PERMEABILITY OF THE SYRIAN HAMSTER PLACENTA TO MANGANOUS IONS DURING EARLY EMBRYOGENESIS D. P. HANLON, T. F. GALE and V. H. FERM Departments of Pharmacology & Toxicology and Anatomy & Cytology, Hanover, New Hampshire 03755, U.S.A.
(Received 13th November 1974) Manganese is an essential trace element in the living system. It is invariably present in its ionic form and is known to participate in a number of biological systems (Cotzias, 1958). Relatively high levels of manganese are required to manifest toxicity. Rats fed massive doses of manganese (1\m=.\73%of the dry diet) show decreased intestinal absorption of calcium and phosphorus (Chornock, Gerrant & Dutcher, 1942). Lambs on a high manganese diet had low liver iron levels and reduced haemoglobin formation (Hartman, Matrone & Wise, 1955). Chronic manganese poisoning, characterized by neurological and psychiatric disorders, is seen among workers handling manganese ores and apparently results from inhalation of manganese oxide dusts (von Oettingen, 1935). Deficiency of the element in animals causes growth retardation, bone abnormalities, degenerative changes in the central nervous and reproductive failure (Cotzias, 1958). A specific example of its importance for the developing embryo is the occurrence of ataxia in the offspring of Mn++-deficient rats (Hurley, Everson & Geiger, 1958). Mutant mice possessing the pallid gene also manifest ataxia which Erway, Hurley & Fraser (1966) found to result from partial or complete absence of otoliths in the inner ear. Hurley and her co\x=req-\ workers showed that a single supplement of Mn++, given specifically on the 14th day of gestation, completely and permanently remedied this condition. Pallid mice also respond to Mn++ supplements but once the critical point in gestation is past the defect cannot be corrected in mutant mice or in the phenocopy rats. These findings strongly suggest that Mn+ + crosses the rodent placental barrier during gestation but so far its passage has not been demonstrated. In this report, we show that the Syrian hamster placenta is permeable to 54Mn++ during the critical stages of organogénesis and have correlated this with our earlier studies of placental transport of metal ions. Timed matings of virgin female hamsters were obtained as described by Ferm (1967). Sufficient carrier-free 54MnCl2 (New England Co.) was added to 2-50 niM-MnCl in demineralized-distilled water to give a solution containing 12-0 /iCi/ml. On Day 8 of gestation, twelve hamsters received an injection into the sublingual vein of 54Mn+ + in doses of 0-5 ml/100 g body weight. On Days 9 and 12 of gestation, six females were killed by chloroform anaesthesia. At h
109
D. P. Hanlon
110
et
al.
autopsy, samples of maternal blood, liver, placenta and
uterus and embryos obtained from each animal. Due to the small size of the entire Day-9 gestation sac, it was not possible to separate the developing chorioallantoic placenta from the proximal segment of the visceral yolk sac and, for this gestation day, the term placenta refers to both the chorioallantoic and yolk sac placenta together. On Day 12, when separation was feasible, the two placentae were handled individually. All embryos in a litter from mothers on Day 9 were pooled for counting because of their small size. Day-12 embryos were counted in groups of four, together with their respective chorioallantoic and yolk sac placentae. Tissues were weighed in previously tared plastic tubes and the radio¬ activity of each sample was determined with a Nuclear Chicago Radiation Analyzer system employing a well-counter. For every sample, sufficient counts were accumulated to reduce the probable error in counting to less than 1 %. Counts per minute were converted to µg Mn+ + by comparing the radio¬ activity of the samples with a 54Mn++ standard prepared from the solution used for injections. Text-figure 1 summarizes the data on the distribution of 54Mn + + in maternal and fetal tissues of the golden hamster following injection of the metal ion on tissues 24 and 96 hr Day 8. Detectable amounts of radioactivity in embryonic after administration of the ion indicate that 54Mn+ + crosses the placental barrier. All of the maternal tissues examined showed uptake of radioactivity. The levels of 54Mn++ decreased in both embryonic and maternal tissues between Day 9 and Day 12. All decreases were statistically significant. were
9th day
^ 12th
day
rfi •S
2
S
0055 0-025
Maternal blood
Maternal liver
Uterus
Allantoic
placenta
m Embryo
Yolk
sac
placenta
Text-fig. 1. The concentrations of S4Mn++ in hamster tissues 24 hr and 96 hr after injection of the isotope. Standard errors for mean values are indicated by the vertical bars. The experimental conditions are described in the text.
111 Permeability of Syrian hamster placenta to Mn+ + Since manganese is essential for normal development, our data showing that the placental barrier is permeable to 54Mn++ during the critical stages of organogénesis in the Syrian hamster were not unexpected. We have previously shown that the placenta is permeable to 65Zn++ (Ferm & Hanlon, 1974a), and 109Cd+ + (Ferm, Hanlon & Urban, 1969) between the 8th and 9th days of gestation, but are unaware of any previous tracer studies relating directly to placental transport of Mn++ in rodents. Our finding is, however, consistent with an earlier report that otolith abnormalities in the offspring of Mn++deficient rats can be prevented by a single supplement of the ion on Day 14 of gestation (Hurley et al., 1958). The disposition and kinetics of Mn+ + in the maternal system can be compared with studies of other rodent species. On Day 9, maternal liver tissues contained 20% of the entire 54Mn++ load injected (assuming the liver is 6% of the total body weight). This is consistent with the data of Maynard & Cotzias (1955) who found that intraperitoneal injection of tracer amounts of the short-lived isotope, 56Mn+ +, resulted in a rapid and extensive uptake of label in liver and other mitochondria-rich tissues of rats. In our study, 54Mn++ also concentrated in the uterus and placenta, but to a lesser degree than in the liver. Those tissues showing greatest uptake of 54Mn++ 24 hr after injection also showed the larger % decrease in radioactivity on Day 12, e.g. the maternal liver lost 70% of its Day-9 content of 54Mn++, while blood levels decreased attributed similar findings by approximately 50%. Britton & Cotzias (1966) in the rat to a rapid change of labelled Mn+ + in parenchymatous organs +
present in the diet. At the dose level used in our Mn++ was neither teratogenic nor embryocidal. Ferm study (136 ^g/100 g), this dose is embryocidal but not teratogenic in that times ten (1972) reports hamsters. The path by which Mn+ + and other divalent metal ions reach embryonic tissues during the critical stages of organogénesis is not known. The allantoic and yolk sac placentae are obvious candidates for this transport process. The finding of Carpenter et al. (1973) that lead ions (210Pb++) concentrate in the visceral wall of the yolk sac placenta between 1 and 4 hr after injection on the 8th day of gestation in the hamster could indicate that this is the more im¬ portant route. Alternatively, the yolk sac may serve as a barrier, offering the ions. The following embryo protection from teratogenic agents by heavy metal + this view. of in The concentration 65Zn+ points support yolk sac is 1-23 times that of embryonic tissues (Ferm & Hanlon, 1974b) while the ratio for 54Mn+ +, using data reported in this paper, is 0-66. Both values are near unity and suggest the absence of any selective storage or barrier effect by the yolk sac placenta for these metal ions. On the other hand, the concentration of the teratogen, 109Cd+ +, is fifty times greater in the yolk sac than in embryonic tissues (Ferm et al., 1969). These data are for Day-12 samples, since yolk sac material on Day 9 is too scanty to assay in the manner used here. Nevertheless, they could reflect the Day-9 conditions and so are worth mentioning. As such, our results imply the existence of a protective mechanism which concentrates heavy metals in the yolk sac placenta, but which does not impede the transport of essential metal ions to embryonic tissues. when stable Mn+
was
112
D. P. Hanlon
et
al.
We are grateful to Mrs Martha L. Hill for her technical assistance and to Mrs Louise White for typing the manuscript. REFERENCES
Britton, A. A. & Cotzias, G. C. (1966) Dependence of manganese turnover on intake. Am. J. Physiol. 211, 203-206. Carpenter, S. J., Ferm, V. H. & Gale, T. F. (1973) Permeability of the golden hamster placenta to inorganic lead: radioautographic evidence. Experientia, 29, 311-313. Chornook, C, Gerrant, . . & Dutcher, R. A. (1942) Effect of manganese on calcification in the growing rat. J. Nutr. 23, 445-458. Cotzias, G. C. (1958) Manganese in health and disease. Physiol. Rev. 38, 502-532. Erway, L., Hurley, L. S. & Fraser, A. (1966) Neurological defect: manganese in phenocopy and prevention of a genetic abnormality of inner ear. Science, N.T. 152, 1766—1768. Ferm, V. H. (1967) The use of the golden hamster in experimental teratology. Lab. Anim. Care, 17,
451-462. V. H. (1972) Teratogenic effects of metal ions on mammalian embryos. Adv. Teratol. 5, 57—75. V. H. & Hanlon, D. P. (1974a) Placental transfer of zinc in the Syrian hamster during early embryogenesis. J. Reprod. Fert. 39, 49—52. Ferm, V. H. & Hanlon, D. P. (1974b) Toxicity of copper salts in hamster embryonic development. Biol. Reprod. 11,97-101. Ferm, V. H, Hanlon, D. P. & Urban, J. (1969) The permeability of the hamster placenta to radio¬ active cadmium. J. Embryol. exp. Morph. 22, 107-113. Hartman, R. H, Matrone, G. & Wise, G. H. (1955) Effect of high dietary manganese on hemoglobin formation. J. Nutr. 57, 429-439. Hurley, L. S., Everson, G. J. & Geiger, J. F. (1958) Manganese deficiency in rats: congenital nature of ataxia. J. Nutr. 66, 309-319. Maynard, L. S. & Cotzias, G. C. (1955) The partition of manganese among organs and intracellular organelles of the rat. J. biol. Chem. 214, 489-495. von Oettingen, W. F. (1935) Manganese: its distribution, pharmacology and health hazards. Physiol. Rev. 15, 175-201.
Ferm, Ferm,
(Received 13th November 1974) Manganese is an essential trace element in the living system. It is invariably present in its ionic form and is known to participate in a number of biological systems (Cotzias, 1958). Relatively high levels of manganese are required to manifest toxicity. Rats fed massive doses of manganese (1\m=.\73%of the dry diet) show decreased intestinal absorption of calcium and phosphorus (Chornock, Gerrant & Dutcher, 1942). Lambs on a high manganese diet had low liver iron levels and reduced haemoglobin formation (Hartman, Matrone & Wise, 1955). Chronic manganese poisoning, characterized by neurological and psychiatric disorders, is seen among workers handling manganese ores and apparently results from inhalation of manganese oxide dusts (von Oettingen, 1935). Deficiency of the element in animals causes growth retardation, bone abnormalities, degenerative changes in the central nervous and reproductive failure (Cotzias, 1958). A specific example of its importance for the developing embryo is the occurrence of ataxia in the offspring of Mn++-deficient rats (Hurley, Everson & Geiger, 1958). Mutant mice possessing the pallid gene also manifest ataxia which Erway, Hurley & Fraser (1966) found to result from partial or complete absence of otoliths in the inner ear. Hurley and her co\x=req-\ workers showed that a single supplement of Mn++, given specifically on the 14th day of gestation, completely and permanently remedied this condition. Pallid mice also respond to Mn++ supplements but once the critical point in gestation is past the defect cannot be corrected in mutant mice or in the phenocopy rats. These findings strongly suggest that Mn+ + crosses the rodent placental barrier during gestation but so far its passage has not been demonstrated. In this report, we show that the Syrian hamster placenta is permeable to 54Mn++ during the critical stages of organogénesis and have correlated this with our earlier studies of placental transport of metal ions. Timed matings of virgin female hamsters were obtained as described by Ferm (1967). Sufficient carrier-free 54MnCl2 (New England Co.) was added to 2-50 niM-MnCl in demineralized-distilled water to give a solution containing 12-0 /iCi/ml. On Day 8 of gestation, twelve hamsters received an injection into the sublingual vein of 54Mn+ + in doses of 0-5 ml/100 g body weight. On Days 9 and 12 of gestation, six females were killed by chloroform anaesthesia. At h
109
D. P. Hanlon
110
et
al.
autopsy, samples of maternal blood, liver, placenta and
uterus and embryos obtained from each animal. Due to the small size of the entire Day-9 gestation sac, it was not possible to separate the developing chorioallantoic placenta from the proximal segment of the visceral yolk sac and, for this gestation day, the term placenta refers to both the chorioallantoic and yolk sac placenta together. On Day 12, when separation was feasible, the two placentae were handled individually. All embryos in a litter from mothers on Day 9 were pooled for counting because of their small size. Day-12 embryos were counted in groups of four, together with their respective chorioallantoic and yolk sac placentae. Tissues were weighed in previously tared plastic tubes and the radio¬ activity of each sample was determined with a Nuclear Chicago Radiation Analyzer system employing a well-counter. For every sample, sufficient counts were accumulated to reduce the probable error in counting to less than 1 %. Counts per minute were converted to µg Mn+ + by comparing the radio¬ activity of the samples with a 54Mn++ standard prepared from the solution used for injections. Text-figure 1 summarizes the data on the distribution of 54Mn + + in maternal and fetal tissues of the golden hamster following injection of the metal ion on tissues 24 and 96 hr Day 8. Detectable amounts of radioactivity in embryonic after administration of the ion indicate that 54Mn+ + crosses the placental barrier. All of the maternal tissues examined showed uptake of radioactivity. The levels of 54Mn++ decreased in both embryonic and maternal tissues between Day 9 and Day 12. All decreases were statistically significant. were
9th day
^ 12th
day
rfi •S
2
S
0055 0-025
Maternal blood
Maternal liver
Uterus
Allantoic
placenta
m Embryo
Yolk
sac
placenta
Text-fig. 1. The concentrations of S4Mn++ in hamster tissues 24 hr and 96 hr after injection of the isotope. Standard errors for mean values are indicated by the vertical bars. The experimental conditions are described in the text.
111 Permeability of Syrian hamster placenta to Mn+ + Since manganese is essential for normal development, our data showing that the placental barrier is permeable to 54Mn++ during the critical stages of organogénesis in the Syrian hamster were not unexpected. We have previously shown that the placenta is permeable to 65Zn++ (Ferm & Hanlon, 1974a), and 109Cd+ + (Ferm, Hanlon & Urban, 1969) between the 8th and 9th days of gestation, but are unaware of any previous tracer studies relating directly to placental transport of Mn++ in rodents. Our finding is, however, consistent with an earlier report that otolith abnormalities in the offspring of Mn++deficient rats can be prevented by a single supplement of the ion on Day 14 of gestation (Hurley et al., 1958). The disposition and kinetics of Mn+ + in the maternal system can be compared with studies of other rodent species. On Day 9, maternal liver tissues contained 20% of the entire 54Mn++ load injected (assuming the liver is 6% of the total body weight). This is consistent with the data of Maynard & Cotzias (1955) who found that intraperitoneal injection of tracer amounts of the short-lived isotope, 56Mn+ +, resulted in a rapid and extensive uptake of label in liver and other mitochondria-rich tissues of rats. In our study, 54Mn++ also concentrated in the uterus and placenta, but to a lesser degree than in the liver. Those tissues showing greatest uptake of 54Mn++ 24 hr after injection also showed the larger % decrease in radioactivity on Day 12, e.g. the maternal liver lost 70% of its Day-9 content of 54Mn++, while blood levels decreased attributed similar findings by approximately 50%. Britton & Cotzias (1966) in the rat to a rapid change of labelled Mn+ + in parenchymatous organs +
present in the diet. At the dose level used in our Mn++ was neither teratogenic nor embryocidal. Ferm study (136 ^g/100 g), this dose is embryocidal but not teratogenic in that times ten (1972) reports hamsters. The path by which Mn+ + and other divalent metal ions reach embryonic tissues during the critical stages of organogénesis is not known. The allantoic and yolk sac placentae are obvious candidates for this transport process. The finding of Carpenter et al. (1973) that lead ions (210Pb++) concentrate in the visceral wall of the yolk sac placenta between 1 and 4 hr after injection on the 8th day of gestation in the hamster could indicate that this is the more im¬ portant route. Alternatively, the yolk sac may serve as a barrier, offering the ions. The following embryo protection from teratogenic agents by heavy metal + this view. of in The concentration 65Zn+ points support yolk sac is 1-23 times that of embryonic tissues (Ferm & Hanlon, 1974b) while the ratio for 54Mn+ +, using data reported in this paper, is 0-66. Both values are near unity and suggest the absence of any selective storage or barrier effect by the yolk sac placenta for these metal ions. On the other hand, the concentration of the teratogen, 109Cd+ +, is fifty times greater in the yolk sac than in embryonic tissues (Ferm et al., 1969). These data are for Day-12 samples, since yolk sac material on Day 9 is too scanty to assay in the manner used here. Nevertheless, they could reflect the Day-9 conditions and so are worth mentioning. As such, our results imply the existence of a protective mechanism which concentrates heavy metals in the yolk sac placenta, but which does not impede the transport of essential metal ions to embryonic tissues. when stable Mn+
was
112
D. P. Hanlon
et
al.
We are grateful to Mrs Martha L. Hill for her technical assistance and to Mrs Louise White for typing the manuscript. REFERENCES
Britton, A. A. & Cotzias, G. C. (1966) Dependence of manganese turnover on intake. Am. J. Physiol. 211, 203-206. Carpenter, S. J., Ferm, V. H. & Gale, T. F. (1973) Permeability of the golden hamster placenta to inorganic lead: radioautographic evidence. Experientia, 29, 311-313. Chornook, C, Gerrant, . . & Dutcher, R. A. (1942) Effect of manganese on calcification in the growing rat. J. Nutr. 23, 445-458. Cotzias, G. C. (1958) Manganese in health and disease. Physiol. Rev. 38, 502-532. Erway, L., Hurley, L. S. & Fraser, A. (1966) Neurological defect: manganese in phenocopy and prevention of a genetic abnormality of inner ear. Science, N.T. 152, 1766—1768. Ferm, V. H. (1967) The use of the golden hamster in experimental teratology. Lab. Anim. Care, 17,
451-462. V. H. (1972) Teratogenic effects of metal ions on mammalian embryos. Adv. Teratol. 5, 57—75. V. H. & Hanlon, D. P. (1974a) Placental transfer of zinc in the Syrian hamster during early embryogenesis. J. Reprod. Fert. 39, 49—52. Ferm, V. H. & Hanlon, D. P. (1974b) Toxicity of copper salts in hamster embryonic development. Biol. Reprod. 11,97-101. Ferm, V. H, Hanlon, D. P. & Urban, J. (1969) The permeability of the hamster placenta to radio¬ active cadmium. J. Embryol. exp. Morph. 22, 107-113. Hartman, R. H, Matrone, G. & Wise, G. H. (1955) Effect of high dietary manganese on hemoglobin formation. J. Nutr. 57, 429-439. Hurley, L. S., Everson, G. J. & Geiger, J. F. (1958) Manganese deficiency in rats: congenital nature of ataxia. J. Nutr. 66, 309-319. Maynard, L. S. & Cotzias, G. C. (1955) The partition of manganese among organs and intracellular organelles of the rat. J. biol. Chem. 214, 489-495. von Oettingen, W. F. (1935) Manganese: its distribution, pharmacology and health hazards. Physiol. Rev. 15, 175-201.
Ferm, Ferm,
