Toxicology, 64 (1990) 89--104 Elsevier Scientific Publishers Ireland Ltd.

Cadmium teratogenicity and its relationship with metallothionein gene expression in midgestation mouse embryos Swapan K. De a, Sudhansu K. Dey b and Glen K. Andrews a,* °Department of Biochemistry and Molecular Biology and bDepartment of Obstetrics-Gynecology and Physiology, University of Kansas Medical Center, Kansas City, Kansas 66103, (U.S.A.) (Received April 16th, 1990 ; accepted June 16th, 1990)

Summary As an approach toward understanding the mechanisms by which cadmium (Cd) exerts its teratogenie effects, the expression and metal regulation of the metallothionein (MT) genes in midgestation mouse embryos were studied by Northern blot and in situ hybridization. Maternal injection of a teratogenic dosage of Cd (50/amoi Cd/kg body wt) did not induce MT mRNA in day 10 (D10) CD-I mouse embryos, whereas zinc (Zn) (50/amol/kg) was an effective inducer. In contrast, Cd was about 10-fold more potent than Zn at rapidly inducing MT mRNA in DI0 embryos incubated in vitro in medium containing micromolar concentrations of these metals. This suggests that following maternal injection, Cd but not Zn is prevented from reaching the DI0 embryo and establishes that the embryonic MT genes are not refractory to metal induction, which might have explained the sensitivity of the embryo to Cd. MT mRNA was detected at high levels only in the extraembryonic membranes of D9 embryos exposed to Cd in vivo. On days 9 and 10, no embryonic cell types contained detectable levels of MT mRNA. This mRNA was detected first at low levels in hepatocytes on D l l , soon after formation of liver and these levels increased dramatically by DI2. Therefore, Cd teratogenicity was not associated with high levels of cell type-specific expression of the MT genes in Cd-sensitive regions of the embryo (neural tube, limb bud), that might have served to target Cd to these cells. Taken together, the results of this study suggest that Cd teratogenicity reflects damage to maternal or extraembryonic tissues. However, the results cannot exclude the possibility that certain cells in the embryo are exceptionally sensitive to low levels of Cd.

Key words: Cadmium teratogenicity; Gene regulation; Metal ions; Metallothionein; Midgestation mouse embryo; In situ hybridization; Zinc.

Introduction

Cadmium (Cd) has well-documented teratogenic and embryotoxic effects in a large variety of species, including man [reviewed by 1,2]. However, the molecular *Address all correspondence to: Dr. G.K. Andrews, Department of Biochemistry and Molecular Biology, WHE 4018, University of Kansas Medical Center, 39th and Rainbow Blvd., Kansas City, KS. 66103, (U.S.A.) 0300-483X/90/$03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

89

basis for these effects is poorly understood. When administered during pregnancy, Cd causes necrosis o f decidual a n d / o r placental tissues [1]. Apparently, in the rodent, the transfer of Cd from the maternal environment into the embryonic environment is limited by retention of this metal by the placenta [reviewed by 3], this restriction in passage of Cd may serve to protect the embryo [1,3,4]. Nonetheless, Cd, when administered during the organogenic period, leads to prolonged anorexia, growth retardation, nephrotoxicity and skeletal, neural tube, limb bud and craniofacial abnormalities [5,6]. An injection of Cd bound to metallothionein (MT) is also teratogenic, but apparently little Cd, released from this metalprotein complex, is detected in the embryo [6] and thus an indirect teratogenic effect of Cd was suggested. However, Cd is teratogenic when directly presented to rat, mouse and chick embryos developing in culture [7--10] and is also embryotoxic to preimplantation mouse, rat, and rabbit embryos in vitro [11--16, reviewed in 17]. Therefore, in the in vivo situation, it is unclear as to whether Cd exerts its teratogenic effects primarily via direct actions on the embryo or via secondary effects, such as yolk sac or placental deficiencies or kidney failure. In the body, most Cd remains bound to metallothioneins; a group of ubiquitous, low molecular weight, cysteine-rich proteins [reviewed by 18--21]. In the mouse, there are two isoforms of MT (MT-I and MT-II) that differ slightly in amino acid sequence and net charge [22]. These genes are usually expressed constitutively at low levels in most adult mammalian organs, but are rapidly induced transcriptionally by micromolar concentrations of heavy metals, such as Cd and Zn. MT genes are also induced by other stimuli, including glucocorticoid hormones and inflammatory agents [19]. Recently, we have shown that from the time of implantation to late in gestation the mouse embryo is surrounded by cells in the deciduum, placenta and visceral yolk sac that accumulate high levels of MT mRNA [4,23]. These findings may serve to explain the accumulation of Cd in these structures and are consistent with the possibility that most Cd is prevented from reaching the embryo. However, it is possible that cell-specific expression of the MT genes in the midgestation mouse embryo, during the teratogenic period (D8 to D10), could serve to sequester, in these cells, Cd that overcomes the decidual/placental/yolk sac barriers. Although nothing is known about expression of MT genes in early to midgestation embryos, the fetal and neonatal rodent liver contains high levels o f MT [reviewed by 24i and striking cell-specific expression of MT has recently been documented in a variety of organs [4,25]. Measurements of Cd suggest low levels in the embryo, and autoradiographic detection demonstrates accumulation in the deciduum, placenta and yolk sac [1,3]. However, Cd detected in the embryo may reflect contamination by maternal serum or extraembryonic tissues and difficulties in fixation of Cd may not allow for accurate determination of cell-specific localization. Furthermore, the presence of Cd in the embryo does not provide evidence for an effect of this metal on embryonic cells. In the present study, the relative levels of MT mRNA in the embryo following an acute in vivo or in vitro exposure to Cd or Zn were measured as a sensitive indicator of a rapid direct effect of these metals on the embryo. Furthermore, cell-specific MT gene expression was determined by in situ hybridization.

90

Materials and methods

Animals Female CD-1 mice (48 days old; Charles River Laboratories) were mated and the gestational age of the embryo calculated from the day of the vaginal plug (designated D1 of gestation). In some experiments, mice were injected subcutaneously with Zn and/or Cd chloride salts or with vehicle alone (normal saline).

Embryo manipulation In experiments analyzing the expression and metal regulation of MT genes in vivo, embryos (D10) were recovered by dissection free from extraembryonic and maternal tissues, 5 h after an injection of Cd, Zn or saline. These samples were quick frozen, and stored at - 8 0 ° C for later extraction of RNA. In order to examine the in vitro effects of metal ions on MT mRNA, D10 embryos were dissected out into phosphate-buffered-saline (PBS). Embryos from several liters (50 --60 per sample) were collected, and incubated for 5 h in DMEM plus 10o70 fetal bovine serum containing various concentrations of ZnCl 2 or CdCl 2. Following incubation, samples were quick frozen for RNA extraction. In order to examine the cell-specific expression of MT genes in the developing embryo, pregnant females, on days 9 through 13, were anesthetized with Avertin and embryos were fixed by perfusion with 4O7oparaformaidehyde in PBS.

Cd teratogenicity Cadmium chloride was injected subcutaneously at various dosages (10--75 /~mol Cd/kg body wt) early on day 10 of pregnancy. Eight animals were included in each group. Four animals from each group were sacrificed on D14 and embryos were examined under a dissecting microscope and numbers of normal and malformed (defective limb bud, open neural tube, craniofacial abnormalities) embryos were recorded. The remaining animals were sacrificed on D17 and the wet weights of embryos were determined and malformation recorded.

Hybridization probes A mouse MT-I cDNA was provided by Dr. R.D. Palmiter (University of Washington, Seattle, WA). This cDNA was inserted into the SP6 vector (Promega Biotech, Madison, WI) and used as a template for the synthesis of 32p or 35S-labeled cRNA probes as described by Melton et a1.[26]. A MT sense strand probe was also synthesized. Probes had specific activities of about 2 x 109 dpm/ /~g.

Isolation of total RATA and Northern blot hybridization RNA was extracted, using a sodium dodecyl sulfate (SDS)-phenol:chloroform procedure, [16], separated by formaidehyde-agarose gel electrophoresis and analyzed by Northern blot hybridization as described previously [4]. In all experiments, duplicate gels were stained with acridine orange to ensure integrity of the RNA sample and to confirm that equal amounts of RNA had been loaded onto each lane. 91

In situ hybridization The details for in situ hybridization have been described previously [4] and were adapted from methods published by Angerer and colleagues [27--30]. Perfusion-fixed embryos were dissected out intact and further fixed in 4070 paraformaldehyde in PBS at 4°C for 2 h. Embryos were paraffin embedded and serially sectioned at 7 /am. Slides containing sections of D9 through D13 embryos were hybridized with a 35S-labeled mouse MT-I cRNA probe. After hybridization, slides were washed to remove the excess probe and treated with RNase A to reduce background and ensure specificity of the remaining hybrids. Hybrids were detected by autoradiography using Kodak NTB-2 liquid emulsion, and a 4 day exposure period. Slides were post-stained lightly in hematoxylin. Results

Cadmium teratogenicity Dose-dependent teratogenic effects of Cd were determined by injection (s.c.) of Cd early on day 10 of pregnancy, followed by examination of embryos for malformations on D14 or for wet weights on D17 (Table I). An injection of 20-30 /amol CdCl2/kg of body weight had no apparent teratogenic effects, but reduced embryo wet weight occurred at the higher dosage. In contrast, an injection of 40--50 tamole CdC12/kg was teratogenic and led to a significant reduction in embryo wet weight. At the higher dosage (50 tamol/kg), about 80°70 of the embryos were malformed, exhibiting open neural tubes, abnormal limb bud development, craniofacial abnormalities and a reduction in somite number. An injection of > 60/amol Cd/kg resulted in embryolethality (data not shown). Metal effects on M T - I gene expression in DIO embryos The effects of heavy metals (Zn and Cd) on MT mRNA levels in the embryo were examined both in vivo and in vitro. Animals were injected early in the

TABLE I D O S E - D E P E N D E N T C A D M I U M T E R A T O G E N I C I T Y O F T H E CD-1 M O U S E EMBRYO Cd' Oamol/kg)

E m b r y o weight b (g)

Malformed ¢ (070)

0 20 30 40 50

0.725 0.700 0.523 0.413 0.406

0 0 2 15 82

aCadmium chloride was injected s.c. at the indicated dosages Oamol C d / k g body wt) early on day 10 of pregnancy, and embryos were examined on day 14 or on day 17. At least four litter (about 50 embryos) per group were examined, bEmbryo weight was determined on day 17. cMalformations included; open neural tube, abnormal limb bud development, craniofacial abnormalities, and reduction in somite number.

92

D

;

t

Zn

50 100

I

I

Cd

50 75 umol/kg

i

J

0

i

f

I

Zn

Cd

50 100 10 20 uM

g

IN V I T R O

Fig. 1. Northern blot analysis o f MT m R N A levels in D10 embryos exposed in vivo or in vitro to c a d m i u m or zinc. DI0 embryos (about 40 per point) were :ollected 5 h after a maternal sc injection of the indicated dosage of metal, or following 5 h o f culture in medium containing the indicated concentrations o f ~etal ions. Total R N A was extracted and analyzed by Northern blot hybridization using a mouse MT-I cRNA probe. Hybrids were detected by autoradiography for 5 h. Duplicate agarose gels were analyzed by acridine staining to ensure that equal a m o u n t s of R N A had been loaded. Injection of a teratogenic Josage of Cd has no effect on embryonic MT m R N A levels, whereas in vitro, Cd was at least a 10-fold more potent inducer o f M T m R N A than was Zn. rhis suggests that Cd is prevented from reaching the embryo in vivo.

~T-

IN V I V O

Visceral yolk sac

Somite

Wall of neural tube

3ay 9

a

O

e

t

¸

Fig. 2. In situ hybridization analysis of cell-specific MT-I gene expression in D9 and D10 mouse embryos. Pregnant mice, on the indicated days of gestation, were anesthetized and embryos were fixed by perfusion with 4°70 paraformaldehyde in PBS. Samples were paraffin embedded, serially sectioned at 7/~m and hybridized in situ for 5 h at 42°C with a 35S-labelled MT-I cRNA probe. RNase A resistant hybrids were detected following 4 days of autoradiography using Kodak NTB-2 liquid emulsion. Slides were poststained lightly in hematoxylin. Hybridization signals (grains in the emulsion) appear as small white dots, whereas large white areas represent refraction of light by debris on the slides. (a) Bright-field and (b) dark-field photomicrographs (magnification is 40 × ) of D9 embryo and extraembryonic membranes isolated from a mother injected 5 h previously with a teratogenic dosage of Cd (50/amol/kg body wt). (c) Bright-field and (d) dark-field photomicrographs (magnification is 40 X ) of a normal D10 embryo.

Liver rudimer

~leural tube_

Gut

Oral cavity

4th VentriclG

Day 10

c~

Day 11

Li v er

Fig. 3. Localization of MT-I mRNA in DI 1 and DI2 mouse embryos by in situ hybridization. Normal embryos, on the indicated days o f gestation, were analyzed by in situ hybridization as described in the legend of Fig. 2. Autoradiography was for 4 days (sections were on the same slide), and the slides were poststained lightly with hematoxylin. Shown here are bright-field (a and d) and dark-field (b, c and e) photomicrographs o f sagittal sections o f normal D11 and D12 embryos. (c) High power view (200 X magnification) of (b) which includes newly formed liver and surrounding tissues. As stated above, only the small white dots are the hybridization signals, large ones are due to refraction o f light by debris on the slide. The red blood cells (RBC) also refract light (b), but are not labeled.

Day 12

morning of D10 with various doses o f Zn or Cd and embryos were collected 5 h after the injection. Northern blot hybridization of total embryonic RNA demonstrated that MT-I mRNA levels were low in D10 embryos (Fig. 1A) and no significant change in MT-I mRNA levels occurred after injection of a teratogenic dosage (50/amol Cd/Kg) of Cd (Fig. 1A). On the other hand, an injection of the equivalent amount of Zn significantly induced MT-I mRNA in the embryo. Although some induction of the embryonic MT-I gene was observed after administration of a higher dose of Cd (75 /amol/kg), this dosage exerted embryotoxic effects within one day following injection, and resulted in maternal lethality within a few days (data not shown). To examine the possibility that the MT genes in the embryo resist induction by Cd, D10 embryos were isolated and incubated in vitro in medium with or without excess heavy metals (Cd or Zn). MT mRNA levels were determined after 5 h of culture (Fig. 1B). Under these conditions, Cd was an effective inducer of embryonic MT gene expression. A comparison of the dose-response for these metals suggests that Cd is about 10-fold more potent than Zn at inducing MT mRNA in the embryo (Fig. 1B). A large induction of MT-I mRNA was noted in the embryo following incubation in medium containing 10 /am Cd. Taken together the above results suggest that little, if any, Cd reaches the D10 embryo following a maternal injection o f this metal.

Cell type-specific MT-I gene expression in midgestational mouse embryos In situ hybridization was used to determine the cellular distribution of MT-I mRNA during normal development of the embryo and following a maternal injection of a teratogenic dose of Cd. Females were injected with a teratogenic dosage of Cd on D9. Embryos were recovered 5 h later and analyzed by in situ hybridization using an MT-I cRNA probe. Control embryos were also processed and examined on the same slide. Examination of multiple sections of many embryos established that only the extraembryonic membrane (visceral yolk sac) contained high levels of MT mRNA following exposure to Cd (Figs. 2a,b). Little labeling was noted in the embryo either before or after Cd injection (normal D9 data not shown). The grains over the embryo in Fig. 2b reflect a higher background in that experiment, but are many-fold less than those found over the extraembryonic membrane and essentially the same as those noted in the control embryos (data not shown). Normal D10 embryos contained only low levels of MT mRNA and no evidence for cell-specific expression of MT genes was obtained (Figs. 2c,d). However, newly formed hepatocytes in D l l embryos apparently contained more MT mRNA than did other cells (Figs. 3a,b,c). Although this was routinely observed in D11 hepatocytes, the results suggest the presence of only low levels of MT-I mRNA. The embryonic red blood cells (RBC) refract light in these dark-field photomicrographs, but are not labelled. In contrast, D12 hepatocytes contained high levels of MT mRNA (Figs. 3d,e) and on D13, the embryonic liver was still the only organ that contained high levels of these transcripts (Figs. 4a,b). Hybridization using the sense strand MT probe did not produce any labeling in sections of embryos (Fig. 4c). These results suggest that high level expression of the MT genes in the embryo is restricted to the hepa-

98

Live

Lun(.

-lear

Tong1

~)ay 13

Fig. 4. Localization of MT-I mRNA in the D13 mouse embryo by in situ hybridization. DI3 embryos were analyzed by in situ hybridization as described in the legend of Fig. 2. Sagittal sections of normal D13 embryo (40 × magnification) hybridized with the antisense MT cRNA probe (a and b) or a sense MT cRNA probe (c). (a) is bright-field and (b) and (c) are dark-field photomicrographs. Pictures shown here were taken to cover the complete developing liver and GI tract.

tocytes. Furthermore, a teratogenic dose of Cd had little or no effect on expression of the embryonic MT genes in the D9 embryo. Discussion

In order to understand more about the mechanisms by which Cd exerts its teratogenic effects, the effects o f the toxic metal Cd and the essential metal Zn on expression of the MT genes in the embryo were analyzed. In a wide variety of cultured cells, as well as in the intact animal, transcription o f the MT genes is rapidly and dramatically induced by these metal ions, and this protein is thought to provide protection from heavy metal toxicity. Cultured cells which overexpress MT, due to preexposure to metals or to amplification o f MT genes, are more resistant to Cd toxicity [31]. On the other hand, lymphoid cells that are more sensitive to Cd toxicity, exhibit a diminished capacity of the MT genes to respond to metal ions, due to the methylation status o f the MT genes [32]. The MT genes in embryos, cultured in vitro, were shown to rapidly respond to micromolar concentrations of metals. These concentrations of Cd and Zn also induce MT genes in a variety of cultured cell types [16,19,20,33]. Thus, the toxic effects of Cd on the embryo do not result from a lack of responsiveness of the embryonic MT genes. In marked contrast, an injection of a teratogenic dosage of Cd did not induce embryonic MT gene expression during the teratogenic period, b u t a Zn injection did. These results strongly suggest that Cd, but not Zn, is prevented from reaching the embryo, at least in high enough concentrations to induce the MT genes. This possibility is consistent with the observations that little radioactive Cd reaches the embryo following a maternal injection of this metal, but instead, the Cd is concentrated in the deciduum, placenta and visceral yolk sac [3]. We have recently reported that these extraembryonic tissues actively express the MT genes [4,23]. High levels of this Cd-binding protein may, therefore, provide a molecular mechanism for the Cd barrier function of these tissues which surround the developing embryo and fetus. Since MT binds to Zn with 10 000fold less avidity than it does with Cd, Zn ions can be readily transferred from MT to other proteins, whereas Cd ions remain tightly bound [19]. This likely accounts for the in vivo induction of the embryonic MT genes by Zn. In the adult mouse, the liver, kidney and pancreas are major sites of synthesis of MT following Cd injection and these organs preferentially accumulate and retain this metal [19,34]. Retention of Cd results in hepato- and nephro-toxicity and pancreatitis [34]. Perhaps these organs can be considered as targets for Cd toxicity by virtue of elevated expression of MT genes. Analysis of the cell-specific expression of MT genes in the embryo, using in situ hybridization, established that MT mRNA is not elevated in the limb bud and neural tube, or in any other cell types in the embryo during the teratogenic period. This suggests that small amounts of Cd which pass the presumed 'barriers' that restrict entry into the embryonic environment, are not targeted by virtue of expression of the MT genes in those regions of the embryo that undergo Cd-induced dysmorphogenesis. Although, changes in metal ion uptake may influence MT gene expression in cultured cells [35], it seems unlikely that increased influx (or lack of efflux) of Cd

100

could account for its targeting to Cd-sensitive regions of the embryo. This would be expected to have led to increased intraceUular concentrations o f Cd and activation of the MT genes, but instead, induction of MT genes was not noted in any D9 embryo cell-types following a maternal injection o f Cd. Cd toxicity must, therefore, be postulated to involve concentrations of this metal that have little or no effect on MT gene transcription. Analysis of the distribution of radioactive Cd suggests that very low levels o f this metal can be detected in mouse and rat embryos [1,3,6,36] and analysis o f Cd toxicity in cultured rat embryos suggests that sub-micromolar concentrations of Cd are embryotoxic in vitro [6]. However, in these instances the level of Cd in the visceral yolk sac was 100-fold greater than that in the embryo [6]. The accumulation o f Cd in the deciduum, placenta and visceral yolk sac leads to dysfunction of these structures. Yolk sac pinocytosis is inhibited by Cd and Cd-induced dysgenesis of the yolk sac may lead to impaired transfer of nutrients to the fetus [8,37]. It has also been suggested that Cd is responsible for the decreased volume of fetal capillaries in the terminal villi of placentae, as well as for an increase of connective tissue around the fetal vessels [38,39]. Altered synthesis o f serum and amniotic fluid proteins by the yolk sac, a n d / o r reduced expression o f growth factors in the deciduum, placenta and yolk sac might also play a role in Cd teratogenicity. These extraembryonic effects of Cd must also occur in whole embryo cultures o f mouse and rat embryos [7,10], because successful morphogenesis requires the presence of the visceral yolk sac, and the developing placenta. The possibility that Cd acts indirectly on the embryo has been proposed previously by other investigators. Levin and Miller [40] suggested that following a direct injection of Cd into the fetus, fetal death resulted from Cd effects on the placenta and not from direct effects on the fetus. More recently, Webb et al.[6] have shown that Cd-MT complexes are teratogenic in vivo, but only very low amounts of Cd are detected in the embryo and fetus. The nephrotoxic effects of Cd-MT may account, in part, for its teratogenic effect. During the course of these experiments the ontogeny o f expression of the MT genes in the liver was also examined. Previous studies have established that the fetal and neonatal rodent liver actively expresses the MT genes [reviewed by 24] and this had been documented as early as D13 of gestation [23]. It is now apparent that the MT genes are apparently first expressed in differentiating hepatocytes on D l l and by D12 the MT mRNA level in these ceils is much greater than in any other embryonic cell types. The mechanisms which regulate expression of MT genes in embryonic hepatocytes are unclear. Both metal ions and glucocorticoids have been suggested to play a role [41,42], but these analyses centered on methods to attenuate the elevated levels normally found in the late gestation fetal and neonatal liver. Determining whether exogenous metals or glucocorticoids can induce premature elevation of MT mRNA levels in the D11 hepatocyte may provide an alternative approach for analysis o f the developmental regulation of these genes. In conclusion, the relative levels of MT mRNA in the embryo after an acute in vivo or in vitro exposure to Cd or Zn were determined as a sensitive indicator of a direct effect of these metals on the embryo. In addition, the cell-specific expres101

sion o f these genes was d e l i n e a t e d in m i d g e s t a t i o n e m b r y o s (D9 t o D13). T h e d a t a suggest t h a t the levels o f C d r e a c h i n g the D10 e m b r y o f o l l o w i n g m a t e r n a l i n j e c t i o n o f a t e r a t o g e n i c dose o f this m e t a l are insufficient to induce M T gene expression, a n d are thus likely to be n o n - t o x i c to m o s t e m b r y o n i c ceils. F u r t h e r m o r e , only the h e p a t o c y t e s actively express the M T genes in n o r m a l m i d g e s t a t i o n m o u s e e m b r y o s ( D I l - - 1 3 ) , w h i c h eliminates the possibility o f C d - t a r g e t i n g v i a e m b r y o n i c cell-specific M T gene expression d u r i n g the t e r a t o g e n i c p er i o d . H o w ever, the possibility that certain cells in the e m b r y o are e x c e p t i o n a l l y sensitive to low levels o f C d , c a n n o t be ruled out.

Acknowledgements This w o r k was s u p p o r t e d by grants f r o m the N I H (ES 04725) to G . K . A n d r e w s an d ( H D 12304) to S.K. Dey. S w a p a n De is f u n d e d by a W e s l e y F o u n d a t i o n S ch o l a r P r o g r a m in C a n c e r R e s e a r c h p o s t d o c t o r a l fellowship. This w o r k was also f u n d e d , in p a r t, by a W e s l e y F o u n d a t i o n G r a n t to G K A an d SKD.

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Cadmium teratogenicity and its relationship with metallothionein gene expression in midgestation mouse embryos.

As an approach toward understanding the mechanisms by which cadmium (Cd) exerts its teratogenic effects, the expression and metal regulation of the me...
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