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Source : MNHN, Paris

AINTES

INTERNATIONAL JOURNAL OF BATRACHOLOGY

June 1996 Volume 14, N° 2

Alytes, 1996, 14 (2): 53-84. Bibliothèque Centrale Muséum Review paper

The amphibian micronucleus test,

a model for in vivo monitoring

of genotoxic aquatic pollution

Laury GAUTHIER !

Centre de Biologie du Développement, UMR 5547 du CNRS, affiliée à l'INSERM,

Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse Cedex, France

This review deals with the experiments carried out over the last decade on

the detection of the genotoxicity in aquatic medium (fresh water) using

amphibian larvae. Three amphibian species have been widely used: two

species of urodeles, the Spanish newt or pleurodele (Fleurodeles walti) and

the Mexican axolotl (Ambystoma mexicanum), as well as one anuran, the

South African clawed frog or common platanna (Xenopus laevis). The protocol

carried out on these amphibians, at the Developmental Biology Center in

Toulouse, France, allows the direct detection of genotoxic agents in the

rearing medium of the animals by measuring the induction level of micronu-

cleated erythrocytes of larvae exposed to pure substances, physical agents,

complex mixtures of substances like drinking and surface water, domestic and

industrial wastes, or any aqueous effluent.

After introducing the test procedure and the rearing conditions of the

animals, the results obtained with the amphibian micronucleus assay are

presented with respect to the chronological order of the different steps leading

to the publication of the standardized method in 1992 and to the state of the

art in the field of in vivo eco-genotoxicology of aquatic media, where

amphibians, thanks to the works reported in this paper, play a major role.

Then, the choice of future development areas in the field is discussed in

terms of validity of the use of amphibians as genotoxicity bioindicators in the

aquatic environment, considering the many advantages of this model and its

complementarity with the commonly used in vitro test-systems.

1. Present address: Groupe de Recherche Pluridisciplinaire sur l'Environnement d'Albi (GRePEA),

Université Paul Sabatier, campus d’Albi, 2 avenue Franchet d’Esperey, BP 95, 81003 Albi Cedex, France.

PARIS

Source : MNHIg Pfris

54 ALYTES 14 (2)

INTRODUCTION

Increased environmental pollution can be attributed to a variety of factors resulting

from new industrial and agricultural technologies together with changes in our way of life.

Moreover, the nature of the pollution itself has become more diverse. Whatever the origin

of the pollution, it tends to find its way into the aquatic environment. Genotoxic

pollutants affect the aquatic ecosystem, and their presence in the water can also have

repercussions on non-aquatic species via food chains or simply from drinking the water.

One should therefore be aware of the hidden risks stemming from potential genotoxic

substances in the aquatic environment. Moreover, a considerable time may elapse between

the action of the mutagenic agent and the outward signs of its effects. The relationship

between cause and effect may thus become obscured.

The mutagenic risk is particularly apparent in prokaryotes, and readily discernible in

plants and animals with a rapid rate of reproduction, although it is often not very

perceptible in plants and animals (including humans) with a slower reproduction rate. It

should be remembered that the manufacture and use of aggressive mutagenic substances

is too recent to allow judgment of effects over a relevant number of generations. The

mutagenicity of an unknown substance is usually evaluated by putting it in contact with

a living system, which is then examined for genetic damage. It is generally agreed that it

is difficult to extrapolate results obtained in one living system to another one, or even from

animal to man. Nevertheless, the commonly used tests, as a first step, are based on

bacteria, such as the Ames test (AMES et al., 1975; MaRON & AMES, 1983). The main

advantages are that such tests can be carried out rapidly and are low in cost. One of the

main drawbacks of these bacterial tests for the detection of genotoxic substances in water

is that they are relatively insensitive, and in general they cannot be used on unconcentrated

water samples (WEAVER et al., 1981).

Ideally, one should evaluate the biological hazards of environmental genotoxic

pollutants in situ. In vivo mutagenicity tests applied to unconcentrated water samples

represent a step in this direction (CHOUROULINKOV & JAYLET, 1989; JAYLET & ZOLL,

1990). They give an indication of the overall genotoxic potential of the water under testing.

One example is the micronucleus test adapted to larvae of amphibians that we developed

over the last decade. The larvae can be reared not only in containers filled with

unconcentrated water samples (laboratory conditions), but also in running water of

various sources (effluents of factories, river water or even drinking water).

In amphibian larvae, as in most eukaryotes, chromosome and genome mutations

result in the formation of micronuclei (fig. 1). These micronuclei are small intracytoplas-

mic masses of chromatin resembling small nuclei (fig. 1A). They are formed from

chromosome fragments or complete chromosomes which have not migrated to a spindle

pole during anaphase (fig. 1C). Therefore, the formation of micronuclei stems from either

chromosome fragmentation or a malfunction of the mitotic apparatus. In the former case,

micronuclei correspond to chromosome fragments that, having lost the centromere, have

been unable to connect with the spindle fibers. In the latter case, they arise from complete

chromosomes lagging at anaphase due to spindle abnormalities. Clastogenic compounds

and spindle poisons both lead to an increase in the number of micronucleated cells.

Source : MNHN, Paris

GAUTHIER 55

Fig. 1. — Aspect of the micronucleated erythrocytes of pleurodele larvae during (B, C) and after (A)

cell division. Larvae have been treated during 12 days with BaP (0.5 ppm) and blood smears

were fixed in methanol (5 minutes), stained with Masson’s acid hemalun (12 minutes) and then

placed under running water for 10 minutes.

A. — Four micronucleated erythrocytes (interphase). After treatment with a strong

genotoxic agent, it is not rare to observe cells containing 4 or more micronuclei (cell on the

right), whereas in the control larvae, most of the micronucleated cells contain only one

micronucleus but rarely two or three.

B. — Erythrocyte of pleurodele during metaphase, including 3 chromosomal fragments.

C. — Erythrocyte of pleurodele during anaphase, in which a chromosomal fragment and

probably a whole chromosome are not incorporated to the spindle.

Source : MNHN, Paris

56 ALYTES 14 (2)

Evans et al. (1959) were the first to suggest counting cells containing micronuclei as

a method for the evaluation of cytogenetic damage. Since then, induction of micronuclei

has been widely used for genotoxicity testing. À detailed description of the micronucleus

test using bone marrow polychromatic erythrocytes from small mammals is given by

Scamip (1976). Results from the micronucleus test and recommendations for its practical

application have been reviewed by HEDDLE et al. (1983).

In the animal kingdom, micronucleus formation has been studied mainly in mammals.

In aquatic vertebrates, micronucleus tests using fish have been carried out on different

species: the mudminnow (Umbra limi) and the brown bullhead (/ctalurus nebulosus)

(METCALFE, 1988), the Eastern mudminnow (Umbra pygmaea) (HOOFTMAN & DE RAAT,

1982), the white croaker (Genyonemus lineatus) (CARRASCO et al., 1990) and Heteropneustes

fossilis (Das & NANDA, 1986). In our hands, a similar test, on red blood cells from three

other fish species (Brachydanio rerio, Cyprinus carpio and Nothobranchius rachowi), did not

lead to statistically significant results (unpublished data).

In 1987, KRAUTER et al. demonstrated micronuclei formation in peripheral eryth-

rocytes of Rana catesbeiana tadpoles after irradiation. They proposed this animal for in

vivo genotoxicity studies. Other amphibian species have been designed to score the level

of micronucleated cells in animals exposed to genotoxic substances in water and to

monitor aquatic genotoxic pollutions in the environment.

This paper has two major purposes: to present the procedure of the micronucleus test

applied to 3 amphibian larvae, the pleurodele, the axolotl and the platanna; and to review

the works done until now on the use of these three amphibian species to evaluate the geno-

toxic impact of chemicals or physical agents and to detect genotoxicity in aquatic media.

MATERIALS AND METHODS

THE AMPHIBIAN MICRONUCLEUS TEST

The test procedure has been established in two species of urodeles, Pleurodeles waltl

(the Spanish newt or pleurodele; family Salamandridae) and Ambystoma mexicanum (the

Mexican axolotl; family Ambystomatidae), as well as in the anuran Xenopus laevis (the

South African clawed frog or common platanna; family Pipidae). These three amphibians

are abundant egg-layers: a female of pleurodele or axolotl can lay up to 1000 eggs at once,

while 2000 or 3000 is not rare in platannas. Their rearing and development are now well

described. The details of the test procedure have been described in the publications

mentioned above, and for urodeles documentation sheets have been published by the

French Standards Institute (ANONyMous, 1987, 1992).

Rearing conditions

Urodeles

Similar methods are used to rear both axolotl and pleurodele larvae. After they are

laid, the eggs are placed in an aquarium. The water is normal tap water filtered through

Source : MNHN, Paris

GAUTHIER 57

active carbon or “ultrapure” water reconstituted with salts. The young larvae eat only live

food. After hatching, the animals are fed on freshly hatched artemia (Artemia salina) or

daphnia (any species). Then the food is switched to Chironomus larvae. Usually this latter

food is subsequently used throughout the experiments. The temperature of the water can

range from 12 to 20°C. Within this range, an increase in temperature increases the growth

rate of the animals. It is therefore possible to change the rate of development of animals

depending on requirements, and several groups at different stages of development can be

studied at the same time. However, the temperature dependence of the mitotic index must

be taken into account. AI treatments are carried out at 20°C. They are always carried out

after a 8-day habituation period at this temperature.

Platanna

Xenopus tadpoles are fed on dehydrated aquarium fish food. The temperature of the

water can range from 18 to 23°C; the growth rate also depends on the rearing temperature.

All treatments are carried out at 22°C after a 6-day habituation period at this temperature.

Treatment stage

The size of the larvae must be large enough to allow an easy taking of blood samples.

They must also be at a stage of intense erythropoiesis with a large number of divisions of

the red blood cells in circulating blood. As the rate of growth is a function of temperature,

and as treatment should be carried out when the larvae are in an identical physiological

state, age cannot be used as a reference. An accurate morphological marker is required.

For the axolotl, we have found that treatment must start when the hind limb buds of the

larvae exhibit slight indentation (onset of formation of the two first digits). For pleurodele,

treatment is started when the hind limbs present four well formed digits with an outline

of the fifth (fig. 2A). In both cases, the mean size of the larvae is around 30 mm (about

6 weeks after laying). They reach 40 mm within the next 10 days. The growth of Xenopus

larvae is quicker and the test can begin 2 weeks after laying. At this time, one must choose

larvae at stage 50 (fig. 2B) of the chronological table of NIEUWKOOP & FABER (1956) (hind

limb bud longer than broad, constricted at base).

Experimental design

The treatment procedure is basically the same for all three species. They are treated

. ja groups of twenty in 5-liter flasks filled with 2 liters of the sample (100 ml of water per

larva). Control groups are reared in purified water. The media are renewed and food is

added every 24 hours. At the end of the treatment period (generally 12 days for pleurodele

and platanna and 10 days for axolotl), the animals are anesthetized with 0.02 % tricaine

methane sulfonate. Blood samples are taken by cardiac puncture into heparinized

micropipettes (20 % solution at 5000 IU/ml). Blood smears are made, then fixed in

methanol and stained with hemalun solution. Slides are examined under the microscope

with an immersion lens (X 100). For each animal, the number of micronucleated red

blood cells per 1000 cells is determined (fig. 2C-D). In these conditions, the frequencies of

micronucleated erythrocytes in the control animals is about 0.5 — 1 % for urodeles and

0.1 % for platanna.

Source : MNHN, Paris

58 ALYTES 14 (2)

Significance of the test

For each group of animals, the results (level of micronucleated red cells per 1000)

obtained for the individual larvae are arranged in increasing order of magnitude and the

medians and quartiles calculated (see example in Table III). In most studies, the statistical

method used to compare the medians is based on the recommandations of MAC GILL et

al. (1978). It consists in determining the theoretical medians of samples of size N (where

N > 7) and their 95 % confidence limits expressed by M + 1.57 x IQR / N'!, where M

= median and IQR = Inter Quartile Range (see example in fig. 3). In these conditions,

the difference between the theoretical medians of the test groups and the theoretical

median of the control group is significant to within 95 % certainty if there is no overlap.

The result is then positive. In the absence of statistical calculations, a result is considered

positive if the two following conditions are satisfied : (1) the median of the treated animals

is twice that of controls; (2) the lowest quartile of the treated animals is above the highest

quartile of the controls. A positive result can be taken for any duration of treatment, but

in general 4 to 8 days is long enough for a strongly clastogenic substance used at maximal

concentration (MC). MC is defined as half the lethal concentration within a period of

6 days.

BACTERIAL TEST-SYSTEMS

This paper relates the results obtained in comparative studies including in vitro test

systems on bacteria: the Ames test, detecting point mutation on Salmonella typhimurium,

the fluctuation test, which is a modification of the Ames test where the compounds under

study are exposed to bacteria in a liquid medium instead of the agar plates used in the

Ames assay, and the SOS chromotest, showing primary DNA damage on Escherichia coli.

Fig. 2. — Amphibian larvae used for the micronucleus assay and corresponding erythrocytes showing

micronucleated cells.

A. — Pleurodele larva at the stage of treatment, ie. stage 53b, according to the

chronological development table of GALLIEN & DUROCHER (1957), or stage 45-46 of the

development table of SH1 & BOUCAUT (1995). At this stage of development, the pleurodele larva

is a small aquatic animal (about 35 mm long). Axolotl larva has the same appearance and the

same size as pleurodele larva at the treatment stage.

B. — Xenopus larvae at the stage of treatment. Left, larva at stage 50 of the chronological

table of development of NIEUWKOOP & FABER (1956), corresponding to the beginning of the test.

Right, larva at stage 54, 12 days later, corresponding to the end of the test. During the testing

period, at 22°C, the animals are growing very quickly, increasing in size from 22-26 mm (st. 50)

to 58-65 mm (st. 54).

C. — Blood smear from a pleurodele larva, stained with Masson’s hemalun, showing red

blood cells with their main nucleus and one micronucleated cell. Pleurodele and axolotl

erythrocytes are big ovoid cells (about 30 um in diameter).

D. — Blood smear from a Xenopus larva, stained with hematoxylin, showing one

micronucleated erythrocyte. Xenopus erythrocytes are smaller than those of urodele species

(about 10 um in diameter).

Source : MNHN, Paris

GAUTHIER 59

Source : MNHN, Paris

60 ALYTES 14 (2)

Principles of the tests

Ames test

The most widely used and validated bacterial reverse-mutation test is that devised by

AMES et al. (1975). The Ames test (also called the Salmonella-mammalian microsome test)

employs strains of Salmonella typhimurium, which are unable to produce their own

histidine. Each of these strains bears a specific defect or mutation in the metabolic

pathway to histidine. Reverse mutations restore the ability to synthesize histidine. These

reverse mutations are induced by specific mutagens. Mutagens that cause base pair

substitutions can be distinguished from those that lead to frameshift mutations by the use

of different strains. If the mutant strain (his-), which is unable to grow in media devoid

of histidine, regains the ability to synthesize histidine after being exposed to an extract,

then the extract is assumed to contain a mutagen.

However, unlike mammals, bacteria lack the necessary oxidative enzyme systems that

metabolize foreign compounds to electrophilic compounds that interact with DNA

(indirect mutagens). Bacterial tests are thus carried out in the presence of a liver

microsomal fraction that contains such enzymes. This is usually prepared by ultracentri-

fugation of a cell homogenate of rat liver. The metabolic activity of this fraction (S9

fraction that contains cytochrome P 450 / P 448 oxidases) is enhanced by prior treatment

of the rats for several days with an inducer (usually Aroclor 1254). The so-called S9-mix

is made up from this S9 fraction that is buffered and supplemented with NADP and

glucose-6-phosphate. A standard procedure has been drawn up for this test. Each test is

carried out several times, both with (detection of indirect mutagens) and without metabolic

activation (detection of direct mutagens).

Fluctuation test

GREEN et al. (1976) have developed another mutagenicity test based on the Ames test

that is both simple and sensitive. The same bacterial strains are exposed to the test

substances in a liquid medium. The auxotrophic mutant strains (around 3.107 bacteria) are

placed in a culture medium containing glucose, a small quantity of histidine and the test

substance. Activation medium (S9) may be added later. An overall volume of 5 ml is

generally employed. Fifty independent cultures in 0.1 ml containing around 5.10% bacteria

are then set up. The small bacterial population minimizes the number of preexisting

revertants. The tubes are incubated at 37°C overnight. The bacteria are allowed to grow

until the supply of histidine is exhausted. This is referred to as the period of auxotrophic

growth. The next day, 2 ml of a selective medium (containing glucose, a pH indicator such

as bromocresol, but without histidine) is added to each culture. The tubes are incubated

for a further 2 or 3 days at 37°C. Only the revertants (prototrophic for histidine) are able

to grow. This leads to a fall in pH, and the tubes turn from blue to yellow. This is referred

to as the phase of prototrophic growth. The number of positive tubes are counted

(prototrophic growth = yellow) in a batch of 50 tubes. Using appropriate statistical

methods, the number of positive tubes are compared between the tubes containing test

substances and the control tubes. The test is replicated a number of times, and similar

controls to those used in the classic Ames test are run in parallel.

Source : MNHN, Paris

GAUTHIER 61

SOS chromotest

The genotoxic effects observed in the bacterial tests are often not direct actions of the

agent. They are often the outward signs of the overall responses of the cell to this action.

In Escherichia coli, DNA-damaging treatments can activate a set of functions known as

the SOS responses. This has been exploited by an operon fusion placing lac Z, the

structural gene for B-galactosidase, under control of the sfiA gene, a SOS function

involved in the inhibition of cell division. A simple and direct colorimetric assay of this

SOS response to DNA damage has been developed, called the SOS chromotest

(QUILLARDET et al., 1982; QUILLARDET & HOFNUNG, 1985). Mutagenicity is evaluated

quantitatively in terms of the SOS-inducing potency. Enzyme activity after incubating the

tester strain in the presence of various amounts of the test compound is measured

colorimetrically. Aliquots of a dilution of exponential phase cultures are placed in glass

test tubes containing the compound to be tested. After 2 hours incubation at 37°C with

shaking, B-galactosidase and alkaline phosphatase activities are assayed.

The classic microsomal activation preparation may also be added to the incubation

mixture. To correct for the inhibition of protein synthesis that may be induced by certain

substances, the strain is made constitutive for synthesis of alkaline phosphatase. This

enzyme is non-inducible by DNA-damaging agents. The ratio of the two activities

(B-galactosidase to alkaline phosphatase) is taken as a measure of the specific activity of

B-galactosidase.

RESULTS OBTAINED WITH THE AMPHIBIAN MICRONUCLEUS TEST

GENOTOXICITY OF X-RAYS AND CHEMICALS

Initially, the sensitivity and dose-response of the micronucleus test were evaluated

with X-ray irradiations as well as known physical clastogenic agents and chemicals

exposure, in pleurodele larvae.

SIBOULET et al. (1984) measured the frequences of micronucleated red blood cells in

the animals 6 days after X-ray irradiation. A dose of 6 rad (relatively weak) leads to a

significant effect. The dose-response is approximately linear up to 150 rad, after which the

slope falls and the maximal effect is reached at 600 rad.

Two years later, JAYLET et al. (1986a) determined the most suitable larval stage for

testing chemicals using pleurodele larvae reared in water containing one of the 4 following

compounds: benzo(a)pyrene (BaP), ethyl methanesulphonate (EMS), diethyl sulphate

(DES) and N-ethyl-N’-nitro-N-nitrosoguanidine (ENNG). Response curves as a function

of treatment duration over a period of 16 days were plotted for 3 different concentrations

of the 4 compounds in order to optimize conditions for a low dose micronucleus test.

The same year, GRINFELD et al. (1986) focused on BaP studies. These authors exposed

pleurodele larvae to different concentrations of the substance for various lengths of time.

Frequencies of micronuclei in circulating erythrocytes were determined at different times

after termination of the treatment. The incidence of micronuclei in larvae kept for 8 days

Source : MNHN, Paris

62 ALYTES 14 (2)

Table I. - Summary of the results obtained for different chemical classes and X-rays

with Pleurodeles waltl (from FERNANDEZ et al., 1993).

Number of Positive Negative

chemicals tested responses responses

Chemical class

Miscellaneous

Amines (aromatic, aliphatic)

Nitroso compounds

Polycyclic carbocycles

N-, S-, O-mustards

Aziridines

Carbamates

D = » RD D U U w

Oxygenated sulfur

Total

X-Rays 12 doses

S3Ssssccecrw

in BaP containing water displayed a marked increase with dose up to 0.075 ppm and a

more gradual one with higher doses, reaching 158 per 1000 at 0.75 ppm. The lowest dose

at which a significant increase could be discerned was 0.01 ppm. Uptake and release was

studied with tritiated BaP. Larvae concentrated BaP rapidly, attaining maximal levels after

12 hours. The ratio of radioactivity in larvae to that in an equivalent volume of

surrounding water was about 200, independent of the amount of BaP added. The marked

bioaccumulation potential of newt larvae partially explains why it is not necessary to

concentrate mutagenic micropollutants in samples of natural or drinking water to detect

genotoxic effects.

Results with 19 organic compounds have been published by FERNANDEZ et al. (1989).

Most of them were known or suspected mutagenic or carcinogenic substances in mammals.

The results were compared with published data from other tests used to detect the

clastogenic or mutagenic properties of chemicals.

In 1993, FERNANDEZ et al. published results obtained with 47 different chemical

compounds, after 12 days or/and 8 days of exposure in pleurodele, axolotl and Xenopus

larvae. The overall results obtained for the different chemical classes tested with pleurodele

are shown in Table I. For comparative purposes, literature data have been collected on

other short-term genotoxicity tests and on long-term carcinogenicity assays in rodents

(Table IT). In this study, the newt micronucleus assay on pleurodele larvae was found to

agree better with the Ames test (bacterial test) (percent concordance with the newt

micronucleus test: 86 %) than with the rodent micronucleus test (71 %).

Source : MNHN, Paris

GAUTHIER 63

Using pleurodele larvae, the genotoxicity of 7 polycyclic aromatic hydrocarbons

(PAHSs) was compared and the influence of UVA irradiation evaluated by FERNANDEZ &

L'HARIDON (1992). The authors classified the PAHSs in the following order of genotoxicity:

benz(a)anthracene (BA) = 7,12-benz(a)anthraquinone > 7,12 dimethyl-benz(a)anthracene

(DMBA) > 9,10-dimethylanthracene; whereas anthracene, 9,10 anthraquinone and

dibenz(a,h)anthracene were not found to be clastogenic. Under lighting conditions (UVA

irradiations for 24 hours) of the rearing media alone (water containing the tested substance),

and then tested on larvae in the dark, BA (50 and 100 ppm) gave rise to clastogenic

products, whereas DMBA (12.5; 25 and 50 ppb) gave no positive response in the test.

More recently, in the same area of research, DJoMo et al. (1995) studied the

genotoxicity of 4 polycyclic hydrocarbons representing a part of the major fraction of

hydrocarbons found in a crude oil. The authors confirmed the strong genotoxic potential

of benzo(a)pyrene, whereas the genotoxicity of naphtalene was weak. In contrast, the two

other compounds tested, anthracene and phenanthrene, gave negative responses in the

newt micronucleus test.

L’HaRIDON et al. (1993) applied the same test system to evaluate the genotoxicity of

amines and/or potential nitrosating agents (nitrite and nitrate) in vivo. The authors

obtained negative results under varying rearing conditions (lighting conditions, pH 8-6-5)

with atrazine, diethanolamine alone or in combination with sodium nitrite or nitrate.

However, the genotoxicity of N-nitrosoatrazine (NAT) at 7.5 and 15 ppm and

N-nitrosodiethanolamine (NDELA) at 12.5-25 and 50 ppm, was demonstrated. In

conclusion, the authors suggested that, at the concentrations used (close to those which

may be encountered in a polluted natural aquatic environment), if NAT or NDELA are

formed, the amounts produced are probably too low to yield a positive response in the

newt micronucleus test.

The cytogenetic effects of mercury compounds have been widely studied in plants,

Drosophila and tissue culture cells, but to our knowledge they have not been evaluated in

vertebrates in vivo. Pleurodele larvae were raised in water containing low concentrations

of methyl mercuric chloride or mercuric chloride (ZoLL et al., 1988). It should be noted

that a low concentration of the two substances (12 ppb) gave a positive result and that

equivalent concentrations in the water of both mercuric compounds led to similar levels

of micronucleated cells. The test gives positive results for concentrations below those often

found in samples of contaminated water (GIRAUD & GuILLET, 1972). Both chromosome

aberrations and abnormalities in cell division were observed in cells from animals treated

with these two substances. Bioaccumulation of both compounds was also evaluated by

determination of mercury levels in the larvae. After 12 days of treatment, concentration

factors (concentration in the organism / concentration in the water) of 1200 and 600 were

found for methyl mercuric chloride and mercuric chloride respectively.

In order to investigate the generality of the micronucleus test in pleurodele, larvae

from another urodele, the axolotl, were reared in water containing either of the

compounds benzo(a)pyrene (BaP) or ethyl methane sulfonate (EMS) (JAYLET et al.,

1986b). The level of micronucleated erythrocytes on blood smears was compared with

control samples from larvae reared in fresh water. The optimum larval stage for this test

system was determined. The effects of the indirect mutagen (BaP) and the direct mutagen

Source : MNHN, Paris

64 ALYTES 14 (2)

(EMS) were found to depend on both dose and exposure to the clastogen. For BaP,

positive results were obtained after 8 days of treatment at a concentration of 0.025 ppm.

After 10 days of treatment at a concentration of 0.1 ppm, numerous micronuclei were seen

(< 25 %). Positive results were also obtained with EMS after 8 days of treatment at a

concentration of 24 ppm. At 62 ppm, positive results were found after 6 days, while at 124

ppm positive results were found after only 4 days. The results with both these agents show

that the axolotl also holds promise as an in vivo test system for the detection of low

concentrations of clastogens in the aquatic environment. This is not very surprising, since

the axolotl is morphologically and biologically rather similar to the pleurodele at both the

embryonic and larval stages, although not of the same family.

The third species used was Xenopus laevis. It differs in a number of respects from the

previous two species, including its feeding behavior. Three different variables, temperature,

stage of larval development and frequency of renewal of the test substance, were

investigated using EMS as the clastogenic compound. In addition, a dose-response curve

was established for BaP in order to determine the limits of sensitivity of the test (VAN

HUMMELEN et al., 1989). With BaP, the lowest concentration (0.03 ppm) gave a negative

response. From 0.06 ppm up to 0.5 ppm, an approximately linear increase in median value

of cells with micronuclei was observed. This linear response indicates that the test is

Table II. - Comparison of qualitative results obtained for different chemical

substances and X-rays in the newt Pleurodeles waltl, with various short-term

genotoxicity and long-term rodent carcinogenicity tests (from FERNANDEZ et

al., 1993). Abbreviations for chemical substances: AO, acridine orange; Aro,

Aroclor 1254; AT, atrazine; BaP, benzo(a)pyrene; BrFo, bromoform; BHA,

butylated hydroxyanisole; CAP, e-caprolactam; ClFo, chloroform; CP,

cyclophosphamide monohydrate; DEIA, diethanolamine; DES, diethyl sulfate;

DMSO, dimethyl sulfoxide; ECH, epichlorhydrin; EtOH, ethanol; EB,

ethidium bromide; EMS, ethyl methane sulfonate; DBE, ethylene dibromide;

ENNG, N-ethyl-N'-nitro-N-nitrosoguanidine; ENU, N-ethyl-N-nitrosourea;

GSH, glutathione; HEMPA, hexamethylphosphoramide; InM, indomethacin;

MeC, mercuric chloride; 3-MC, 3-methylcholanthrene; MMeC, methyl

mercuric chloride; MCA, monochloramine; NAT, N-nitrosoatrazine; NDEIA,

N-nitrosodiethanolamine; PB, phenobarbital; Py, pyrene; NaF, sodium

fluoride; NaOCI, sodium hypochlorite; NaNO,, sodium nitrate; NaNO,,

sodium nitrite; STS, sodium thiosulfate; TPA, 12-O-tetradecanoylphorbol-13-

acetate; o-TOL, o-toluidine; TCAH, trichloroacetaldehyde hydrate; BA,

benz(a)anthracene; CAF, caffeine; CAN, Captan; CARB, Carbaryl; COL,

colchicine; DMBA, 7,12-dimethylbenz(a)anthracene; ETI, ethyleneimine; FA,

formaldehyde; N-CARB, N-nitrosocarbaryl. Abbreviations for results: +,

positive; (+), weakly positive; -, negative; ?, inconclusive; L.P, limited

positive; L.N, limited negative; E.E, equivocal evidence; +/-, different results

depending on the authors or on the cell types; A, aneuploidy; P, polyploidy.

Source : MNHN, Paris

GAUTHIER 65

Rodents

Newt Carcinome

MN test

MN test in rodents

+ +

+/P (Hum)

+/-'PIA

a. From UPTON et al. (1984).

b. Percent concordance with newt micronucleus test.

Source : MNHN, Paris

66 ALYTES 14 (2)

reliable, although the lower frequencies of cells with micronuclei at doses above 0.5 ppm

are probably accounted for by a lower rate of growth of the larvae exposed to high doses

of BaP. In fact, above BaP concentrations of 1 ppm the larvae eat less and grow more

slowly than the larvae in the other samples. The mitotic index is thus lower and, since the

production of micronuclei depends on cell division, a fall in the mitotic index results in a

decrease in the number of red blood cells with micronuclei. It is worth noting that a similar

phenomenon occurs for the same concentrations in axolotl and pleurodele with BaP.

Al these results demonstrate the sensitivity and the reliability of the test for known

genotoxic agents experimentally added to the rearing water. It was important to find out

whether the amphibian micronucleus test was also applicable to real-world situations.

GENOTOXICITY IN DRINKING WATER

Using pleurodele larvae, JAYLET et al. (1986c, 1987) evaluated the mutagenic activity

in drinking water taken directly from the tap supplying the laboratory. Groups of larvae

were reared in tap water, while control animals were reared in tap water filtered over sand

and active carbon, to remove micropollutants. Seven separate tests carried out in samples

of tap water taken at different times throughout the year gave positive results of varying

degree depending on the time of the year (see results in Table III and fig. 3). The authors

concluded that this test was therefore able to detect clastogens in normal drinking water

and that it could be used for quality control of drinking water during the various stages

in the treatment of raw water. The aim of these studies was to determine whether the test

was sufficiently sensitive to be used directly on drinking water samples without prior

extraction or concentration of the micropollutants. To our knowledge, this was the first

description of a test system using an aquatic vertebrate for the detection of potential

clastogens in samples of tap water.

To explain the positive results obtained in these previous experiments, various

hypothesis have been formulated. Genotoxic micropollutants in drinking water can come

from a variety of origins. Various possibilities suggest themselves which are not mutually

exclusive. The micropollutants may be (1) residual chlorine from the chlorination processes

used for water disinfection, (2) substances produced by the action of chlorine on organic

matter forming halogenated organic compounds, or (3) even substances present in the raw

water.

(1) In order to examine the first possibility, further experiments were carried out.

GAUTHIER et al. (1989) evaluated mutagenic activity of chlorinated and monochlo-

raminated water devoid of all organic matter on pleurodele larvae. The level of

micronuclei in erythrocytes was compared between a group of larvae reared for 12 days

in chlorinated reconstituted ultrapure water treated with sodium hypochlorite and a

control group reared in just the reconstituted water. Sodium hypochlorite was added when

both the food and medium were changed each day. Chlorine levels of 0.125 and 0.25 ppm

led to significant elevations of micronuclei. The possibility of indirect effects of chlorine

through chemical interactions with the food were also investigated, using the following

scheme: larvae were left for 3 hours in chlorinated reconstituted ultrapure water and then

placed in non-chlorinated water. Food was only introduced when they were transferred to

Source : MNHN, Paris

Table III. - Results of tests on laboratory tap water (expressed as the number of micronucleated cells per thousand) carried out on

pleurodele larvae, over a 8-month period between October 1985 and May 1986 (from JAYLET et al., 1987). +, positive result.

12.10.85 24.10.85 04.12.85 31.01.86 04.03.86 21.03.86 17.05.86

10 24.10.85 to 12.11.85 to 16.12.85 to 19.02.86 to 20.03.86 10 15.04.86 to 23.05.86

Filtered Tap Filtered Tap Filtered Tap Filtered Tap Filtered Tap Filtered Tap Filtered Tap

tapwater water tpwatr water tapwatr Water tpwaer Waler lapwaler Waler tapwater Water tpwater water o

Lower extreme 2 14 4 5 1 5 0 1 Q 4 0 2 0 2 ë

Lower quartile 6.5 20 5.5 14 5 10 2 5 2 10 1 3 4 7 sl

Median 9 31 7 23 7 13 3 6.5 4 12.5 c 4 7 12.5 El

Upper quartile 13.5 45 9° a 11.5 17 4 8 5 19 3 4.5 8 16.5

Upper extreme 21 64 15 105 20 29 7 15 14 36 4 12 12 42

Mean 10.5 343 T4 3141 868 14.1 3.1 12 4.1 14.7 21 43 605 14.05

Results + + + + + + +

Animal number 10 13 15 17 19 19 10 10 20 20 20 20 20 20

Source : MNHN, Paris

68 ALYTES 14 (2)

= 30

2 10!

1 2 3 4 5 6 7

Fig. 3. — Histograms of median values obtained for various tests on laboratory drinking water

samples. In black, tests using drinking water filtered over sand and carbon; in white, tests over

the same period using non-filtered tap water. Error bars indicate 95 % confidence limits. Date

of tests: N° 1, 12.10.85 to 24.10.85; N° 2, 24.10.85 to 12.11.85; N° 3, 04.12.85 to 16.12.85; N°

4, 31.01.86 to 19.02.86; N° 5, 04.03.86 to 20.03.86; N° 6, 21.03.86 to 15.04.86; N° 7, 17.05.86 to

23.05.86 (from JAYLET et al., 1987).

the non-chlorinated water. This procedure was repeated for 12 consecutive days. Control

larvae were reared in non-chlorinated water throughout this period. In this case, results

were also positive when the larvae were exposed for only 3 hours to the chlorine (0.2 ppm

for 12 days) in the absence of food. The same experiment was carried out with

monochloramine instead of sodium hypochlorite. The level of micronuclei increased with

increasing concentration of monochloramine (0.05, 0.1 and 0.15 ppm) although only the

0.15 ppm concentration gave a statistically significant response. The results of these

experiments indicated that free chlorine and monochloramine were responsible for the

clastogen effect observed in newt larvae.

GAUTHIER et al. (1990) studied dechlorination effects of sodium hyposulfite (Na,S,0;)

on the genotoxic potential of chlorinated water with pleurodele larvae. The animals were

reared both in reconstituted water and in reconstituted water containing 0.22, 0.56 and

1.12 ppm of chlorine (sodium hypochlorite). Chlorinated water samples were then fully

dechlorinated by adding increasing concentrations of sodium hyposulfite (respectively 1,

2.5 and 5 ppm). For each test, the dechlorination was controlled by colorimetric dosage

Source : MNHN, Paris

GAUTHIER 69

of the residual chlorine. The same concentrations of sodium hyposulfite have been tested

with the newt micronucleus test. In these experiments, no positive responses were

observed, neither in dechlorination experiments nor in the water containing sodium

hyposulfite alone. The authors concluded that sodium hyposulfite, a substance that is

widely used in water treatment plants to control the concentration of chlorine in drinking

water, was not genotoxic on pleurodele larvae. Moreover, dechlorination of water

chlorinated with hypochlorite amounts, normally leading to clastogenic effects in the newt,

led to the elimination of the genotoxicity.

These results confirmed previous results observed in water treatment plants by

GAUTHIER et al. (1988) showing that pleurodele larvae reared in water samples taken at

different treatment steps generally gave no positive responses after dechlorination with

sodium hyposulfite, whereas positive responses were observed in chlorinated water samples

partially dechlorinated by physical techniques.

Since ozone is also used to disinfect water (ozone has been increasingly used in

Europe in view of its oxidizing, bleaching and deodorizing properties), JAYLET et al.

(19904) carried out other experiments in which pleurodele larvae were reared in river water

containing different concentrations of ozone. After determination of the ozone demand in

water samples of the river Seine, the following ozone concentrations were tested: 1/4 of the

ozone demand in the raw water, 1/2, the overall and twice the ozone demand. The authors

showed that treatment of Seine river water with low concentrations of ozone (correspon-

ding to 1/4 of the ozone demand) led to a genotoxic effect, whereas ozonation at higher

doses had no significant effect on the level of micronuclei induced. They concluded that

ozonation of surface water with low doses of ozone may lead to genotoxic effects which

are abolished by higher doses of ozone, thus confirming other results obtained with in

vitro test systems (VAN Hoor, 1982; BOURBIGOT et al., 1986; KooL & HRUBEC, 1986).

(2) According to our second hypothesis, genotoxic micropollutants may be substances

produced by the action of chlorine on organic matter present in the raw water.

In order to examine this possibility, LECURIEUX et al. (19954) studied the genotoxic

effects of six halogenated acetonitriles identified in chlorinated waters (monochloro-,

dichloro-, trichloro-, monobromo-, dibromo- and bromochloroacetonitrile), with three

short-term assays: the SOS chromotest (QUILLARDET & HOFFNUNG, 1985; MARZIN et al.,

1986; Xu et al., 1989), the Ames fluctuation test (MARON & AMEs , 1983; HUBBARD et al.,

1984) and the amphibian micronucleus test on pleurodele larvae. In this study, clastogenic

effects on peripheral blood erythrocytes of the animals were detected for all the six

haloacetonitriles tested. The authors noted two structure-activity relationships. The

genotoxic activity of haloacetonitriles containing bromine substituants appeared higher

than the corresponding chlorinated acetonitriles and the clactogenic activity of the

chlorinated acetonitriles increased with the number of chlorine substituents.

In the same way, LECURIEUX et al. (1995b) evaluated the genotoxicity of four trihalo-

methanes (chloroform, bromodichloromethane, chlorodibromomethane and bromoform).

The newt micronucleus assay detected a clastogenic effect on peripheral blood erythrocytes

of pleurodele larvae for bromodichloromethane and bromoform. The authors noted that

the presence of bromine substituent(s), generally led to significant genotoxic activity.

Source : MNHN, Paris

70 ALYTES 14 (2)

(3) Our last hypothesis on the origin of the genotoxic effects observed in drinking

water suggested the responsibility of pollutants and/or micropollutants present in the raw

water before treatment. It was important to find out whether the amphibian micronucleus

test could be relevant to in vivo studies using natural waters taken directly from the

aquatic medium.

MONITORING GENOTOXIC POLLUTION IN THE AQUATIC ENVIRONMENT

There are two different origins for water contamination. It may be due either to the

presence of living organisms, viruses, bacteria and parasites, or it may have chemical

origins. Within chemical contaminants, it is useful to distinguish solid particles in

suspension, inorganic soluble and organic compounds, and radioactive substances. The

genetic impact of pollutants corresponds to the overall effect of the various contaminants

which interact within the aquatic environment and are all very unstable. Attempts to get

a definition of the genotoxic load of polluted waters (waste waters, industrial effluents,

etc.), were most often based on short-term bacterial assays, such as the Ames test,

performed either directly in the waste water or after preconcentration of the organics.

These assays performed directly in water often yield negative or ambiguous results

(WEAVER et al., 1981). An additional problem is that loss of genotoxicity may occur when

the sample is filtered for sterilization. Testing of concentrates will limit the scope of a

survey to that part of the organic matter that can be recovered by concentration

techniques. Many of the problems encountered with the in vitro assays may be

circumvented with direct testing in aquatic organisms. In the past ten years, a number of

tests were developed, either with plants or with aquatic animals (see the reviews of:

CHOUROULINKOV & JAYLET, 1989; JAYLET & ZOLL, 1990; JAYLET et al., 1990b; ZoLL. et al.,

1990; Goper et al., 1993), which can potentially be used to assess the genotoxic potency

of a waste water. The advantages are clear: there is no need to concentrate a sample or

to sterilize it, and the tests can be carried out with intact animals, taking into account

uptake and elimination, internal transport and metabolism.

Until now, few attempts have been made to incorporate in vivo genotoxicity assays

in the quality assessment of effluents. A first comparison between different approaches,

both from the technical point of view (sensitivity, practical use) and from the economic

side, has been made by VAN DER GAAG et al. (1990). In order to make a comparison

between several approaches for the assessment of genotoxic agents in wastewater, the

authors characterized an industrial effluent with three different in vivo assays for

genotoxicity, including sister chromatid exchange (SCE) induction in the fish Nothobran-

chius rachowi (VAN DER GAAG & VAN DE KERKHOFF, 1985; VAN DE KERKHOFF & VAN

DER GAAG, 1985), and the formation of micronuclei in the amphibian Pleurodeles waltl

and in the mussel Mytilus edulis (MAJONE et al., 1987; BRUNETTI et al., 1988; SCARPATO et

al., 1990). The same effluent was tested in the Ames test and in the SOS chromotest. The

genotoxicity assays were also performed in XAD-extracts of this effluent, as well as in the

flow-through of the XAD columns. A number of routine chemical parameters have been

determined in the waste water and in the effluents of the filter and of the XAD columns.

Water samples were taken from an effluent of a biological waste water treatment unit

receiving effluents from various petrochemical industries.

Source : MNHN, Paris

GAUTHIER 71

An 80-liter sample of waste water was taken at the discharge point of the biological

waste water treatment plant. Twenty liters were used for direct in vivo testing and 15 liters

were sterilized with gamma rays (25 kGy) for the tests on bacterial genotoxicity assays. To

check the effect of sterilization, part of this sample was assayed with in vivo test systems.

In order to assess the effectivity of methods that concentrate pollutants from the water,

45 liters of the sample were filtered over a 1 um glass fiber filter, which was then passed

over XAD 4, first at ambient pH and subsequently at pH 2 (according to the technique

of Noorpsy et al., 1983). The organics were eluted from the filter and the XAD columns,

and the final samples concentrated in ethanol. Part of the sample was collected after

filtration and XAD pH 7 adsorption for testing in vivo and in vitro. After XAD pH 2

adsorption, the sample was neutralized with NaOH before testing.

Physico-chemical analysis before and after treatment showed that filtration of the

industrial effluent affected neither the concentration of dissolved organic carbon nor the

concentration of the various organohalides measured. Passing the industrial effluent

through the XAD column at pH 7 only retained 25 % of the dissolved organic carbon;

however it did retain the majority of the organohalides. Chromatography of the pH 7

XAD concentrate showed the presence of a large range of compounds which were

lipophilic to moderately hydrophilic. The outflow from the first XAD column at pH 7

contained 75% of the dissolved organic carbon and 50% of the CaOX fraction

(organohalides absorbed on carbon), but almost the entire EOX fraction (extractable

organohalides) was retained by the resin. Adsorption on the second XAD column at pH

2 retained a further 23 % of the dissolved organic carbon of the sample and 30 % of the

CaOX fraction. Analysis by HPLC showed a predominance of moderately hydrophylic

compounds in this fraction. Finally, after the various treatments, the industrial effluent

still contained more than 50 % of the dissolved organic carbon and about 30 % of the

CaOX fraction.

Genotoxic effects were observed in waste water dilutions with the SCE assay with

Nothobranchius rachowi and the micronucleus assays with Pleurodeles waltl and Mytilus

edulis. The increase in SCE frequency and in incidence of micronucleated cells in

pleurodele was still highly significant at the lowest concentration tested of 32 ml/l of

dilution water. In the mussel, a significant increase only occurred at the highest

concentration tested (100 ml/l). Filtration did not affect the in vivo genotoxicity. A

significant amount of the genotoxic effect observed in the SCE assay was present in the

extract from a 1 um glass fiber filter that was placed before the XAD columns. The

genotoxicity in the SCE assay was not, however, altered by this filtration, suggesting that

a part of the potential genotoxic effect of this effluent was not directly biologically

available. XAD adsorption removed a major amount of the mutagenicity. Both in vivo

tests (SCE in the fish and MN in amphibian), however, still detected a significant

genotoxic effect in the flow-through of the XAD pH 2 column. The more hydrophilic part

of the genotoxic activity could amount 15 to 40% of the biologically available

mutagenicity in this sample, according to the estimates from the SCE and micronucleus

assays. Gamma-irradiation sterilization significantly reduced the genotoxic activity of the

waste water in the SCE assay and in the amphibian micronucleus test.

The bacterial mutagenicity assays only detected effects in the different organic

Source : MNHN, Paris

72 ALYTES 14 (2)

concentrates. Direct testing was carried out only in gamma-ray sterilized samples, but

effects were not observed in the Ames test or in the SOS chromotest.

For the authors, this pilot study was a first clear proof that the analytical chemical

techniques used to monitor waste waters only cover a part of the organic components that

can be a potential risk to health: 30 to 50 % of the genotoxic effect present in the dissolved

fraction of this specific waste water was not recovered on XAD. The yield is even lower

if one accounts for the substantial amount of mutagenicity that was found in the filter

extract, a non soluble fraction that was not directly biologically available. Although the

genotoxic substances in this fraction are not directly taken up by aquatic animals, it is

highly probable that these compounds will finally settle in sediments, and become available

for bioconcentration in the long run if they are not degraded. Furthermore, in this study,

the authors demonstrated that sterilization with gamma rays reduced the genotoxic effect

of the waste water sample in both the SCE and micronucleus assay. These results introduce

an uncertain aspect about those of the bacterial assays: the direct tests were carried out

in gamma-ray treated waste water, and did not detect any mutagenicity at all. Considering

the effectivity of in vivo test systems to detect genotoxic compounds in the industrial

effluent studied, the authors concluded that the sensitivity of the in vivo assays was higher

than that of the bacterial tests. The SCE assay and the micronucleus test could be carried

out directly in dilutions of the sample, while a concentrate had to be made of the organics

to assess the effect in the Ames test and the SOS chromotest. Furthermore, the costs and

duration of the in vivo assays, if carried out on a routine basis, are not very different from

those of bacterial assays.

Following this pilot study, GAUTHIER et al. (1993) published the results of a first

environmental study using the micronucleus test on pleurodele larvae to detect the

genotoxicity of waste waters and industrial effluents. In this study, pleurodele larvae were

exposed to various types of industrial waste waters in order to estimate the ability of the

micronucleus test system to reveal the genotoxic potency of polluted waters. The first test

carried out on effluent from wool and leather industries (tannery effluent) showed that the

newt micronucleus test was clearly able to demonstrate the genotoxicity of river water

contaminated by this type of waste. Furthermore, negative results obtained with water

samples taken at the same point and tested at the same concentration after that a large

anti-pollution campaign has been in operation in the tanning and wool industries (waste

reduction and purification) underlined the ability of the newt micronucleus test to play a

potential role in assessing optimization of waste water treatment. A series of experiments

carried out with an oil-refinery waste gave positive responses on the effluent sampled

directly at the outlet, but not on effluent taken 300 m downstream. It could therefore be

assumed that the genotoxic substances contained in the discharge have been diluted too

much to bring about a positive result in the newt micronucleus test. It is also suggested

that antagonistic relationships exist between certain substances contained in the waste

water and substances present in the water receiving the waste (for example binding to

humic matters or suspended solids). Furthermore, on the same effluent, induction of

micronuclei was also observed when the animals were exposed to the samples for 8 days

instead of 12. At the dilution of 250 ml/l, for both test durations, a positive response of

similar amplitude was obtained, demonstrating that shorter test durations may be possible

without substantial changes in the response. In the same study, other genotoxicity tests

Source : MNHN, Paris

GAUTHIER 73

carried out directly on a Dutch effluent taken in the river Rhine and on XAD extracts of

the industrial effluent, also gave a direct demonstration of the genotoxic activity of waste

from the petrochemical industry. The authors concluded that the experiments carried out

with the newt micronucleus assay on industrial effluent of various origins underlined the

ability of this method to detect the anthropogenic genotoxicity of polluted natural waters

and to evaluate directly the impact of aquatic contamination on the exposed ecosystems.

The simplicity of the test could allow its use in routine monitoring of, for example, the

contamination of a river and in the evaluation of programs intended to reduce pollution.

The newt micronucleus test was therefore considered as a welcome additional tool to help

in decision making by the organizations responsible for supervising the discharge of waste

into watercourses.

More recently, GODET (1994) compared the genotoxic potential of 8 effluents, using

the Ames test and the newt micronucleus assay on pleurodele larvae. He obtained positive

responses with 6 industrial effluents from various origins (metallurgy, chemistry) on

pleurodele larvae and with the Ames test applied to metallic waste samples and to organic

extracts of a paper mill effluent. Trying to identify genotoxic pollutants responsible of the

positive responses observed with the newt micronucleus assay in industrial effluents

containing Fe, Cr II, Cr VI, and Zn, the author focused on the study of these metallic

constituants on pleurodele larvae. Thus, Fe concentrations of 0.6 and 12.5 mg/l led to

positive responses, whereas concentrations of 2.5 and 1 mg/l for Cr III and Cr VI

respectively led only occasionally to positive responses. Previous genotoxic effects

observed on pleurodele larvae with industrial effluents containing different mixtures of

metallic pollutants were explained, considering the different types of effluents tested, either

by the predominent effect of one of the metallic pollutants (Fe for example), or by the

combined action of two of the metallic ions present in the effluent (Fe III and Cr VI). In

conclusion, the author suggested the use of the sensitive newt micronucleus assay to reveal

the possible synergistic effects of pollutants mixtures in water. He also proposed a new

strategy to study the genotoxic potential of effluents, combining the Ames test and the

amphibian micronucleus assay on pleurodele larvae, and suggested to modify the test

procedure to optimize the detection of genotoxic effects in effluents and to help the

development of this assay in a routine use.

In the current use of the amphibian micronucleus assay to evaluate genotoxic impact

of waste water discharged in the environment, other types of effluents have been recently

studied by GAUTHIER (1996). In the framework of an environmental study on the impact

of roadway runfall effluents, the genotoxicity of 6 runfall samples has been eval-

uated during 6 different rainfall periods over a year, on pleurodele larvae. The author

observed positive responses in animals reared in 3 different effluents taken from the

sampling site (a water retention tank receiving runfall effluents of a characterized roadway

section). For each positive effluent, dose-effect responses have been obtained. Physico-

chemical analysis of the water samples have been carried out simultaneously to the

genotoxicity assays, in order to try to correlate the genotoxic effects observed with the

presence of some chemical substances. Even if it is not possible to conclude definitely

from the data obtained, part of the genotoxic effects observed could probably be

attributed to the high level of polycyclic aromatic hydrocarbons measured in the 3 positive

effluents (from 84 to 188 ng/l compared to < 30 ng/l in the non-genotoxic water samples).

Source : MNHN, Paris

74 ALYTES 14 (2)

AIl the environmental studies reported previously have been carried out on pleurodele

larvae. Until now, few studies of environmental interest have been devoted to Xenopus

laevis, in spite of its rearing advantages and ease of use.

In 1987, LEHMAN & MILTENBURGER proposed the use of Xenopus larvae to study

cytogenetic effects of toxicants in water. Micronucleus inducing effects of a waste water

were analysed in poly- and monochromatic erythrocytes, with regard to exposure time.

One of the strongest effects was observed in larvae exposed to the waste water during their

entire larval development (3 months). Although these exposure conditions were incompat-

ible with a routine use of a Xenopus micronucleus assay, these results were the first, from

our knowledge, to report the use of platanna larvae for monitoring the genotoxic potential

of a waste water.

In our laboratory, ZoLL-MOREUX (1995) developed a test procedure on Xenopus

larvae inspired from the protocol of the newt micronucleus assay, that can be applied to

environmental complex mixtures. This author compared the results obtained with the

amphibian micronucleus test, using pleurodele and platanna larvae, on 5 chemicals of

environmental interest and on 5 waste waters from various origins (urban waste and

different industrial effluents). Four of the 5 tested effluents were found genotoxic with the

Xenopus micronucleus test, whereas only 3 were genotoxic in the newt micronucleus assay.

Both organisms are considered quite equally sensitive (with a small advantage to Xenopus

larvae) and the test procedures are similar. In conclusion, the author suggested that, even

if it may be presumptuous to substitute the platanna for the pleurodele micronucleus assay

in environmental genotoxicity studies (because of the lack of data), the undeniable

advantages of the frog provide a good prospect for the use of this animal in future

evaluation of genotoxic effects in the aquatic environment.

MECHANISMS INVOLVED IN THE FORMATION OF MICRONUCLEI IN AMPHIBIANS

Parallel to the in vivo detection of genotoxic effects observed with pure substances in

water or with complex mixtures, several studies have been carried out, in our laboratory,

to help the comprehension of the mechanisms of action involved in the formation of the

micronucleated red blood cells observed in pleurodele larvae.

In 1987, FERNANDEZ & JAYLET demonstrated the antioxidant protection of

the 2(3)-tert-butyl(4)hydroxyanisole (BHA), currently used as a food additive (E 320),

against the clastogenic effects of BaP in pleurodele larvae. BHA was added to the

water at concentrations of 0.5, 1 and 1.5 ppm. It reduced the clastogenic effects of BaP

in the test larvae, with the most marked effect at a concentration of 0.5 ppm. In order to

explain the results observed, the authors assumed that in the newt BHA influences various

stages in the metabolic transformation and/or detoxication mechanisms of BaP. So, it was

likely that metabolites of BaP and secondary reaction products (e.g. oxygenated free

radicals) were responsible for the chromosome damage, leading to the appearance of

micronuclei. BHA could thus act by attenuating the formation of these reactive

intermediates or perhaps preventing them reaching their target(s) (DNA or mitotic

apparatus).

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GAUTHIER 75

Two years later, MARTY et al. (1989) investigated the effects of the indirect acting

mutagen BaP, involving the action of hepatic cytochrome P 450 dependent monooxygen-

ases, to explain the mechanisms controlling the formation of the active metabolites in newt

larvae. The authors demonstrated that the pleurodele is capable of metabolizing BaP

into hydroxylated products in a manner not so different from mammals or other aquatic

organisms, of conjugating them with molecules implicated in the formation of polar

derivatives to achieve their excretion and to respond to inducers of the

3-methylcholanthrene (3-MC) type. However, despite the metabolizing capacity in the

pleurodele, accumulation of active metabolites of BaP can occur and is suggested as the

main cause of the clastogenic effect of the compound.

MarTy et al. (1992), working on the enzymatic activity of cytochrome P 450 forms

induced in pleurodele after pretreatment by 3-MC or phenobarbital, showed that the newt

is characterized by a lower level of hepatic cytochrome P 450-dependent activity than the

rat. Variations of enzymatic activities according to sex and season were observed. Specific

activities in newt were characterized by an almost complete insensitivity to induction by

phenobarbital pretreatment. On the other hand, pretreatment by 3-MC resulted in an

increase in the metabolism of several hydroxycoumarin and resorufin derivatives, similar

to the effects observed in rat liver.

DISCUSSION

WHICH SPECIES IS THE BEST SUITED?

From the results presented above, it is possible to compare the performances of the

different amphibian species used in the micronucleus assay to detect genotoxic substances

in water. For the three species, the optimal treatment duration is quite similar and rearing

the breeders does not present any particular problem. Urodele larvae eat exclusively live

prey, whereas Xenopus tadpoles can be fed on dehydrated aquarium fish food. The interval

between egg laying and testing is 6 weeks for urodeles and only 2 weeks for platanna,

giving a technical advantage to the latter species. However, recording micronucleated cells

is somewhat easier in urodeles than in platanna, since red blood cells and micronuclei are

larger in the former ones. Nevertheless, recording micronuclei for all three species is more

straightforward than in the rodent micronucleus test. Pleurodele and Xenopus tests differ

in sensitivity depending on the compound considered. For example, for cyclophosphamide

the detection threshold is 0.5 ppm in pleurodele (FERNANDEZ et al., 1989) and 5 ppm in

platanna. Similarly, for BaP the test is positive with 0.025 ppm in pleurodele (FERNANDEZ

et al., 1989) and 0.06 ppm for platanna (VAN HUMMELEN et al., 1989). Conversely the

detection threshold for methyl mercury is 2.5 ppb in platanna and 12 ppb in pleurodele

(ZoL et al., 1988).

AIl three species can demonstrate the genotoxicity of both direct and indirect

mutagens (i.e. those requiring metabolic activation). Among the three species used for the

test, it is not currently possible to state which is best suited for detection of genotoxic

Source : MNHN, Paris

76 ALYTES 14 (2)

substances in water. Nevertheless, recent work conducted by ZoLL-MoREUX (1995) with

platanna on industrial effluents, together with the easy rearing conditions of the larvae,

promise a future development of this amphibian to study the genotoxic activity of natural

waters.

AMPHIBIANS: A SENSITIVE MODEL?

Al our previous investigations on pure substances added to the rearing medium of the

larvae have shown that the micronucleus test in amphibian is a sensitive and valuable

model for the detection of genotoxic compounds in water. Various factors contribute to

the high sensitivity of our biological model. The larvae strongly accumulate pollutants

from the surrounding medium. This has been well demonstrated for compounds such as

methyl mercury (ZoLL et al., 1988) and BaP (GRINFELD et al., 1986; MARTY et al., 1989).

Studies carried out with various waste waters taken directly at industrial or urban sites

confirm the ability of the amphibian micronucleus assay to reveal the presence of

genotoxic substances discharged in the aquatic environment, even after dilution. In most

cases studied, dose-related responses of the induction of micronucleated erythrocytes were

observed. Furthermore, induction of micronuclei was also observed with test durations

shorter than 12 days, without substantial changes in the response (GAUTHIER et al., 1993).

The sensitivity of the method allows direct testing of water samples without the

requirement for extraction or concentration of the micropollutants (techniques required

for most in vitro studies) leading to the preparation of extracts which are not

representative of the initial water samples (VAN DER GAAG et al., 1990). Because of the

high sensitivity of our test system, genotoxic potential of drinking water has been detected.

Water samples taken directly from the tap or taken at different treatment steps in water

treatment plants were found genotoxic, whereas other test systems probably would not

have revealed any mutagenic activity.

Presently, the amphibian micronucleus assay using pleurodele larvae is the only test

system dedicated to an aquatic vertebrate and sufficiently sensitive to detect the genotoxic

potential in drinking water samples.

IN VIVO AGAINST IN VITRO TEST-SYSTEMS?

We have shown (VAN DER GaAAG et al., 1990) that the techniques of sterilization,

extraction and concentration of organic micropollutants in water required for in vitro

studies lead to the preparation of extracts which are not representative of the initial water

sample. Moreover, the proportion of the genotoxicity arising from chemical pollutants

transformed or neoformed during extraction, compared to that of the original sample,

cannot be evaluated. These problems can be circumvented by the use of in vivo tests giving

a direct evaluation of the genotoxicity of dilute effluents without the requirement for

extraction or concentration of the micropollutants. Apart from this undeniable advantage,

these in vivo tests present others which are also of considerable interest. Firstly, they use

aquatic species which are exposed directly to the water to be tested. Also the species used

are phylogenetically closer to man than are bacteria. Finally, the chromosomal damage is

Source : MNHN, Paris

GAUTHIER TT

evaluated in whole organisms and thus takes into account any interaction that might occur

between micropollutants as well as phenomena of bioavailability and the overall

metabolism of the animal. The responses obtained not only express the complex

phenomena taking place in the whole organism, but also the interactions between the

organism and its environment. Moreover, in vitro tests are not suited for detection of

indirectly acting mutagens. To get around this problem, metabolic-activating systems are

added to the bacterial culture. These are generally in the form of a microsomal preparation

from rat liver, but can lead to problems in the interpretation of the results.

Nevertheless, the use of in vitro assays (tissue culture systems and bacterial tests)

presents some other advantages. They are generally less time-consuming and expensive

than the in vivo tests. They represent suitable early warning systems, although they cannot

supplant the in vivo methods for more accurate evaluation of risks. Moreover, they score

complementary endpoints of those of in vivo test systems. So, it is more and more

proposed to associate in vitro and in vivo detection in a “test battery”, able to reveal the

various types of mutations (genic, chromosomal and genomic), to assess the majority of

the genotoxic fraction in the water samples studied. For example, integration of the

amphibian micronucleus test in a test battery for quality control of the water would help

to the evaluation of risks to human health, as well as to the protection of aquatic

ecosystems. The most commonly used tests, at present, are the in vitro test systems using

bacteria, as they are both cheap and quick, and the in vivo test systems like the mouse

micronucleus test, as it is well developed worldwide. However, this latter is not well

adapted to aquatic environmental studies and was found less sensitive than the newt

micronucleus assay for the detection of some carcinogens in water (LECURIEUX et al.,

1992). In many cases, the risk from genotoxic agents in water is a hidden one. Although

mutagenic micropollutants may not be lethal, they may alter the natural equilibrium in

subtle ways or even sterilize fragile species. An enhancement in the rate of mutation may

also accelerate the process of evolution, leading to a domination of certain species to the

detriment of others, although here the precise risk is hard to estimate. In any event,

extrapolation of the tests’ results to the real-word situation represents an exercise in

judgment.

FUTURE DEVELOPMENT AREAS

Presently, one of the research axes developed in our laboratory deals with

fundamental preoccupations. Recent research (unpublished results) has shown the

possibility to detect aneugenic effects of some genotoxic substances using the amphibian

micronucleus assay. Knowing the close relations between aneuploidy and the initiation of

cancers, it seems important to be able to detect the presence of these specific genotoxic

substances in the aquatic environment. It is clear that the future development of the

amphibian micronucleus test will find its way in environmental research. Up to now, the

pilot studies carried out with this test system on waste waters of various origins underline

the ability of this method to detect the anthropogenic genotoxicity of polluted natural

waters and to evaluate directly the impact of aquatic contamination on the ecosystems

exposed. The simplicity of the test could allow its use in routine monitoring of, for

example, the contamination of a river and in the evaluation of programs of installations

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78 ALYTES 14 (2)

intended to reduce pollution. However, in its current form, this in vivo test system remains

a relatively heavy and costly technique, limiting its general diffusion in spite of its ease of

use. For this reason, it seems now necessary to improve the feasibility of the test by

shortening the exposure time of the larvae and helping the scoring of the micronucleated

cells in analyzing the blood smears with a computer. We are therefore trying to develop

in our laboratory an automated method of scoring the micronucleated red blood cells with

a computerized imaging system. Another line of research consists in the extension of the

application field of the amphibian micronucleus assay. Up to now, only the aquatic

compartment has been evaluated using this method. However, it is well known that

genotoxic pollutants, even if they transit in the aquatic compartment, often find their way

in soils or sediments. For instance, the results obtained by VAN DER GAAG et al. (1990)

indicate that part of the genotoxicity in natural water samples was absorbed onto

particulate matter and would only become available to biota during intensive contact, such

as may occur in sediment. In this respect, the genotoxicity may have a serious toxicological

relevance in sedimentation areas. To explore the genotoxicity of contaminated sediments

or soils, using our laboratory test system, new approaches have to be perfected (new

exposure conditions, lixiviation of soil samples).

AMPHIBIANS: WELCOME GENOTOXICITY BIOINDICATORS?

Finally, all the experiments carried out over the last decade on the induction of

micronuclei in the red blood cells of pleurodele, axolotl and platanna larvae confirm our

initial choice of these animals as efficient laboratory detectors of the hidden risks in the

aquatic environment. Up to now, few studies have reported the use of amphibians as real

in situ bioindicators of the genotoxic potential of natural waters. Some trials have been

undertaken on other aquatic vertebrates, like fishes, representing a potential material for

the setting of the micronucleus test, but at the present time the fish micronucleus assay

does not seem capable of constituting a routine test to study the genotoxicity of water

(HOOFTMAN & VINK, 1981; HOOFTMAN & DE RAAT, 1982; CARRASCO et al., 1990).

Programs designed to protect the environment require methods for evaluating the

genotoxic hazards to which the various populations constituting aquatic ecosystems are

exposed. Since such hazards are water-borne, laboratory tests need to be developed that

assess directly the genotoxicity of water, effluents and substances or preparations to

aquatic organisms. However, the in vivo tests on aquatic organisms are not yet widely

accepted, they require more backing to play a major role. They can already be useful at

this time, for instance by including them in Toxicity Reduction Evaluation (TRE) such as

they are advised or prescribed already by different governmental agencies (ANONYMOUS,

1989, 1990; VAN DER GAAG, 1991).

DECLINING AMPHIBIAN POPULATIONS: A WAY OF EXPLANATION?

The populations of many amphibian species, in various habitats worldwide, appear to

be in severe decline (Wake et al., 1991). There is no known single cause for the declines,

but their widespread distribution suggests involvement of both global and local agents

(BLAUSTEIN et al., 1994b; Dugois, 1994), among which habitat destruction, conversion of

Source : MNHN, Paris

GAUTHIER 79

agricultural lands from traditional uses, introduction of predators and competitors,

pollution from pesticides (BERRILL et al., 1993, 1994), mining and logging, acid

precipitation (PouGH & WiLsoN, 1977; TOME & PouGH, 1982; Cook, 1983), increased levels

of ultraviolet irradiation (BLAUSTEIN et al., 1994a), consumption by humans and global

climate change.

In any way, the increasing presence of genotoxic agents in many natural aquatic

media could be considered as a factor contributing to the decline of amphibian

populations. Indeed, the mutagenic risk may affect any cell in the organism. Mutation in

a somatic cell may trigger a process leading to carcinogenesis. Mutagenic agents also exert

their action on germ cells. If the toxicity is severe and many cells are affected, there may

be a lowered or even temporary loss in fertility. If a gamete with a genetic anomaly

contributes to the formation of a zygote, the disorder created in the hereditary material

may be serious enough to lead to the death of the embryo. Although a genetic anomaly

that is compatible with survival of the organism frequently leads to an immediately

obvious anomaly in the phenotype, the effect may be differed and only become apparent

in future generations. This is often the case with equilibrated chromosomal rearrangements

and recessive mutations.

Until now, few studies have been carried out to monitor the behavior of our three

test-organisms after exposure. GRIENFELD et al. (1986) observed an increase in frequency

of micronuclei in pleurodeles larvae 6 days after the beginning of exposure (12 to 48 hours)

to BaP (0.5 ppm) and a progressive decrease of the micronucleated cells from day 6 to day

12. The decrease could be interpreted as the result of several phenomena: the progressive

dilution of the micronucleated erythrocytes with normal cells, the destruction of the

micronucleated cells or of the micronuclei in the damaged cells. What about the real

impact of exposure to genotoxic substances in amphibian erythrocytes with other exposure

times, in other cellular types, especially those involved in the reproduction, in the whole

organism and on the population itself?

At this time, there is no clear evidence to answer these questions on our

test-organisms, because of the lack of data. Most of the work remains to be done and will

constitute the challenge for batrachologists and toxicologists over the next decades, as

expressed by WakE (1991): ‘“‘Amphibians may serve usefully as bioindicators, organisms

that convey information on the state of health of environments. How to read the message

and what to do about it, are timely challenges to scientists and to the public.”

RÉSUMÉ

Cet article constitue une revue des travaux réalisés depuis une dizaine d’années sur la

détection du pouvoir génotoxique des milieux aquatiques (eaux douces) à l’aide de larves

d'amphibiens. Trois espèces ont été plus particulièrement utilisées pour réaliser ces études.

Il s’agit de deux urodèles, le pleurodèle (Pleurodeles waltl) et l'axolotl (Ambystoma

mexicanum), ainsi que d’un anoure, le xénope d’Afrique du Sud (Xenopus laevis). La

technique mise au point sur les larves de ces trois espèces d'amphibiens au Centre de

Biologie du Développement de Toulouse, France, permet de révéler directement la

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80 ALYTES 14 (2)

présence de substances génotoxiques dans l’eau d’élevage des animaux en mesurant le

niveau d’induction de micronoyaux dans les érythrocytes des larves exposées à des

substances pures, à des agents physiques ou à des mélanges complexes de substances, tels

que des eaux de boisson, des eaux de surface, des rejets industriels et domestiques, ou tout

effluent aqueux.

Après une présentation du protocole des essais et des conditions d’élevage des

animaux, les résultats obtenus avec le test des micronoyaux sur les larves d'amphibiens

sont rapportés en respectant le déroulement chronologique des différents événements qui

ont abouti, d’une part à la publication de l'essai sous sa forme normalisée en 1992, et

d'autre part aux orientations actuelles des recherches menées dans le domaine de

l’écotoxicologie génétique in vivo des milieux aquatiques, où les amphibiens, grâce

à l’ensemble des travaux rapportés dans le présent article, tiennent une place prépon-

dérante.

Le choix des orientations de travail futures est ensuite discuté en termes de validité de

l'utilisation des amphibiens comme bioindicateurs de génotoxicité en milieu aquatique,

considérant les nombreux avantages de ce modèle et sa complémentarité avec les tests in

vitro qui sont les plus utilisés dans ce domaine.

ACKNOWLEDGEMENTS

This paper is dedicated to the memory of Professor André JAYLET, who was the first to carry out

and to develop the amphibian micronucleus assay. Actually, the newt micronucleus test is also named

the Jaylet test. I would like to thank Dr. V. FERRIER, manager of the group of Environmental Genetic

Toxicology in the Developmental Biology Center of Toulouse, where most of the work described in

this article has been performed. I am very greatful to all my coworkers in the laboratory who designed

and developed the amphibian micronucleus test system over the years, and namely Ms. M.

FERNANDEZ, J. MARTY, C. ZoLL-MOREUX and Mrs. J. E. DJOMO and J. L'HARIDON. I am also greatly

indebted to Mr. Y. LÉvI and Mr. M. A. VAN DER GAAG for their help in field work.

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Source : MNHN, Paris

Alytes, 1996, 14 (2): 85-100. 85

Morfologia de los oocitos en diplotene

de Melanophryniscus stelzneri

(Weyenbergh, 1875) (Anura, Bufonidae)

Maria Fernanda RocA & Dinorah Diana ECHEVERRIA

Facultad de Ciencias Exactas y Naturales (UBA), Departamento de Cencias Biolôgicas,

Laboratorio de Vertebrados, 1428 Buenos Aires, Argentina

Five stages of the oogenesis, with eight substages of the diplotene oocyte

development, are identified applying histological and transmission electron

microscopy techniques. Based on the internal morphology of the oocytes, the

following features were taken into account: yolk vesicles, yolk platelets,

cortical granules and pigment appearance, morphological variations of the

nuclear envelope and nucleoli.

INTRODUCCION

La distribuciôn geogräfica de las especies del género Melanophryniscus Gallardo, 1961

se halla restringida a Sudamérica. Cri (1980) mencioné la existencia de cuatro subespecies

de M. stelzneri, de las cuales M. s. stelzneri habita en las sierras de Cérdoba y San Luis.

Es frecuente hallar ejemplares de M. s. stelzneri activos durante el dia, luego de fuertes

Iluvias, cerca de arroyos o pequeños cuerpos de agua de zonas serranas, entre los 900 y

2.000 m. La época de reproducciôn de la especie abarca desde mediados de octubre a fines

del verano (GALLARDO, 1987).

Debido a lo restringido de su distribuciôn y a que estos animales son comercializados

como mascotas, se hallan entre las especies que la IUCN (Uniôn Internacional para la

Conservaciôn de la Naturaleza) y la FVSA (Fundacion Vida Silvestre Argentina)

coinciden en Ilamar raras (especies sujetas a riesgo) y comercialmente amenazadas,

respectivamente. La extracciôn de estos animales de su medio ambiente, Ilevada a cabo en

forma indiscriminada, podria afectar, por ejemplo, la abundancia y distribucion de las

poblaciones de M. s. stelzneri en los ambientes serranos.

El estudio de la oogénesis en los anuros ha sido objeto de numerosas investigaciones,

que en su mayoria han destacado la variacién morfolgica de los oocitos en el ovario

adulto durante el periodo reproductivo de la especie. La caracterizaciôn histolégica de los

oocitos del ovario se ha efectuado en diversas especies. Se pueden mencionar, por ejemplo,

los trabajos de BOUIN (1901) en Rana sylvatica, KING (1908) en Bufo lentiginosus, ALLENDE

(1938), VALDEZ TOLEDO & PisAN (1980) y ECHEVERRIA (1988) en Bufo arenarum, KEMP

Source : MNHN, Paris

86 ALYTES 14 (2)

(1953) en Rana pipiens, IWASAWA (1969) en Rana ornativentris, DUMONT (1972) en Xenopus

laevis, LAMOTTE et al. (1973) en Nectophrynoides occidentalis, y IWASAWA et al. (1987) en

Rana nigromaculata.

El proceso mâs evidente de la maduracion de los oocitos es la vitelogénesis. Se han

propuesto distintos modelos para explicar la formaciôn del vitelo. Tanto WALLACE (1985)

como DUMONT (1972) han expresado que el principal proceso de adquisicin de vitelo en

los oocitos de Xenopus laevis es la micropinocitosis. OPRESKO et al. (1980) propusieron un

modelo de compartimentalizaciôn endocitica de las proteinas en oocitos de X. laevis. En

varias especies de anfibios se ha descrito la formacion de inclusiones cristalinas dentro de

las mitocondrias en oocitos que fueron interpretadas como progenitoras de los cristales

formados dentro de las plaquetas (WARD, 1962; KARASAKI, 1963; WALLACE & KARASAKI,

1963; LANZAVECCHIA, 1968; WALLACE & DUMONT, 1968; MASssOvER, 1971; WALLACE et al.,

1972). Años después, WaRD et al. (1985) concluyeron que las formaciones cristalinas no

eran precursoras del vitelo. En oocitos jovenes se ha observado la presencia de cuerpos

multivesiculares (LANZAVECCHIA, 1968) que han sido relacionados con la sintesis endégena

de vitelo (WALLA£ÇE, 1985). VILLECCO et al. (1992) han observado en Ceratophrys cranwelli

la formaciôn de cuerpos multivesiculares como precursores de las plaquetas vitelinas, que

se originan de la fusién de endosomas.

Es una caracteristica sobresaliente de los oocitos de anfibios la presencia de una gran

cantidad de nucléolos en ciertas etapas del crecimiento. Estos nucléolos son formados a

partir de genes del ADN ribosomal, que se hallan amplificados en los oocitos de anfibios

(BROWN & DawiD, 1968; AMALDI et al., 1973; SCHEER & DABAWALLE, 1985).

En Rana temporaria y Bufo arenarum, al cabo de tres años los oocitos finalizan su

desarrollo y la hembra alcanza la madurez sexual (GRANT, 1953; ECHEVERRIA, 1988).

VALDEZ TOLEDO & PisanN6 (1980) han convenido en clasificar las fases de la oogénesis de

Bufo arenarum en cinco estadios. Se pueden distinguir cuatro estadios inmaduros

(previtelogénesis, vitelogénesis temprana, vitelogénesis tardia y oocito en auxocitosis) que

conducen al estado final postvitelogénico de oocito ovulable (ECHEVERRIA, 1988).

El propésito de este trabajo radica en caracterizar los estadios de la oogénesis en

M. s. stelzneri como primeros pasos hacia el conocimiento de la biologia reproductiva de

la especie en cuestiôn que, sumado a otras investigaciones, permitirian implementar una

buena y eficiente estrategia de conservacion de M. s. stelzneri.

MATERIAL Y MÉTODOS

Se capturaron 17 hembras de Melanophryniscus stelzneri en las localidades de Tanti,

provincia de Cordoba y El Trapiche, provincia de San Luis, en los meses de octubre,

diciembre y febrero.

Los animales fueron adormecidos con éter y luego colocados con el abdomen abierto

en distintos fijadores.

La muestra const de trozos de ovario de aproximadamente 5 mm x 5 mm de

espesor y de oocitos aislados de cada una de las hembras.

Source : MNHN, Paris

RocA & ECHEVERRIA 87

A fin de conocer el aspecto general de los oocitos se realizé el examen con

microscopio estereoscopico. Se implementaron dos técnicas complementarias.

TÉCNICAS HISTOLOGICAS GENERALES (SEGÜN ECHEVERRIA, 1988)

— Liquidos fijadores: formol al 10 %; solucion de Bouin; liquido de Zenker.

— Inclusién: en parafina 56-58°.

— Técnicas de coloracién e histoquimicas (DRURY & WALLINGTON, 1967; PEARSE,

1972): hematoxilina - eosina; tricromico de Masson; coloracion de Mann; reaccion del

äcido peryédico Schiff (PAS) - hematoxilina; PAS - azul Alciän (pH 3,5); PAS - diastasa;

verde de metilo-pironina.

TÉCNICAS DE MICROSCOPIA ELECTRÔNICA DE TRANSMISION

Fijaciôn con glutaraldehido 3 % en buffer cacodilato de sodio 0,1 M (pH 7,2-7,4);

refijacion con tetréxido de osmio 2 %; coloraciôn en bloque con acetato de uranilo 2 %;

deshidrataciôn en alcoholes de graduaciôn ascendente, con liquido intermediario 6xido de

propileno; impregnaciôn e inclusién en Polybed.

Se realizaron cortes de 500 À que se recogieron en grillas y se colorearon

sucesivamente con solucion de Reynolds (acetato de uranilo al 2 %, citrato de plomo)

segün REYNOLDS (1963, fide MERCER & BIRBECK, 1979), y nitrato de bismuto en soluciôn

de tartrato de sodio.

Se han establecido los estadios de desarrollo de los oocitos segün ECHEVERRIA (1988),

teniendo en cuenta diferentes caracteristicas anatomicas e histolégicas que se mencionan

en la Tabla I.

Los estadios de la oogénesis se identificaron a partir de las observaciones de los

oocitos desprovistos de la teca externa y de la serosa, y se dividieron en subestadios para

facilitar las observaciones del progreso de la oogénesis.

RESULTADOS

ESTADIO 1. PREVITELOGÉNESIS

Son los oocitos de tamaño mäs pequeño (subtipo a), entre 22 y 50 um de diametro.

Se ubican por fuera de la teca externa, cubiertos por la serosa que envuelve al ovario.

Presentan un citoplasma transparente a través del cual se observa el nücleo ocupando gran

parte de la célula. El contenido citoplasmätico, de apariencia granular, es levemente

basofilo. El nücleo es esférico y central, con la membrana lisa. En la periferia del nücleo

se disponen prominentes nucléolos esferoidales y de contorno irregular, en un nümero que

Source : MNHN, Paris

Tabla 1. - Caracteres morfolégicos y métricos para la identificaciôn de los estadios del desarrollo de los oocitos de Melanophryniscus stezneri en cortes

histolégicos. A, anulares; B, bipartitos; C, central; E, esferoidales; G, plegada; L, lisa; O, ondulada; $, saculada; P, polarizado; V, vacuolados:

+, presencia; #, escasos y aislados; *, ordenados; -, ausencia.

Nücleo Citoplasma

Diémetro Membrana

inimo- M Gränul teli

En Posiciôn embrana | Nniécios Vitelo Pigmento | dr

méximo k

nuclear corticales

22-50 c L E : 2 = _ a

50-150 c L E a à : : Ce

150-270 c o E 3 . ; ”. si

a 270-310 c s B u 5 : + 5

F ê

b 310-350 ce s B + . ; + 8

a 350-650 c A,B,E + +4 +

3 b 650-740 c s ByE + + +4 &

e 750-900 P leve s ByV + P+ +4 +

4 900-1200 P G EyV P+ P+ ++ + |

5 1200-1400 P G EyV + + ++ + |

Source : MNHN, Paris

ROCA & ECHEVERRIA 89

varia entre cinco y diez. El nucleoplasma presenta densas granulaciones intensamente

coloreadas que permiten asociarlas con el estado de diplotene temprana de la profase de

la meiosis (fig. 1). Rodeando a cada oocito, en contacto con la membrana celular se

disponen las células foliculares de nücleo prominente.

A partir de los 50 um de diämetro los oocitos se hallan rodeados por las tecas. En los

oocitos cuyos diâmetros estän comprendidos entre los 50 y 150 im (subtipo b), la basofilia

del citoplasma se ve incrementada notablemente. El nücleo presenta en la zona periférica

una mayor cantidad de nucléolos. A medida que el diämetro celular aumenta, los

nucléolos tienden a disminuir el volumen. La membrana nuclear manifiesta leves

ondulaciones. Ocupando todo el volumen del nücleo se observan los cromosomas en

diplotene temprano (fig. 2). Aumentan las células foliculares alrededor de los oocitos.

Los oocitos que presentan un diämetro comprendido entre los 150 y 270 um (subtipo

c), se observan levemente opacos. En los cortes histolôgicos el citoplasma evidencia zonas

densas, continuas o irregulares alrededor de nücleo, con gran afinidad por los colorantes

bâsicos, que denominamos zonas basôfilas del citoplasma. El nücleo conserva la posiciôn

central. La membrana nuclear presenta ondulaciones que se hacen mâs notorias a medida

que aumenta el tamaño del oocito. En la zona cortical del nücleo es evidente el aumento

del nümero de los nucléolos esferoidales. Los cromosomas que pierden su afinidad por los

colorantes bäsicos pueden observarse en el ârea central del nücleo. La membrana vitelina

comienza a visualizarse como una delgada y discontinua envoltura PAS positiva rodeando

a los oocitos, por debajo de las células foliculares.

ESTADIO 2. VITELOGÉNESIS TEMPRANA

Los oocitos que inician el proceso de vitelogénesis y cuyos diämetros estän

comprendidos entre 270 y 310 um (subtipo a) se caracterizan por un color blanco opaco.

El citoplasma exhibe vesciculas en la zona periférica, que dan reaccién PAS positiva, y

contactan con la membrana celular (figs. 3-4). Zonas basôfilas se disgregan alrededor del

nücleo. La vesicula germinal manifiesta una membrana excesivamente saculada que se

proyecta hacia el citoplasma. Los nucléolos se disponen ocupando la mayor parte del

nücleo. Son evidentes nucléolos bipartitos, constituidos por dos zonas de distinta afinidad

por el colorante. Las zonas menos densas pueden repetirse en la estructura del mismo

nucléolo y disponerse alternadamente en forma lineal o como brotes sobre la esfera mâs

densa, generalmente de mayor volümen (fig. 5). La membrana vitelina continüa su

desarrollo envolviendo totalmente al oocito.

Los oocitos de color ocre claro con un diâmetro que oscila entre los 310 y 350 um

conforman el subtipo b. Las observaciones histolôgicas ponen de manifiesto la presencia

de vitelo de apariencia granular en la zona cortical del oocito, que se destaca del resto del

citoplasma por su gran afinidad por los colorantes âcidos (fig. 6). Se ha estudiado con

MET esta zona del oocito, pudiéndose identificar pequeñas plaquetas vitelinas y vesiculas

cuyo contenido central es de forma irregular (premelanosomas, segûn DUMONT, 1972),

dispersas en el citoplasma. La membrana oocitaria manifiesta un gran desarrollo de

microvellosidades que se proyectan hacia la membrana vitelina.

Source : MNHN, Paris

90 ALYTES 14 (2)

Fig. 1. — Oocito de estadio la. S, serosa; T, teca interna; Te, teca externa. Tricromico de Masson.

Escala: 20 um.

Fig. 2. — Vista general de oocito de estadio 1h. F, nücleo de célula folicular. Tricrémico de Masson.

Escala: 30 pm.

Fig. 3. — Vista general de oocito de estadio 24. Triängulos blancos, citoplasma periférico. B, zonas

basôfilas. Tricromico de Masson. Escala: 100 um.

Fig. 5. — Nucléolos bipartitos en el nücleo de un oocito de estadio 2a. Verde de metilo-pironina.

Escala: 20 pm.

Fig. 8. — Nucléolos en anillo. Azul de metileno. Escala 20 pm.

Source : MNHN, Paris

ROCA & ECHEVERRIA 91

Fig. 4. — Citoplasma periférico de oocito de estadio 2a con zonas irregulares PAS positivas (sagitas

negras cortas) adyacentes a la membrana vitelina (sagita negra larga). T, teca interna; Te, teca

externa. PAS - azul Alciän. Escala: 20um.

Fig. 6. — Secciôn de un oocito de estadio 2h con una banda periférica de vitelo (V). B, zonas

basôfilas; N, nücleo; P, plaquetas vitelinas de un oocito adyacente en vitelogénesis tardia.

Tricrémico de Masson. Escala: 50 pm.

Fig. 7. — Oocito de estadio 3a. Plaquetas vitelinas (P) invadiendo el citoplasma (C). N, nücleo.

Coloraciôn de Mann. Escala: 50 um.

Fig. 9. — Corte semifino de un oocito de estadio 3a coloreado con azul de metileno. F, nücleo de

células foliculares; G, grânulo cortical; I, pigmento; M, membrana vitelina; P, plaquetas

vitelinas. Escala: 20 um.

Source : MNHN, Paris

92 ALYTES 14 (2)

ESTADIO 3. VITELOGÉNESIS ACTIVA

A partir de los 350 y hasta 650 um, el vitelo se encuentra organizado en plaquetas

vitelinas, ocupando las dos terceras partes del citoplasma (subtipo a). A medida que

progresa la vitelogénesis se observa un gradiente en la forma y tamaño de las plaquetas;

las mâs pequeñas se ubican en la zona subcortical de la célula, mientras que las mâs

internas son de mayor tamaño. El citoplasma sin plaquetas queda restringido a una franja

perinuclear (fig. 7). Las vesiculas PAS positivas, localizadas en la zona mäs externa de la

banda de vitelo, aumentan en nümero y tamaño. El nücleo conserva las caracteristicas del

estadio anterior y los nucléolos se hallan distribuidos en todo el volumen nuclear; aparecen

nucléolos con distinta morfologia. Se identifican tres tipos: nucléolos bipartitos; nucléolos

en forma de anillo, constituidos por cuentas, que se observan con frecuencia en la zona

mäs central (fig. 8); y nucléolos esferoidales simples periféricos. En la zona cortical del

citoplasma se evidencia la presencia de gränulos de pigmento y de gränulos corticales de

2,5 um, cercanos a la membrana celular (figs. 9-10); éstos son esféricos y su contenido es

homogéneo.

Los oocitos cuyo diämetro oscila entre los 650 y 740 um se caracterizan por presentar

un color castaño en toda su superficie (subtipo b). El citoplasma se halla invadido por

plaquetas vitelinas, las cuales alcanzan el nücleo. La franja perinuclear de citoplasma

basôfilo queda reducida a un anillo de grosor variable inmerso en el vitelo. Los gränulos

de pigmento se distribuyen homogeneamente en toda la regiôn cortical de la célula

(fig. 11). La disposiciôn de los nucléolos se modifica. Los nucléolos bipartitos se agrupan

en el centro del nücleo y nucléolos esferoidales se localizan en la periferia.

A partir de los 750 um de diämetro, comienza a distinguirse un desplazamiento

gradual del pigmento (subtipo c). Las plaquetas vitelinas de mayor tamaño se ubican en

el centro del hemisferio vegetativo, por debajo de la vesicula germinal. Los gränulos

corticales se hallan distribuidos en una amplia franja de citoplasma cortical entre las

plaquetas vitelinas. El nücleo migra hacia la zona mâs pigmentada del oocito, el hemisferio

animal. La morfologia del mismo se modifica de esférica a oval, y su eje mayor se dispone

paralelo al ecuador del oocito. En la zona periférica del nücleo aparecen nucléolos

esféricos de aspecto vacuolar o vacuolados (segün DUMONT, 1972) que contienen äreas de

material de menor afinidad por los colorantes bäsicos.

ESTADIO 4. VITELOGÉNESIS TARDIA

Estos oocitos presentan una visible polarizacion del pigmento. El hemisferio animal,

castaño oscuro, contrasta con el hemisferio vegetativo de color castaño claro. El diâmetro

està comprendido entre los 900 y 1200 um. La figura 12 muestra una vista general de un

corte de un oocito de estadio 4. El citoplasma estä totalmente ocupado por plaquetas

vitelinas que se distribuyen segün un gradiente de tamaño. Alrededor del nücleo son

evidentes las de menor tamaño. La membrana nuclear manifiesta una serie de pliegues

hacia el interior del nücleo que se hacen mäs numerosos y profundos hacia el lado del

hemisferio vegetativo. Los nucléolos periféricos se observan con mayor frecuencia entre los

pliegues de la membrana nuclear. En el centro se agrupan nucléolos esferoidales de menor

Source : MNHN, Paris

RoCA & ECHEVERRA 93

Fig. 10. — Regiôn cortical de un oocito en estadio 3a. C, membrana citoplasmätica; F, nücleo de

célula folicular; G, gränulo cortical; 1, pigmento; M, membrana vitelina; P, plaqueta vitelina.

Aumento: 7000 x.

tamaño y de apariencia homogénea. Los cromosomas comienzan a visualizarse en la zona

central al aumentar su afinidad por los colorantes bäsicos. El nücleo presenta un halo

subnuclear de material basôfilo y PAS positivo, en intimo contacto con los pliegues de la

membrana. El tratamiento con diastasa produce una reacciôn PAS negativa. Con MET se

observa que estä constituido por mitocondrias y un material electro-opaco que podria

asociarse con glucogeno y ribosomas (fig. 13). Los gränulos corticales se disponen

ordenados debajo de la membrana plasmätica. Presentan un diämetro aproximado de

3um. En el hemisferio animal, los grânulos de pigmento se hallan por debajo de los

grânulos corticales.

ESTADIO 5. OOCITO OVULABLE O MADURO

En estos oocitos, cuyos diämetros oscilan entre 1200 y 1400 um, se acentüa la

diferencia de coloraciôn entre el hemisferio animal y el vegetativo. El hemisferio animal,

Source : MNHN, Paris

94 ALYTES 14 (2)

Fig. 11. — Vista general de oocito de estadio 3b. Pigmento (1) distribuido en la regiôn cortical y banda

de material perinuclear basôfilo (triängulos blancos). Hematoxilina de Harris - eosina. Escala:

200 um.

Fig. 12. — Vista general de un oocito de estadio 4. Polo animal (PA) con pigmento. H, halo

subnuclear; PV, polo vegetativo. Hematoxilina de Harris - eosina. Escala: 300 um.

Source : MNHN, Paris

ROCA & ECHEVERRIA 95

Fig. 13. — Citoplasma subnuclear de oocito de estadio 4. K, mitocondrias; M, membrana nuclear.

Aumento: 22500 x.

Fig. 14. — Regiôn cortical del polo animal de un oocito de estadio 5. D, complejo de Golgi; G,

gränulo cortical, I, grânulos de pigmento; K, mitocondrias; M, membrana vitelina; P, plaqueta

vitelina. Aumento: 6000 x.

Source : MNHN, Paris

9%6 ALYTES 14 (2)

de color castaño oscuro, presenta una zona circular en el polo animal con menos

pigmento. La disposiciôn de las plaquetas vitelinas en el citoplasma es semejante a la del

estadio 4. En el hemisferio vegetativo, las plaquetas alcanzan una longitud mäxima de

10 um. El anillo subnuclear persiste en estos oocitos. En el nücleo se observa una

reducciôn del nümero de nucléolos. Los nucléolos periféricos aumentan de tamaño

alcanzando un diämetro de 9 um. En este estadio los cromosomas son poco visibles con

las técnicas de coloracién utilizadas. Los gränulos corticales conservan la forma esférica,

se hallan situados por debajo de la membrana celular y alcanzan un diâmetro de 4 um. Las

microvellosidades oocitarias se hallan retraidas y el espacio perivitelino se ve aumentado

(fig. 14).

DisCUSIÔN

Para establecer los estadios de la oogénesis se han utilizado distintos criterios. En

Rana pipiens, estän basados principalmente en el tamaño del oocito o en la cantidad y

distribucién del vitelo y del pigmento (GRANT, 1953; Kemp, 1953), aunque otros autores

utilizaron solamente la morfologia de los cromosomas (DURYEE, 1950). En Xenopus laevis,

la identificacién de los estadios se basa en el aspecto externo de los oocitos y en las

observaciones histolôgicas de los mismos (DUMONT, 1972; CALLEN, 1984). En este trabajo

se han considerado los caracteres morfolégicos externos e internos de los oocitos,

integrando las diferentes propuestas aludidas, que permite establecer cinco estadios de la

oogénesis con ocho subestadios que se sintetizan en la Tabla I. Los cinco estadios se

consideran suficientes y convenientes para establecer el curso de la oogénesis.

El pigmento ha sido considerado como un producto catabélico y comienza a aparecer

cuanto mäs activo es el metabolismo del oocito (VALDEZ TOLEDO & PisAN6, 1980). En Bufo

arenarum, la formacién del pigmento se evidencia en el estadio de vitelogénesis primaria,

distribuido en todo el citoplasma (VALDEZ TOLEDO & PiSAN6, 1980; ECHEVERRIA, 1988). La

acumulacién de la melanina en oocitos de Xenopus laevis se incrementa durante los

estadios de vitelogénesis temprana y tardia hasta el estadio V (DUMONT, 1972). En

Melanophryniscus stelzneri, el pigmento es escaso y de color castaño a diferencia de las

especies anteriormente mencionadas, en las cuales éste es negro y abundante. El pigmento

aparece al comienzo del estadio de vitelogénesis activa distribuido homogeneamente en el

citoplasma cortical. À partir del estadio 4 se acumula en el hemisferio animal, siendo

caracteristica la presencia de la mancha polar en el polo animal en el estadio 5.

El inicio del proceso de vitelogénesis se evidencia en Melanophryniscus stelzneri en el

estadio de vitelogénesis temprana (2.a) con la aparicion de pequeñas vesiculas en la zona

cortical del citoplasma. Las plaquetas vitelinas se organizan al finalizar la vitelogénesis

temprana (estadio 2.b), ubicandose en la periferia de la célula. Todas las plaquetas

vitelinas son homogéneas independientemente de su ubicaciôn y tamaño, es decir, no se

distingue en ellas un cuerpo principal cristalino como se describe en las especies estudiadas

(WISCHNITZER, 1957). DUMONT (1972) y OPRESKO et al. (1980) proponen que el aumento

de tamaño de las plaquetas vitelinas es ocasionado por la fusion de vesiculas endociticas

a las plaquetas ya formadas. En M. stelzneri, se ha podido observar con microscopio

Source : MNHN, Paris

RoCA & ECHEVERRIA 97

electrénico de transmisiôn grupos de pequeñas plaquetas que se contactan; este hecho

podria interpretarse como un proceso de fusiôn. El crecimiento de las plaquetas

posiblemente se produzca a expensas del material presente en el citoplasma.

La mayoria de los autores asocia la aparicién de las vesiculas de vitelo y de los

grânulos corticales con formaciones PAS positivas y menciona que la aparicin de éstas

se produce en forma simultänea.

En Rana pipiens, Xenopus laevis y Bufo arenarum, los gränulos corticales se observan

por primera vez en el cortex del oocito, durante el estadio de vitelogénesis temprana o

primaria (WarD & WarD, 1968; DUMONT, 1972; VALDEZ TOLEDO & PisAN6, 1980).

BALINSKY & Davis (1963) describieron a los gränulos corticales como cuerpos con matriz

granular de aspecto homogéneo, de 2um de diâmetro que aparecen durante la

vitelogénesis primaria. En M. stelzneri, se evidencian durante la etapa de vitelogénesis

activa, a la vez que se observa un aumento de los complejos de Golgi y de las vesiculas

de vitelo. Las observaciones con microscopio electrénico de transmisiôn mostraron

similitudes en cuanto al aspecto de la sustancia que compone los gränulos corticales pero,

con respecto al tamaño de los mismos, no se hallaron coincidencias respecto de las especies

aludidas (KEMP & IsTOK, 1967; ANDREUCCETTI & CAMPANELLA, 1980), siendo los gränulos

corticales de M. stelzneri los mäs desarrollados.

Si bien la actividad ovärica de las hembras adultas de X. laevis y B. arenarum no son

equivalentes (CALLEN et al., 1986; ECHEVERRIA & GONZALEZ, 1994), se debe destacar que

M. stelzneri comparte, con las especies mencionadas, los patrones generales del ciclo de la

vitelogénesis. En los oocitos de M. stelzneri, una banda de material basdfilo perinuclear se

hace evidente al final de la previtelogénesis y en los oocitos en vitelogénesis temprana, y

un halo subnuclear de caracteristicas basôfilas se halla en los oocitos maduros. Una banda

discontinua de material citoplasmätico similar a la hallada en M. stelzneri ha sido puesta

de manifiesto en Bufo lentiginosus (KING, 1908) y en B. arenarum (ECHEVERRIA, 1974, 1987)

con técnicas histolégicas generales y reacciones histoquimicas para condrioma. TOURTE et

al. (1984) confirman por primera vez en X. laevis la presencia de mitocondrias en el

citoplasma agrupadas en una corona perinuclear. Cabe señalar que X. laevis y M. stelzneri

presentan mitocondrias agrupadas por fuera de la membrana nuclear que se denominan

corona (“crown”, ToURTE et al., 1984) y halo subnuclear respectivamente. El halo

subnuclear contiene, ademäs, gränulos de glucogeno que podrian motivar la respuesta

positiva a la reaccién de PAS, mientras que las zonas basôfilas del citoplasma de

M. stelzneri, anälogas a la corona perinuclear de X. /aevis, no reaccionan con PAS.

Tanto el nümero como la morfologia de los nucléolos de los oocitos de M. stelzneri

presentan variaciones en el transcurso del desarrollo. En oocitos muy jovenes y en los

maduros, los nucléolos presentan una forma esferoidal, pero en estadios intermedios, éstos

pueden adoptar distintas morfologias: bipartitos, anulares y vacuolados. Se han descrito

nucléolos en forma de collar y de anillo en diferentes especies de anfibios como Plethodon

cinereus (MACGREGOR, 1965), Ambystoma mexicanum (CALLAN, 1966) y Notophthalmus

viridescens (LANE, 1967), siendo poco frecuente la descripcién de estos nucleélos en los

anuros. En Bufo arenarum, ECHEVERRÏA (1980) los describe con una morfologia semejante

a los que hemos hallado en M. stelzneri. LANE (1967) y VAN GANSEN & SCHRAM (1972)

propusieron que la variacién morfolégica aludida es producida por cambios en la

Source : MNHN, Paris

98 ALYTES 14 (2)

distribucién en el ADN y por la segregaciôn de los constituyentes granulares y fibrilares

del nucléolo respectivamente. Los nucléolos bipartitos hallados en M. stelzneri son

equivalentes a los nucléolos vacuolares descritos en B. arenarum por ECHEVERRiA (1980).

Se utilizé el término bipartito debido a que presentan por lo menos dos partes con distinta

afinidad por los colorantes bâsicos, a fin de distinguirlos de los nucléolos vacuolados

hallados en X. laevis por DUMONT (1972) y en M. stelzneri, que contienen areas de material

con menor afinidad por los colorantes bâsicos.

RESUMEN

En hembras maduras de Melanophryniscus stelzneri (N = 17) de las Sierras de

Cordoba (Tanti) y de San Luis (El Trapiche), se identifican cinco estadios de la oogénesis

con ocho subestadios (subtipos) estructurados sobre la base de la apariciôn de vesiculas de

vitelo, plaquetas vitelinas, gränulos corticales y de pigmento, variaciones morfolôgicas de

la membrana nuclear y de los nucléolos.

AGRADECIMIENTOS

A la Dra. Gladys N. PELLERANO de la Câtedra de Histologia Animal, Facultad de Ciencias

Exactas y Naturales de la Universidad de Buenos Aires, por la lectura critica del manuscrito, a la Lic.

Luisa E. FIORITO por su contribuciôn con material de Tanti, Cérdoba, al Dr. Mario RIVERO por el

apoyo técnico en el uso del microscopio electrénico, y al Sr. GONZALEZ del Servicio de Microscopia

del Depto. de Ciencias Biolôgicas (FCEN, UBA).

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Corresponding editor: Alain DuBois.

BIBL. DU

MUSÉUM

PARIS

Sourèg SMAHN, Paris

AUNVTES

International Journal of Batrachology

published by ISSCA

EDITORIAL BOARD FOR 1996

Chief Editor: Alain Dugois (Laboratoire des Reptiles et Amphibiens, Muséum national d'Histoire

naturelle, 25 rue Cuvier, 75005 Paris, France)

Deputy Editor: Janalee P. CALDWELL (Oklahoma Museum of Natural History, University of Oklahoma,

Norman, Oklahoma 73019, U.S.A.).

Editorial Boar -Louis ALBARET (Paris, France); Ronald G. ALniG (Mississippi State University,

U.S.A.); Emilio BALLETTO (Torino, Italy); Alain COLLENOT (Paris, France); Günter GOLLMANN (Wien,

Austria); Tim HALLIDAY (Milton Keynes, United Kingdom); W. Ronald HevEr (Washington,

U.S.A.); Walter HôDL (Wien, Austria); Pierre JoLY (Lyon, France), Masafumi Maïsui (Kyoto,

Japan); Jaime E. Péraur (Mérida, Venezuela); J. Dale RoBERTS (Perth, Australia); Ulrich SINSCH

(Koblenz, Germany); Marvalee H. Wake (Berkeley, U.S.A.).

Technical Editorial Team (Paris, France): Alain DuBois (texts) Roger BoUR (tables): Annemarie

Ouen (figures).

Index Editors: Annemarie OuLer (Paris, France); Stephen J. RICHARDS (Townsville, Australia)

GUIDE FOR AUTHORS

Alytes publishes original papers in English, French or Spanish, in any discipline dealing with

amphibians. Beside articles and notes reporting results of original research, consideration is given for

publication to synthetic review articles, book reviews, comments and replies, and to papers based upon

original high quality illustrations (such as color or black and white photographs), showing beautiful or rare

species, interesting behaviors, etc.

The title should be followed by the name(s) and addresses) of the author(s). The text should be

typewritten or printed double-spaced on one side of the paper. The manuscrit should be organized as

follows: English abstract, introduction, material and methods, results, discussion, conclusion, French or

Spanish abstract, acknowledgements, literature cited, appendix.

Figures and tables should be mentioned in the text as follows: fig. 4 or Table IV. Figures should not

exceed 16 x 24 cm. The size of the lettering should ensure its legibility after reduction. The legends of figures

and tables should be assembled on a separate sheet. Each figure should be numbered using a pencil.

References in the text are to be written in capital letters (BOURRET, 1942; GRAF & POLLS PELAZ, 1989;

INGER et al., 1974). References in the literature cited section should be presented as follows:

BoURRET, R., 1942. — Les Batraciens de l'Indochine. Hanoï, Institut Océanographique de l'Indochine: i-x

+ 1-547, pl. I-IV.

GRAF, J.-D. & POLLS PELAZ, M., 1989. — Evolutionary genetics of the Rana esculenta complex. In: R. M.

Dawey & J. P. BoGART (eds.), Evolution and ecology of unisexual vertebrates, Albany, The New York

State Museum: 289-302.

INGeR, R. F., Vois, H. K. & Voris, H. H., 1974. — Genetic variation and population ecology of some

Southeast Asian frogs of the genera Bufo and Rana. Biochem. Genet., 12: 121-145.

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