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Effects of methylmercuric chloride administered to pregnant rats during the preimplantation period
ECOTOXICOLOCY
AND
ENVIRONMENTAL
SAFETY
9, 189-195 (1985)
Effects of Methylmercuric Chloride Administered to Pregnant during the Preimplantation Period ERMINIOGIAVINI,
CLAUDIO VISMARA, ANDMARIA
Rats
LUISA BROCCIA
University of Milan, Department of Biology. Via Celoria 26.20133 Milan, Italy Received April 17. I984 Female rats were treated on Day 1. 2, 3, 4, or 5 of pregnancy with 5 mg/kg methylmercuric chloride ip. Some dams were killed on Day 5 of gestation in order to evaluate toxic effects on the early embryos or blastocysts. The remaining rats were sacrificed at the end of pregnancy to verify any embryofetotoxic and teratogenic effects. Unlike what has been found by other authors with in vitro experiments, no clear toxic effects in the blastocyst on Day 5 of pregnancy were evident. A slight embryofetotoxic effect was found when the females were killed at the end of pregnancy. 0 1985 Academic Press, Inc.
INTRODUCTION The high sensitivity of the mammalian embryo to the embryotoxic activity of organic mercury has been fully documented by extensive research into different animal species.When administered during the organogenetic period, methylmercuric chloride (MMC) was proved to be teratogenic and embryolethal in mice (Fujita et al., 1979; Spyker and Smithberg, 1972; Su and Okita, 1976a), rats (Fujita et al., 1978; Nolen et al., 1972) hamsters (Harris et al., 1972), and cats (Khera, 1973). The major malformations found among rodents were palatoschisis, edema, hydrocephalus, and skeletal malformations. In all the speciesstudied, serious lesions to the central nervous system, and notably to the cerebellum, have been reported (Khera, 1973; Khera and Tabacova, 1973). MMC toxic activity is particularly evident during both fetal and perinatal periods, accompanied by behavioral alterations (Su and Okita, 1976b). Recent studies have shown that MMC can affect the preimplantation mammalian embryos. Matsumoto et al. ( 1982) have observed a reduction in the number of blastomeres, an inhibited growth of the inner cell mass, and the collapsing of the blastocoel in mouse blastocysts cultured in vitro for 24 hr with MMC above 2 X lop5 A4. Matsumoto and Spindle (1982) have reported an arrest in development and the collapsing of the blastocoel after in vitro culture of early mouse embryos with l-2 pA4 MMC. These results bring up the problem of the effects of mercury exposure in the early stage of pregnancy, all the more so because these results are partly borne out by observations of Pritchard et al. (1982) according to which exposure to inorganic mercury before and during pregnancy would entail a considerable preimplantation loss in female rats. Since other heavy metals, and in particular Cu and Pb, have also been found to be toxic to the mammalian embryo at the preimplantation stage (Giavini et al., 1980; Jacquet et al., 1976). we have decided to check the MMC effect in vivo on the preimplantation rat embryos by using a particularly high dosage. 189
0147-6513/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
190
GIAVINI,
VISMARA, TABLE
EFFECTOF MATERNAL
AND BROCCIA 1
MMC TREATMENT(~ AT IMPLANTATION(DAY
mg/kg)~~ 5)
RAT EMBRYOS
Day of treatment Control Mated females Pregnant females No. collected blastocysts (mean f SD) No. of blastomeres (mean -C SD) Zona-free blastocysts (%) Degenerated blastocysts (W) Mitotic index (%) Frequency of micronuclei (%0)
1
2
3
4
19 19 184 9.6 * 2.8
15 13 140 10.7 f 3.1
13 13 133 10.2 + 3.3
14 13 127 9.7 f 2.9
15 13 122 9.3 f 3.4
38.2 ii 5.1 3.8
35.1 rt 7.1 5.7
39.0 + 6.1 3.7
36.7 f 7.5 10.2*
37.9 f 8.3 24.5**
3.2 9.4
3.5 7.9
3.7 10.5
3.9 8.3
0.8 9.4
0.3
0.8
0.2
1.0
2.8*
l P i 0.05. ** P < 0.01.
MATERIALS
AND
METHODS
Virgin albino female rats of Sprague-Dawley CD-1 strain (Charles River, Calco, Italy) with an initial weight of 180 f 20 g were used. They were housed in a controlled environment (12 hr light-darkness, temperature 22 +- 2°C 60% relative humidity, and food and water ad libitum). After a 2-week period of acclimatization, the females were mated with males of proven fertility, from 6 PM to 9 PM. The day that a positive vaginal smear was observed was counted as Day 1 of pregnancy. Preimplantation study. The mated females were divided into five groups and treated ip on Day 1, 2, 3, or 4 of gestation with 5 mg/kg of MMC (98% purity, Carlo Erba, Italy) dissolved in distilled water (10 mg/lO ml). Females of the control group were treated with an equal volume of distilled water during Days l-4 of pregnancy. All females were treated with 1 mg/kg of colchicine ip at 1 PM on the fifth day of gestation; after 2 hr they were killed by ether overdose and their uteri were removed. The blastocysts were collected in watch glassesby flushing the uterine horns with buffered saline (1 ml/horn) and their numbers were recorded. The blastocysts were accurately observed under a phase-contrast microscope to show any morphological anomaly or degenerative phenomena. Subsequently, preparations of individual blastocysts were made according to Tarkowski’s method (Tarkowski, 1966). Blastomeres and mitosis counting were performed on these preparations. All the females were weighed on Days 1 and 5 of pregnancy and on the day of treatment. Postimplantation study. The mated females, divided into six trial groups, were treated on Day 1, 2, 3, 4, or 5 of pregnancy with the same doses and the same procedure as those of the preimplantation experiment. Females of the control group were treated with distilled water during the first 5 days of pregnancy. Weights were taken on the day of treatment as well as on the lst, 5th, 15th, and 2 1st days of pregnancy, on which day females were killed and necropsied to determine major organ-related toxicity. Their liver and kidneys were removed and prepared for
PREIMPLANTATION
EFFECTS OF MMC
191
histologic examination. Uteri were exposed and observations were made as to number and position of live, dead, and reabsorbed fetuses, external examination, and weight of live fetuses. Fifty percent of live fetuses were fixed in Bouin’s fluid and subsequently examined according to the free-hand razor sectioning technique of Wilson ( 1965) for investigating visceral malformations. The remainder were processedby the Alizarin staining technique (Staples and Schnell, 1964) for skeletal examination. The number of corpora lutea was recorded. The uteri that did not show any implant signs were colored with a solution of 10% ammonium sulfide to show any early reabsorption. The degree of ossification reached was assessed according to Aliverti et al. (1979). Tlhe data were analyzed by analysis of variance or a 2 X 2 contingency x2 test. In all analysesa probability of P < 0.05 was accepted as significant. RESULTS
Preimplantation
Study
The results of the observations on blastocysts collected on the fifth day of pregnancy are given in Table 1. MMCI treatment does not alter any of the parameters indicative of preimplantation toxicity. In particular, there was no variation in the number of blastocystsfound, no increasein the frequency of abnormal or degenerated blastocysts, or a variation in the average number of blastomeres. During blastomere counting, micronucleus-like structures were observed in some samples. This is a phenomenon that we had noticed in previous studies and interpreted as being nuclear fragments indicating a possible clastogenic activity. If this were true, this effect would only be remarkable in embryos treated on the fourth day pc. The mitotic index remains at the control values in all groups treated. The groups treated on the third and fourth days of pregnancy had a slight increase in the number of zona-free blastocysts.
Postimplantation
Study
The only clear sign of toxicity caused to pregnant females has been a minor weight gain during the preimplantation period among the animals that were treated on the first, second, and third days pc. Results stemming from the study conducted with killing at the end of pregnancy essentially bear out the results obtained with killing on the fifth day of pregnancy (Table 2). A slight increasein the preimplantation loss appeared only with treatment on the second day of pregnancy, while the postimplantation loss was significantly higher with treatments on the fifth day. The reduction in average weight of live fetuses, found with treatment on the first day, is due to the appearance of stunted fetuses in two litters. No increase in major malformations was found among all treated groups, as against control groups, even though a significant increase in skeletal variations was observed among the groups treated on the Days 1, 3, and 5 of pregnancy (Table 3).
DISCUSSION In spite of our using an extremely high dosagelevel, our results are in substantial contradiction with those reached by other authors who have studied the toxicity of
192
GIAVINI,
VISMARA,
AND
BROCCIA
om
sd V 4 l
Ondulated ribs anomalies
* P < 0.05.
No. of ossified Stemebrae Caudal vertebrae Metacarpals
Visceral Renal pelvis dilatation Hydroureter Skeletal Extra ribs Asymmetrical sternebrae Emisternebrae Vertebrae bipartite Stemebrae bipartite
Minor
Vascular ring VSD Skeletal Reduced skull ossification
Right aortic arch
Major malformations Visceral
3
5.3 -c 0.4 2.9 + 0.5 3.4 +- 0.3
0
l/93 0 1 12/89 10 2 :,
0
A l/89 1
1
2193
Control
5.1 f 0.5 2.9 + 0.7 3.4 -t 0.3
6/58* 3 3 18/58* 4 5 7 1 1
1
0 l/58 0
0
O/58
1
5.3 f 0.6 3.1 + 0.6 3.5 + 0.3
4156 0 4 12/55 2 6 5 0 0
0
0 3155 3
0
O/56
2
INCIDENCEOF FETALMALFORMATIONSANDANOMALIES
TABLE
5.2 It_ 0.5 2.7 + 0.4 3.5 f 0.3
2165 1 1 19/64* 5 7 8 6 0
0
0 l/64 1
0
O/65
3
Day of treatment
5.1 It- 0.5 2.6 +- 0.5 3.5 z?c0.3
6/51* 0 6 10/50 4 4 2 1 0
0
0 0;o 0
0
o/5 1
4
5.0 It_ 0.2* 2.9 _+ 0.3 3.2 f 0.1
27,\3* 7 8 8 8 0
l/77 0
0
01 o/73 0
0
l/77
5
% z 5
5 3ctl
g
$ 5 5
2
2
194
GIAVINI.
VISMARA,
AND
BROCCIA
Hg during the preimplantation period. In particular. we have not found any of the embryotoxic effects shown by Matsumoto and Spindle ( 1982) and by Matsumoto et al. (1982) in mouse embryos cultured in vitro with organic mercury. A number of discrepancies have also been found with the results recently obtained by Pritchard et al. (1982). In this case, however, the females were treated with a prolonged administration of inorganic mercury that was started before mating, so that it is possible to postulate an effect on the female hormonal balance responsible for both reduced ovulation and probably inhibition of implantation. In our case, the treatment performed does not affect these parameters dramatically, even though the increase in preimplantation loss found with treatments applied on the second day pc might indicate a particular sensitivity of the rat embryo to MMC at this stage of development. On the whole, our results do not show any particular sign of toxicity to the blastocyst. Instead, they bring out a slight embryofetotoxic effect that is particularly apparent during the postimplantation period with an increase in the frequency of skeletal variations, as well as a slight increase in the postimplantation loss. These results, obtained with a particularly high dosage level, are far from those we might have expected on the basis of the data given by Matsumoto and Spindle (1982) and Matsumoto et al. (1983) and further underline the notable differences existing between tests in vitro and in vivo.
REFERENCES ALIVERTI, V., BONANOMI. L., GIAVINI, E., LEONE, V. G., AND MARIANI, L. (1979). The extent of fetal ossification as an index of delayed development in teratogenic studies on the rat. Teratology 20, 237242. FUJITA, M., FUJIMOTO, T., AND HIRATA, S. (1978). Embryotoxic effects of methylmercuric chloride administered to mice and rats during organogenesis. Teratology 18, 353-366. FUJITA, M., FUJIMOTO, T., AND KIJOF’UJI, E. (1979). Teratogenic effects of a single administration of methyl mercuric chloride in mice. Acta Anat. 104, 356-362. GIAVINI, E., PRATI, M., AND VISMARA, C. (1980). Effects of cadmium, lead and copper on preimplantation rat embryos. Bull. Environ. Contam. Toxicol. 25, 702-706. HARRIS, S. B.. WILSON, J. G., AND PRINTZ, R. H. (1972). Embryotoxicity of methylmercuric chloride in golden hamster. Teratology 6, 139- 142. JACQUET, P., LEONARD, A., AND GERBER, G. B. (1976). Action of lead on early divisions of the mouse embryo. Toxicology 6, 129- 132. KHERA, K. S. (1973). Teratogenic effects of methylmercury in the cat: Note of the use of this species as a model for teratogenicity studies. Teratology 8, 293-304. KHEFW, K. S., AND TABACOVA, S. A. (1973). Effects of methylmercuric chloride on the progeny of mice and rats treated before or during gestation. Food Cosmet. Toxicol. 11, 245-254. MATSUMOTO, N., KATAYAMA, S., AND KUBO, H. (1982). Toxic effects of methylmercuric chloride (MMC) and mercuric chloride (MC) on the development of early mouse embryos in vitro. Teratology 26, 26A. MATSUMOTO, N., AND SPINDLE, A. (1982). Sensitivity of early mouse embryos to methylmercury toxicity. Toxicoi. Appl. Pharmacol. 64, 108- 117. NOLEN, G. A., BUCHLER, E. V., GEIL, R. G., AND GOLDENTHAL, E. 1. (1972). Effects of trisodium nitrilotriacetate on cadmium and methyl mercury toxicity and teratogenicity in rats. Toxicol. Appi.
Pharmacol. 23,222-237. PRITCHARD, A. L., MCANULTY, P. A., COLLIER, M. J., AND TESH, J. M. (1982). mercury on fertility and survival in utero in the rat. Teratology 26, 20A.
The effects of inorganic
PREIMPLANTATION
EFFECTS OF MMC
195
SPYKER, J. M., AND SMITHBERG, M. (1972). EXects of methylmercury on prenatal development in mice. Teratology
5, I8 I- 190.
STAPLES, R. E., AND SCHNELL, V. L. (1964). Refinements in rapid technic in the KOH Alizarin red S method for fetal bone. Stain Technol. 39, 61-63. Su, M. G., AND OKITA, G. T. (1976a). Embryocidal and teratogenic effects of methylmercury in mice. Toxicol.
Appl.
Pharrnacol.
38, 207-2
16.
SW, M. G.. AND OKITA, G. T. (1976b). Behavioral effects on the progeny of mice treated with methylmercury. Toxicol. Appl. Pharmacol. 38, 195-205. TARKOWSKI, A. K. (1966). An air-drying method for chromosome preparations from mouse eggs. Cytogenetics
5, 394-400.
WILSON, J. G. (1965). Methods for administering agents and detecting malformations in experimental animals. In Teratology: Principles and Techniques (J. G. Wilson and J. Warkany, eds.), pp. 262-277. Univ. of Chicago Press, Chicago.
AND
ENVIRONMENTAL
SAFETY
9, 189-195 (1985)
Effects of Methylmercuric Chloride Administered to Pregnant during the Preimplantation Period ERMINIOGIAVINI,
CLAUDIO VISMARA, ANDMARIA
Rats
LUISA BROCCIA
University of Milan, Department of Biology. Via Celoria 26.20133 Milan, Italy Received April 17. I984 Female rats were treated on Day 1. 2, 3, 4, or 5 of pregnancy with 5 mg/kg methylmercuric chloride ip. Some dams were killed on Day 5 of gestation in order to evaluate toxic effects on the early embryos or blastocysts. The remaining rats were sacrificed at the end of pregnancy to verify any embryofetotoxic and teratogenic effects. Unlike what has been found by other authors with in vitro experiments, no clear toxic effects in the blastocyst on Day 5 of pregnancy were evident. A slight embryofetotoxic effect was found when the females were killed at the end of pregnancy. 0 1985 Academic Press, Inc.
INTRODUCTION The high sensitivity of the mammalian embryo to the embryotoxic activity of organic mercury has been fully documented by extensive research into different animal species.When administered during the organogenetic period, methylmercuric chloride (MMC) was proved to be teratogenic and embryolethal in mice (Fujita et al., 1979; Spyker and Smithberg, 1972; Su and Okita, 1976a), rats (Fujita et al., 1978; Nolen et al., 1972) hamsters (Harris et al., 1972), and cats (Khera, 1973). The major malformations found among rodents were palatoschisis, edema, hydrocephalus, and skeletal malformations. In all the speciesstudied, serious lesions to the central nervous system, and notably to the cerebellum, have been reported (Khera, 1973; Khera and Tabacova, 1973). MMC toxic activity is particularly evident during both fetal and perinatal periods, accompanied by behavioral alterations (Su and Okita, 1976b). Recent studies have shown that MMC can affect the preimplantation mammalian embryos. Matsumoto et al. ( 1982) have observed a reduction in the number of blastomeres, an inhibited growth of the inner cell mass, and the collapsing of the blastocoel in mouse blastocysts cultured in vitro for 24 hr with MMC above 2 X lop5 A4. Matsumoto and Spindle (1982) have reported an arrest in development and the collapsing of the blastocoel after in vitro culture of early mouse embryos with l-2 pA4 MMC. These results bring up the problem of the effects of mercury exposure in the early stage of pregnancy, all the more so because these results are partly borne out by observations of Pritchard et al. (1982) according to which exposure to inorganic mercury before and during pregnancy would entail a considerable preimplantation loss in female rats. Since other heavy metals, and in particular Cu and Pb, have also been found to be toxic to the mammalian embryo at the preimplantation stage (Giavini et al., 1980; Jacquet et al., 1976). we have decided to check the MMC effect in vivo on the preimplantation rat embryos by using a particularly high dosage. 189
0147-6513/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.
190
GIAVINI,
VISMARA, TABLE
EFFECTOF MATERNAL
AND BROCCIA 1
MMC TREATMENT(~ AT IMPLANTATION(DAY
mg/kg)~~ 5)
RAT EMBRYOS
Day of treatment Control Mated females Pregnant females No. collected blastocysts (mean f SD) No. of blastomeres (mean -C SD) Zona-free blastocysts (%) Degenerated blastocysts (W) Mitotic index (%) Frequency of micronuclei (%0)
1
2
3
4
19 19 184 9.6 * 2.8
15 13 140 10.7 f 3.1
13 13 133 10.2 + 3.3
14 13 127 9.7 f 2.9
15 13 122 9.3 f 3.4
38.2 ii 5.1 3.8
35.1 rt 7.1 5.7
39.0 + 6.1 3.7
36.7 f 7.5 10.2*
37.9 f 8.3 24.5**
3.2 9.4
3.5 7.9
3.7 10.5
3.9 8.3
0.8 9.4
0.3
0.8
0.2
1.0
2.8*
l P i 0.05. ** P < 0.01.
MATERIALS
AND
METHODS
Virgin albino female rats of Sprague-Dawley CD-1 strain (Charles River, Calco, Italy) with an initial weight of 180 f 20 g were used. They were housed in a controlled environment (12 hr light-darkness, temperature 22 +- 2°C 60% relative humidity, and food and water ad libitum). After a 2-week period of acclimatization, the females were mated with males of proven fertility, from 6 PM to 9 PM. The day that a positive vaginal smear was observed was counted as Day 1 of pregnancy. Preimplantation study. The mated females were divided into five groups and treated ip on Day 1, 2, 3, or 4 of gestation with 5 mg/kg of MMC (98% purity, Carlo Erba, Italy) dissolved in distilled water (10 mg/lO ml). Females of the control group were treated with an equal volume of distilled water during Days l-4 of pregnancy. All females were treated with 1 mg/kg of colchicine ip at 1 PM on the fifth day of gestation; after 2 hr they were killed by ether overdose and their uteri were removed. The blastocysts were collected in watch glassesby flushing the uterine horns with buffered saline (1 ml/horn) and their numbers were recorded. The blastocysts were accurately observed under a phase-contrast microscope to show any morphological anomaly or degenerative phenomena. Subsequently, preparations of individual blastocysts were made according to Tarkowski’s method (Tarkowski, 1966). Blastomeres and mitosis counting were performed on these preparations. All the females were weighed on Days 1 and 5 of pregnancy and on the day of treatment. Postimplantation study. The mated females, divided into six trial groups, were treated on Day 1, 2, 3, 4, or 5 of pregnancy with the same doses and the same procedure as those of the preimplantation experiment. Females of the control group were treated with distilled water during the first 5 days of pregnancy. Weights were taken on the day of treatment as well as on the lst, 5th, 15th, and 2 1st days of pregnancy, on which day females were killed and necropsied to determine major organ-related toxicity. Their liver and kidneys were removed and prepared for
PREIMPLANTATION
EFFECTS OF MMC
191
histologic examination. Uteri were exposed and observations were made as to number and position of live, dead, and reabsorbed fetuses, external examination, and weight of live fetuses. Fifty percent of live fetuses were fixed in Bouin’s fluid and subsequently examined according to the free-hand razor sectioning technique of Wilson ( 1965) for investigating visceral malformations. The remainder were processedby the Alizarin staining technique (Staples and Schnell, 1964) for skeletal examination. The number of corpora lutea was recorded. The uteri that did not show any implant signs were colored with a solution of 10% ammonium sulfide to show any early reabsorption. The degree of ossification reached was assessed according to Aliverti et al. (1979). Tlhe data were analyzed by analysis of variance or a 2 X 2 contingency x2 test. In all analysesa probability of P < 0.05 was accepted as significant. RESULTS
Preimplantation
Study
The results of the observations on blastocysts collected on the fifth day of pregnancy are given in Table 1. MMCI treatment does not alter any of the parameters indicative of preimplantation toxicity. In particular, there was no variation in the number of blastocystsfound, no increasein the frequency of abnormal or degenerated blastocysts, or a variation in the average number of blastomeres. During blastomere counting, micronucleus-like structures were observed in some samples. This is a phenomenon that we had noticed in previous studies and interpreted as being nuclear fragments indicating a possible clastogenic activity. If this were true, this effect would only be remarkable in embryos treated on the fourth day pc. The mitotic index remains at the control values in all groups treated. The groups treated on the third and fourth days of pregnancy had a slight increase in the number of zona-free blastocysts.
Postimplantation
Study
The only clear sign of toxicity caused to pregnant females has been a minor weight gain during the preimplantation period among the animals that were treated on the first, second, and third days pc. Results stemming from the study conducted with killing at the end of pregnancy essentially bear out the results obtained with killing on the fifth day of pregnancy (Table 2). A slight increasein the preimplantation loss appeared only with treatment on the second day of pregnancy, while the postimplantation loss was significantly higher with treatments on the fifth day. The reduction in average weight of live fetuses, found with treatment on the first day, is due to the appearance of stunted fetuses in two litters. No increase in major malformations was found among all treated groups, as against control groups, even though a significant increase in skeletal variations was observed among the groups treated on the Days 1, 3, and 5 of pregnancy (Table 3).
DISCUSSION In spite of our using an extremely high dosagelevel, our results are in substantial contradiction with those reached by other authors who have studied the toxicity of
192
GIAVINI,
VISMARA,
AND
BROCCIA
om
sd V 4 l
Ondulated ribs anomalies
* P < 0.05.
No. of ossified Stemebrae Caudal vertebrae Metacarpals
Visceral Renal pelvis dilatation Hydroureter Skeletal Extra ribs Asymmetrical sternebrae Emisternebrae Vertebrae bipartite Stemebrae bipartite
Minor
Vascular ring VSD Skeletal Reduced skull ossification
Right aortic arch
Major malformations Visceral
3
5.3 -c 0.4 2.9 + 0.5 3.4 +- 0.3
0
l/93 0 1 12/89 10 2 :,
0
A l/89 1
1
2193
Control
5.1 f 0.5 2.9 + 0.7 3.4 -t 0.3
6/58* 3 3 18/58* 4 5 7 1 1
1
0 l/58 0
0
O/58
1
5.3 f 0.6 3.1 + 0.6 3.5 + 0.3
4156 0 4 12/55 2 6 5 0 0
0
0 3155 3
0
O/56
2
INCIDENCEOF FETALMALFORMATIONSANDANOMALIES
TABLE
5.2 It_ 0.5 2.7 + 0.4 3.5 f 0.3
2165 1 1 19/64* 5 7 8 6 0
0
0 l/64 1
0
O/65
3
Day of treatment
5.1 It- 0.5 2.6 +- 0.5 3.5 z?c0.3
6/51* 0 6 10/50 4 4 2 1 0
0
0 0;o 0
0
o/5 1
4
5.0 It_ 0.2* 2.9 _+ 0.3 3.2 f 0.1
27,\3* 7 8 8 8 0
l/77 0
0
01 o/73 0
0
l/77
5
% z 5
5 3ctl
g
$ 5 5
2
2
194
GIAVINI.
VISMARA,
AND
BROCCIA
Hg during the preimplantation period. In particular. we have not found any of the embryotoxic effects shown by Matsumoto and Spindle ( 1982) and by Matsumoto et al. (1982) in mouse embryos cultured in vitro with organic mercury. A number of discrepancies have also been found with the results recently obtained by Pritchard et al. (1982). In this case, however, the females were treated with a prolonged administration of inorganic mercury that was started before mating, so that it is possible to postulate an effect on the female hormonal balance responsible for both reduced ovulation and probably inhibition of implantation. In our case, the treatment performed does not affect these parameters dramatically, even though the increase in preimplantation loss found with treatments applied on the second day pc might indicate a particular sensitivity of the rat embryo to MMC at this stage of development. On the whole, our results do not show any particular sign of toxicity to the blastocyst. Instead, they bring out a slight embryofetotoxic effect that is particularly apparent during the postimplantation period with an increase in the frequency of skeletal variations, as well as a slight increase in the postimplantation loss. These results, obtained with a particularly high dosage level, are far from those we might have expected on the basis of the data given by Matsumoto and Spindle (1982) and Matsumoto et al. (1983) and further underline the notable differences existing between tests in vitro and in vivo.
REFERENCES ALIVERTI, V., BONANOMI. L., GIAVINI, E., LEONE, V. G., AND MARIANI, L. (1979). The extent of fetal ossification as an index of delayed development in teratogenic studies on the rat. Teratology 20, 237242. FUJITA, M., FUJIMOTO, T., AND HIRATA, S. (1978). Embryotoxic effects of methylmercuric chloride administered to mice and rats during organogenesis. Teratology 18, 353-366. FUJITA, M., FUJIMOTO, T., AND KIJOF’UJI, E. (1979). Teratogenic effects of a single administration of methyl mercuric chloride in mice. Acta Anat. 104, 356-362. GIAVINI, E., PRATI, M., AND VISMARA, C. (1980). Effects of cadmium, lead and copper on preimplantation rat embryos. Bull. Environ. Contam. Toxicol. 25, 702-706. HARRIS, S. B.. WILSON, J. G., AND PRINTZ, R. H. (1972). Embryotoxicity of methylmercuric chloride in golden hamster. Teratology 6, 139- 142. JACQUET, P., LEONARD, A., AND GERBER, G. B. (1976). Action of lead on early divisions of the mouse embryo. Toxicology 6, 129- 132. KHERA, K. S. (1973). Teratogenic effects of methylmercury in the cat: Note of the use of this species as a model for teratogenicity studies. Teratology 8, 293-304. KHEFW, K. S., AND TABACOVA, S. A. (1973). Effects of methylmercuric chloride on the progeny of mice and rats treated before or during gestation. Food Cosmet. Toxicol. 11, 245-254. MATSUMOTO, N., KATAYAMA, S., AND KUBO, H. (1982). Toxic effects of methylmercuric chloride (MMC) and mercuric chloride (MC) on the development of early mouse embryos in vitro. Teratology 26, 26A. MATSUMOTO, N., AND SPINDLE, A. (1982). Sensitivity of early mouse embryos to methylmercury toxicity. Toxicoi. Appl. Pharmacol. 64, 108- 117. NOLEN, G. A., BUCHLER, E. V., GEIL, R. G., AND GOLDENTHAL, E. 1. (1972). Effects of trisodium nitrilotriacetate on cadmium and methyl mercury toxicity and teratogenicity in rats. Toxicol. Appi.
Pharmacol. 23,222-237. PRITCHARD, A. L., MCANULTY, P. A., COLLIER, M. J., AND TESH, J. M. (1982). mercury on fertility and survival in utero in the rat. Teratology 26, 20A.
The effects of inorganic
PREIMPLANTATION
EFFECTS OF MMC
195
SPYKER, J. M., AND SMITHBERG, M. (1972). EXects of methylmercury on prenatal development in mice. Teratology
5, I8 I- 190.
STAPLES, R. E., AND SCHNELL, V. L. (1964). Refinements in rapid technic in the KOH Alizarin red S method for fetal bone. Stain Technol. 39, 61-63. Su, M. G., AND OKITA, G. T. (1976a). Embryocidal and teratogenic effects of methylmercury in mice. Toxicol.
Appl.
Pharrnacol.
38, 207-2
16.
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