Placental transmission of mercury to the fetal rat

TOXICOLOGY

AND APPLIED PHARMACOLOGY

Placental

Transmission

22, 649-654 (1972)

of Mercury

to the Fetal Rat1

N. E. GARRETT, R. J. BURRISSGARRETT~ AND J. W. ARCHDEACON Department of Physiology and Biophysics, The Medical Center, University of Kentucky, Lexington, Kentucky 40506 Received November 4,197l

Placental Transmission of Mercury to the Fetal Rat. GARRETT, N. E., R. J. BURRISS, and ARCHDEACON, J. W. (1972). Toxicol. Appl. Phurmucol. 22,649-654. 203HgC12was administered to the Sprague-Dawley pregnant rat (day 16) by intracardiac injection, and after 30 min measurement was made of uptake of the isotope at placental and fetal sites. Both the chorioallantois and the visceral yolk sac concentrated mercury, but there was little transmission of the metal to the fetus. Fetal and placental accumulations were also monitored after depriving the visceral yolk sac of 1 possible source of mercury, secretions in the uterine lumen. This was accomplished by cutting the uterine mucosa and allowing it to retract to the base of the chorioallantois. Retraction produced no significant effect on mercury uptake by fetal and placental tissues.From in vitro experiments the absorptive capacity of the visceral yolk sac for mercury on a unit weight basis was shown to be about 10 times greater than that of the chorioallantois. Sodium selenite (1 mM) stimulated about 100% increase in mercury accumulation in both tissues.Intracellular partitioning studies after in vivo administration of 203Hg showed that the metal was taken up prominently in the fraction containing the stroma, indicating a strong placental cell surface binding affinity for mercury. GARRETT,

The placenta has a dual transport function in that it facilitates the passage of some materials to the fetus while acting as a barrier to other materials. There is little information available about the mechanisms involved in this discriminatory function. The present study has been conducted in the pregnant rat on day 16 to determine (1) the

extent to which the placenta transmits mercury to the fetus; (2) whether mercury, if present in fluids in the uterine lumen, can be transmitted by the visceral yolk sac to the

fetus; (3) the subcellular distribution of mercury in placental cells; and (4) the effects of sodium arsenite, sodium citrate and sodium selenite on in vitro mercury uptake by placental tissues. There is evidencethat the placenta has a discriminating transport function for nonessential elements. Schulert et al. (1969) studied rat placental uptake of nuclides, including 203Hg. They found that the placenta had a retentive filtering action on nonessentialelements,including mercury, and that the more mature fetus was better protected from these elementsthan the early fetus. ’ This work was supported by U.S. Public Health Service Grant HD05016 and NIH Predoctoral Fellowship, Training Grant FOl-DE41063 to R.J.B.G. ’ Present address: College of Pharmacy, The Medical Center, University of Kentucky. Copyright 0 1972 by Academic Press, Inc. 649 All rights of reproduction in any form reserved.

650

GARRETT,

BURRISS GARRETT,

AND ARCHDEACON

Certain chemical compounds may exert a protective action, inhibiting mercury accumulation by the fetus. Parizek et al. (1969) reported that sodium selenite increases the retention of mercury in the mother and decreasesmarkedly its passageto the fetus. Selenite did not cause a significant change in *03Hg content of the placenta but increased the concentration of the isotope in the maternal liver. METHODS

Gravid Sprague-Dawley rats (Holtzman Co., Madison, Wisconsin) on day 16 of gestation were the experimental animals. The sperm positive date indicated by the suppliers was designated day 1 of pregnancy. Parturition occurred on day 22. Anesthesia was maintained with sodium pentobarbital. After the animal was positioned on a warming pad, the uterine horns were exteriorized through a ventral midline incision from the symphysis pubis to the epigastric region. Approximately 4 of the fetal-placental units were “exposed” as follows. At each implantation site the uterine wall was incised antimesometrically and allowed to invert and retract spontaneously to the sides and base of the chorioallantois. Thus the endometrial mucosa was no longer in contact with the visceral yolk sac which in the rat underlies the endometrium on day 16. The remaining fetal-placental units served as controls. The uterine horns were supported and covered with paper tissue wipes wetted with sodium chloride. Each rat received by careful intracardial injection 1 ml of rat serum which had been allowed to stand 1 hr at 23°C with 2 &i of *03HgC1,(specific activity 5.6 mCi/mg; New England Nuclear). At 30 min after injection the exposed and control fetal-placental units were resected. The chorioallantois, visceral yolk sac and fetus with amnion were separated, weighed and counted for radioactivity with a NaI(TI) crystal well-type scintillation counter (NuclearChicago). Paper tissues surrounding the horns contained negligible radioactivity. The chorioallantois and visceral yolk sac were treated as follows to obtain data on intracellular distribution of mercury. Thirty minutes after intracardial injection of *03HgC12the structures were removed from the uterine horns, radiomonitored and then frozen. They were later thawed, minced and homogenized in isotonic NaCl in a Teflon homogenizer (2000 rpm, 10 complete strokes). All operations were performed at 2-5°C. The homogenate was centrifuged in the International Model PR-2 refrigerated centrifuge and the Beckman Model L-2 ultracentrifuge, as outlined in the flow chart in Fig. 1. The fractions were designated mitochondrial (P& microsomal (P4) and supernatant (S,). In in vitro experiments the fetal-placental unit was removed from the implantation site and the components separated. The chorioallantois was cut into about 10 slices by razor blade, but the visceral yolk sac was left intact. The tissues were placed separately into tared flasks containing modified Ringer-Tris solution (130 mM NaCl, 3 mM KCl, 1 mM CaCl,, 19 mM Tris[2-amino-2-(hydroxymethyl)-I,3 propanediol], 0.02 % glucose, pH adjusted to 7.4 with HCl. Each flask was weighed and any additional compound (sodium selenite, 1 mM; sodium arsenite, 5 mM; sodium citrate, 5 mM) was added at this time. All flasks were agitated in a water bath shaker 10 min at 37°C. Mercury was added as 2 $Zi of *03HgC12with nonlabeled HgC12so that the final concentration of the metal was 0.92 pg/ml in a total flask volume of 10 ml. The tissues were incubated 1 hr and measurement ofZo3Hg uptake was made at 30 and 60 min. After removal from the flask

PLACENTAL

651

MERCURY TRANSMISSION

Is eLkK%L 2 IOmin , s Mom*9 -160min,

3

‘54

FIG. 1. Flow chart for intracellular fractionation of chorioallantoic and visceral yolk sac tissues. P and S refer to homogenate,pellet and supernatant,respectively.Fractions cited in text are mitochondrial (P6), microsomal (P4) and supematant (S,). H,

the tissues were washed 5 times with 10 ml aliquots of the modified Ringer-Tris solution

before radioactive counting. RESULTS

The bar graph in Fig. 2 represents radioactive counts of 203Hgon a unit weight basis in the components of control and exposed fetal-placental units. Uptake of 203Hgwas not changed significantly by retraction of the endometrial envelope. The strong barrier maintained by the placenta against mercury transmission to the fetus was manifested, The low radioactive counts were identical (0.016 x lo4 counts/min/g or 0.023 rig/g) in exposed and control fetuses. Mercury uptake in the control chorioallantois and visceral yolk sac was 1.68 rig/g and 0.83 rig/g, respectively. On a unit weight basis the chorioallantoic to fetal count ratio was approximately 75.

2.5 I I

n Control Exposed

Chorioollantoir

Yolk

Sac

Fetus

FIG. 2. 703Hgaccumulation in control and exposedfetal-placentalcomponentson a unit wet weight basis in in vivo studies. Bars represent SD from the mean (n = 12).

652

GARRETT,BURRISS GARRETT,AND

ARCHDEACON

TABLE 1 INTRACELLULAR PARTITIONING OF 203H~ IN THE RAT CHORIOALLANTOIS AND VISCERAL YOLK SAC 30 MIN AFTER MATERNAL INTRACARDIAL INJECTION’

Exp. No.* 1 2 3 1 2 3

Stroma,cell nuclei, debris, Mitochondria Microsomes etc. Homogenate (PC4 (Pa) 26,270 32,120 30,815

Chorioallantois 7,768 (29 “/,) 3,118 (11%) 9,878 (30 %) 5,248 (16 %) 8,907 (28 %) 4,700 (15 %I

2,083 531 664

Visceral yolk sac 582 (27%) 90 (4 %I 105 (19 %) 35 (6%) 138 (20%) 61 (9%)

992 (3 %I

1,628 (5 %I 1,965 (6 %I 32 (1%) 0 (0%) 28 (4%)

Supernatant cw 13,351 (50%) 14,564 (45 %)

12,849(41%) 1,241 (59 %) 283 (53%) 410 (61%)

a Data expressed as counts/min and % of homogenate count. c Tissue obtained from 6 structures.

The homogenate and partitioning data for 3 chorioallantoic and visceral yolk sac experiments are shown in Table 1. Radioactive counts are expressed also as % of homogenate counts. The visceral yolk sac homogenate activity was much lower than chorioallantoic activity. A larger % of the radioactive counts was in the supernatant (S,) of the visceral yolk sac than in the chorioallantois, whereas chorioallantoic mitochondria and microsomes had higher counts. In vitro uptake of mercury on a unit weight basis by the chorioallantoic and yolk sac tissues is presentedgraphically in Fig. 3a, b. The values are expressedas total mercury,

/’ No-SehM)

30 -Min

,/

/I

60 Min

. 3 . ‘O’Hg accumulation in chorioallantoic (CA) and visceral yolk sac (YS) tissues on a unit wet weight basis after addition of chemical compounds in in vitro studies. Bars represent SD from the mean (generally 5 experiments). FIG

PLACENTAL

MERCURY

TRANSMISSION

653

radiolabeled and unlabeled. The visceral yolk sac had a much greater affinity for the metal: in an equal time it accumulated 10 times more mercury than the chorioallantois. Addition of sodium selenite (1 mM) approximately doubled mercury concentration in each tissue. Sodium arsenite (5 mM) stimulated 203Hg uptake in each tissue but not as markedly as selenite.Sodium citrate (5 mM), a metal chelator, was moderately effective in inhibiting mercury binding to chorioallantoic tissue. Under control conditions, averagerates of mercury uptake (ng/mg/min) for the visceral yolk sacwere 8.9 from 0 to 30 min and -0.1 from 30 to 60 min while the corresponding rates for the chorioallantois were 0.82 and 0.11, respectively. DISCUSSION

The results demonstrated clearly that the rat chorioallantois and visceral yolk sac took up and accumulated radioactive mercury from maternal blood. However, these structures restricted passageof the metal: either they had low ability to transmit mercury to the fetus, or the fetus resisted uptake, a property it may have acquired during development, as Schulert et al. (1969) observed with nonessential elements. The higher 203Hgradioactive counts in the chorioallantois, in contrast to the visceral yolk sac, shown in Fig. 2 and Table 1, reflect the strategic anatomic position of the chorioallantois in the maternal circulation. Apparently the isotope was initially presentedto and entrappedby the chorioallantois, the main route for materials in maternal blood to reach the fetus. The in vitro data in Fig. 3 demonstrate that the visceral yolk sac on a unit weight basis can accumulate mercury much better than the chorioallantois under similar exposureto the metal. Also it is evident that both the chorioallantois and visceral yolk sac accumulated much more mercury in vitro than in the in vivo study. When 1 mM sodium selenitewas added in vitro the effect was to raise the level of 203Hg in both chorioallantois and yolk sac. As stated earlier, Parizek et al. (1969)found that selenite exerted a protective action against passageof mercury to the fetus. However, they did not find that the mercury level in the placenta in vivo was raised. Conceivably the rat visceral yolk sac on day 16 of gestation could accumulate mercury, if presentin uterine secretions,and transmit it to the fetus. In the yolk sacplacenta the visceral wall is separatedfrom the parietal wall by a fluid-filled region. The decidua capsularis,through which maternal blood flows, lies above the parietal wall (Nylander, 1953). On day 16 the parietal wall and the capsularis rupture, following which the visceral yolk sac lies directly beneaththe endometrial mucosa (Wislocki and Dempsey, 1945).The results of our experimentsin which the uterine wall was retracted about the fetal-placental unit argue strongly against mercury being in uterine secretionsor being absorbed by the visceral yolk sac from these secretions on day 16. The rather high concentrations of ‘03Hg in the intracellular fraction containing the stroma suggestsa strong binding affinity of the placental cell surface for mercury. Demis and Rothstein (1955) concluded that the binding of mercury to rat diaphragm has 2 rate constants : a fast constant followed by a slow one. Calculation of the rates of mercury uptake in our experiments indicates a similar 2 step process in binding of mercury by placental cells. Our experiments show that inorganic mercury was well representedin all subcellular compartments except the microsomes,in which only relatively small amounts of the metal were found. Norseth (1968) studied intracellular distribution in the rat liver cell after a single injection of mercuric chloride. The metal

654

GARRETT,

BURRISS GARRETT,

AND ARCHDEACON

accumulated in all cell fractions; however, the uptake was transitory in mitochondria and microsomes, whereas lysosomes and peroxisomes retained mercury. In conclusion, the placenta seemingly constitutes a strong barrier to transmission of mercury to the fetus. However, McClain and Becker (1970) have shown that small amounts of lead transported across the placenta can cause teratogenic effects in rats. Under our experimental conditions concentrations of mercury in the chorioallantois and the visceral yolk sac were approximately 73 and 36 times greater, respectively, than in the fetus, indicating a strong retentive ability of these structures for mercury. Nevertheless we cannot conclude that this barrier function gives adequate protection to the fetus from toxic effects of mercury. REFERENCES DEMIS,D. J., and ROTHSTEIN, A. (1955).Relationship of the cell surfaceto metabolism.XII. Effect of mercury and copper on glucoseuptake and respiration of rat diaphragm. Amer. J. Physiol. 180, 566-574.

R. M., and BECKER, B. A. (1970).Placentaltransport and teratogenicityof lead in rats and mice. Fed. Proc., Fed. Amer. Sot. Exp. Biol. 29, 347. NORSETH, T. (1968). The intracellular distribution of mercury in rat liver after a single injection of mercuric chloride. Biochem. Pharmacol. 17, 581-593. NYLANDER, G. (1953). On the placental transfer of iron, an experimental study in the rat. Acfa Physiol. Stand. 29, Suppl. 107, l-105. PARIZEK, J., BABICKY, A., OSTADALOVA, I., and PAVILK, L. (1969). Effect of seleniumcompounds on the cross-placentalpassageof *03Hg.In : Radiation Biology of the Fetaland Juvenile Mammal (M. R. Sikov and D. D. Mahlum, eds.), U.S.A.E.C., Division of Technical Information, pp. 137-143. SCHULERT, A. R., GLASSER, S. R., STANT, E. G., JR., BRILL, A. B., KOSHAKJI, R. P., and MANSOUR, M. M. (1969). Development of placental discrimination among homologous elements.In : Radiation Biology of the Fetal and Juvenile Mammal, (M. R. Sikov and D. D. Mahlum, eds.), U.S.A.E.C., Division of Technical Information, pp. 145-152. WISLOCKI, G. B., and DEMPSEY, E. W. (1945).Histochemicalreactionsof the endometriumin pregnancy.Amer. J. Anat. 77, 365-403. MCCLAIN,