.95 + 0.38

5.29 + 0.16

U/mgPt

SOD

22.53 *

0.20

45.09 ÷ 0.55

Failed

23.87 ± 0.11

Failed

Failed

28.12 ÷ 1.11

6.27 + 0.44

Failed

11,03 * 0.15

leukocytes

mU/mgPt

POD

Mononuclear

+

0.12

4.55 ÷ 0.01

9.62 + 0.61

17.19 ± 0.02

13.42 ÷ 0.45

16.33 ÷ 0.87

17,75 + 1.13

3.77

4.63 ÷ 0.68

7.54 + 0.11

4.52 * 0.34

mU/mgPt

CAT

7.9

8.3

8.3

28.5

+ 0.3

+ 0.3

+ 0.1

* 0.1

9 . 4 0 ÷ 0.1

5 . 7 0 ± 0.1

2 . 8 2 ± 0.2

2 . 2 6 + 0.1

1 . 7 4 ± 0.2

2 . 8 5 + 0.1

>U/mgPt

POD

Plasma

Measurements of CAT, POD and SOD activities in whole blood and different blood fractions and chromosomal sensitivity of human lymphocytes to BLM expressed as frequency of dicentric chromosomes per 100 cells (Donors 1-10)

0

Dicentric frequency per 10O cells

Table l a

0.57 + 0.01

0.38 ± 0.01

0.41 ± 0.01

0.46 ± 0.01

0.31 ÷ 0.01

0.18 ÷ 0.01

0.15 ± 6.01

0.15 ± 0.01

0.38 + 0.02

0.24 ± 0.02

U/mgPt

SOD

100

5

Failed Failed

0

10

100

200

0

5

15

17

100

200

12

3

19

0

10

0

100

200

7

5

13

0

10

O

100

200

13

18

0

10

18

25

O

100

200

5

Failed

0

10

0

100

200

18

6

13

O

10

O

100

260

9

2

11

0

10

0

100

200

16

5

11

O

10

0

200

0

10

O

lO0

200

14

Failed

0

10

20

19

18

17

16

15

14

13

12

11

20.21 ± 0.14

19.02 ÷ 0.13

11.65 + 0.51

15.43 + 0.14

45.28 ÷ 1.45

46.63 ± 1.05

17.90 ÷ 0.43

18.03 + 0.50

10.01 + 0,40

15.55 ÷ 0.24

U/mgHb

6

CAT

(#g/ml)

Donor

BLM

Blood

244.8 ~

204.1 +

214.6 *

172.0 ÷

6.1

9.3

14.3

3.6

419.6 ÷ 13.2

384.4 ÷ 33.3

2 1 6 . 0 *- 1 5 . 0

225.0 ÷ 11.7

540.0 + 23.0

901.0 + 38.6

~U/mgHb

POD

Whole

÷ 0.18

3.48 ± 0.10

4.08 + 0.03

4.11

3.82 ± 0.05

3.27 ÷ 0.02

3,68 ÷ 0.06

2.55 ± 0.06

2.28 ± 0.05

1.34 ÷ 0.03

1.54 + 0.01

U/mgHb

SOD

21.72 ± 0.63

18.07 ± 0.25

10.75 + 0.06

15.31 * 0.30

48.68 ± 1.83

45.61 ± 1.04

19.01 ÷ 0.30

17.58 + 0.35

11.86 + 0.64

13.37 ± 0.78

U/mgHb

CAT

1.6

2.3

3.8

340.3 ± 11.1

290.9 + 17.0

514.2 ± 11.1

314.3 ÷

365.9 + 16.4

523.6 + 20,5

92.9 +

102.6 +

635.0 + 32.9

843.3 + 42.0

~U/mgHb

POD

Erythrocytes

2.04 + 0.03

1.52 + 0.03

1.66 + 0.04

2.89 ± 0.07

3.22 ÷ 0.01

3.04 * 0.03

2.87 ÷ 0,02

1.36 + 0.04

1.82 + 0.02

1.89 + 0.09

U/mgHb

SOD

3.96 ¢ 0.30

5.13 ± 0.40

2.67 ÷ 0,30

2.88 + 0.16

2.83 + 0.08

2.31 + 0.09

5.79 ± 1.04

3.44 ÷ 0.40

2.00 + 0.20

2.58 + 0.16

U/mgPt

CAT

1.72 ÷ 0.13

1.06 + 0.02

4 . 7 2 -* 0 . 0 2

5.73 ÷ 0.02

3.44 * 0.15

1.13 + 0.10

3.50 + 0.09

1.35 ± 0.12

0.27 + 0.03

0.58 + 0.04

5.58 + 0.30

9.06 ± 0.24

4.96 ÷ 0.31

U/mgPt

SOD

9.80 ~ 0.31

13.23 ÷ 0.41

4.70 + 0.22

7.70 ± 0 . 3 1

4.53 + 0.23

6.20 ¢ 0.02

10.07 + 0,10

leukocytes

mU/mgPt

POD

Mononuclear

42.50 ± 1.12

34.46 ÷ 1.67

59.2 + 4.02

29.77 ÷ 2.42

4 0 . 0 0 + 3.0

19.80 ± 1.68

25.51 + 2.90

Failed

38.50 + 1.43

27.49 ± 2.62

mU/mgPt

CAT

40,5 ± 2.6

2 7 . 5 + 2.5

6 8 . 9 ± 3.5

3 2 . 8 * 3.7

4 9 . 4 + 1.6

3 5 . 8 * 1.9

7.3 + 0.3

2 9 . 1 ± 1.1

3 7 . 2 * 3.1

4 7 . 6 + 2.4

~U/mgPt

POD

Plasma

Measurements of CAT, POD and SOD activities in whole blood and different blood fractions and chromosomal sensitivity of human lymphocytes to BLM expressed as frequency of dicentric chromosomes per 100 cells (Donors 11-20)

0

Dicentric frequency per 1 0 0 cells

Table lb

0.41 + 0.01

0.45 ± 0.01

0.58 + 0.01

0 . 3 5 ± 0.01

0.67 ÷ 0.02

0.29 * 0.01

0.46 + 0.01

0.34 ± 0.01

0.51 + 0.02

0 . 1 6 ± O.Ol

U/mgPt

SOD

CO

Chromosomal Sensitivity to Bleomycin and Antioxidant Enzymes

Table 2

Enzyme CAT

Correlation coefficients (r) between the yield of BLM-induced chromosome aberrations in lymphocytes (dicentrics per 100 cells) and the antioxidant enzyme activities in whole blood and different blood fractions analyzed

BLM

WB

E

PL

MNL

10 100

-0.23 -0.18

-0.14 0.01

-0.30 -0.52 ~

0.23 0.06

200

-0.15

-0.03

10

0.35

POD

100 200 10

0.06 0.25 -0.53"

SOD

100 200

-0.33 -0.59 ~

-0.36

0.14

-0.18

0.49 °

-0.20 0.17 -0.71 ~

-0.53 ° -0.21 -0.43

0.35 0.19 0.51"

-0.50 ~ -0.51"

-0.48 ° -0.56 U

0.29 0.10

0.17

Abbreviations: BLM,bleomycin(#g/ml);WB, whole blood;E, erythrocytes;

PL, plasma; MNL, mononuclear leukocytes; CAT. catalase; POD, peroxidoses; SOD, superoxide dismutases. "p -~ 0.05. ~p < 0.01.

Table 4

137

Correlation coefficients (r) between the activity of CAT, POD, and SOD observed in whole blood and in each blood fraction studied Red cells

CAT Whole blood POD Whole blood SOD Whole blood

r p r p r p

= = = = =

0.95 0.000 0.85 0.000 0.58 0.007

Plasma r p r p r p

= = = = = =

0.28 0.24 0.66 0.001 0.38 0.10

Mononuclear leukocytes r p r p r p

= = = = = =

0.18 0.46 -0.22 0.35 -0.48 0.06

Abbreviations: CAT, catalase; POD, peroxidases; SOD, superoxide dismu-

rases.

correlated with the activity of these enzymes in red cells. Moreover, the activity of POD in whole blood showed a direct correlation not only with its concentration in red cells but also with their levels in plasma.

DISCUSSION correlation between frequency of dicentrics (10 gg/ml BLM) and the levels of POD and SOD in mononuclear leukocytes (p -< 0.05) (Table 2). No significant correlation between the yield of dicentrics and the activity of CAT in whole blood, red cells, or plasma was observed. Additional support to the inverse correlation between SOD activity and the chromosome sensitivity to BLM was obtained by analyzing the average of dicentrics in the five individuals showing the highest and the lowest SOD levels in whole blood. Table 3 shows that for each BLM dose, the average of dicentrics was higher in the group having the lowest level of SOD. We also determined the correlation between CAT, POD, and SOD activities in whole blood and in each blood fraction (Table 4). A linear regression analysis showed that the activities of CAT and SOD in whole blood were positively

Table 3

Average of dicentrics i n d u c e d by BLM in the five i n d i v i d u a l s showing the highest (H) and the lowest (L) SOD levels in whole blood

BLM (~g/ml) 10 100 200

Average of dicentrics H L H L

4.40 15.60 13.20 21.20

-+ -+ -+ -+

0.60 b 5.14 2.24 5.27

H L

1 7 . 0 0 +- 1.05 2 9 . 4 0 -+ 6.14

Average of SOD leveF H L H L

4.02 1.44 4.09 1.51

-+ -+ -+ -+

0.22 0.06 0.19 0.O7

H L

3.83 +- 0.12 1.59 -+ 0.09

Abbreviations: BLM, bleomycin;SOD, superoxide dismutases. " SOD acitivity is in U/mg of hemoglobin.Because of sporadic failure in dicentricscorings(Tablesla and b), the fiveindividualsshowingthe highest and lowest enzyme levels are not always the same for each BLM dose. t' Mean -+ SD.

We report here a negative correlation between the frequency of BLM-induced dicentrics and the levels of SOD in whole blood, erythrocytes and plasma, and the levels of CAT and POD in plasma. On the other hand, we found a positive correlation between the frequency of BLM-dependent chromosomal damage and the activity of SOD and POD in m o n o n u c l e a r blood cells. The Papworth test indicates that the majority of individuals studied showed a marked intercellular variability in the chromosomal response to BLM (Z > 2). Very likely this variability tends to blur the correlations analyzed. Therefore, it seems reasonable to assume that the relationships obtained are an underestimation of the real correlations existing between AOE levels and BLM-induced chromosomal damage. Nordenson et al. [20] were the first to establish a correlation between AOE activity and the yield of chromosome damage i n d u c e d by active oxygen species in h u m a n lymphocytes. These authors found that the addition of CAT or CAT plus SOD to lymphocyte cultures decreased the chromosome damage i n d u c e d by y-ray exposure. Later, Lipecka et al. [21] reported an ample i n t e r i n d i v i d u a l variability in SOD activities from normal h u m a n lymphocytes and red cells and found that the chromosome damage induced by ionizing radiation had a negative correlation with SOD levels in lymphocytes. This finding from Lipecka et al. is controversial with our finding of a positive correlation between BLM-induced dicentrics and SOD activity in mononuclear blood cells. These differences however, may be ascribed to the different clastogenic agent used in the experiments. Ionizing radiation induces chromosome damage mainly through the production of free radicals originating in the degradation of cellular water and organic matter. DNA degradation occurs very fast (~1 × 10-6 second), and, because of the high energy of ionizing radiation, the damage in the

138

DNA m o l e c u l e is at r a n d o m with a ratio of 1/20 double to single-strand breaks [22]. DNA degradation by BLM requires the intercalation of the planar bithiazole moiety of BLM in G-rich areas of the DNA molecule. Upon c o m p l e x i n g with dioxygen and divalent metal ions (mainly Fe + 2), BLM induces DNA nicks by preferential attacking of p y r i m i d i n e nucleotides adjoining the guanidyl-3-phosphate at the site of B L M - D N A intercalation [23, 24]. Although the primary m e c h a n i s m of DNA strand breakage by BLM is not yet well understood, there are indications that free radicals may be i n v o l v e d in the origin of the DNA damage [25, 26]. The maximal damage to the DNA in the cellular chromatin occurs in about 5-15 minutes after the initiation of BLM treatments [18], the ratio of double- to single-strand breaks is 1/9 [27, 28], and the DNA sensitivity to BLM is m o d u la te d by the level of cytosine methylation [15, 16], the organization of the chromatin [17, 18], and the intracellular level of BLMhydrolases [29]. Because the intrinsic m e c h a n i s m of BLM damage to the cellular DNA is not yet fully understood, it is difficult at the present time to provide a simple explanation for the influence of AOE in this damage. Yet the finding of a correlation between c h r o m o s o m a l sensitivity to BLM and AOE activity is in itself interesting, because it suggests that by determining the levels of these enzymes in a given cell population, it w o u l d be possible to predict the chromosomal sensitivity of these cells to BLM. The authors wish to thank Mr. Livio De Rossi (Laboratorios Dr. Gador, Argentina) for providing bleomycin samples, the Blood Bank of Buenos Aires Province (Argentina) for providing blood samples, and Lic. Miguel Reigosa and Mr. Juan Padr6n for technical assistance. This work was supported by grants from the Consejo Nacional de Investigaciones Cientificas y T6cnicas (CONICET) and the Comisi6n de Investigaciones Cientificas de la Provincia de Buenos Aires (C1C) from Argentina, and from the Sigrid Juselius Foundation and the Finnish Academy of Sciences from Finland.

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