PROCEEDINGS OF THE SOCIETY FOR EXPERIMENTAL BlOLOGY AND MEDICINE
156, 68-71 (1977)
Passage of Cadmium Across the Perfused Guinea Pig Placenta’ (39877)
BRUCE J. KELMAN
AND
BIRGITTA K. WALTER
Comparative Animal Research Laboratory, Oak Ridge, Tennessee 37830
There is extensive documentation in the again so that gestational age was known literature which implicates cadmium as a within 24 hr. The animals were from an causative or contributing agent in human inbred colony at the Comparative Animal diseases including renal dysfunction, osteo- Research Laboratory in which full-term malacia, and hypertension. Very little is pregnancies last approximately 68 days. All known, however, about chronic exposures of the guinea pigs used were approximately to low levels of cadmium such as we might 60 days pregnant with a range between 59 find in cadmium-polluted environments. and 61 days. Since background levels of cadmium are The perfusion technique used in these exsteadily increasing with growing usage of the periments is similar to one previously demetal, it seems especially important to gain scribed (11). A schematic diagram of the more knowledge of the effects of cadmium perfusion system is shown in Fig. 1. The on mammalian systems (1). pregnant dam was anesthetized with a comThe widespread belief that the placenta bination of sodium pentobarbital injected ip constitutes an “effective barrier” against the and Innovar-Vet injected im. The anesthetransfer of cadmium at low concentrations tized dam, tied to a plastic rack in a supine to the developing fetus is based primarily on position, was placed in a glass tray containreports of low accumulations of cadmium in ing mammalian Ringer’s solution that covfetuses of several species (1-5). For exam- ered the lower legs and part of the posterior ple, at 12-days gestation fetuses from rats abdomen. The Ringer’s solution and perfuspreviously dosed with 0 .l-0.6 mg of Cd/kg ing solution were maintained at 39” during accumulated between 0.0001 and 0.0095% each experiment. of the maternal dose, while placentas accuThe lateral abdominal wall was incised, mulated 0.063-0.206% (6). Yet high doses one uterine horn was exteriorized, and an of cadmium (0.88-30 mg/kg maternal body incision was made through the uterine wall weight) have been teratogenic or fetotoxic and amniotic sac, exposing one or two feto hamsters (7, 8), rats (4), and mice (9), as tuses. The uterus, placentas, and fetuses well as the cause of fetal death by a peculiar were immersed in the Ringer’s solution. necrosis in the fetal vessels of the placenta Connective tissue surrounding the umbilical (10). Despite existing literature, only lim- vessels of one fetus was stripped away, the ited data are avialable on cadmium trans- vessels were tied off near the fetus, and an port across the placenta since most studies umbilical artery was cannulated with a hubof cadmium transfer to the fetus are con- less needle connected to polyethylene tubfounded by an inability to separate fetal ing. The arterial cannula was attached to uptake and placental transport. We have Tygon tubing and passed through a peristalexamined transport of cadmium in the ma- tic pump and then to a reservoir placed in ternal-fetal direction across the perfused the water bath (11). The venous cannula placenta of the guinea pig, a species with was elevated to a height that maintained the placental structure histologically similar to venous pressure at approximately its normal man. level (12). Outflow from the placenta Materials and methods. Strain 13 guinea passed through the venous cannula and was pigs were bred, allowed to farrow, and bred collected in 3-ml portions. After injection of 100 pCi of llsCd (sp act 0.42 mCi/mg) and Research sponsored by the U.S. Energy Research 5 pCi of tritiated water into the maternal and Development Administration under Contract No. jugular vein, 0.75-ml maternal blood samples were drawn from the carotid cannula at EY-76-C-05-0242 with the University of Tennessee. 68 Copyright 0 1977 by the Society for Experimental Biology and Medicine All rights reserved,
Downloaded from ebm.sagepub.com at UNSW Library on July 18, 2015
ISSN 0037-9727
69
TRANSPLACENTAL MOVEMENT OF CADMIUM
10-min intervals; five to nine samples were collected in each experiment. Immediately after collection, maternal blood samples were centrifuged and an aliquot of plasma was obtained. Perfusion pressure, maternal cardiac rate, ECG, blood pressure, and respiratory rate were monitored continuously during each perfusion; if any of these measurements varied abnormally, the data from that animal were discarded. Perfusion rates were varied between 1.15 and 3.50 ml/min. Perfusion samples were collected from 15 min after injection of radiocadmium so that sampling during the initial rapid decrease in maternal plasma radiocadmium was avoided. After a single pass, the perfusate was analyzed for radiocadmium and tritium. The perfusing solution consisted of sterile, filtered guinea pig serum with a p H of 7.35. Radiochemical analyses
I 1 Perfusate
Tr it iat ed Water Rodiolobeled C a d m i u m Matern*al Circulation
c
Peristaltic Pump
mbilical rteries
Umbilical Vein
Collection Tubes
FIG. 1. Schematic diagram of the perfusing system.
were made on triplicate samples of maternal plasma , whole blood, and perfusing solution. Clearance of tritiated water and radiolabeled cadmium from maternal blood was calculated by the following formula: Clearance = ( P / M ) R , where clearance is milliliters of maternal plasma containing an amount of radiocadmium or tritium equal to that entering the perfusate per minute (milliliters per minute), and P is the tritium or radiocadmium concentration (microcuries per milliliter) measured in the perfusate. For tritium, M is the average tritium concentration (microcuries per milliliter) as determined from a series of linear approximations of a graph of maternal tritium concentration versus time. For radiocadmium, M is the average radiocadmium concentration (microcuries per milliliter) of maternal plasma during the collection time of each sample as determined from a series of monoexponential approximations of maternal radiocadmium concentrations versus time. R is the rate of flow of the perfusion sample (milliliters per minute). Results. A total of 145 perfusion samples was collected from 10 placentas in six dams. Clearances of radiolabeled cadmium, presented in Table I , correlated significantly with perfusion rate in 9 out of 10 perfusions. Included in Table I is an estimate of radiocadmium clearance (+SE) from each perfusion at a flow rate of 2.5 ml/min. This flow
TABLE I . RELATIONSHIPS BETWEEN CADMIUM CLEARANCE AND PERFUSION RATE.
Dam 1 1 2 2 3 3 4 5 6 6 a
Placenta aa 1 0.187 2 -0.056 1 0.043 2 0.099 1 -0.122 2 0.065 1 0.128 1 -0.109 1 0.069 2 0.049
Y =a
ba 0.044 0.130 0.025 0.018 0.289 0.410 0.030 0.164 0.159 0.036
B0.05
156, 68-71 (1977)
Passage of Cadmium Across the Perfused Guinea Pig Placenta’ (39877)
BRUCE J. KELMAN
AND
BIRGITTA K. WALTER
Comparative Animal Research Laboratory, Oak Ridge, Tennessee 37830
There is extensive documentation in the again so that gestational age was known literature which implicates cadmium as a within 24 hr. The animals were from an causative or contributing agent in human inbred colony at the Comparative Animal diseases including renal dysfunction, osteo- Research Laboratory in which full-term malacia, and hypertension. Very little is pregnancies last approximately 68 days. All known, however, about chronic exposures of the guinea pigs used were approximately to low levels of cadmium such as we might 60 days pregnant with a range between 59 find in cadmium-polluted environments. and 61 days. Since background levels of cadmium are The perfusion technique used in these exsteadily increasing with growing usage of the periments is similar to one previously demetal, it seems especially important to gain scribed (11). A schematic diagram of the more knowledge of the effects of cadmium perfusion system is shown in Fig. 1. The on mammalian systems (1). pregnant dam was anesthetized with a comThe widespread belief that the placenta bination of sodium pentobarbital injected ip constitutes an “effective barrier” against the and Innovar-Vet injected im. The anesthetransfer of cadmium at low concentrations tized dam, tied to a plastic rack in a supine to the developing fetus is based primarily on position, was placed in a glass tray containreports of low accumulations of cadmium in ing mammalian Ringer’s solution that covfetuses of several species (1-5). For exam- ered the lower legs and part of the posterior ple, at 12-days gestation fetuses from rats abdomen. The Ringer’s solution and perfuspreviously dosed with 0 .l-0.6 mg of Cd/kg ing solution were maintained at 39” during accumulated between 0.0001 and 0.0095% each experiment. of the maternal dose, while placentas accuThe lateral abdominal wall was incised, mulated 0.063-0.206% (6). Yet high doses one uterine horn was exteriorized, and an of cadmium (0.88-30 mg/kg maternal body incision was made through the uterine wall weight) have been teratogenic or fetotoxic and amniotic sac, exposing one or two feto hamsters (7, 8), rats (4), and mice (9), as tuses. The uterus, placentas, and fetuses well as the cause of fetal death by a peculiar were immersed in the Ringer’s solution. necrosis in the fetal vessels of the placenta Connective tissue surrounding the umbilical (10). Despite existing literature, only lim- vessels of one fetus was stripped away, the ited data are avialable on cadmium trans- vessels were tied off near the fetus, and an port across the placenta since most studies umbilical artery was cannulated with a hubof cadmium transfer to the fetus are con- less needle connected to polyethylene tubfounded by an inability to separate fetal ing. The arterial cannula was attached to uptake and placental transport. We have Tygon tubing and passed through a peristalexamined transport of cadmium in the ma- tic pump and then to a reservoir placed in ternal-fetal direction across the perfused the water bath (11). The venous cannula placenta of the guinea pig, a species with was elevated to a height that maintained the placental structure histologically similar to venous pressure at approximately its normal man. level (12). Outflow from the placenta Materials and methods. Strain 13 guinea passed through the venous cannula and was pigs were bred, allowed to farrow, and bred collected in 3-ml portions. After injection of 100 pCi of llsCd (sp act 0.42 mCi/mg) and Research sponsored by the U.S. Energy Research 5 pCi of tritiated water into the maternal and Development Administration under Contract No. jugular vein, 0.75-ml maternal blood samples were drawn from the carotid cannula at EY-76-C-05-0242 with the University of Tennessee. 68 Copyright 0 1977 by the Society for Experimental Biology and Medicine All rights reserved,
Downloaded from ebm.sagepub.com at UNSW Library on July 18, 2015
ISSN 0037-9727
69
TRANSPLACENTAL MOVEMENT OF CADMIUM
10-min intervals; five to nine samples were collected in each experiment. Immediately after collection, maternal blood samples were centrifuged and an aliquot of plasma was obtained. Perfusion pressure, maternal cardiac rate, ECG, blood pressure, and respiratory rate were monitored continuously during each perfusion; if any of these measurements varied abnormally, the data from that animal were discarded. Perfusion rates were varied between 1.15 and 3.50 ml/min. Perfusion samples were collected from 15 min after injection of radiocadmium so that sampling during the initial rapid decrease in maternal plasma radiocadmium was avoided. After a single pass, the perfusate was analyzed for radiocadmium and tritium. The perfusing solution consisted of sterile, filtered guinea pig serum with a p H of 7.35. Radiochemical analyses
I 1 Perfusate
Tr it iat ed Water Rodiolobeled C a d m i u m Matern*al Circulation
c
Peristaltic Pump
mbilical rteries
Umbilical Vein
Collection Tubes
FIG. 1. Schematic diagram of the perfusing system.
were made on triplicate samples of maternal plasma , whole blood, and perfusing solution. Clearance of tritiated water and radiolabeled cadmium from maternal blood was calculated by the following formula: Clearance = ( P / M ) R , where clearance is milliliters of maternal plasma containing an amount of radiocadmium or tritium equal to that entering the perfusate per minute (milliliters per minute), and P is the tritium or radiocadmium concentration (microcuries per milliliter) measured in the perfusate. For tritium, M is the average tritium concentration (microcuries per milliliter) as determined from a series of linear approximations of a graph of maternal tritium concentration versus time. For radiocadmium, M is the average radiocadmium concentration (microcuries per milliliter) of maternal plasma during the collection time of each sample as determined from a series of monoexponential approximations of maternal radiocadmium concentrations versus time. R is the rate of flow of the perfusion sample (milliliters per minute). Results. A total of 145 perfusion samples was collected from 10 placentas in six dams. Clearances of radiolabeled cadmium, presented in Table I , correlated significantly with perfusion rate in 9 out of 10 perfusions. Included in Table I is an estimate of radiocadmium clearance (+SE) from each perfusion at a flow rate of 2.5 ml/min. This flow
TABLE I . RELATIONSHIPS BETWEEN CADMIUM CLEARANCE AND PERFUSION RATE.
Dam 1 1 2 2 3 3 4 5 6 6 a
Placenta aa 1 0.187 2 -0.056 1 0.043 2 0.099 1 -0.122 2 0.065 1 0.128 1 -0.109 1 0.069 2 0.049
Y =a
ba 0.044 0.130 0.025 0.018 0.289 0.410 0.030 0.164 0.159 0.036
B0.05
