Topical and intratumoral photodynamic therapy with 5-aminolevulinic acid in a subcutaneous murine mammary adenocarcinoma

Topical and intratumoral photodynamic therapy with 5-aminolevulinic acid in a subcutaneous murine mammary

adenocarcinoma

Adriana Casasa, HaydeÂe Fukudaa, Roberto Meissb, Alcira M. del C. Batllea,*

aCentro de Investigaciones sobre Por®rinas y Por®rias (CIPYP) FCEyN (University of Buenos Aires) and CONICET, Ciudad Universitaria,

PabelloÂn II, 2do piso, (1428)Capital Federal, ArgentinabDepartamento de PatologõÂa, I.E.O., Academia Nacional de Medicina, Las Heras 3092, (1425)Capital Federal, Argentina

Received 4 July 1998; received in revised form 1 March 1999; accepted 1 March 1999

Abstract

One of the most promising substances used in photodynamic therapy (PDT) is 5-aminolevulinic acid (ALA), which induces

endogenous synthesis and accumulation of porphyrins in malignant cells. In this paper we have shown that both topical and

intratumoral administration of ALA in a subcutaneously implanted mammary carcinoma produced a signi®cant synthesis of

porphyrins and subsequent sensitization to laser light. Porphyrin accumulation was greater when ALA was administered

intratumorally and tumour/normal skin porphyrin concentration ratios were higher compared with topical application. Irradia-

tion was optimal between 2 and 3 h after topical application of 50 mg of a 20% ALA cream and 2±4 h after intratumoral

administration of 30 mg ALA/cm3. The pattern of tumour response evaluated as the delay of tumour growth was similar

following either route of drug administration. Applications of PDT were performed once, twice or three times in the study. The

response to successive applications was constant for the same tumour, indicating that no resistance was acquired. Microscopic

analysis showed both induction of foci of necrosis and haemorrhage, morphological features of apoptotic cells and total absence

of cellular immune response. This paper reports on PDT with topical ALA in a subcutaneous carcinoma leading to tumour

growth delay. These ®ndings may have great relevance in the treatment of cutaneous metastasis of mammary carcinomas.

q 1999 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: d-Aminolevulinic acid; Photodynamic therapy (PDT); Subcutaneous transplantable tumour; Topical application; Intratumour

injection

1. Introduction

Photodynamic therapy (PDT) is a cancer treatment

based on the accumulation of a porphyrin-related

photosensitizer in tumour cells, and their subsequent

destruction on exposure to visible light. Singlet

oxygen species are produced, causing damage to

membranes and organelles, leading to cell death and

tumour ablation [10]. One of the most promising

substances for PDT is 5-aminolevulinic acid (ALA),

a haem precursor which induces endogenous accumu-

lation of porphyrins, mainly protoporphyrin IX

(PpIX), in malignant tissues.

Biological membranes are considered as critical

targets for cell killing by PDT; damage to the vascular

endothelium resulting in tissue/tumour ischaemia is

Cancer Letters 141 (1999) 29±38

0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved.

PII: S0304-3835(99)00079-8

* Corresponding author at: Viamonte 1881 10A, 1056 Buenos

Aires, Argentina. Fax: 1 54-1-8117447.

E-mail address: [email protected] (A.M. del C. Batlle)

an additional mechanism leading to tumour necrosis

[24]. PDT has also been shown to induce apoptosis in

vivo [28] and in vitro [9]; however, the apoptotic

response to PDT seems to depend on both the photo-

sensitizer and the cell line [18].

Due to its water solubility ALA can be given either

by intravenous injection [12], oral administration [8],

topically [13], and there are a few reports on its intra-

tumoral (i.t.) application [3,6,7]. In topical PDT

limited penetration of ALA and inadequate distribu-

tion of induced porphyrins might be a possible draw-

back for this modality. Recently, Peng et al. [20]

demonstrated that the systemic administration of

ALA led to more selective and homogeneous accu-

mulation of ALA-induced porphyrins in the tumour.

Clinical trials have already been performed using

topically [2,26] or systemically [15] applied ALA,

with good results. Topical ALA-PDT has also been

shown to be effective in the treatment of premalignant

conditions such as keratoses and in psoriasis [1,25].

Experimental models have shown that i.t. injection

was more effective than or at least equally effective

to the other two routes of ALA administration [3,6,7].

We have shown that the i.p. and i.t. injection of

ALA could produce a signi®cant synthesis of

porphyrins in the mammary transplantable adenocar-

cinoma M2 [6].

In the present study we investigated the effects of

laser irradiation on the growth of a subcutaneous

implanted tumour after topical or i.t. application of

ALA. The amount of ALA leading to the highest

accumulation of porphyrins in the tumour and the

time point of the highest tumour/skin ratio of

porphyrin levels, to attain optimum ef®ciency of

PDT were determined. Tumour response was evalu-

ated following tumour growth pro®les, delay growth

indexes and light microscopy examination of PDT-

treated areas.

2. Materials and methods

2.1. Chemicals

ALA was purchased from Sigma Chemical Co., St.

Louis, MO. All other chemicals were of analytical

grade.

2.2. Animals

Male BALB/c mice, 12 weeks old, weighing 20±25

g were used. They were provided with food (Purina 3,

Molinos RõÂo de la Plata) and water ad libitum. A

mammary adenocarcinoma [23] (M2, Hospital

Roffo, Buenos Aires) was propagated by serial trans-

plantation into male BALB/c mice. Non-necrotic

tumour material for inoculation was obtained by

sterile dissection of large ¯ank tumours. A 1-mm3

sample of macroscopically viable tumour, which is

equal to approximately 2 £ 105 cells, was injected

s.c. under the dorsal ¯ank of each mouse. The take

rate of the tumours following transplantation was

nearly 100%. Under these controlled conditions the

implant size did not vary by more than 10%. Animals

were treated in accordance with guidelines established

by the Animal Care and Use Committee of the Argen-

tine Association of Specialists in Laboratory Animals

(AADEALC), in full accord with the UK Guidelines

for the Welfare of animals in Experimental Neoplasia

[27].

2.3. Preparation and administration of ALA

The hydrochloric salt of ALA was dissolved in

sterile water (pH 6.5) at a concentration of 100 mg/

ml and used immediately for i.t. administration. For

topical application ALA was prepared daily as a 10,

20, 30 or 40% formulation in a cream (Genargen

Argentinaw).

2.4. Pharmacokinetics of ALA-induced porphyrins in

tissues

Tumours with a volume of approximately 400±450

mm3 were used for pharmacokinetic studies. For time-

response kinetics mice were given either 20 mg of

ALA per cm3 of tumour tissue intratumorally, or 50

mg of a 20% ALA cream by topical application. At

different times between 1 and 24 h after ALA admin-

istration (®ve mice for each point) animals were sacri-

®ced.

For dose-response kinetics, 50 mg of a cream

containing 10, 20, 30 or 40% ALA were applied topi-

cally on tumours of the same uniform size rubbing

over the whole surface of the tumour during a period

of 60 s. For the i.t route, 10, 20, 30, 40 and 80 mg

ALA/cm3 tumour were injected (®ve mice for each

A. Casas et al. / Cancer Letters 141 (1999) 29±3830

point). Animals were killed 3 h after topical adminis-

tration and 4 h after i.t. injection.

The skin overlying the tumour (SOT) was carefully

removed from the tumour itself. Samples of normal

skin (NS) previously shaved were excised from the

contralateral ¯ank of the tumour-bearing mice.

2.5. Tissue extraction of porphyrins

The tissue samples were homogenized in a 4:1 solu-

tion of ethyl acetate/glacial acetic acid mixture. The

homogenates were centrifuged for 30 min at 3000 £ g,

and the supernatants shaken with an equal volume of

5% HCl [5]. Extraction with HCl was repeated until

negative ¯uorescence in the organic layer. Porphyrins

were spectrophotometrically determined in the

aqueous fraction measuring absorbances at 380, 430

nm and the Soret band [22].

2.6. Laser and irradiations

A rhodamine dye laser (Model DL30, Oxford

Lasers) pumped by a copper vapour laser (CU15A,

Oxford Lasers) tuned to 630 nm was used. The light

was focused into a 400-mm diameter optical ®bre and

the cut end of the ®bre was positioned to provide a

3.5-cm diameter light spot, producing a treatment area

of uniform intensity. The output power from the ®bre

was measured with a power meter (Model LM-

100XL, Coherent, Auburn, CA) before each applica-

tion. Total doses of 97 J/cm2 were delivered using a

¯uence rate of 80 mW/cm2 over 20 min. Light doses

up to 100 J/cm2 do not cause additional hyperthermic

effects, which may in¯uence the ef®cacy of PDT;

therefore, higher light doses were not used (Casas et

al., unpublished results).

2.7. Treatment protocol for ALA-based PDT

Eight days after implantation, when tumours

reached the appropriate size of 70±100 mm3, the

SOT was shaved and a 20% ALA cream was topically

applied. Ten days after implantation, when tumours

reached the size of 180±200 mm3, they were intratu-

morally injected with 30 mg ALA/cm3 tumour.

Animals were anaesthetized by i.p. injection of 70

mg/kg ketamine hydrochloride (Parke Davis, Argen-

tina, S.A.) and 6 mg/kg xylazine (RompuÂnw, Bayer

Argentina S.A.) and lesions were super®cially irra-

diated. During treatment normal tissue surrounding

the tumour was shielded with a piece of black plastic,

leaving exposed a peritumoral margin of 3 mm. Body

temperature was monitored with a rectal probe (Tini-

talk II, Gemini Dataloger). Normal shaved skin was

exposed to both ALA and light for histological exam-

ination.

2.8. Assessment of tumour response

The longer (l) and shorter (w) perpendicular axes

and height (h) of each tumour were determined with

callipers prior to or after PDT daily up to day 25 after

implantation to evaluate the delay of tumour growth.

Tumour volume was calculated using the formula

l £ w £ h £ 0:5, where 0.5 is a correction factor

empirically determined [21]. To assess the response

to treatment two indexes were de®ned: D24 (tumour

volume 24 h after PDT/tumour volume before PDT)

and D48 (tumour volume 48 h after PDT/tumour

volume before PDT).

2.9. Histological studies

Prior to and 4, 9, 24, 48 and 72 h, up to 6 days after

PDT, samples of tumour with its surrounding skin and

samples of the NS were excised. They were extended,

sliced, ®xed in 10% buffered formalin, embedded in

paraf®n, sectioned, stained with haematoxylin and

eosin and examined by light microscopy.

The presence of tumour tissue, necrosis and cells

with morphological features of apoptosis was evalu-

ated. Epidermal and dermal damage, vascularity

changes and presence of lymphocytic in®ltration

were also investigated.

2.10. Statistical analysis

The unpaired t-test was used to establish the signif-

icance of differences between groups. Differences

were considered statistically signi®cant when

P , 0:05.

A. Casas et al. / Cancer Letters 141 (1999) 29±38 31

3. Results

3.1. Kinetics of porphyrin biosynthesis in tumour,

normal skin and skin overlying the tumour after

topical or intratumoral administration of ALA

Fig. 1 shows the kinetics of porphyrin accumulation

after topical application of ALA. The highest amount

of porphyrins (0.6 mg/g tissue) was found in the

tumour tissue 2±3 h after administration of ALA,

then concentration decreased rapidly. The ALA-

induced porphyrin accumulation in NS (0.4 mg/g

tissue) was highest 90 min after ALA application. A

delay in peak levels of porphyrins was observed in the

SOT, reaching a maximum concentration of 1.1 mg/g

tissue, 8 h after ALA application.

When ALA was given intratumorally (Fig. 2), the

highest amount of tumoral porphyrins (2 mg/g tissue)

was found between 2 and 4 h after drug administra-

tion.

Normal and tumour-overlying skin showed a

A. Casas et al. / Cancer Letters 141 (1999) 29±3832

Fig. 1. Concentration of ALA-induced porphyrins in tumour,

normal skin and skin overlying the tumour as a function of time

after topical application of ALA cream. At different times after

tumour topical application of 50 mg of 20% ALA cream, tissues

were excised and porphyrins extracted as detailed in Section 2. Each

data point represents the average of ®ve determinations. Error bars

show standard deviations.

Fig. 2. Concentration of ALA-induced porphyrins in tumour,

normal skin and skin overlying the tumour as a function of time

after intratumoral administration of ALA. At different times after

intratumoral injection of 20 mg/cm3 ALA, tissues were excised and

porphyrins extracted as detailed in Section 2. Each data point repre-

sents the average of ®ve determinations. Error bars show standard

deviations.

similar pattern of porphyrin synthesis. Maxima were

observed at 2 and 4 h after ALA injection (1.2 and 1

mg/g tissue, respectively).

After reaching the peak, the porphyrin concentra-

tion declined rapidly near to basal levels in tumour

and NS by 24 h, in either route of ALA administration,

but not in SOT tissue. Tumour to NS porphyrin

concentration ratios were approximately 3 between

2 and 3 h after topical application of ALA and nearly

2 between 4 and 6 h after i.t. administration. Conver-

sely, tumour/SOT porphyrin values were higher for

the i.t. route, reaching a maximum of nearly 3

between 4 and 6 h after, compared to 0.86 for the

topical application at the same interval.

3.2. ALA-induced accumulation of porphyrins

The amount of porphyrins generated in tumour

after topical application reached a plateau with a

20% ALA cream (Fig. 3). SOT showed almost an

identical pattern, although the amount of porphyrins

was higher.

Normal skin porphyrins peaked at 30% ALA, and

total accumulation was always lower than tumoral

porphyrins. Tumour to NS and SOT porphyrin

concentration ratios reached their maximum levels

after 20% ALA (2.64 and 0.89, respectively).

When ALA was i.t. injected (Fig. 4), the maximum

accumulation of porphyrins was found with a 30-mg

ALA/cm3 injection. Both NS and SOT exhibited a

saturation pattern above 40 mg ALA/cm3. The

tumoral levels of ALA-induced porphyrins were

always higher than those of NS and SOT. At 30 mg

ALA/cm3, tumour to NS and SOT ratios were also

maximal (2.7 and 2.4, respectively).

3.3. Effectiveness of ALA-based PDT in delaying

tumour growth

The effectiveness of ALA-induced porphyrins as

photosensitizers for PDT was determined by assessing

the extent of tumour growth after one, two and three

PDT applications.

Two response indexes were de®ned, measuring the

A. Casas et al. / Cancer Letters 141 (1999) 29±38 33

Fig. 3. Porphyrin accumulation in tissues after topical application

of various concentrations of ALA. Three hours after application of

50 mg of a cream containing 10, 20, 30 or 40% ALA, tumour (X),

normal skin (P) and skin overlying the tumour (O) were excised

and porphyrins extracted as detailed in Section 2. Each data point

represents the average of ®ve determinations. Error bars show stan-

dard deviations.

Fig. 4. Porphyrin accumulation in tissues after intratumoral admin-

istration of various concentrations of ALA. Four hours after injec-

tions of 10, 20, 30, 40 and 80 mg ALA/cm3 tumour tissue, tumour

(X), normal skin (P) and skin overlying the tumour (O) were

excised and porphyrins extracted as detailed in Section 2. Each

data point represents the average of ®ve determinations. Error

bars show standard deviations.

ratios between tumour volumes before and after treat-

ment, since the rapid growth of M2 tumour does not

allow the evaluation of survival or of tumour doubling

times.

D24 and D48 indexes for both topical and intratu-

moral ALA-PDT are shown in Table 1. After a single

application both indexes were lower than those of the

untreated tumours, indicating that if not a complete

reduction, a delay of tumour growth occurs. After two

and three PDT applications similar indexes were

obtained and no signi®cant differences between either

route of ALA administration were evidenced (data not

shown).

Irradiation on ALA injected intratumorally

appeared to induce a greater reduction in tumour

volume earlier. An index of 0.75 was observed at 24

h and 0.94 at 48 h, but differences between times were

not statistically signi®cant.

In order to compare the response to repetitive PDT,

tumour growth curves with the same D24 index were

used (Figs. 5 and 6). Animals received either one, two

or three successive treatments of topical and intratu-

moral ALA-PDT, respectively.

A single application induced a clear tumour growth

delay for both topical and intratumoral ALA admin-

istration. A second PDT treatment applied 48 h later

and a third one, applied 6 days later induced a further

delay in tumour growth. It is noteworthy that tumour

A. Casas et al. / Cancer Letters 141 (1999) 29±3834

Table 1

Tumour response indexes after a single application of ALA-PDTa

D24 (XÅ � ) D48(XÅ � )

Control (n � 7) 1.71 ^ 0.84 2.56 ^ 1.10

Topical ALA (n � 12) 0.96 ^ 0.39b 0.87 ^ 0.65c

i.t. ALA (n � 5) 0.75 ^ 0.21d 0.94 ^ 0.44b

a As a measurement of tumour response to topical and i.t. ALA-

PDT, two indexes were de®ned: D24 and D48 (see Section 2). D24

and D48 control indexes were calculated as volume ratios between

day 10 and days 9 and 8, respectively, after implantation of tumours

in non-treated mice. Results are presented as means ^ standard

deviations.b **P , 0:01 compared to the corresponding control.c *P , 0:007 compared to the corresponding control.d ***P , 0:03 compared to the corresponding control.

Fig. 5. Time-dependence of tumour growth after topical ALA-PDT.

Tumours with an initial volume of 70±100 mm3 were topically

treated with 20% ALA cream and 3 h later they were exposed to

630 nm light at a ¯uence rate of 97 J/cm2. Animals were treated with

one (P), two (O) or three (B) doses of ALA-PDT. Arrows indicate

the day of treatment. X, mean of six control curves (neither ALA

nor light). Growth curves of tumours with the same response

indexes are represented (D24 � 0:70).

Fig. 6. Time-dependence of tumour growth after intratumoral

ALA-PDT. Tumours with an initial volume of 180±200 mm3

were intratumorally injected with 30 mg ALA/cm3 tumour and 4

h later they were exposed to 630 nm light at a ¯uence rate of 97 J/

cm2. Animals were treated with one (P), two (O) or three (B) doses

of ALA-PDT. Arrows indicate the day of treatment. X, mean of six

control curves (neither ALA nor light). Growth curves of tumours

with the same response indexes are represented (D24 � 0:80).

height was the axis most dramatically reduced after

PDT.

The D24 and D48 indexes were always similar for

the same tumour after the ®rst, second or third PDT

dose.

The application of ALA or light alone did not cause

any measurable effects on tumour growth.

In all cases core body temperature throughout laser

treatment decreased from 33 to 298C during irradia-

tion, due to anaesthesia.

3.4. Macroscopical analysis and histological studies

Normal skin exhibited neither macroscopic nor

microscopic changes with PDT treatment. The same

results were observed for tumour or skin exposed to

either ALA or light alone at all times analyzed.

After 24 h of ALA-based PDT, administered by

either route, macroscopically necrotic zones, gross

tumour volume reduction, ulceration and eschar

formation were induced.

At 0, 4 and 9 h after treatment no microscopic

changes in either tumour tissue or its overlying skin

were observed. At 24 h preserved tumour tissue with

scanty foci of necrosis and cells with apoptotic images

up to a depth of 4 mm from the epidermis were seen in

both i.t. and topically ALA-PDT treated samples (Fig.

7). Necrotic cells with vacuolization of the cytoplasm,

pycnotic appearance of the nucleus and loss of cellu-

larity were seen. Epidermal with slight keratosis,

dermal oedema and vascular dilation in the SOT

were also observed.

At 48±72 h there was an expansion of tumour in®l-

trating the dermis and epidermis with keratinization

and sloughing of the SOT. Haemorrhagic foci and

large areas of necrosis and of apoptotic cells in tumour

tissue were also seen.

From day 5 after PDT, connective ®brovascular

tissue surrounding the tumour, rich in neo-formed

vessels with wall enlargement, vascular stasis and

congestion was developed.

No lymphocytic in®ltration was ever found in any

microscopic studies performed. Non-shielded peritu-

moral skin of mice, having been treated three times

with topical ALA-PDT, remained free of macrosco-

pically visible tumour for 10 days, and exhibited hair

basophilic, amorphous destruction inside the follicle

which correlated with visible alopecia.

A. Casas et al. / Cancer Letters 141 (1999) 29±38 35

Fig. 7. Morphological images of apoptotic cells after ALA-based PDT treatment. Histological section of tumour treated 24 h after topical ALA-

PDT, showing condensation of an eosinophilic cytoplasm and a basophilic nuclei. Cap-like condensed chromatin at one nuclear pole appear in

some cells. Similar images were observed for the i.t. route 48 and 72 h after treatment. None of these features were obtained in control tissues.

4. Discussion

It has already been demonstrated that porphyrins

can be synthesized and accumulated in different

tissues such as tumour and skin of mice given i.t.

injection of ALA [6]. Here, we have shown that

topical or intratumoral administration of ALA in a

subcutaneously implanted mammary carcinoma

induced a signi®cant synthesis of porphyrins and a

consequent sensitization to laser light.

Although complete remissions were not achieved

with the present light dose regimen, periods up to 10

days free of macroscopically visible tumour were

sometimes observed both with the i.t. and topical

modes of ALA application (data not shown).

For both routes of ALA administration a sort of

saturation pattern was observed in the porphyrin-

synthesizing capacity of the tissues, indicative of the

existence of particular regulatory mechanisms in the

metabolism of ALA-induced porphyrins in tumour-

bearing animals [17]. It is noteworthy that this ALA

dose-response saturation in tumour is evidenced when

the drug is administered by either route. It was

achieved with 50 mg of 20% ALA cream, which

corresponds to a total amount of 25 mg ALA/cm3

tumour, and with 30 mg ALA/cm3 in the case of i.t.

application.

The total amount of porphyrins in tumour tissue

was ®ve times higher when ALA was i.t. injected,

and tumour/SOT porphyrin concentration ratios

were higher. However, no signi®cant differences in

the actual ef®cacy of photosensitization were

observed, in agreement with the results of Cairnduff

et al. [3] for an intradermally located murine adeno-

carcinoma. These ®ndings may be explained by either

the existence of a saturating amount of ALA-formed

porphyrins necessary to produce the photodynamic

damage [14] or alternatively by an uneven distribution

of porphyrins in different tumour layers which even-

tually result in a similar or equivalent amount of

porphyrins directly exposed to light in either route

of ALA administration.

According to tumour/skin porphyrin concentration

ratios and total amounts of porphyrins accumulated in

tumour tissue, irradiation would be optimal between 2

and 3 h after topical application of 20% ALA cream

and 2±4 h after intratumoral administration of 30 mg

ALA/cm3.

Maximal accumulation of porphyrins was observed

3 h after topical ALA application, in agreement with

Henderson et al. [11] and Peng et al. [19], who found

that ¯uorescence of ALA-induced porphyrins in

murine adenocarcinomas peaked between 3 and 5 h

post-topical application.

Induced anoxia by vascular damage does not seem

to lead to an impairment of repetitive-PDT ef®cacy,

since the same tumour response is maintained along

successive treatments. It appears that continuous

applications of PDT would photosensitize surviving

cells, which could recover their oxic stage by means

of the neo-formed vessels provided by the

surrounding ®brovascular reparation tissue. Another

interesting hypothesis is that proposed by Moan and

Sommer [16], who stated that the anoxic tumour cells

are inactive in secondary reactions, probably due to

nutrient deprivation caused by breakdown of the

circulatory system.

Unlike most currently used cancer therapies, the

response to PDT treatment was constant for the

same tumour, indicating that no resistance was

acquired.

As a minor drawback, hair loss in mice surviving 10

days after the third PDT treatment was observed. This

was also addressed by Divaris et al. [4] after exposure

of ALA i.p. injected animals to white light as a conse-

quence of a high porphyrin concentration in the hair

follicle. The ®nding of alopecia in only these long-

surviving animals indicated that almost a week is

necessary to develop follicle damage in addition to

the application of three doses of PDT.

The absence of a cellular immune response after

ALA-PDT is also noticeable, as is the presence of

morphological features of apoptotic cells. These

apoptotic cells may derive either from direct tumour

cell killing or from vascular damage.

This paper reports on the topical ALA-PDT appli-

cation in a subcutaneous carcinoma leading to subse-

quent tumour growth delay. These ®ndings may have

great relevance in the treatment of cutaneous metas-

tasis of mammary carcinomas. Indeed, lack of resis-

tance of this mammary adenocarcinoma to repetitive

ALA-PDT applications encourages the use of ALA-

based photodynamic therapy as a promising alterna-

tive in the routine treatment of tumours resistant to

cytostatic drugs.

A. Casas et al. / Cancer Letters 141 (1999) 29±3836

Acknowledgements

This research was supported by grants from the

Argentine National Research Council (CONICET),

the Association for International Cancer Research

(AICR)-UK and the Alberto J. Roemmers Foundation,

Argentina. The authors are very grateful to Victoria

Castillo for her skilful technical assistance. A.M. del

C.B. and H.F. hold the posts of Superior and Associate

Researchers at the CONICET. A.C. is a CONICET

Research Fellow.

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