ENVIRONMENTAL RESEARCH 51, 83-90 (1990)
Uptake and Distribution of Cd in the Ovaries, Adrenals, and Pituitary in Pseudopregnant Rats: Effect of Cd on Progesterone Serum Levels K A T A L I N PAKSY, M I K L r S N.~RAY, BERTALAN VARGA, IMRE KISS,
G~OR FOLLY, AND GYORGYUNOV~RY National Institute of Occupational Health, P.O. Box 22, Budapest 1450, Hungary Received November 1, 1988 Pseudopregnant (PSP) rats were treated with 3.5 or 7.0 mg/kg body wt of CdC12 on Day 1 of PSP sc. In the lower dose Cd content of the ovaries (luteal and nonluteal tissues), adrenals, pituitary, and blood on Days 1, 2, 5, 8, 10, and 12, and in the higher dose that of luteal and nonluteal tissue on Days 2 and 5 of PSP were determined with atomic absorption spectrophotometry. A rapid incorporation into the corpora lutea was measured on Day 1 and Day 2 of PSP followed by a decrease of Cd content toward the end of PSP whereas the nonluteal tissue, adrenals, and pituitary accumulated Cd gradually until the fifth to 10th day, respectively. Progesterone (P) serum levels were measured with RIA in the blood collected daily from the jugular vein following administration of 3.5 to 7.0 mg/kg body wt of CdC12 sc on Day 1 or Day 8 of PSP. The serum levels of P remained unchanged when CdC12 was administered on Day 1 of PSP; however, 7.0 mg/kg body wt CdC12 given on Day 8 of PSP induced a significant decrease in serum levels of P. It is supposed that the regressing luteal tissue is more sensitive to the toxic effects of Cd than the developing one. © 1990Academic Press, Inc.
INTRODUCTION Cd is known to induce permanent sterility of male laboratory rodents: damaging the capillary network in testicles it destroys the germinal epithelium (reviewed by Setchell, 1978). In females the toxic effect of Cd depends on the continuously changing function of the ovary throughout the lifetime. In anovulatory animals, which are prepuberal (Kar, 1959) or in persistent estrus (Parizek et al., 1968), Cd causes dramatic but transient vascular lesions. In adult cycling female rats (Parizek et aL, 1968) and mice (Suter, 1985) no fertility impairment was proven, while in hamsters (Saksena and Salmonsen, 1983) and rabbits (Saksena, 1982) a pronounced but temporary infertility by disturbance of ovulation and egg transport occurred following a single Cd injection. A single sc CdC12 dose partially inhibited the ovulation and caused dose-dependent changes in the luteal function lowering the basal secretion rate of progesterone (P) and decreasing the response to hCG stimulus (PaLsy et al., 1989). The aim of the present work was to study the Cd uptake and accumulation of ovary (luteal and nonluteal tissue) and adrenal and pituitary gland. Furthermore in a separate experiment the effects of Cd on blood levels of P were determined in pseudopregnant rats.
MATERIALS AND METHODS Animals The experiments were performed on mature female CFY rats (LATI, G6d6116, 83 00130351/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
84
PAKSY ET AL.
Hungary) weighing 220-260 g. They were kept under controlled environmental conditions with a 12-hr light/12-hr dark photoperiod. Free access was provided to standard laboratory pellets and tap water. The ovarian cycle was checked daily by vaginal smear. In animals, having at least three regular cycles, pseudopregnancy (PSP) was induced by mechanical stimulation of the cervix uteri in the afternoon of proestrus and in the morning of estrus.
Experiment I; Cd Determinations Rats were treated with 3.5 or 7.0 mg/kg body wt CdCI 2 sc (CdC12 x 2.5 H 2 0 , REANAL) or 0.9% NaCl solution (1.0 ml/kg body wt) on Day 1 of PSP. Two hours after CdCI 2 administration, or on Days 2, 5, 8, 10, and 12 of PSP under pentobarbital anesthesia (40 mg/kg body wt) blood was taken from the aorta. Two milliliter blood samples were kept at 4°C for the determination of Cd concentration in the whole blood. Ovaries, adrenal glands, and the pituitary gland were removed and trimmed. Ovaries were separated into two parts: luteal tissue [corpora lutea (CL) of PSP] and nonluteal tissue (follicles + stroma). On Days 1 and 2 of PSP corpora lutea from the former cycle(s) also were put into the luteal tissue sample. The wet weight of the organs was measured and they were stored separately at -20°C until analysis. In the group treated with 7 mg/kg CdC12 the procedure was confined to the luteal and nonluteal tissue on Days 2 and 5 of PSP. Samples were digested in 1.0 M HNO 3 for 24 hr at room temperature. Following centrifugation the supernatant was subjected to direct analysis with Zeeman background correction atomic absorption spectrophotometer (Z 5000 AAAS - HGA 500 - AS 40 system, Data System 10, Perkin-Elmer). The method was certified by NBS 1577a bovine liver reference material. The background Cd levels in control tissues during PSP ranged as follows: luteal tissue, 0.0203-0.0323; nonluteal tissues, 0.0100-0.0247; adrenals, 0.0056-0.0113; pituitary, 0.0154-0.0399 ng/mg wet organ weight; blood, 0.0013-0.0043 ng/pJ.
Experiment H: Progesterone Determinations On Day 1 or Day 7 of PSP under light ether narcosis the right jugular vein was exposed and a specially prepared indwelling polyethylene cannula was inserted into the fight atrium for removal of blood samples. The collecting end of the cannula was fixed dorsally and filled with heparin to prevent coagulation. The animals were treated sc with 3.5 or 7.0 mg/kg body w CdC12 or 1.0 ml/kg 0.9% NaC1 solution on Day 1 or Day 8 of PSP, 2 hr before the first blood collection. Blood samples of 0.6-ml volume were taken and centrifuged, and sera were stored frozen (-20°C) until P determination by RIA (Thorneycroft and Stone, 1972). Erythrocytes were resuspended in 0.5 ml of 0.9% NaC1 solution and reinjected after the next blood collection. On Day 7 or 14 of PSP rats were anesthetized with pentobarbital (40 mg/kg body wt), ovaries were excised and trimmed, and then under a dissecting microscope luteal and nonluteal tissues were separated and their wet weights measured.
UPTAKE AND EFFECT OF Cd ON OVARY
85
Statistics Analysis of variance and Dunn (Cd-tissue content) or Dunnett tests (P levels after In transformation) were used for statistical evaluation. RESULTS
Experiment I: Cd Uptake, Distribution, and Accumulation in Endocrine Organs As expected during PSP, there was a significant rise in luteal tissue weights in the control animals. A rise in that of Cd-treated rats also could be observed, on Days 5 and 8; however, luteal tissue weights were lower than that of the controls. An increase in adrenal weights of Cd-treated rats on Day 1 (2 hr after the administration) and on Day 12 of PSP could be observed. The weights of pituitary gland and nonluteal tissue did not change (Table 1). After treatment with 3.5 mg/kg CdC12, comparing the Cd concentration in blood (ng/Ixl) with that in the organs, it can be seen that all the tissues sequestered Cd and accumulated it. Although a marked difference in the partition of Cd in the selected organs is apparent, there was a significant difference in the Cd concentration between the two ovarian compartments. A high initial accumulation rate was characteristic for the luteal tissue with a decreasing tendency toward the end TABLE 1 CHANGES IN THE WEIGHTS OF ENDOCRINE ORGANS IN PSEUDOPREGNANT RATS TREATED WITH 3.5 mg/kg OF CdC12 ON DAY 1 OF PSEUDOPR£GNANCY
Ovary (mg) Nonluteal Pituitary( m g ) Adrenal(mg) Days of pseudopregnancy Control CdCI2 Control CdC12 Control CdC12 Control CdClz Luteal
1
2
5
8
10
12
a Means -+ SEM. * P < 0.05. ** P < 0.01.
10.974 0.51 (9) 14.76 0.87 (9) 21.49 1.46 (8) 23.06 1.01 (5) 21.26 1.39 (8) 21.38 1.44 (9)
11.66 0.32 (7) 16.29 3.49 (7) 18.02" 1.05 (9) 17.83"* 1.17 (7) 21.09 1.82 (8) 22.63 1.41 (7)
39.25 3.06 (9) 39.53 4.32 (9) 38.01 3.01 (8) 33.78 1.98 (5) 33.34 1.49 (8) 31.16 2.19 (9)
31.25 3.42 (7) 39.29 4.86 (7) 39.47 2.82 (9) 32.43 2.54 (8) 35.64 1.98 (8) 33.56 2.18 (7)
11.39 0.41 (11) 12.18 0.95 (8) 12.18 1.04 (8) 12.12 1.04 (6) 11.16 0.70 (8) 11.19 0.45 (10)
11.81 0.53 (7) 11.76 0.51 (8) 10.89 0.57 (9) 10.99 0.36 (7) 11.44 0.55 (8) 12.26 0.46 (7)
55.84 1.89 (11) 53.31 2.47 (8) 53.46 1.39 (8) 56.03 2.86 (6) 55.21 3.38 (8) 51.65 1.31 (10)
64.36* 3.12 (7) 57.84 3.55 (8) 50.17 2.51 (9) 52.74 3.55 (8) 55.54 3.56 (8) 63.09** 2.76 (7)
86
PAKSY ET AL.
of PSP, while in the nonluteal tissue Cd accumulation started with a low level rising gradually until the fifth day. Adrenals and pituitary accumulated Cd in a manner similar to that of nonluteal tissue. In the adrenals and nonluteal tissue the accumulation of Cd reached its maximum on Day 5 of PSP, and in the pituitary gland on Day 10. At Day 12 Cd content in all the tissues started to decline (Fig. 1). In the group treated with 7.0 mg/kg CdC12, Cd levels were as follows: luteal tissue; 4.59 --- 0.54 and 3.06 +- 0.39 ng/mg; nonluteal tissue, 3.51 -+ 0.44 and 3.56 - 0.27 ng/mg on Days 2 and 5 of PSP, respectively (the values are means -+ SE, n = 5-6/group). These data comparing to those seen at the lower dose (Fig. 1) demonstrate that Cd uptake into the ovarian tissues is dose dependent and the distribution between luteal and nonluteal tissues on Days 2 and 5 of PSP is also similar: The luteal tissue on Day 2 contains more Cd than the nonluteal.
Experiment H. Effect o f CdCl 2 on Serum Levels o f P There was no significant change in the body weights of Cd-treated animals compared to the controls. In the group treated with 7.0 mg/kg CdC12 no gain in body weights on Day 7 and Day 14 of PSP was seen. In the Cd-treated groups a dose-dependent decreasing tendency was observed in the weights of the corpora Ed ng/mg wet tissue
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FIo. 1. Accumulation and partition of Cd in the ovary (luteal and nonluteal tissues), adrenals, and pituitary gland or in the blood of pseudopregnant rats. Arrow indicates the injection of CdCI2. Means -+ SEM. / / , number of animals for each point. Background Cd levels in controls are given under Materials and Methods.
87
UPTAKE AND EFFECT OF Cd ON OVARY
lutea on Day 7 and on Day 14 respectively but the number of corpora lutea remained unchanged (data not shown). During the luteal development from Day 1 to Day 7 (treatment on Day 1) P levels of serum increased to about five fold in control rats. In the Cd-treated rats the rise was somewhat lower but significant dose dependency could not be proved (Fig. 2A). In the course of luteal regression (from Days 8 to 14 of PSP, treatment on Day 8) on Day 8 maximal P levels characteristic for this day of PSP were found in the control animals. From this day a rapid decrease in the P levels until the 14th day could be shown representing already the regression of corpora lutea. In the animals treated with 7.0 mg/kg of CdCI 2 diminution of P serum levels was observed, which proved to be significant from Day 8 to Day 10 of PSP. The P levels on Day 8 in this group were as low as P levels on Day 10 in the control rats (Fig.
2B). DISCUSSION In the present work we have demonstrated that following a single sc CdCI 2 injection corpora lutea of PSP displayed a heavier initial incorporation compared to that of the nonluteal tissue, adrenal and pituitary glands. In whole-body autoradiographic studies carried out in pregnant golden hamsters, Cd has been shown to accumulate in the corpora lutea as well (Dencker, 1975). The nonluteal tissue, the adrenals, and the pituitary gland accumulated Cd slower and these levels never reached the initial uptake levels of the luteal tissue. The uptake of Cd in various organs depends on the form in which the metal is circulating in blood (Jin et al., 1986). In rodents Cd, soon after its injection, binds to albumin, mercaptalProgesterone I nglmt
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FIG. 2. Changes in the progesterone serum levels of pseudopregnant rats having indwelling jugular vein catheter and treated with CdCI 2 (arrows) on Day 1 (A) or on Day 8 (B) of pseudopregnancy. Means - SEM. Number of experiments in parentheses. *P < 0.05 (compared to the control).
88
PAKSY ET AL.
bumin (Suzuki et al., 1986), or other high-molecular-weight components of the blood (reviewed by Arvidson and Tj~ilve, 1986). These low-stability Cd-protein complexes cannot readily be filtered through the glomeruli (Abel et al., 1987) but may penetrate the fenestrated capillaries in the luteal tissue as they do the highly fenestrated vessels of peripheral ganglions (Arvidson and Tj~lve, 1986). Due to the well-known high luteal blood flow (Janson et al., 1981; Varga et al., 1985) and the exceptionally high permeability of luteal blood capillaries to plasma proteins (Morris and Sass, 1966), higher quantities of Cd may reach the luteal tissue. The extremely high metabolism of the cells (Swarm and Bruce, 1987), the initial hyperplasia until the end of Day 1 (Ichikawa et al., 1987) followed by hypertrophy of luteal cells, and the angiogenetic process may promote the incorporation of Cd into the luteal and endothelial cells. In tissues Cd binds to tissue proteins resulting in the formation of Cd-protein complexes with low stability contants (Sumi et al., 1987). These findings are in accord with our results concerning the higher initial uptake of Cd into the luteal tissue. One possibility to explain the rapid decline in the Cd content of CL after Day 2 of PSP can be that by this time most of the Cd is transferred from its initial macromolecular binding sites to metallothionein (MT) (Scheuhammer et al., 1985). The MT-Cd complex has a high stability constant (Sumi et al., 1987). In this bound form or in the form of a complex, Cd eventually may not be so easily trapped by tissues as it is in the free form or in the form of protein complexes of low stability constants. On the other hand the volume of CL is about doubled during PSP in control animals (Table 1) (Norjavaara et al., 1987); that is why the relative Cd content of luteal tissue decreases. Cd treatment counteracts this process, as Cd induces a decreasing tendency in CL weights. The high amount of Cd accumulated in the luteal tissue during the first half of PSP seems not to be closely related to P secretion. The rise in blood levels of P in rats, treated with 3.5 or 7.0 mg/kg body wt CdC12 on Day 1 of PSP, runs somewhat lower but parallel with the controls. It is known that around the time of Cd injections (10-12 hr following ovulation), the formation of fresh CL is nearly completed (Ichikawa et al., 1987). Apparently, once luteinization occurred Cd treatment did not significantly interfere with the P production of luteal cells although in the weight of luteal tissue a decreasing tendency was observed. These results are confirmed by the evidence that Cd given 3 days after mating did not affect pregnancy and circulating P concentration in rabbits (Saksena, 1982). Contrary to the relative insensitivity of luteal function to Cd during the first half of PSP, steroidogenesis during the second part proved to be markedly influenced, 7.0 mg/kg CdC12 administered on Day 8 of PSP decreased serum levels of P significantly from Days 8 through 12. Although ovarian uptake of Cd given on Day 8 of PSP has not been determined, on the basis of Experiment I, we supposed a heavy incorporation in the luteal and nonluteal tissue. Cd incorporation has been reported in the CL of pregnant golden hamsters on Day 8 of pregnancy after sc injection of Cd (Dencker, 1975). It is known that blood flow of corpora lutea on Days 6-11 of PSP is rather high in rats (Norjavaara et al., 1987), which may additionally promote the Cd incorporation into the luteal ovary. Our results seem to confirm in vivo (Chellmann et al., 1985) and in vitro (Fischer, 1985) data which support the idea that it is not as much the amount of Cd taken up by cells but
UPTAKE AND EFFECT OF Cd ON OVARY
89
cellular sensitivity to Cd that is responsible for its toxic effect. The mechanism of how Cd interferes with steroidogenesis is unclear. Besides the possible involvement of the hypothalamo-pituitary-ovarian system, partial ischemia due to injury of the ovarian vessels, as it has been described previously (Kar et al., 1959; Parizek et al., 1968), cannot be excluded; however, in PSP rats no dramatic change in the vascular bed could be detected in the gross morphology of CL. Moreover 48 hr after Cd administration no significant changes in the ovarian venous outflow occurred (Paksy et al., 1989). Evidence has been accumulated that Cd may interact with bivalent cations, e.g., Zn (Waalkes and Poirier, 1985) and Ca essential for normal cell functions. Ca has been reported to play an important role in the regulation of ovarian steroidogenesis (Tsang and Carnegie, 1983). The changes in the adrenal weights during PSP shows that CdC12injection may serve as a stressor. However, the toxic effects of Cd accumulated in the pituitary and adrenal glands have not been studied. Our results show that luteal and nonluteal tissues of pseudopregnant rat ovary bind Cd but that there are differences in the uptake and accumulation between the luteal and nonluteal tissues. It seems that Cd does not severely influence the development of CL; however, it may facilitate the luteal regression. ACKNOWLEDGMENTS Thanks go to Mrs. l~va Erdei and Mr. Gy. Koch for their excellent technical assistance. The study supported by the Hungarian National Research Foundation (OTKA 14/161t/86).
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REFERENCES Abel, J., H6hr, D., and Schurek, H. J. (1987). Renal handling of cadmium and cadmiummetallothionein: Studies on the isolated perfused kidney. Arch. Toxicol. 60, 370-375. Arvidson, B., and Tj~ilve, H. (1986). Distribution of 1°9-Cd in the nervous system of rats after intravenous injection. Acta Neuropathol. 69, 111-116. Chellmann, G. J., Saikh, Z. A., Baggs, R. B., and Diamond, G. L. (1985). Resistance to Cd-induced necrosis in testes of inbred mice: possible role of methallothionein-like cadmium-bindingprotein. Toxicol. Appl. Pharmacol. 79, 511-523. Dencker, L. (1975). Possible mechanisms of cadmium fetotoxicity in golden hamsters and mice: Uptake by the embryo, placenta and ovary. J. Reprod. Fertil. 44, 461-471. Fischer, A. B. (1985). Factors influencing cadmium uptake and cytotoxicity in cultured cells. Xenobiotica 15, 751-757. Ichikawa, S., Uchino, S., and Hirata, Y. (1987). Lymphatic and blood vasculature of the forming corpus luteum. Lymphology 20, 73-83. Janson, P. O., Damber, J. E., and Ax6n, C. (1981). Luteal blood flow and progesterone secretion in pseudopregnant rabbits. J. Reprod Fertil. 63, 491-497. Jin, T., Nordberg, G. F., and Nordberg, M. (1986). Uptake of cadmium in isolated kidney cells. Influence of binding form and in vivo pretreatment. J. Appl. Toxicol. 6, 697-400. Kar, A. B., Das, R. P., and Karkun, J. N. (1959). Ovarian changes in prepuberal rats after treatment w i t h cadmium chloride. Acta Biol. Med. Get. 3, 372-399. Morris, B., and Sass, M. B. (1966). The formation of lymph in the ovary. Proc. Soc. London B 164, 577-591. Norjavaara, E., Olofsson, J., Gafvels, M., and Selstam, G. (1987). Redistribution of ovarian blood flow after injection of human chorionic gonadotropin. Endocrinology 120, 107-114. Paksy, K., Varga, B., Horv~ith, E., and Ungv~iry, Gy. (1989). Acute effects of CdC12 on the ovulation and hormone secretion of ovary in oestrous rats. Reprod. Toxicol. 3.
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Parizek, J., Ostadalova, I., Benes, I., and Pitha, J. (1968). The effect of a subcutaneous injection of cadmium salts on the ovaries of adult rats in persistent oestrus. J. Reprod. Fertil. 17, 55%562. Saksena, S. K. (1982). Cadmium: Its effects on ovulation, egg transport and pregnancy in the rabbit. Contraception 26, 181-192. Saksena, S. K., and Salmonsen, R. (1983). The effects of cadmium chloride on ovulation and on induction of sterility in the female Golden hamster. Biol. Reprod. 29, 249-256. Scheuhammer, A. M., Onosaka, S., Rodgers, K., and Cherian, M. G. (1985). The interaction of zinc and cadmium in the synthesis of hepatic metallothionein in rats. Toxicology 36, 101-108. Setchell, B. P. (1978). Naturally occurring and induced dysfunctions of the testis. In "The Mammalian Testis" (P. B. Setchell, Ed.), pp. 393-410. ELEK, London. Sumi, Y., Suzuki, T., and Suzuki, K. T. (1987). Autoradiographic demonstration of cadmium not bound to metallothionein using 14C-labeled thiazolo-naphtol. Histochemistry 87, 327-329. Suter, K. E. (1985). Studies on the dominant lethal and fertility effects of the heavy metal compounds methylmercuric hydroxide, mercuric chloride and cadmium chloride in male and female mice. Mutat. Res. 30, 365-374. Suzuki, K. T., Sunaga, H., Kobayashi, E., and Shimajo, N. (1986). Mercaptalbumin as a selective cadmium binding protein in rats serum. Toxicol. Appl. Pharmacol. 86, 466--473. Swann, R. T., and Bruce, N. W. (1987). Oxygen consumption, carbon dioxide production and progestagen secretion in the intact ovary of the day-16 pregnant rat. J. Reprod. Fertil. 80, 599-605. Thorneycroft, I. H., and Stone, S. C. (1972). Radioimmunoassay of serum progesterone in women receiving oral contraceptive steroids. Contraception 5, 12%146. Tsang, B. K., and Carnegie, J. A. (1983). Calcium requirement in the gonadotropic regulation of rat granulosa cell progesterone production. Endocrinology 113, 751-757. Varga, B., Horwith, E., Folly, G., and Stark, E. (1985). Study of luteinizing hormone-induced increase of ovarian blood flow during estrous cycle in the rat. Biol. Reprod. 32, 480--488. Waalkes, M. P., and Poirier, L. A. (1985). Interactions of cadmium with interstitial tissue of the rat testes. Biochem. Pharmacol. 34, 2513-2518.
Uptake and Distribution of Cd in the Ovaries, Adrenals, and Pituitary in Pseudopregnant Rats: Effect of Cd on Progesterone Serum Levels K A T A L I N PAKSY, M I K L r S N.~RAY, BERTALAN VARGA, IMRE KISS,
G~OR FOLLY, AND GYORGYUNOV~RY National Institute of Occupational Health, P.O. Box 22, Budapest 1450, Hungary Received November 1, 1988 Pseudopregnant (PSP) rats were treated with 3.5 or 7.0 mg/kg body wt of CdC12 on Day 1 of PSP sc. In the lower dose Cd content of the ovaries (luteal and nonluteal tissues), adrenals, pituitary, and blood on Days 1, 2, 5, 8, 10, and 12, and in the higher dose that of luteal and nonluteal tissue on Days 2 and 5 of PSP were determined with atomic absorption spectrophotometry. A rapid incorporation into the corpora lutea was measured on Day 1 and Day 2 of PSP followed by a decrease of Cd content toward the end of PSP whereas the nonluteal tissue, adrenals, and pituitary accumulated Cd gradually until the fifth to 10th day, respectively. Progesterone (P) serum levels were measured with RIA in the blood collected daily from the jugular vein following administration of 3.5 to 7.0 mg/kg body wt of CdC12 sc on Day 1 or Day 8 of PSP. The serum levels of P remained unchanged when CdC12 was administered on Day 1 of PSP; however, 7.0 mg/kg body wt CdC12 given on Day 8 of PSP induced a significant decrease in serum levels of P. It is supposed that the regressing luteal tissue is more sensitive to the toxic effects of Cd than the developing one. © 1990Academic Press, Inc.
INTRODUCTION Cd is known to induce permanent sterility of male laboratory rodents: damaging the capillary network in testicles it destroys the germinal epithelium (reviewed by Setchell, 1978). In females the toxic effect of Cd depends on the continuously changing function of the ovary throughout the lifetime. In anovulatory animals, which are prepuberal (Kar, 1959) or in persistent estrus (Parizek et al., 1968), Cd causes dramatic but transient vascular lesions. In adult cycling female rats (Parizek et aL, 1968) and mice (Suter, 1985) no fertility impairment was proven, while in hamsters (Saksena and Salmonsen, 1983) and rabbits (Saksena, 1982) a pronounced but temporary infertility by disturbance of ovulation and egg transport occurred following a single Cd injection. A single sc CdC12 dose partially inhibited the ovulation and caused dose-dependent changes in the luteal function lowering the basal secretion rate of progesterone (P) and decreasing the response to hCG stimulus (PaLsy et al., 1989). The aim of the present work was to study the Cd uptake and accumulation of ovary (luteal and nonluteal tissue) and adrenal and pituitary gland. Furthermore in a separate experiment the effects of Cd on blood levels of P were determined in pseudopregnant rats.
MATERIALS AND METHODS Animals The experiments were performed on mature female CFY rats (LATI, G6d6116, 83 00130351/90 $3.00 Copyright © 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.
84
PAKSY ET AL.
Hungary) weighing 220-260 g. They were kept under controlled environmental conditions with a 12-hr light/12-hr dark photoperiod. Free access was provided to standard laboratory pellets and tap water. The ovarian cycle was checked daily by vaginal smear. In animals, having at least three regular cycles, pseudopregnancy (PSP) was induced by mechanical stimulation of the cervix uteri in the afternoon of proestrus and in the morning of estrus.
Experiment I; Cd Determinations Rats were treated with 3.5 or 7.0 mg/kg body wt CdCI 2 sc (CdC12 x 2.5 H 2 0 , REANAL) or 0.9% NaCl solution (1.0 ml/kg body wt) on Day 1 of PSP. Two hours after CdCI 2 administration, or on Days 2, 5, 8, 10, and 12 of PSP under pentobarbital anesthesia (40 mg/kg body wt) blood was taken from the aorta. Two milliliter blood samples were kept at 4°C for the determination of Cd concentration in the whole blood. Ovaries, adrenal glands, and the pituitary gland were removed and trimmed. Ovaries were separated into two parts: luteal tissue [corpora lutea (CL) of PSP] and nonluteal tissue (follicles + stroma). On Days 1 and 2 of PSP corpora lutea from the former cycle(s) also were put into the luteal tissue sample. The wet weight of the organs was measured and they were stored separately at -20°C until analysis. In the group treated with 7 mg/kg CdC12 the procedure was confined to the luteal and nonluteal tissue on Days 2 and 5 of PSP. Samples were digested in 1.0 M HNO 3 for 24 hr at room temperature. Following centrifugation the supernatant was subjected to direct analysis with Zeeman background correction atomic absorption spectrophotometer (Z 5000 AAAS - HGA 500 - AS 40 system, Data System 10, Perkin-Elmer). The method was certified by NBS 1577a bovine liver reference material. The background Cd levels in control tissues during PSP ranged as follows: luteal tissue, 0.0203-0.0323; nonluteal tissues, 0.0100-0.0247; adrenals, 0.0056-0.0113; pituitary, 0.0154-0.0399 ng/mg wet organ weight; blood, 0.0013-0.0043 ng/pJ.
Experiment H: Progesterone Determinations On Day 1 or Day 7 of PSP under light ether narcosis the right jugular vein was exposed and a specially prepared indwelling polyethylene cannula was inserted into the fight atrium for removal of blood samples. The collecting end of the cannula was fixed dorsally and filled with heparin to prevent coagulation. The animals were treated sc with 3.5 or 7.0 mg/kg body w CdC12 or 1.0 ml/kg 0.9% NaC1 solution on Day 1 or Day 8 of PSP, 2 hr before the first blood collection. Blood samples of 0.6-ml volume were taken and centrifuged, and sera were stored frozen (-20°C) until P determination by RIA (Thorneycroft and Stone, 1972). Erythrocytes were resuspended in 0.5 ml of 0.9% NaC1 solution and reinjected after the next blood collection. On Day 7 or 14 of PSP rats were anesthetized with pentobarbital (40 mg/kg body wt), ovaries were excised and trimmed, and then under a dissecting microscope luteal and nonluteal tissues were separated and their wet weights measured.
UPTAKE AND EFFECT OF Cd ON OVARY
85
Statistics Analysis of variance and Dunn (Cd-tissue content) or Dunnett tests (P levels after In transformation) were used for statistical evaluation. RESULTS
Experiment I: Cd Uptake, Distribution, and Accumulation in Endocrine Organs As expected during PSP, there was a significant rise in luteal tissue weights in the control animals. A rise in that of Cd-treated rats also could be observed, on Days 5 and 8; however, luteal tissue weights were lower than that of the controls. An increase in adrenal weights of Cd-treated rats on Day 1 (2 hr after the administration) and on Day 12 of PSP could be observed. The weights of pituitary gland and nonluteal tissue did not change (Table 1). After treatment with 3.5 mg/kg CdC12, comparing the Cd concentration in blood (ng/Ixl) with that in the organs, it can be seen that all the tissues sequestered Cd and accumulated it. Although a marked difference in the partition of Cd in the selected organs is apparent, there was a significant difference in the Cd concentration between the two ovarian compartments. A high initial accumulation rate was characteristic for the luteal tissue with a decreasing tendency toward the end TABLE 1 CHANGES IN THE WEIGHTS OF ENDOCRINE ORGANS IN PSEUDOPREGNANT RATS TREATED WITH 3.5 mg/kg OF CdC12 ON DAY 1 OF PSEUDOPR£GNANCY
Ovary (mg) Nonluteal Pituitary( m g ) Adrenal(mg) Days of pseudopregnancy Control CdCI2 Control CdC12 Control CdC12 Control CdClz Luteal
1
2
5
8
10
12
a Means -+ SEM. * P < 0.05. ** P < 0.01.
10.974 0.51 (9) 14.76 0.87 (9) 21.49 1.46 (8) 23.06 1.01 (5) 21.26 1.39 (8) 21.38 1.44 (9)
11.66 0.32 (7) 16.29 3.49 (7) 18.02" 1.05 (9) 17.83"* 1.17 (7) 21.09 1.82 (8) 22.63 1.41 (7)
39.25 3.06 (9) 39.53 4.32 (9) 38.01 3.01 (8) 33.78 1.98 (5) 33.34 1.49 (8) 31.16 2.19 (9)
31.25 3.42 (7) 39.29 4.86 (7) 39.47 2.82 (9) 32.43 2.54 (8) 35.64 1.98 (8) 33.56 2.18 (7)
11.39 0.41 (11) 12.18 0.95 (8) 12.18 1.04 (8) 12.12 1.04 (6) 11.16 0.70 (8) 11.19 0.45 (10)
11.81 0.53 (7) 11.76 0.51 (8) 10.89 0.57 (9) 10.99 0.36 (7) 11.44 0.55 (8) 12.26 0.46 (7)
55.84 1.89 (11) 53.31 2.47 (8) 53.46 1.39 (8) 56.03 2.86 (6) 55.21 3.38 (8) 51.65 1.31 (10)
64.36* 3.12 (7) 57.84 3.55 (8) 50.17 2.51 (9) 52.74 3.55 (8) 55.54 3.56 (8) 63.09** 2.76 (7)
86
PAKSY ET AL.
of PSP, while in the nonluteal tissue Cd accumulation started with a low level rising gradually until the fifth day. Adrenals and pituitary accumulated Cd in a manner similar to that of nonluteal tissue. In the adrenals and nonluteal tissue the accumulation of Cd reached its maximum on Day 5 of PSP, and in the pituitary gland on Day 10. At Day 12 Cd content in all the tissues started to decline (Fig. 1). In the group treated with 7.0 mg/kg CdC12, Cd levels were as follows: luteal tissue; 4.59 --- 0.54 and 3.06 +- 0.39 ng/mg; nonluteal tissue, 3.51 -+ 0.44 and 3.56 - 0.27 ng/mg on Days 2 and 5 of PSP, respectively (the values are means -+ SE, n = 5-6/group). These data comparing to those seen at the lower dose (Fig. 1) demonstrate that Cd uptake into the ovarian tissues is dose dependent and the distribution between luteal and nonluteal tissues on Days 2 and 5 of PSP is also similar: The luteal tissue on Day 2 contains more Cd than the nonluteal.
Experiment H. Effect o f CdCl 2 on Serum Levels o f P There was no significant change in the body weights of Cd-treated animals compared to the controls. In the group treated with 7.0 mg/kg CdC12 no gain in body weights on Day 7 and Day 14 of PSP was seen. In the Cd-treated groups a dose-dependent decreasing tendency was observed in the weights of the corpora Ed ng/mg wet tissue
I
3.5 mg/kg CdCt2 so.
I
3.0
// 2.5
pifuifary
A /8/
2,0
/ ~ T
..//
/"
~
~
~
"
~ --~
C /8/
1.5
/
"~A tufeat
o /7/
1,0
T
0,5
brood i
2
5
8
1'0
1'2
d(iysofpseudopregnoncy Significonce -~ p< 0.05 • *p,:O.01
A~'-C * A--~D.x-~ B--C* B--D**
A---D -~ B---D* C~--D *
A--~B* B---C *xB ~ - D -x:
A--~C A---D B~-~C B--~O
* * ~ *
A---C * A4--D * B---C ~
A'-~C* A "-~D*-~ B---C* C~-'D *
C---D **
FIo. 1. Accumulation and partition of Cd in the ovary (luteal and nonluteal tissues), adrenals, and pituitary gland or in the blood of pseudopregnant rats. Arrow indicates the injection of CdCI2. Means -+ SEM. / / , number of animals for each point. Background Cd levels in controls are given under Materials and Methods.
87
UPTAKE AND EFFECT OF Cd ON OVARY
lutea on Day 7 and on Day 14 respectively but the number of corpora lutea remained unchanged (data not shown). During the luteal development from Day 1 to Day 7 (treatment on Day 1) P levels of serum increased to about five fold in control rats. In the Cd-treated rats the rise was somewhat lower but significant dose dependency could not be proved (Fig. 2A). In the course of luteal regression (from Days 8 to 14 of PSP, treatment on Day 8) on Day 8 maximal P levels characteristic for this day of PSP were found in the control animals. From this day a rapid decrease in the P levels until the 14th day could be shown representing already the regression of corpora lutea. In the animals treated with 7.0 mg/kg of CdCI 2 diminution of P serum levels was observed, which proved to be significant from Day 8 to Day 10 of PSP. The P levels on Day 8 in this group were as low as P levels on Day 10 in the control rats (Fig.
2B). DISCUSSION In the present work we have demonstrated that following a single sc CdCI 2 injection corpora lutea of PSP displayed a heavier initial incorporation compared to that of the nonluteal tissue, adrenal and pituitary glands. In whole-body autoradiographic studies carried out in pregnant golden hamsters, Cd has been shown to accumulate in the corpora lutea as well (Dencker, 1975). The nonluteal tissue, the adrenals, and the pituitary gland accumulated Cd slower and these levels never reached the initial uptake levels of the luteal tissue. The uptake of Cd in various organs depends on the form in which the metal is circulating in blood (Jin et al., 1986). In rodents Cd, soon after its injection, binds to albumin, mercaptalProgesterone I nglmt
I
I •
•
,,-
• 0,9 % NoCt
o.... t~
~) 3,5mg/kgCdC[ 2 D 7,Omg/kg CdCt2
50
4O 3O
2O 15-
107.5.
3) 5-
(7)
i
{
~
;.
~
6
~
I
8
g
1'0
il
5
1'3 1~. days
FIG. 2. Changes in the progesterone serum levels of pseudopregnant rats having indwelling jugular vein catheter and treated with CdCI 2 (arrows) on Day 1 (A) or on Day 8 (B) of pseudopregnancy. Means - SEM. Number of experiments in parentheses. *P < 0.05 (compared to the control).
88
PAKSY ET AL.
bumin (Suzuki et al., 1986), or other high-molecular-weight components of the blood (reviewed by Arvidson and Tj~ilve, 1986). These low-stability Cd-protein complexes cannot readily be filtered through the glomeruli (Abel et al., 1987) but may penetrate the fenestrated capillaries in the luteal tissue as they do the highly fenestrated vessels of peripheral ganglions (Arvidson and Tj~lve, 1986). Due to the well-known high luteal blood flow (Janson et al., 1981; Varga et al., 1985) and the exceptionally high permeability of luteal blood capillaries to plasma proteins (Morris and Sass, 1966), higher quantities of Cd may reach the luteal tissue. The extremely high metabolism of the cells (Swarm and Bruce, 1987), the initial hyperplasia until the end of Day 1 (Ichikawa et al., 1987) followed by hypertrophy of luteal cells, and the angiogenetic process may promote the incorporation of Cd into the luteal and endothelial cells. In tissues Cd binds to tissue proteins resulting in the formation of Cd-protein complexes with low stability contants (Sumi et al., 1987). These findings are in accord with our results concerning the higher initial uptake of Cd into the luteal tissue. One possibility to explain the rapid decline in the Cd content of CL after Day 2 of PSP can be that by this time most of the Cd is transferred from its initial macromolecular binding sites to metallothionein (MT) (Scheuhammer et al., 1985). The MT-Cd complex has a high stability constant (Sumi et al., 1987). In this bound form or in the form of a complex, Cd eventually may not be so easily trapped by tissues as it is in the free form or in the form of protein complexes of low stability constants. On the other hand the volume of CL is about doubled during PSP in control animals (Table 1) (Norjavaara et al., 1987); that is why the relative Cd content of luteal tissue decreases. Cd treatment counteracts this process, as Cd induces a decreasing tendency in CL weights. The high amount of Cd accumulated in the luteal tissue during the first half of PSP seems not to be closely related to P secretion. The rise in blood levels of P in rats, treated with 3.5 or 7.0 mg/kg body wt CdC12 on Day 1 of PSP, runs somewhat lower but parallel with the controls. It is known that around the time of Cd injections (10-12 hr following ovulation), the formation of fresh CL is nearly completed (Ichikawa et al., 1987). Apparently, once luteinization occurred Cd treatment did not significantly interfere with the P production of luteal cells although in the weight of luteal tissue a decreasing tendency was observed. These results are confirmed by the evidence that Cd given 3 days after mating did not affect pregnancy and circulating P concentration in rabbits (Saksena, 1982). Contrary to the relative insensitivity of luteal function to Cd during the first half of PSP, steroidogenesis during the second part proved to be markedly influenced, 7.0 mg/kg CdC12 administered on Day 8 of PSP decreased serum levels of P significantly from Days 8 through 12. Although ovarian uptake of Cd given on Day 8 of PSP has not been determined, on the basis of Experiment I, we supposed a heavy incorporation in the luteal and nonluteal tissue. Cd incorporation has been reported in the CL of pregnant golden hamsters on Day 8 of pregnancy after sc injection of Cd (Dencker, 1975). It is known that blood flow of corpora lutea on Days 6-11 of PSP is rather high in rats (Norjavaara et al., 1987), which may additionally promote the Cd incorporation into the luteal ovary. Our results seem to confirm in vivo (Chellmann et al., 1985) and in vitro (Fischer, 1985) data which support the idea that it is not as much the amount of Cd taken up by cells but
UPTAKE AND EFFECT OF Cd ON OVARY
89
cellular sensitivity to Cd that is responsible for its toxic effect. The mechanism of how Cd interferes with steroidogenesis is unclear. Besides the possible involvement of the hypothalamo-pituitary-ovarian system, partial ischemia due to injury of the ovarian vessels, as it has been described previously (Kar et al., 1959; Parizek et al., 1968), cannot be excluded; however, in PSP rats no dramatic change in the vascular bed could be detected in the gross morphology of CL. Moreover 48 hr after Cd administration no significant changes in the ovarian venous outflow occurred (Paksy et al., 1989). Evidence has been accumulated that Cd may interact with bivalent cations, e.g., Zn (Waalkes and Poirier, 1985) and Ca essential for normal cell functions. Ca has been reported to play an important role in the regulation of ovarian steroidogenesis (Tsang and Carnegie, 1983). The changes in the adrenal weights during PSP shows that CdC12injection may serve as a stressor. However, the toxic effects of Cd accumulated in the pituitary and adrenal glands have not been studied. Our results show that luteal and nonluteal tissues of pseudopregnant rat ovary bind Cd but that there are differences in the uptake and accumulation between the luteal and nonluteal tissues. It seems that Cd does not severely influence the development of CL; however, it may facilitate the luteal regression. ACKNOWLEDGMENTS Thanks go to Mrs. l~va Erdei and Mr. Gy. Koch for their excellent technical assistance. The study supported by the Hungarian National Research Foundation (OTKA 14/161t/86).
was
REFERENCES Abel, J., H6hr, D., and Schurek, H. J. (1987). Renal handling of cadmium and cadmiummetallothionein: Studies on the isolated perfused kidney. Arch. Toxicol. 60, 370-375. Arvidson, B., and Tj~ilve, H. (1986). Distribution of 1°9-Cd in the nervous system of rats after intravenous injection. Acta Neuropathol. 69, 111-116. Chellmann, G. J., Saikh, Z. A., Baggs, R. B., and Diamond, G. L. (1985). Resistance to Cd-induced necrosis in testes of inbred mice: possible role of methallothionein-like cadmium-bindingprotein. Toxicol. Appl. Pharmacol. 79, 511-523. Dencker, L. (1975). Possible mechanisms of cadmium fetotoxicity in golden hamsters and mice: Uptake by the embryo, placenta and ovary. J. Reprod. Fertil. 44, 461-471. Fischer, A. B. (1985). Factors influencing cadmium uptake and cytotoxicity in cultured cells. Xenobiotica 15, 751-757. Ichikawa, S., Uchino, S., and Hirata, Y. (1987). Lymphatic and blood vasculature of the forming corpus luteum. Lymphology 20, 73-83. Janson, P. O., Damber, J. E., and Ax6n, C. (1981). Luteal blood flow and progesterone secretion in pseudopregnant rabbits. J. Reprod Fertil. 63, 491-497. Jin, T., Nordberg, G. F., and Nordberg, M. (1986). Uptake of cadmium in isolated kidney cells. Influence of binding form and in vivo pretreatment. J. Appl. Toxicol. 6, 697-400. Kar, A. B., Das, R. P., and Karkun, J. N. (1959). Ovarian changes in prepuberal rats after treatment w i t h cadmium chloride. Acta Biol. Med. Get. 3, 372-399. Morris, B., and Sass, M. B. (1966). The formation of lymph in the ovary. Proc. Soc. London B 164, 577-591. Norjavaara, E., Olofsson, J., Gafvels, M., and Selstam, G. (1987). Redistribution of ovarian blood flow after injection of human chorionic gonadotropin. Endocrinology 120, 107-114. Paksy, K., Varga, B., Horv~ith, E., and Ungv~iry, Gy. (1989). Acute effects of CdC12 on the ovulation and hormone secretion of ovary in oestrous rats. Reprod. Toxicol. 3.
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