Stage dependent susceptibility to lead in Bufo arenarum embryos

Environmental Pollution 63 (1990) 239-245

Stage Dependent Susceptibility to Lead in Embryos

Bufo arenarum

C. S. P6rez-Coll & J. H e r k o v i t s Instituto de Bioiogia de la Reproduccion y Desarrollo Embrionario, Universidad Naeional de Lomas de Zamora, C.C. 95, 1832 Lomas de Zamora (B), Argentina (Received ii January 1989; accepted 25 September 1989)

A BSTRA C T The stage dependent susceptibility to lead in amphibian development was studied by exposing Bufo arenarum embryos during neurulae, neuromuscular activity and gill circulation stages for twenty hours to I ppm Pb 2 +. Survival, malformations and behavioral disorders were evaluated. The embryonic susceptibility to lead was markedly stage dependent. The survival at the neuromuscular activity stage was approximately half that of the other two periods; concerning malformations, the gill circulation stage was the least sensitive. The observed malformations consisted of failed closure of neural tube, hydropsy, small and o'lindered tails, reduced body size and incurvations in the body axis. Some alterations occurred in all experimental groups and therefore were considered non-dependent on the period of treatment. In all experimental embryos, neurological disorders such as trembles and loss of equilibrium were observed.

INTRODUCTION The toxicological effects o f lead include developmental alterations such as reduced litter size, retarded growth, delayed development, reduced body size and eventually death in mammalian embryos (Goyer, 1981). Particularly deleterious effects of this heavy metal on neurological development of infants have been reported (Moore, 1980). Considering that these disorders 239 Environ. Pollut. 0269-7491/90/$03.50 © 1990 Elsevier Science Publishers Ltd, England. Printed in Great Britain

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also affect amphibian embryos (P~rez-Coll et al., 1988)--normal components of continental aquatic ecosystems--we employed Bufo arenarum embryos as a model to describe the effects of lead on certain stages of development. Although amphibian embryos are commonly used to evaluate the effects of aquatic pollutants (Bando, 1976; Cook, 1977), the stage dependent susceptibility to teratogenic agents has not usually been studied (Wilson, 1972; Herkovits & Fernfindez, 1979). The main aim of this work was to compare the effects of lead, when administered for the same length of time, during naurulae, neuromuscular activity or gill circulation stages for the same length of time. Survival, malformations and behavioral disorders of treated embryos are reported.

MATERIAL AND METHODS Ovulation was induced by injecting a suspension of homologous hypophysis into adult Bufo arenarum. Oocytes were fertilized in vitro and jelly coats were removed with 2% thioglycolic acid, pH 7.2. Batches of 60 embryos were treated from: (a) the end of gastrulation (S. 12) (Del Conte & Sirlin, 1951); (b) the onset of neuromuscular activity (S. 18); and (c) the gill circulation (S.20), in each case during twenty hours with 1 ppm Pb 2÷ in 10% Holtfreter solution (HS). After this period the embryos were maintained in HS until the development was completed. Controls were untreated embryos developed in HS without additions. Experiments were carried out at 20 _ I°C. The effects were observed with a Wild stereoscopic microscope, evaluating survival, malformations and behavioral disorders. Individuals of each group were prepared for scanning electron microscopy (SEM) (Herkovits, 1977) and observed in a Jeol JSM-35 CF scanning electron microscope operated at 10kw. The results obtained were statistically analyzed by using X2 test.

RESULTS Table 1 shows the survival and malformation data of embryos from the three different stages. Treated embryos from the end of gastrulation (S.12) reached the caudal bud stage (S.17) synchronously with control embryos, and at that time, the survival as well as the embryo structure were only slightly altered. However, 24h later, 43% of the embryos exhibited malformations and at the end of development (S.25), there were almost 80% of malformed embryos. Concerning malformations, 9% of altered embryos

Susceptibility to lead in Bufo arenarum embryos

241

TABLE I

Survival and Malformation Data ofBufo arenarum Embryos Treated With I ppm Pb 2÷ from Different Stages During 20 h. The Observation Times are Referred to the Beginning of the Treatment Treatment from

Condition

No. o f embryos

t (h) 24

48

72

96

No. No. No. No. No. No. No. No. S V ° M F b S V ° M F b SV" M F b S V ° M F ~

Late gastrulae (S.12) Neuro-muscular activity (S.18) Gill circulation (S.20)

Control Experimental Control Experimental Control Experimental

180 180 180 180 180 180

180 0 180 174" 15 162 180 0 180 129 120 93 180 0 180 174" 12 156

15 70 0 81 0 51

180 159 ! 80 81 180 144

6 180 126 159 0 180 74 81 0 180 68 144

6 i 26 0 74 0 68

° Number of surviving embryos. b Number of malformed surviving embryos. * Z2p < 0-01. The remaining data were significant at the level of 0-005. failed to close the n e u r a l t u b e (Table 2). In 4 7 % o f the embryos, small a n d cylindered tails as well as incurved b o d y axis were observed, while 5 8 % o f the individuals exhibited partially developed fins a n d gills. There were h y d r o p i c e m b r y o s in 2 3 % o f the cases, a n d in 100% o f individuals the average size was r e d u c e d by 20%. In a few e m b r y o s , difficulties in h a t c h i n g were observed, often leading to d e a t h (not tabulated). A t the onset o f n e u r o m u s c u l a r activity, all e m b r y o s exhibited trembles. T h e susceptibility to lead was h i g h e r f r o m the n e u r o m u s c u l a r activity stage (S.18) o n w a r d s . In these e m b r y o s , m o r t a l i t y TABLE 2

Types of Malformations Observed in Bufo arenarum Embryos Exposed to Lead Treatment from

Late gastrulae (S.12) Neuromuscular activity (S.18) Gill circulation (S.20)

Alteration (%)

1

2

3

4

5

6

7

9

23 50

47 25

58 100 100

47 100 25

58 100 100

100 100 100

Key: 1. failed closure ofthe neural tube: 2. hydropsy: 3. small and cylindered tail: 4. partially developed fins plus irregular borders: 5. incurved body axis: 6. partially developed gills: and 7. reduced body size.

Fig. i. A. Panoramic view ofa Bufo arenarum embryo treated with I ppm Pb" ÷ from neuromuscular activity stage (S.I 8) during 20 h and fixed at gill circulation stage {S.20). Notice the partially developed gills (G), reduced body size, underdeveloped fin {F) and incurved body axis (IJ ( x 24). B. Control embryo fixed at S.20 ( x 24). C. Embryo from the group illustrated in A fixed at the end of development ~S.25). Observe the severe incurvation in the body axis (I), hydropsy (H) and the partially developed fin (F) with irregular borders ( x 24). D. Control embryo fixed at S.25 ( x 20).

q~

b.J

IxJ

Susceptibility to lead in Bufo~arcnarUmembryos

243

(almost 30%) and malformations (more than 90%) were significantly higher at 24h than with other treatments. Finally, at 72h, mortality and malformations reached the highest values, being 45 % and 91%, respectively (Table 1). Fifty percent of altered embryos were hydropic individuals (Fig. 1, C and D); 25% of altered embryos were hydropic individuals (Fig. 1, C and D); 25% of malformed embryos had small and cylindered tails, while in 100% of the cases there were partially developed fins with irregular borders, incurved body axis, underdeveloped gills and reduced body size (Table 2, Fig. 1, A and B). Neuromuscular development was also noticeably affected because embryos remained quiet, with the ventral side up or laterally inclined, producing brief and weak movements when stimulated. Embryos treated from gill circulation stage (S.20) showed similar survival to embryos treated from S.12. Malformations such as underdeveloped fins (100%), incurvated body axis (25%), partially developed gills (100%) and reduced body size (100%) were observed (Table 2). Regarding behavioral disturbances, the effects were similar to those obtained in embryos treated from S.18. Although the fin of these embryos was more narrow, it was proportionally well-developed. These embryos exhibited blisters in the ectodermal surface, but these alterations disappeared 24 h later. The SEM studies of control as well as experimental embryos showed the normal pattern of glandular and ciliated cells in the epithelium.

DISCUSSION Lead, like other heavy metals, interferes with the normal development of vertebrates (Mas & Arola, 1985; Narbaitz, 1985; Wide, 1985), including amphibians (P6rez-Coll et al., 1988). The survival of treated embryos is markedly stage-dependent, the neuromuscular activity stage being approximately twice as sensitive to lead than neurulae or gill circulation periods. Regarding malformations, the gill circulation stage was the least sensitive, probably because the organogenic processes are already advanced at that time. In addition, a qualitative difference between the three experimental groups was identified, consisting in the fact that in embryos treated from the late gastrulae stage, the effects were usually detectable only after the end of the lead treatment. This difference could be due to a reduced uptake of lead during this period (because of the presence of the viteline membrane and/or a limited epithelial permeability), while on the other hand the cellular activities during this period could be less sensitive to lead action. Concerning abnormal development, the failure in closure of the neural tube was also observed in treatments with another heavy metal--cadmium--in mammalian (Schmid

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C. S. Pbrez-Coil, J. Herkovits

et al., 1985) as well as in amphibian embryos (Keino, 1973; P6rez-Coll et al.,

1985). These effects, as reported for cadmium exposure, may be related to histopathological changes on neuroepithelium (Sippel et al., 1983). The blisters and distortion of body cavities in embryos exposed to lead might be due to disturbances of the osmoregulatory mechanism, for instance inhibition of N a - K ATPase (Vallee & Ulmer, 1972), or by means of the antinatriuretic effect of Pb (Powers & Foulkes, 1985). Some effects such as reduced body size and incurved body axis were reported for many physicochemical teratogenic agents (Rosenthal & Alderdice, 1976; Herkovits & Fernhndez, 1979) including lead in continuous treatments (P6rez-Coll et ai., 1988), and can therefore be considered as non-specific effects. According to the present results, they are also non stage-dependent. Independent of the stage of treatment, the neurological disorders such as trembles, swimming and equilibrium alterations, were present, confirming the well known neurotoxic action of lead. From a biochemical point of view, lead effects may be related to a dramatic inhibition in the increase of total proteins, D N A and RNA, as well as to an overall decrease of cellular activities resulting from the inhibition of key metabolic processes (Marion & Denizeau, 1983). In addition, lead binds sulphydryl groups of proteins, accounting for distortion of the polypeptide chain which results in the inhibition of many systems (Eisinger, 1978). Concerning the controls of the embryos treated from gastrulae stage, the diminution in the number of affected individuals from 72 h onwards may be due to a spontaneous recovery process as was previously described (Herkovits & Faber, 1978).

ACKNOWLEDGEMENTS We are grateful to Aria Maria Martinez for secretarial assistance. This study was supported by grants from Comisi6n de Investigaciones Cientificas de la Provincia de Buenos Aires (CIC) and Consejo Nacional de Investigaciones Cientificas y T~cnicas (CONICET), Argentina.

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