* Biology Department, Winona State University, Winona, Minnesota 55987;
Biological Sciences Department, Benedictine University, Lisle, Illinois 60532;
Department of Natural Sciences, Dominican University, River Forest, Illinois 60305; and
Biosciences Division, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, Illinois 60439
Received July 17, 2002; accepted October 18, 2002
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ABSTRACT |
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Key Words: metallothionein; cadmium; pregnancy; gestation; lactation; mice.
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INTRODUCTION |
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Four major isoforms of MT have been identified in mammals, with the predominant and best characterized being MT1 and MT2 (Chan and Cherian, 1993; Masters et al., 1994
). The MT3 and MT4 have been found primarily in brain (Palmiter et al., 1992
) and certain stratified squamous epithelia, including stomach and tongue (Quaife et al., 1994
), respectively. The MT1 and MT2 proteins are considered functionally similar (Chan and Cherian, 1993
). Hepatic concentrations of these latter two MT isoforms are detectable in rat fetal liver around gestation day 16 and increase into the neonatal period (Chan and Cherian, 1993
).
Previous work in this and other laboratories has shown that MT gene expression and MT protein concentrations are increased in specific tissues of female mice and rats during pregnancy and lactation as a result of normal physiological changes that occur during those periods (Shimada et al., 1997; Solaiman et al., 2001
). Induction of MT in the placenta occurs during gestation and has been suggested to prevent transfer of cadmium from mother to fetus during this time (Goyer et al., 1992
; Itoh et al., 1996
; Lau et al., 1998
; Petersson and Oskarsson, 2000
). Sequestration of cadmium in maternal mammary tissue also occurs during late gestation and throughout lactation (Bhattacharyya et al., 1981
, 1982
; Floris et al., 2000
; Lucis et al., 1972
; Petersson and Oskarsson, 2000
), though results indicate that this cadmium is bound to high-molecular-weight proteins rather than to MT (Lucis et al., 1972
).
Pregnant and lactating female animals (also called by the animal breeders term, "dams") absorb and retain substantially more dietary cadmium than do their nonpregnant counterparts (Bhattacharyya et al., 1982, 1986
, Floris et al., 2000
). However, only a small fraction of the cadmium is passed from dams to offspring: in cadmium-transfer studies in which pregnant mice were chronically exposed to tracer levels of 109Cd in drinking water, fetal cadmium concentration was much lower than maternal levels, and only about 0.01% of the 109Cd dose ingested by the dam was transferred to each 21-day-old pup during lactation (Whelton et al., 1993
). Cadmium pathways in maternal and neonatal animals have been hypothesized to reflect their changing MT concentrations, but this hypothesis has not been directly tested.
The current investigation was designed to determine the effects of maternal and neonatal MT on (1) the tissue distribution of orally absorbed cadmium during pregnancy and lactation and (2) the transfer of cadmium to offspring via milk. Concentrations of Cd and MT were determined in both generations of MT1- and MT2-normal (MTN) and MT1- and MT2-knockout (MT1,2KO) mice (Masters et al., 1994; Michalska and Choo, 1993
), following 109Cd exposure via drinking water. By using tracer amounts of 109Cd, cadmium pathways were studied in the absence of exogenous MT-inducing agents, making this study as relevant as possible to humans exposed to environmental levels of cadmium. These results demonstrate that some of the pathways previously proposed to involve binding of Cd to MT in a particular tissue are in fact MT-independent.
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MATERIALS AND METHODS |
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Experimental protocol.
Female MTN and MT1,2KO mice, 36 of each strain, aged between 55 and 62 days, and weighing 19.7 ± 0.2 g (MTN) and 19.0 ± 0.2 g (MT1,2KO; mean ± SE), were bred with male mice of the same age and strain (MTN or MT1,2KO). One male was housed with three females beginning at 2 h into the animals dark period on the first day of mating. Females were checked for vaginal plugs 8 h later on the day of mating, and at daily intervals thereafter. On the day each mouse showed a vaginal plug (day of conception), it was placed individually in a plastic cage.
Each dam was provided deionized water containing 109CdCl2 at a concentration of 0.15 ng Cd/ml (74 nCi 109Cd/ml; specific activity, 490 nCi 109Cd /ng Cd; Amersham, Burlington, MA). This very low cadmium concentration was chosen to avoid influencing the natural pattern of MT induction in the MTN dams during gestation and lactation (Sauer et al., 1998; Solaiman et al., 2001
). Water and 109Cd consumption were separately determined for each dam by weighing each water bottle and measuring 109Cd concentration when it was full and again when it was replaced with a fresh 109Cd-water bottle, thus avoiding dosage errors due to adherence of Cd ions to glass water bottles and rubber stoppers. Radiation safety protocols were supervised by the Environmental Safety and Health Division of Argonne National Laboratory.
There were four experimental groups of pregnant dams for each MT gene type (MTN and MT1,2KO), with 89 plug-positive females/group. In addition, six MTN and six MT1,2KO female mice of the same age, weight, and strain were kept out of the mating protocol to serve as nonpregnant (NP) controls. Day 0 of gestation (GD 0) was the day the vaginal plug occurred. Day 19 (GD 19) was the day of delivery, and the following day, Day 20, was the first day of lactation (LD 1). Experimental groups were designated GD 7, GD 14, GD 17, and LD 11, indicating pregnant mice continuously exposed to 109Cd-water for 7, 14, or 17 days of gestation or 30 days of a combined pregnancy and lactation period (GD 0LD 11), respectively. The six MTN and six MT1,2KO nonpregnant control mice were exposed continuously to 109Cd-water for 20 days, comparable to the GD 17 group. Litters were adjusted to five pups per dam on LD 1 or LD 2. For all groups of mice, 109Cd-water was replaced by deionized water 24 h prior to their sacrifice to allow for clearance of oral 109Cd from the gastrointestinal tract. For the LD 11 group, each dam with her pups was placed in a stainless steel metabolism cage 48 h before sacrifice, to allow for collection of pup feces on LD 10 and LD 11. Pup feces were clearly observed by LD 8 and easily separable from the much larger dam feces.
Following each exposure period, mice were sacrificed by ip injection (230 mg/kg bw) of sodium pentobarbital (as NembutalTM, Abbott Laboratories, Abbott Park, IL). Animals were sacrificed near the end of their active (dark) period. The following tissues were removed, weighed, and analyzed for 109Cd content: liver, kidney, blood (collected by cardiac puncture in heparinized tubes), mammary tissue, placenta and fetuses (for GD 14/17 dams), pups and pup organs (at LD 11), duodenum (first 5 cm distal to the pylorus), jejunum (next 10 cm), ileum (remaining small intestine), and remaining gastrointestinal tract. (Each section of small intestine from mouse dams was rinsed with ice-cold phosphate-buffered saline. For pups, the intestinal tract was separated into stomach, duodenum, and remaining intestinal tract, including the large intestine.) Pup duodena were rinsed as above, but not the stomachs or remaining intestinal tract. Organs and tissues were immediately subdivided into two approximately equal, weighed portions for the 109Cd measurements or MT analyses. Tissues for which MT content was to be measured were immediately frozen in liquid nitrogen and transferred to a 70°C freezer for storage and analysis.
109Cd determination in mouse tissues.
Tissues were homogenized with a glass homogenizer and Teflon pestle in 5 volumes of 5% sulfosalicylic acid. The sulfosalicylate acidified the tissues, allowing for release of 109Cd from cellular components. Aliquots of the tissue homogenates were suspended in Ready Safe liquid scintillation cocktail (Beckman Instruments, Inc., Fullerton, CA) and counted in a Packard Model 2200CA Liquid Scintillation Analyzer (Packard Instrument Company, Downers Grove, IL). The 109Cd was measured by the activity of its 0.16 Mev Auger electron emission. All 109Cd values were corrected for decay back to the first day of the experiment. Samples with low amounts of 109Cd were counted for long times to acquire a total of at least 1000 counts (< 3% RSD). Because of differences in amounts of 109Cd ingested by the dams (Table 1), the values of 109Cd radioactivity in the dam and pup tissues reported in Figures 1
and 2
and Table 7
were adjusted to represent equivalent cadmium intake levels. For example, the 109Cd values for the MT1,2KO pup samples were divided by 1.2 to account for the 1.2-fold greater consumption of 109Cd by the L11 MT1,2KO versus MTN dams (11.8 versus 10.0 µCi/mouse). This approach corrected for any increase in dam or pup 109Cd content due solely to increased 109Cd intake by the dam.
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Validation of the MT determination.
Because preliminary measurements indicated low MT concentrations in the experimental tissues, additional MT determinations were made to validate the assay procedure and to demonstrate the ability of this strain of MTN mice to produce MT in response to Cd exposure. Four days after gavage administration of 200 µg CdCl2/mouse, the same MT assay was performed upon livers, kidneys, and duodena from NP female mice of the same age and strain as the MTN mice in this study.
Statistics and data analysis.
Comparisons involving more than two groups were made by ANOVA and Fischers least significant difference test (LSD). Comparisons between two groups of direct cadmium content measurements were made using Students t-test. The assumption of unequal variances was used for comparisons of data points. Comparisons between ratios of cadmium content in liver and kidney were made using both the unpaired t-test with Welchs correction and the Mann-Whitney non-parametric test. Differences in data points with a p value of < 0.05 were considered statistically significant.
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RESULTS |
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The relative amounts of 109Cd in liver versus kidney (Table 6) differed between the two strains of mice during gestation and lactation. For the MTN mice on GD 7, the ratio of liver 109Cd/kidney 109Cd was 5.2 (Table 6
). As gestation continued, this ratio gradually decreased, reaching 3.1 on GD 17 (Table 6
). By LD 11, the amount of 109Cd in the kidney was nearly equivalent to that in the liver, and the liver/kidney 109Cd content ratio (1.4) was similar to that in the MTN NP mice (1.6). In contrast, in the MT1,2KO dams, liver/kidney 109Cd ratios were close to 10 throughout gestation and lactation and, after GD 7, were significantly higher in MT1,2KO dams than in MTN dams (Table 6
). This significantly greater deposition of cadmium in liver versus kidney in MT1,2KO dams was also observed in the NP controls (Table 6
).
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The amount of 109Cd transferred from both MTN and MT1,2KO dams to pups during 11 days of lactation (0.0470.062% ingested dose) was much greater than that transferred to fetuses during 17 days of gestation (Table 4). Furthermore, on LD 11, the 109Cd content of a litter of MT1,2KO pups was not significantly different from that of a litter of MTN pups (Table 4
). There was, however, a three-fold higher level of 109Cd in the mammary tissue of MT1,2KO dams than MTN dams on L11 (Table 4
). The 109Cd in mammary tissue on GD 7 and GD 14 was in range of that in the NP20 controls and was similar in both mouse strains (0.0010.004% ingested dose).
109Cd in Pup Tissues
As was observed in dams, the pup intestines contained a major fraction of the 109Cd present in the whole animal (Table 7, Fig. 2
). In addition, this distribution was essentially the same for MTN and MT1,2KO animals (109Cd in intestine = 89% of that in whole pup for MTN; 84% for MT1,2KO; Table 7
). The milk-filled stomachs also had a very similar level of Cd in both types of mouse. Less than 1% of pup total body 109Cd was in the pup feces for both MTN and MT1,2KO mice (Table 7
).
Although there was a tendency for somewhat higher 109Cd levels in all tissues of the MT1,2KO pups, the strain difference was statistically significant only for the liver (Fig. 2) and whole pup minus intestinal tract (Table 7
). The amount of Cd in the kidney of pups of both types of mice was close to the limit of detection (Fig. 2
). However, long count times indicated that, as for the dams, the ratio of liver/kidney 109Cd content was significantly higher for the MT1,2KO than MTN pups (Fig. 2
, Table 6
).
Metallothionein Assay Applied to Cadmium-Gavaged Metallothionein-Normal Mice
MT concentrations in the tissues of MTN mice four days after cadmium gavage (200 µg/mouse), were 94 ± 12 (liver), 43 ± 5 (kidney), and 142 ± 18 (duodenum) µg/g wet weight of tissue (mean ± SE, n = 5).
Metallothionein Concentrations in MTN Mice during Pregnancy and Lactation
In MTN dams, liver MT concentrations were significantly higher than in NP20 controls on GD 7 and increased to a peak of 14.8 µg MT/g on GD 14 (Table 8); by LD 11, liver MT was at a concentration similar to that in the nonpregnant mouse (2.6 µg MT/g). In pups on LD 11, the liver MT concentration was about the same as in the GD 7 dams and higher than in the LD 11 dams. In contrast, kidney MT concentrations remained low (
1 µg MT/g) throughout gestation and lactation in dams, and were more than 10-fold higher in LD 11 pups (Table 8
). Duodenal MT concentrations in the dam were higher than in the jejunum and ileum; on GD 7 they were significantly higher than in the NP20 controls and rose steadily and significantly until LD 11. MT concentrations in the duodena of LD 11 pups were significantly higher than in the LD 11 dams. Placental and duodenal MT concentrations were similar to one another on GD 14, but placental MT decreased significantly by GD 17. Although mammary MT concentrations were low, they were three-fold higher on LD 11 than in the NP mice (1.0 versus 0.3 µg MT/g, respectively, Table 8
).
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DISCUSSION |
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Pathways of Cd Absorption in Dams
MT-independent cadmium absorption from intestinal lumen to small intestine cells.
Oral cadmium at low concentrations was sequestered by intestinal epithelium, primarily duodenum, as observed by others in MT wild-type animals (Lind and Wicklund-Glynn, 1997; Min et al., 1991
). Our results demonstrate that this cadmium pathway was independent of MT in both NP females and mouse dams (Fig. 1
, Table 3
).
Cadmium absorption into intestinal epithelial cells recently has been shown to utilize the divalent metal ion transport system, DMT1, which facilitates an MT-independent pathway for intestinal iron absorption (Picard et al., 2000). Others have connected low iron status with increased intestinal absorption of cadmium in pregnant humans (Akesson et al., 2002
). In our study, cadmium uptake by small intestinal tissue increased during pregnancy and lactation. These results might be expected if intestinal iron absorption increased in dams during that period (Akesson et al., 2002
)
MT-dependent release of cadmium from small intestine cells to blood.
The approximately 5- to 10-fold lower amount of cadmium in nonintestinal internal organs than in small intestine tissue (Table 5) indicates that release of cadmium to blood across the basolateral side of intestinal cells is restricted. The biochemical forms of cadmium passing from intestinal cells to blood might differ according to the MT status of those cells, resulting in an MT-dependent pattern of cadmium distribution in tissues other than the GI tract.
However, an MT-independent cadmium transport pathway was observed during the first seven days of low-level cadmium exposure of dams via drinking water. At GD 7, nonintestinal internal organs of the MTN and MT1,2KO dams each contained 0.18% of ingested cadmium, with a similar high liver/kidney ratio (Table 6). It has been proposed that a large fraction of cadmium released early to blood is transported to liver (Friberg et al., 1986
; Jonah and Bhattacharyya, 1989
), probably complexed to albumin (Lucis et al., 1972
) or to erythrocytes (Dawson and Ballatori, 1995
) and independent of MT (Bhattacharyya et al., 2000
; Frazier, 1984
; Liu et al., 2001
; Min et al., 1991
; Ohta and Cherian, 1991
).
An MT-dependent pattern of cadmium distribution emerged as pregnancy and lactation progressed. Increasing MT levels in duodena of MTN dams (Table 8) were associated with two-fold lower levels of 109Cd in all nonintestinal tissues compared to MT1,2KO mice by LD 11 (Table 5
), and a steadily declining proportion of 109Cd in liver/109Cd in kidney, (Table 6
). The increasing maternal intestinal MT may have favored Cd distribution to kidney (Lau et al., 1998
; Ohta and Cherian, 1991
), due to specific release of the Cd-MT complex from duodenum or liver to kidney, as suggested by a number of investigators (Dorian et al., 1995
; Jonah and Bhattacharyya, 1989
; Min et al., 1991
, 1992
; Ohta and Cherian, 1991
).
Pathways of Cd Absorption and Distribution in Pups
Nearly all the 109Cd in pups (8590%) was retained in intestinal tissues on LD 11 and, as in the dams, the capacity to synthesize MT had no significant effect on this pathway (Figs. 1 and 2
, Tables 5
and 7
). Less than 1% of the oral 109Cd obtained from milk passed through the pups intestines to feces by LD 11, indicating that the neonatal mouse pups gastrointestinal epithelium absorbed virtually all milk-derived Cd, independent of MT production (Table 7
). This gastrointestinal sequestration by suckling pups has been reported by Sasser and Jarboe (1977)
and Whelton et al.(1993)
. The fact that this pathway does not require MT is shown here for the first time.
Unlike dams, pups transferred 1015% of the milk-derived 109Cd across the gastrointestinal tract to nonintestinal tissues (Table 7, 109Cd in whole pup minus intestinal tract versus whole pup). Because absorption of CdCl2 from small intestines to blood of adult mice was less than 1% in this (Table 5
) and other studies (Bhattacharyya et al., 1981
, 1986
; Whelton et al., 1993
), we have also demonstrated that both the MTN and MT1,2KO nursing pups absorbed milk-derived Cd with high efficiency compared with the adult. However, 1020% of the Cd in small intestinal pools passed to internal organs in both adults and pups (Table 5
, last two columns), indicating that basolateral membrane transport was similar in adults and pups and that age-dependent differences were seen only in the percentage of oral Cd retained by the small intestines.
The MT1,2KO pups retained almost twice as much 109Cd in nonintestinal tissue as MTN pups (Table 7), again reflecting the restriction by MT of Cd transfer from intestines to blood. An exception to this MT-dependent decrease was in pup kidneys, which showed very low Cd levels in both strains (Fig. 2
). The nearly five-fold higher 109Cd level in liver and eight-fold higher ratio of 109Cd in liver/109Cd in kidney in MT1,2KO pups (Fig. 2
, Table 6
) again illustrate the high efficiency of Cd transport to liver in the absence of MT.
Pathways of Cd Transfer from Dams to Pups
MT in placentas of MTN dams was associated with a reduction of 109Cd transferred to fetuses, which by GD 17 was 100-fold lower than in MT1,2KO fetuses (Table 4). Previous reports also suggest that placental MT facilitates transplacental transport of zinc and copper but restricts movement of toxic metals like cadmium (Goyer et al., 1992
; Itoh et al., 1996
). A striking feature remains, however, that cadmium movement from maternal blood to fetus was extremely low during gestation in both mouse types, demonstrating that MT was not the main barrier to transplacental Cd transport. Cd clearance from blood, which was extremely rapid in dams of both strains (Fig. 1B
), may have been a determining factor in restricting this transfer pathway.
Accumulation of cadmium in maternal mammary tissue during gestation and lactation has been reported by a number of workers (Bhattacharyya et al., 1982; Floris et al., 2000
; Lucis et al., 1972
; Petersson and Oskarsson, 2000
). The measurements of 109Cd in whole pups, however, indicate little or no role for MT in restricting cadmium transfer via milk. Although absolute magnitudes of 109Cd in mammary tissues as well as 109Cd content of blood were higher in MT1,2KO dams than in MTN dams, 109Cd levels in whole pups were similar, including the milk-filled stomachs, reaffirming that MT1,2KO dams did not provide their pups a significantly greater amount of 109Cd via their milk than did MTN dams (Tables 4
and 7
).
Roles of Other Metallothionein Isoforms
For completeness, the other isoforms of mouse MT should be discussed here, because, although total MT concentrations were very low in the MT1,2KO mouse tissues tested (Table 8), MT3 and MT4 were not specifically evaluated. However, Palmiter and coworkers (1992)
have shown that the cadmium-binding capacity of small intestine tissue did not increase in MT1,2KO mice, even after ip injection of 15 µmol Cd/kg, although MT3 detected in brain did respond to exogenous Cd. The MT4 isoform is believed to play a role in differentiation of stratified squamous epithelium (Quaife et al., 1994
). The whole body of MT1,2KO pups did contain significantly more 109Cd than that of MTN pups (Table 7
), but no evidence exists in our data (Table 8
, MT1,2KO mouse tissues) or that of others (Liu et al., 1996a
; Quaife et al., 1994
) to indicate that MT3 or MT4 induction might be a compensatory mechanism in the MT1,2KO animal.
In summary, the results of this study indicate that some aspects of cadmium absorption are independent of MT, including (1) early uptake and sequestration of oral cadmium by the duodenum, (2) early transfer of 109Cd from intestine to internal organs, and (3) nearly total uptake and sequestration of milk-derived cadmium by pup intestinal tract. The presence of MT in the dams duodenum at pregnancy-induced levels appears to (1) restrict the movement of oral Cd across the gastrointestinal tract to blood by a factor of two to three, (2) reduce transfer of 109Cd to liver, mammary, and placental tissues, and (3) in conjunction with hepatic MT, enhance transfer of 109Cd to kidney. 109Cd distribution in nonintestinal organs of the pups was similarly influenced by MT, although net absorption of cadmium from intestinal lumen to pup organs was proportionally higher than in dams. The functions of the divalent metal transport system in cadmium uptake, the biochemical mechanism for restricted cadmium transport through the basolateral side of intestinal epithelium, and the role of MT in this latter process all require additional study.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed. Fax: (630) 252-5517. E-mail: mhbhatt{at}anl.gov.
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