* Department of Chemistry, Eastern Regional Research Center, ARS U.S. Department of Agriculture, 600 East Mermaid Lane, Philadelphia, Pennsylvania 19118;
Biosciences Division, Argonne National Laboratory, Argonne, Illinois 60439; and
Department of Natural Sciences, Dominican University, 7900 West Division Street, River Forest, Illinois 60305
Received August 17, 2000; accepted October 23, 2000
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ABSTRACT |
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Key Words: metallothionein; cadmium exposure; environmental cadmium; lactation; liver; kidneys; duodenum; mouse.
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INTRODUCTION |
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Cadmium is a widely distributed environmental pollutant (Friberg et al., 1986). Environmental exposure levels of cadmium are relatively low, with daily intake ranging from 4 to 60 µg in humans (Friberg et al., 1974
, 1986
; Tahvonen, 1996
). However, the cumulative retention of cadmium, due to its long biological half-life in mammalian systems (Bhattacharyya et al., 1986
; Friberg et al., 1986
; Goyer, 1991
), makes this metal one that demands close scrutiny as a potential environmental health concern. Cadmium exposure has been implicated as one cause for the outbreak of Itai-Itai disease in Japan (Nogawa et al., 1990
). This disease is characterized by osteomalacia and renal tubular dysfunction, and it affects mainly postmenopausal women with a history of multiple child-bearing. The particular susceptibility of this population to Itai-Itai disease may be partially attributed to increased maternal absorption of dietary cadmium during pregnancy and lactation (Wang et al., 1994
; Webster, 1988
). In addition to the renal and bone responses, cadmium has more recently been linked to carcinogenic and teratogenic changes in tissues (Copras and Antonio, 1998; Mahalik et al., 1995
; Piasek and Laskey, 1999
; Waalkes and Oberdörster, 1990
; Webb et al., 1988
). On the basis of these and other studies, cadmium is considered a human carcinogen in the occupational setting (Waalkes and Oberdörster, 1990
; U.S. OSHA, 1992
).
The mechanisms for increased intestinal absorption and retention of both essential and toxic metal ions during pregnancy and lactation are still under study, but metal-binding proteins, including MT, clearly influence metal absorption pathways (Chan et al., 1993; Chmielnicka and Sowa, 1996
; Suzuki et al., 1990
; Webb, 1986
). Because MT plays an important role in the binding and metabolism of zinc and copper, as well as cadmium, in all developmental stages (Borghesi and Lynes, 1996
; Cherian and Chan, 1993
; Clarkson, 1995
; Kägi, 1993
; Kern et al., 1981
; Liu et al., 1996
), and has been shown to be elevated in pregnancy (Chan and Cherian, 1993
; Chan et al., 1993
; Chmielnicka and Sowa, 1996
; Payan et al., 1990
; Suzuki et al., 1990
), we have examined whether the concentrations of this protein show change in maternal tissues during lactation. Analyses for MT include gel filtration chromatography and hemoglobin-binding assay techniques, utilizing the protein's low molecular weight (
6000 Da), characteristic elution from Sephadex G-50/-75 gel filtration columns (Ve [elution volume]/Vo [void volume]
1.82.3), and relative heat stability (Eaton and Cherian, 1991
; Suzuki, 1992
). Possible regulation of cellular MT at the MT mRNA level is explored through Northern blotting techniques.
In the current study, changes were examined in maternal MT levels in tissues from lactating mouse dams in the absence of any external stimulus. In addition, we investigated the binding of trace amounts of 109Cd to MT in mouse tissues after oral administration of carrier-free 109Cd to evaluate the role of MT in Cd pathways during lactation. Concentrations of MT mRNA were determined in livers from mid-lactating and post-lactating mouse dams with no cadmium exposure. The results demonstrate that increases in MT protein and mRNA concentrations occur in mouse dams as a physiological response to lactation. In addition, this MT is an important part of the mechanism of handling low levels of oral cadmium by the lactating dam.
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MATERIALS AND METHODS |
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Lactating dams were assigned to 1 of 3 groups of 10 animals each. These groups were designated L 13, L 20, and W 5, indicating the dams were to be sacrificed on L 13, L 20, or day 5 after weaning (W 5), respectively. To relate the results of this study to our previous one, which demonstrated increased absorption of cadmium on specific days of lactation (Bhattacharyya et al., 1981), one subgroup of mice was treated according to the same fasting and gavage procedures used in our earlier study. Thus, half of the mice from each group were fasted overnight (18 h), gavaged with 0.2 ml of double-deionized water, and sacrificed 3 days later, as described previously (Bhattacharyya et al., 1981
). The other 5 animals in each group served as nongavage controls and were not fasted or gavaged. This protocol was carried out to investigate the possibility that the gavage procedure and the stress of overnight fasting, particularly in lactating dams, might itself have induced MT, as suggested by Bremner and Davies (1975) and Oh and co-workers (1978).
Two additional groups of mice were the nonpregnant (NP) controls. A zero-time NP group was sacrificed on the same day that mating of animals in the lactating groups was initiated. To track possible changes in MT concentrations with age in the young adults used in this study, a second NP control group was sacrificed 37 days later. (With the 6-day mating period employed, delivery dates spanned a week's time, and dams with early delivery dates were assigned to the L-20 group. Consequently, the 37-day NP mice were intermediate in age (±2 days) between the L-13 and L-20 animals.) Each NP control group was also subdivided into gavage and nongavage subgroups, as above.
Mice were sacrificed by intraperitoneal injection with pentobarbital (12 mg/mouse, ip, Nembutal, Abbott Laboratories, Abbott Park, IL). Liver, kidneys, duodenum, and jejunum (defined as the first 5 cm and the next 10 cm of the small intestine, respectively) were removed. The lumens of the small intestinal segments were thoroughly rinsed with10 mM Tris HCl, pH 7.4, in 0.9% NaCl. The duodenum and jejunum were immediately placed on an ice-cold stainless steel plate. Each segment was cut longitudinally, spread open, and the mucosal layer scraped off by using the sharp edge of a microscope slide, leaving behind only the transparent muscle layer. All tissue samples were immediately frozen inside a cryogenic tube by immersion in liquid nitrogen and stored at 20°C. These intestinal tissues were later homogenized for determination of MT by Cd/hemoglobin assay, as described below. Tissue homogenates and supernatants were prepared in cold, 10 mM Tris HCl buffer containing 5 mM 2-mercaptoethanol (2-ME) (TME buffer), at pH 7.4 or 8.6, as specified in the particular procedure.
Determination of metallothionein by Cd/hemoglobin assay.
The MT content of clear supernatants (10,000 x g, 20 min, 4°C) from tissue homogenates was analyzed by a modified version of the Cd/hemoglobin assay (Eaton and Cherian, 1991). Because of concern for the stability of MT (Minkel et al., 1980
), 2-ME was added as an SH-protector. The 2-ME did not appreciably increase the assay background (data not shown). To assure accuracy of the Cd/hemoglobin assay, preliminary assays were carried out with each tissue homogenate to determine the amount of tissue that gave MT values in the linear portion of the tissue dilution curve. Samples were stored overnight at 4°C in buffer with 5 mM 2-ME prior to the final assay. This method of storage was found necessary in order to retain the full amount of MT in the samples. To confirm the Cd-binding protein as MT, the final Cd/hemoglobin assay supernatant was chromatographed, as below.
The 109Cd content of the final supernatant was used to calculate the MT concentration in the tissue, according to Eaton and Cherian (1991). A well-type gamma-ray spectrometer (Beckman Gamma 310 or Beckman Gamma 8000) was used to measure 109Cd. The counting efficiency of the 88 KeV gamma radiation of 109Cd was 7478% as determined with a 109Cd reference source (New England Nuclear, Boston, MA). Radiation safety protocols were supervised by the Environmental Safety and Health Division of Argonne National Laboratory.
Preparation of cytosol fractions after oral 109Cd administration during lactation.
L-10 and nonpregnant B6CF1/Anl mice analogous to those described above were fed by gavage with 66 ng of cadmium as 109CdCl2 (86 µCi per mouse; carrier-free 109Cd, 1.3 Ci/mg; New England Nuclear, Boston, MA) in 0.2 ml water after an overnight fast. Three days later (L 13 for lactating dams), the mice were sacrificed and samples of duodenum and liver, the major early deposition sites for orally administered cadmium, were taken as described above. Tissue samples were stored at 20°C until use. To prepare cytosol fractions, frozen tissues were homogenized in 4 volumes of 10-mM TME buffer, pH 8.6. The homogenate was centrifuged at 105,000 x g for 90 min at 4°C. A portion of the 109Cd-containing cytosol was chromatographed on a Sephadex G-75 column. The remainder was heat-treated once (60°C, 2 min), centrifuged (40,000 x g for 20 min) to remove heat-labile precipitates, and re-chromatographed on Sephadex G-75, to determine whether gentle heating might change the chromatographic profile.
Gel filtration column chromatography.
Cd-binding molecules present in the supernatant fractions obtained from Cd/hemoglobin assays, as well as in the 109Cd-containing cytosol fractions obtained after 109Cd gavage, were evaluated by gel filtration chromatography on Sephadex G-75 (Sigma Chemical, St. Louis, MO). Columns (2.5 x 100 cm) were equilibrated and eluted with TME buffer, pH 8.6. The volume of each sample applied to the columns was 3 ml. Elution was performed at a constant flow rate of 10 ml/h; eluant was collected in 1.5-ml fractions. Chromatographic fractions containing 109Cd were analyzed as above. Molecular weight markers were used to calibrate the columns (Sigma Chemicals).
In addition, standard mouse liver MT was produced by stimulating male B6CF1/Anl mice (129 days old, 2628 g) with a series of subcutaneous injections of CdCl2, according to the schedule of Tsunoo et al. (1978). The induced liver was first analyzed by Sephadex chromatography, as above, using atomic absorption spectroscopy to monitor MT complexed to Cd (data not shown). The induced MT concentration was then determined by the Eaton and Cherian (1991) method and the 109Cd-containing supernatant from this assay was also separated on Sephadex. Both elution profiles had a peak with Ve/Vo = 2.12.3, consistent with published values for MT (Suzuki, 1992). This chromatographic position of induced MT was used to evaluate whether the chromatographic elution pattern of 109Cd in tissues from the lactating mice was consistent with its being bound to MT.
Northern analysis of MT mRNA concentrations in tissues of mouse dams.
Female CF1 mice, 95 days old and weighing 2630 g, were bred, housed, and litters normalized as described above for the B6CF1 mice. This strain was chosen because B6CF1/Anl mice were no longer available, and our results had shown that increases in cadmium absorption and retention during pregnancy and lactation were essentially the same at the end of lactation in B6CF1 and CF1 mouse dams continuously exposed to low levels of cadmium in drinking water (Bhattacharyya et al., 1982, 1986
). Mouse dams were analogous to the nongavage groups above (no Cd, gavage, or fasting protocol). Mice (3 dams/group) were sacrificed at either L 13 or W 5, to observe the relationship of MT mRNA levels to hepatic MT protein concentrations during lactation. The mouse liver MT mRNA, used as a comparison standard in the Northern blots, was induced in CF1 female NP mice (141 days old, 2832 g) by a single subcutaneous injection of 250 µg CdCl2. Mice were sacrificed 56 h after injection. At sacrifice, livers were rapidly frozen at 70°C. RNA was extracted by the method of Chirgwin and coworkers (1979) as modified by Cathala et al. (1983). Ethanol-precipitated RNA samples were stored at 20°C before electrophoresis. Gel electrophoresis in 1% formaldehyde-agarose was carried out as described by Woloschak et al. (1990). Fluorescence of ethidium bromide-labeled 18s rRNA bands on completed gels was used to document equal RNA loading in wells. After blotting onto nylon membranes, the separated RNA was hybridized to a 1500-base-pair genomic DNA probe for human MT-II that hybridizes to the mRNA of both MT-I and MT-II from mouse (Karin and Richards, 1982
).
Statistical analyses.
Statistical differences were determined by analysis of variance (ANOVA) followed by the least-significant-difference (LSD) test.
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RESULTS |
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MT in Tissues of Lactating and Non-Pregnant Mice as Determined by Cd/Hemoglobin Assay
As shown in Table 1, significant increases in MT protein concentration occurred in the tissues of mouse dams during lactation. In L-13 animals, the increases were statistically significant (p < 0.05) in liver (4.3-fold), kidneys (2.0-fold), and duodenum (2.4-fold) above those of the NP controls of similar age. By L 20, the MT concentrations of these tissues decreased slightly, but still represented 2-fold increases (p < 0.05) over those of the NP mice. MT increases in the small intestine were specific to the duodenum; concentrations in the jejunum samples from L-13 and L-20 dams were not significantly greater than those of the age-appropriate NP controls. Finally, the increases of MT concentration in affected tissues were specific to the lactation period; by 5 days after weaning, MT concentrations in tissues of the dams returned to levels similar to those of the NP controls.
Gel Filtration of Final Supernatants from Cd/hemoglobin Assay
Gel filtration chromatography of standard MT obtained from the liver cytosol of male mice injected with non-radioactive CdCl2 (to induce MT protein) resulted in the recovery of cadmium in a low-molecular-weight peak at Ve/Vo = 2.2 (Fig. 1A). The gel filtration profiles of the final supernatants from the Cd/hemoglobin assays for specific groups are also presented. Final supernatants from NP liver samples (Fig. 1D
) showed that the main peak of 109Cd chromatographed in the same position as standard mouse MT (Ve/Vo = 2.2). For the L-13 (Fig. 1E
) and L-20 (Fig. 1F
) liver samples, the predominant 109Cd peak was higher in radioactivity than for the NP sample and again co-chromatographed with standard MT, with minor peaks appearing at Ve/Vo values of 1.0 (void volume) and 1.65.
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Northern Blot Analyses of RNA from Mid-Lactating and Post-Lactating Mouse Liver
Standard RNA samples extracted from the livers of Cd-treated mice showed a prominent RNA band that hybridized with the MT DNA probe in the Northern-blot analyses (Fig. 2, lane 1). Corresponding bands of MT mRNA were identified in Northern blots (Fig. 2
) of RNA samples extracted from the livers of individual L-13 (lanes 24) and W-5 (lanes 57) mouse dams. Densitometric measurements of the autoradiograms showed that the mean liver MT mRNA abundance in L-13 dams (percentage intensity of +Cd standard: 58 ± 13%, mean ± SE, n = 3) was 6-fold higher (p < 0.01) than in dams 5 days after weaning the pups (9.6 ± 4%).
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For mouse dams on L-13, the amount of 109Cd deposited in the liver after oral 109Cd increased significantly (Compare ordinate scales of Figs. 3A vs. 3C.). Chromatographic profiles showed that 109Cd in the L-13 liver cytosol was almost exclusively associated with a peak at Ve/Vo = 1.7 (Fig. 3C
). Cytosolfrom the L-13 duodenum (Fig. 3E
) contained 91% of the recovered 109Cd in the region typical of MT (Ve/Vo = 2.3). When these L13 cytosols from liver and duodenum were heated to 60°C as above, 84 and 83% of the cytosolic 109Cd, respectively, remained soluble in the supernatant. Chromatography of these heat-stable supernatants showed that all of the soluble 109Cd in L-13 liver gave an elution profile typical of standard MT (Ve/Vo = 2.1; Fig. 3D
), and 94% of the L-13 duodenum 109Cd retained the same MT peak at Ve/Vo = 2.3 (Fig. 3F
).
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DISCUSSION |
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The results of the present study demonstrate that concentrations of MT, the major cadmium-binding protein in higher organisms, did indeed increase significantly in the liver, kidneys, and duodenum of mouse dams during mid-lactation (Table 1). Because the dams in this study were not administered any agent, such as cadmium, the stimuli for the observed rises in tissue metallothionein levels remain to be explored. The progesterone-induced increases in MT synthesis and Cd toxicity observed in rat-liver cells in vitro by Shimada and coworkers (1997) provide one example of MT expression being under hormonal control.
The increases in MT concentrations reported in this study were specifically associated with the lactation period. Once dams entered the post-lactation period (W 5), MT concentrations returned to levels comparable to those found in NP controls (Table 1) and the MT mRNA levels decreased (Fig. 2
). Although the lactation-induced increase in liver MT concentration shown here was accompanied by a 2-fold increase in the weight of the dam's liver (Bhattacharyya et al., 1981
), the striking decrease in MT after weaning (Table 1
) occurred in the absence of any decrease in liver weight (Bhattacharyya et al., 1986
). The latter study (1986), which used low cadmium concentrations similar to those in this study, further demonstrates that the increased amount of cadmium taken up and retained by the liver of the dam during pregnancy and lactation is not released from that tissue during a 4-week period after weaning, in spite of the precipitous post-weaning declines in MT protein and MT mRNA concentrations demonstrated by the data reported here.
In addition to being lactation-induced, the increase in MT levels also was tissue-specific. Liver and kidney, the main organs that concentrate cadmium in mammals (Bhattacharyya et al., 1981, 1986
; Chan and Cherian, 1993
; Webb, 1986
), exhibited large lactation-dependent MT increases (Table 1
). Duodenum, but not jejunum, also showed an increase in MT levels during lactation. The correspondence of the latter MT changes with our earlier finding of increased cadmium retention in duodenum, but not jejunum, of lactating dams after low-level, oral cadmium administration (Bhattacharyya et al., 1981
) indicate that the changes in MT protein reported here do correlate with changes in the uptake and retention of dietary cadmium by the dam's intestine during lactation. Although previous reports indicated stressful conditions can cause MT increases in rats (Bremner and Davies, 1975
; Oh et al., 1978
), our data (Table 1
) show the fasting and gavage treatment of both NP mice and lactating dams in the gavage subgroups did not induce additional MT synthesis.
Furthermore, our results demonstrate the binding and cytosolic retention by liver and duodenum of trace amounts of administered cadmium (Fig. 3), indicating that the lactation-induced MT would participate in the molecular pathways for handling cadmium at low levels of oral intake such as those experienced by humans environmentally exposed to cadmium in diet. The trace amounts of very high specific activity 109Cd (66 ng Cd per mouse; equivalent to 3 µg/kg) administered by gavage to mouse dams during lactation, is in the range of the daily intake of environmental dietary cadmium in humans (0.051 µg/kg; Friberg et al., 1974, Groten et al., 1994), and because circulating Cd-MT is deposited rapidly and nearly exclusively in the kidney (Cherian et al., 1978
; Nordberg and Nordberg, 1975
), the increased concentrations of Cd-MT in the dam duodenum and liver provide credible sources for the increased movement of cadmium to dam kidneys that occurs during pregnancy and lactation (Bhattacharyya et al., 1981
, 1982
; Chan and Cherian, 1993
; Chan et al., 1993
; Payan et al., 1990
; Suzuki et al., 1990
).
The results of a number of studies, including ours, indicate that the presence of MT in a given tissue does not cause that tissue to initially take up more cadmium from the circulation. That is, endogenous MT does not appear to attract cadmium to a given tissue. For example, in the study of Liu and Klaassen (1996), the significantly elevated concentrations of MT in the tissues of MT-I transgenic mice altered neither the extent of intestinal absorption nor the organ distribution of orally administered cadmium compared to wild-type mice. Furthermore, Liu et al. (1996) demonstrated that the initial pattern of cadmium distribution in the various tissues of mice after parenteral administration was the same, independent of whether the mice were wild-type or were totally deficient in MT1 and MT2. In the latter study, however, cadmium was released from tissues and excreted from the bodies of the MT-deficient mice more rapidly than from the wild-type. Similarly, previous experiments have shown that, after a single dose of cadmium administered by gavage during mid-lactation, the percentage of the administered dose present in the liver at 72 h was not significantly greater in the dam than in nonpregnant controls (Bhattacharyya et al., 1981). Only when cadmium was administered continuously in drinking water over 42 daysa duration potentially long enough to reflect increases in tissue retention half-timewere cadmium amounts and concentrations in the liver greater in dams than in the nonpregnant mice (Bhattacharyya et al., 1982
). These results and those of others (Liu and Klaassen, 1996
; Liu et al., 1996
) support the hypotheses that (1) endogenous MT can increase the biological half-time of cadmium retention in tissues, but (2) the initial rate of uptake of cadmium by the tissues appears to be independent of endogenous concentrations of MT.
Because the Cd/hemoglobin assay method can detect Cd-binding molecules other than MT (Waalkes and Perantoni, 1986), some of which might increase during lactation, the final supernatants from these assays were further analyzed by gel filtration (Figs. 1B1F
). Results confirmed that from 70100% of the 109Cd measured in the Cd/hemoglobin assays did chromatograph in the same position as standard MT (Fig. 1
). Furthermore, our measurements of MT mRNA in liver (Fig. 2
), showing corresponding changes in MT mRNA concentrations in mid-lactating and post lactating mice, provide additional support to our interpretation that the Cd/hemoglobin analyses did measure true changes in MT protein in the lactating dam (Table 1
, Figs. 13
).
Aggregation of the cysteine-rich MT polypeptide may have been the cause of at least some of the observed Cd bound to higher-molecular-weight proteins in the gel filtration analyses. For example, virtually all of the Cd in the elution profile of the liver cytosol from L-13 dams migrated at a position corresponding to an MT dimer (Ve/Vo 1.7; co-chromatographing with molecular weight standard = 12.4 kD) (Fig. 3C
). Because the major difference between the L-13 liver cytosol sample and the liver supernatant from the Cd/hemoglobin assay (Ve/Vo =2.2) was the heating step used in the latter assay, gentle heating at 60°C was tested for its ability to shift the L-13 liver cytosol peak to that of the MT monomer (Fig. 1A
). After heating, 90% of the recovered 109Cd was indeed associated with a peak having a Ve/Vo typical of MT (Figs. 1A and 3D
). A somewhat similar shift of cadmium-binding peaks in gel filtration was observed by Chan et al. (1993) after plasma samples from pregnant rats were heated to 95°C for 5 min. Similarly, when the final supernatants from the Cd/hemoglobin assays of liver (Figs. 1E and 1F
), kidney (Fig. 1B
), and duodenum (Fig. 1C
) were separated by gel filtration, varying amounts of 109Cd were bound to higher-molecular-weight fractions. In this case, reducing the duodenum fractions (Ve/Vo = 1.3) with 2-ME and re-chromatographing them caused nearly all the 109Cd to be associated with a typical MT peak. Whether this type of aggregation, which has been demonstrated in vitro (Tang et al., 1999
), is one that occurs or has physiological significance in vivo is not currently known.
Measurements of MT mRNA provide information about the level of control of the MT increase observed during lactation. While our results in the mouse clearly showed significantly increased MT protein levels in the liver, kidneys, and duodenum of mid-lactating mouse dams, Andersen et al. (1983) reported in the rat that hepatic MT mRNA concentrations, as measured by in vitro translation, rose sharply just prior to parturition and fell to a low level throughout lactation. Our demonstration of an abrupt fall of MT protein to levels comparable to non-pregnant controls by 5 days after weaning, as well as a corresponding drop of liver MT mRNA between L 13 and W 5 (Fig. 2), indicate that neither molecule persists long after the cessation of the stimulus of lactation in the mouse. More data are needed to reconcile these differences and determine whether there may be a species difference between mouse and rat in effects of lactation on MT protein and MT mRNA concentrations. Ours are the first results, however, to clearly demonstrate striking corresponding increases in both MT protein and MT mRNA concentrations during lactation that fall abruptly to low levels after cessation of lactation.
In summary, mid-lactating mouse dams that were free of exogenous Cd showed appreciably increased MT concentrations in the cytosols of liver, kidneys, and duodenum, but not jejunum, as measured by Cd/hemoglobin radioassay, gel filtration analyses, and, indirectly, by m-RNA analyses. This lactation-induced MT was shown to form a Cd-MT complex in the dam tissues after intestinal absorption of small amounts of cadmium during lactation. The increases in MT appeared to be induced by physiological changes associated with lactation, because post-lactational dams showed abrupt decreases in MT and in MT mRNA levels. Additional investigations are required to demonstrate whether these MT changes might play a role in pathways for handling essential metals such as copper and zinc in the dam. However, the MT changes reported here appear to explain results of previous studies showing altered cadmium uptake and tissue distribution in pregnant and lactating mice continuously exposed to very low concentrations (0.1 ppb) of cadmium. (Bhattacharyya et al., 1981, 1982
, 1986
; Whelton et al., 1993
). In the latter mouse model, increases in cadmium intestinal absorption and tissue retention, combined with lactation-induced increases in overall diet consumption, resulted in 6- to 8-fold increases in the kidney cadmium concentrations of mouse dams by the end of lactation, compared to those of the nonpregnant controls. Because diet is the major source of environmental exposure to cadmium in humans, female humans while nursing might show analogous increases in intestinal absorption and renal accumulation of dietary cadmium, with resultant increases in the susceptibility of multiparous females to the adverse effects of long-term cadmium exposure.
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ACKNOWLEDGMENTS |
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NOTES |
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