Pharmacology & Toxicology 1992, 71, 19-23.
The Influence of Mercury on the Secretion of Human Chorionic Gonadotropin in Superfused Young Placental Tissue W. Y. Boadi’, R. Shurtz-Swirski’, E. R. Barnea’, J. Urbaeh’, J. M. Brandes’ and S. Yannai’*
‘Department of Food Engineering and Biotechnology, Technion - Israel Institute of Technology, Haifa 32000, ’Department of Obstetrics and Gynecology, Rambam Medical Center, and the Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion - Israel Institute of Technology, Haifa 31096, and 3FetoPlacental Endocrine Unit, Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa 31096, Israel (Received August 19, 1991; Accepted December 13, 1991) Abstract: The effect of mercury (Hg), as HgCI,, in levels ranging from 0.75 to 12 pg/ml medium, on the secretion of human chorionic gonadotropin (hCG) was examined in first-trimester placental explants, after 6 or 24 hr incubation, employing both static and dynamic systems (the latter by superfusion). Later the unbound Hg was washed for 45 min. with fresh medium devoid of Hg, followed by superfusion with the latter medium for 75 min., during which time samples were collected for hCG assay. For the superfusion experiments the parameters used for evaluating the hCG secretion pattern were: mean peak amplitude, pulse frequency and the area under the hCG secretion curve (AUC). The results observed after 24 hr incubation indicate that in the dynamic system the hCG secretion increased significantly, and this increase was dose-dependent. There was also a dose-related increase in mean total hCG secreted by the explants exposed to Hg. Maximal hCG secretion was observed after 24 hr exposure of explants to 6 pg of the metal/ml. Both the mean peak amplitude and AUC parameters showed a statistically-significant increase for this dose level. At 12 pgiml, the pulsatile secretion of hCG decreased, but the value for the mean hCG secretion was still higher than that observed for 0.75 and 3 pg/ml. After 6 hr incubation, however, there were no significant changes from the control, as judged by all of the above parameters. The levels of hCG secreted by the explants into the media in the static system were not significantly different from their respective controls, for both incubation periods and Hg levels. These results may reflect an altered de n o w synthesis, or increased production of hCG granules, resulting in enhanced hCG secretion, brought about by exposure to Hg.
The after-effects of exposure to all forms of mercury (Hg) are serious. Depending on the form (Hg++or CH3Hg+,the latter being the most toxic form of the metal) and dose exposed to, one can develop a CH,Hg+-induced neuropathy or Hg++-induced nephropathy (Clarkson & Marsh 1982). It has been known for a long time that all Hg compounds are highly toxic to the growing foetus. Observations following maternal CH,Hg+ poisoning in Japan (Murckami 1971) revealed that the human foetus is particularly susceptible. Studies using Hg+ show that this inorganic form seems to be absorbed more by placental tissue than by the foetus. In rats and mice as much as 75 times more radiolabeled Hg is found in placentae than in foetuses per unit weight (Garrett et al. 1972). Thus, Hg appears to induce placental injury and to alter placental function in the rodent and human. The placenta is a unique endocrine organ which secretes, among other hormones, human chorionic gonadotropin (hCG). hCG has been shown to be a major marker of placental function during the first trimester (Simpson & MacDonald 1981). Unlike other placental hormones, hCG secretion follows a peculiar pattern, increasing for the first eight weeks of gestation, thereafter reaching a plateau. After 11 weeks, the level of hCG decreases and remains low until term (Kaplan & Grumbach 1978). Until recently most +
* To whom correspondence should be directed
studies on hCG regulation were carried out at term, using static cultures. It has recently been reported that hCG secretion by superfused placental explants in the first trimester is pulsatile and resembles the pattern seen in vivo (Owens et al. 1981). It is modulable by various hormones, such as gonadotropin releasing hormone (GnRH) and progesterone (Barnea & Kaplan 1989), opioid peptides (Barnea et al. 1991a) and growth factors (Barnea et al. 1990). However, we are not aware of any reports indicating the effect of toxic metals on the secretion of this hormone. This may be of relevance in view of the present-day occupational and environmental exposure of the general population to Hg. The purpose of the present study was to investigate the influence of Hg+ on the secretion of hCG in the superfused first-trimester human placenta. +
Materials and Methods Tesr chemicals. hCG kit MAIA clone was purchased from Serono (Rehovot, Israel). Mercury (as HgCI,) was purchased from Merck. Antibiotic solution (containing 10,000 units of penicillin/ml, 10 mg of streptomycin/ml and 25 pg of amphotericin/ml) and Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/l glucose but without L-glutamate, were purchased from Biological Industries, Kibbutz Beit Haemek, Israel. Placenta preparation. Five (7 to 9 week old) placentae were obtained by appropriate consent after elective pregnancy terminations per-
20
W.Y. BOADI ET A L .
formed at the Rambam Medical Center. Placentae were from healthy women, who did not use any medication and were nonsmokers. Tissues were rinsed copiously in cold sterile saline (0.9% NaCI) to remove the blood. They were further rinsed with DMEM containing 2% antibiotic solution. Incubation. The procedure employed was as previously reported (Barnea & Kaplan 1989; Barnea ef a/. 1990). In short, explants (50-70 mg wet weight) were dissected out from each placenta under sterile conditions and incubation was carried out in 6-well Corning dishes (Bibby Science Products, England), with each well containing 2 ml of the DMEM and 1% antibiotic solution, with 0, 0.75, 1.5, 3, 6 and 12 pg Hg (as HgCI,) per ml. Explants (6 to 8 for each dose level) were incubated for 6 or 24 hr in a C0,-Auto-Zero-Haraeus incubator (U.S.A.) in an atmosphere of 95"/;1 air and 5% CO,, at 37 , At the end of incubation, the media from the static cultures were collected and stored at -20" until assay. The tissue was saved for protein analysis. The viability status of the explants, was tested by the following parameters, both at the start and at the end of each incubation period: a. Glucose consumption, indicated by its disappearance rate from the media, by the enzymatic procedure described in Sera-Pak Kit (Miles, England). b. Lactate dehydrogenase activity in the media (used as a parameter for evaluating tissue viability by Roth (1981), as described by Zimmerman & Weinstein (1956). Superfirsion. The method used was as recently published (Barnea & Kaplan 1989; Barnea et a/. 1990). Briefly, a superfusion apparatus (Accusyst, Endotronics, St. Paul, MN, U.S.A.) with a multichannel peristaltic pump and fraction collector (Model 272, ISCO, Durham, NC, U.S.A.) were used to study the short-term dynamics of hCG secretion. Following incubation as described above, the explants (i.e., the control and treated samples) were placed into the culture chambers and a DMEM solution containing HEPES (18 mM) was washed (in order to remove all remaining free Hg) through in an atmosphere of 95% air and 5% CO,, at 37 for 45 min. Later, the effluent was discarded and the explants were further superfused for 75 min.. during which 1 ml samples from each of the treated and control tissue samples of the effluents were collected by the fraction collector every 2.4 min., for hCG determination. In each experiment, one chamber served as control and the remaining five were used for the treated samples. Subsequently, the samples were stored at - 2 0 until assay.
0.00 0.75
1.50
3.00 6.00 12.00
Hg DOSE (pg/ml) Fig. 1. P-hCG secretion by 7-9 weeks trophoblast explants incubated for 6 hr in static cultures with various concentrations of Hg. Data are expressed as mIU/mg protein k S.D.
concentrations, are shown in figs. 1 and 2, respectively. The data represent the results for 5 placentae, with 6 to 8 explants from each, for every Hg dose or control. The results indicate that Hg did not have any effect on hCG secretion during any of the two culturing periods. Full viability of the placental explants was retained throughout the entire superfusion period, as judged by all the viability parameters used. Superfusion. The data given in fig. 3 (panels A to E) show the effect of incubating explants in a medium containing Hg for 24 hr, followed by superfusion, on the pulsatile pattern of hCG secretion. The effect of Hg on the hCG secretion was dosedependent in the range 0.75 to 6 pg/ml (P
The Influence of Mercury on the Secretion of Human Chorionic Gonadotropin in Superfused Young Placental Tissue W. Y. Boadi’, R. Shurtz-Swirski’, E. R. Barnea’, J. Urbaeh’, J. M. Brandes’ and S. Yannai’*
‘Department of Food Engineering and Biotechnology, Technion - Israel Institute of Technology, Haifa 32000, ’Department of Obstetrics and Gynecology, Rambam Medical Center, and the Rappaport Family Institute for Research in the Medical Sciences, Faculty of Medicine, Technion - Israel Institute of Technology, Haifa 31096, and 3FetoPlacental Endocrine Unit, Rappaport Family Institute for Research in the Medical Sciences, Technion - Israel Institute of Technology, Haifa 31096, Israel (Received August 19, 1991; Accepted December 13, 1991) Abstract: The effect of mercury (Hg), as HgCI,, in levels ranging from 0.75 to 12 pg/ml medium, on the secretion of human chorionic gonadotropin (hCG) was examined in first-trimester placental explants, after 6 or 24 hr incubation, employing both static and dynamic systems (the latter by superfusion). Later the unbound Hg was washed for 45 min. with fresh medium devoid of Hg, followed by superfusion with the latter medium for 75 min., during which time samples were collected for hCG assay. For the superfusion experiments the parameters used for evaluating the hCG secretion pattern were: mean peak amplitude, pulse frequency and the area under the hCG secretion curve (AUC). The results observed after 24 hr incubation indicate that in the dynamic system the hCG secretion increased significantly, and this increase was dose-dependent. There was also a dose-related increase in mean total hCG secreted by the explants exposed to Hg. Maximal hCG secretion was observed after 24 hr exposure of explants to 6 pg of the metal/ml. Both the mean peak amplitude and AUC parameters showed a statistically-significant increase for this dose level. At 12 pgiml, the pulsatile secretion of hCG decreased, but the value for the mean hCG secretion was still higher than that observed for 0.75 and 3 pg/ml. After 6 hr incubation, however, there were no significant changes from the control, as judged by all of the above parameters. The levels of hCG secreted by the explants into the media in the static system were not significantly different from their respective controls, for both incubation periods and Hg levels. These results may reflect an altered de n o w synthesis, or increased production of hCG granules, resulting in enhanced hCG secretion, brought about by exposure to Hg.
The after-effects of exposure to all forms of mercury (Hg) are serious. Depending on the form (Hg++or CH3Hg+,the latter being the most toxic form of the metal) and dose exposed to, one can develop a CH,Hg+-induced neuropathy or Hg++-induced nephropathy (Clarkson & Marsh 1982). It has been known for a long time that all Hg compounds are highly toxic to the growing foetus. Observations following maternal CH,Hg+ poisoning in Japan (Murckami 1971) revealed that the human foetus is particularly susceptible. Studies using Hg+ show that this inorganic form seems to be absorbed more by placental tissue than by the foetus. In rats and mice as much as 75 times more radiolabeled Hg is found in placentae than in foetuses per unit weight (Garrett et al. 1972). Thus, Hg appears to induce placental injury and to alter placental function in the rodent and human. The placenta is a unique endocrine organ which secretes, among other hormones, human chorionic gonadotropin (hCG). hCG has been shown to be a major marker of placental function during the first trimester (Simpson & MacDonald 1981). Unlike other placental hormones, hCG secretion follows a peculiar pattern, increasing for the first eight weeks of gestation, thereafter reaching a plateau. After 11 weeks, the level of hCG decreases and remains low until term (Kaplan & Grumbach 1978). Until recently most +
* To whom correspondence should be directed
studies on hCG regulation were carried out at term, using static cultures. It has recently been reported that hCG secretion by superfused placental explants in the first trimester is pulsatile and resembles the pattern seen in vivo (Owens et al. 1981). It is modulable by various hormones, such as gonadotropin releasing hormone (GnRH) and progesterone (Barnea & Kaplan 1989), opioid peptides (Barnea et al. 1991a) and growth factors (Barnea et al. 1990). However, we are not aware of any reports indicating the effect of toxic metals on the secretion of this hormone. This may be of relevance in view of the present-day occupational and environmental exposure of the general population to Hg. The purpose of the present study was to investigate the influence of Hg+ on the secretion of hCG in the superfused first-trimester human placenta. +
Materials and Methods Tesr chemicals. hCG kit MAIA clone was purchased from Serono (Rehovot, Israel). Mercury (as HgCI,) was purchased from Merck. Antibiotic solution (containing 10,000 units of penicillin/ml, 10 mg of streptomycin/ml and 25 pg of amphotericin/ml) and Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/l glucose but without L-glutamate, were purchased from Biological Industries, Kibbutz Beit Haemek, Israel. Placenta preparation. Five (7 to 9 week old) placentae were obtained by appropriate consent after elective pregnancy terminations per-
20
W.Y. BOADI ET A L .
formed at the Rambam Medical Center. Placentae were from healthy women, who did not use any medication and were nonsmokers. Tissues were rinsed copiously in cold sterile saline (0.9% NaCI) to remove the blood. They were further rinsed with DMEM containing 2% antibiotic solution. Incubation. The procedure employed was as previously reported (Barnea & Kaplan 1989; Barnea ef a/. 1990). In short, explants (50-70 mg wet weight) were dissected out from each placenta under sterile conditions and incubation was carried out in 6-well Corning dishes (Bibby Science Products, England), with each well containing 2 ml of the DMEM and 1% antibiotic solution, with 0, 0.75, 1.5, 3, 6 and 12 pg Hg (as HgCI,) per ml. Explants (6 to 8 for each dose level) were incubated for 6 or 24 hr in a C0,-Auto-Zero-Haraeus incubator (U.S.A.) in an atmosphere of 95"/;1 air and 5% CO,, at 37 , At the end of incubation, the media from the static cultures were collected and stored at -20" until assay. The tissue was saved for protein analysis. The viability status of the explants, was tested by the following parameters, both at the start and at the end of each incubation period: a. Glucose consumption, indicated by its disappearance rate from the media, by the enzymatic procedure described in Sera-Pak Kit (Miles, England). b. Lactate dehydrogenase activity in the media (used as a parameter for evaluating tissue viability by Roth (1981), as described by Zimmerman & Weinstein (1956). Superfirsion. The method used was as recently published (Barnea & Kaplan 1989; Barnea et a/. 1990). Briefly, a superfusion apparatus (Accusyst, Endotronics, St. Paul, MN, U.S.A.) with a multichannel peristaltic pump and fraction collector (Model 272, ISCO, Durham, NC, U.S.A.) were used to study the short-term dynamics of hCG secretion. Following incubation as described above, the explants (i.e., the control and treated samples) were placed into the culture chambers and a DMEM solution containing HEPES (18 mM) was washed (in order to remove all remaining free Hg) through in an atmosphere of 95% air and 5% CO,, at 37 for 45 min. Later, the effluent was discarded and the explants were further superfused for 75 min.. during which 1 ml samples from each of the treated and control tissue samples of the effluents were collected by the fraction collector every 2.4 min., for hCG determination. In each experiment, one chamber served as control and the remaining five were used for the treated samples. Subsequently, the samples were stored at - 2 0 until assay.
0.00 0.75
1.50
3.00 6.00 12.00
Hg DOSE (pg/ml) Fig. 1. P-hCG secretion by 7-9 weeks trophoblast explants incubated for 6 hr in static cultures with various concentrations of Hg. Data are expressed as mIU/mg protein k S.D.
concentrations, are shown in figs. 1 and 2, respectively. The data represent the results for 5 placentae, with 6 to 8 explants from each, for every Hg dose or control. The results indicate that Hg did not have any effect on hCG secretion during any of the two culturing periods. Full viability of the placental explants was retained throughout the entire superfusion period, as judged by all the viability parameters used. Superfusion. The data given in fig. 3 (panels A to E) show the effect of incubating explants in a medium containing Hg for 24 hr, followed by superfusion, on the pulsatile pattern of hCG secretion. The effect of Hg on the hCG secretion was dosedependent in the range 0.75 to 6 pg/ml (P
