* Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599;
Developmental Biology Branch, Reproductive Toxicology Division, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711;
Miami Valley Laboratories, Procter and Gamble Company, Cincinnati, Ohio 45252; and
Departments of Nutrition and Internal Medicine, University of California, Davis, California 95616
Received December 23, 2002; accepted March 11, 2003
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ABSTRACT |
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Key Words: lipopolysaccharide; embryolethality; metallothionein; metallothionein null mice; zinc deficiency.
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INTRODUCTION |
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Metallothionein, a cysteine-rich cytosolic protein, which binds metals of physiological importance, is thought to regulate metal distribution and to serve a protective role in heavy metal detoxification (Kagi, 1991). MT is present in most tissues, including kidney and placenta, but is most abundant in liver and is classically induced by metals (particularly cadmium) as well as organic compounds, endogenous hormones (Klaassen and Lehman-McKeeman, 1989
), and cytokines (Dunn et al., 1987
). Increased maternal hepatic MT synthesis following exposure to a variety of toxicants has been shown to produce a secondary embryonal zinc (Zn) deficiency in rats (Daston et al., 1991
, 1994
; Taubeneck et al., 1994b
). In this scenario, an increase in maternal hepatic MT results in sequestration of Zn and a subsequent reduction in the amount of plasma Zn available for transfer to the embryo. Maternal plasma Zn is the major Zn source for the conceptus, and Zn is essential for normal embryo/fetal development (Keen, 1992
; Keen and Hurley, 1989
).
Daston and coworkers (1994) treated pregnant rats with alpha-hederin, inducing substantial hepatic MT synthesis, and determined hepatic and plasma Zn concentrations and systemic distribution of a pulse dose of 65Zn after treatment. APR induction was evidenced by decreased iron and Zn, and increased copper, alpha 1-acid glycoprotein, and ceruloplasmin in maternal plasma, along with a dosage-related increase in maternal hepatic MT. In addition, a causal role for secondary embryonal Zn deficiency was clearly demonstrated. 65Zn distribution to tissues was decreased except that to the maternal liver, which was increased twofold over controls, and transfer of 65Zn to the conceptus was significantly decreased. Sera collected from donor rats 2 h after alpha-hederin treatment supported normal rat embryo development in vitro. In contrast, sera collected 18 h post alpha-hederin exposure (maximum hepatic MT induction) did not support normal development; however, supplemental Zn added to the 18 h post alpha-hederin-treated serum restored embryonic development.
We have demonstrated that LPS treatment causes a rapid elevation of serum TNF- in pregnant CD-1 mice (Leazer et al., 2002
). Taubeneck et al. (1994a)
examined the effects of TNF-
on maternal and embryonic Zn metabolism in C3HeB/FeJ mice (chosen for their well-characterized cytokine production and endotoxin sensitivity) and found TNF-
to be developmentally toxic. These researchers postulated that the developmental toxicity of TNF-
in this mouse strain was, in part, modulated by maternal Zn status. This report stated that (1) maternal TNF-
treatment in the mouse was teratogenic; (2) TNF-
induced maternal hepatic MT and resulted in increased maternal, and decreased embryonic, Zn concentrations; (3) maternal Zn status modulated the teratogenicity of TNF-
; and (4) the teratogenicity to embryos cultured in serum from TNF-
-treated rats could be ameliorated by the addition of Zn. Given that LPS induces the synthesis of MT, and our previous findings that LPS resulted in induction of TNF-
, we hypothesized that maternal MT-induced secondary embryo Zn deficiency is a principal contributor to the embryotoxicity of LPS in CD-1 mice.
To test this hypothesis we determined the extent to which LPS induced MT synthesis in pregnant CD-1 mice and assessed the effects of LPS on tissue Zn concentrations. Zn supplementation was employed in an attempt to ameliorate the embryo toxicity of LPS, and MT I-II null mice were used to further investigate the role of maternal MT in manifestations of LPS developmental toxicity.
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MATERIALS AND METHODS |
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Metallothionein assessment.
Maternal hepatic MT was measured with the cadmium-hemoglobin radioassay. 109CdCl2 (specific activity of 125 mCi/mg) was added to aliquots of liver cytosol from LPS-treated mice to saturate MT. These samples were incubated at room temperature for 15 min, followed by denaturation at 80°C for 5 min and centrifugation at 20,000 x g for 20 min. The supernatant fraction was applied to a Sephadex G-75 column (60 cm length x 2.5 cm diameter) equilibrated with 10 mM Tris-acetate buffer (pH 7.4 at 4°C) and eluted with the same buffer at a rate of 30 ml/h. Eighty fractions were collected at 10-minute intervals, and radioactivity was counted by gamma scintillation spectroscopy. The UV absorbance spectrum from 250 to 300 nm of the fraction containing the major Cd-binding peak was measured in a scanning spectrophotometer (Beckman DU-50, Fullerton, CA). The identity of the metal-binding protein in liver cytosol as MT was confirmed by resistance to heat denaturation, chromatographic characteristics, and UV spectral properties. Values for MT are expressed as µg/g liver, assuming a molecular weight of 7000 daltons and a Cd-binding capacity of 7 g-atoms/mol MT (Onosaka and Cherian, 1982).
Tissue Zn analysis.
Maternal hepatic and plasma Zn was measured by flame atomic emission spectroscopy. Maternal liver and plasma samples were acid digested with 12 N Ultrex nitric acid overnight. Samples were then heat digested for durations specific to tissue type and appropriately diluted. Zn (wavelength 213.8 nm) was read on an axial torch ICP-Atomic Emission Spectrophotometer (ICP-AES) (Tracescan, Thermo Jerrel Ash). Standards were made from certified atomic absorption standards (Fisher Scientific) by addition of known volumes of standard to 0.1 N nitric acid. To determine the concentrations of the samples, each sample was read 3 times for a period of 3 s at a reference of 950 mH, a nebulizer pressure of 30 psi, and a pump rate of 110 r.p.m. Recovery of trace elements by this method is 98102% (Clegg et al., 1981).
Statistics.
The litter was considered the unit for statistical comparisons. A one-way analysis of variance with the general linear models procedure of SAS (Cary, NC) (SAS, 1990) was used to determine differences among all dose groups. Pairwise t-tests with the least-squares means procedure of SAS (Cary, NC) (SAS, 1990
) were used to test the difference between each dose group and the control. Liver Zn, plasma Zn, and MT concentrations were analyzed by SAS Proc Mixed random effects model. Data are expressed as mean ± SE, and p
0.05 was considered statistically significant.
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RESULTS |
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DISCUSSION |
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Acute LPS exposure increased MT synthesis in the maternal liver, and liver Zn concentrations were concomitantly increased significantly, indicating Zn redistribution. Zn supplementation studies were carried out to address the potential contribution of secondary Zn deficiency to LPS embryolethality. Administration of Zn as a pretreatment on gd 8 or simultaneous to the LPS treatment on gd 9 was designed to supply a Zn bolus to the maternal circulation to compensate for a possible Zn deficiency induced by LPS. However, these Zn supplementations were not protective against LPS embryolethality. Zn coadministration with LPS on gd 9 resulted in an elevated incidence of embryo resorption compared to LPS alone. Although maternal Zn redistribution does occur following LPS exposure, these data suggest that Zn deficiency does not have a primary role in acute LPS embryolethality in mice. Similar results were observed in experiments on the role of Zn in LPS-induced embryolethality in rabbits by Pitt et al. (1997). These investigators found a high incidence of embryo resorptions following maternal LPS exposure on either gd 8 or gd 10, and also observed a striking elevation of maternal hepatic MT (~30-fold) as well as a drop in maternal plasma Zn within 24 h of LPS treatment. Cotreatment with Zn oxide on gd 8 did not reduce the incidence of LPS-induced resorptions, while cotreatment on gd 10 partially improved outcome, reducing the resorption incidence by 44%. Our treatment of CD-1 mice with LPS produced a much smaller induction of MT (~twofold) than did treatment of rabbits by Pitt et al. (1997)
.
Because we found a substantial increase in hepatic MT in LPS-treated CD-1 mice, we further investigated the role of MT in LPS-induced developmental toxicity by comparing the sensitivity to LPS in MT deficient mice and wild-type controls. If MT is a mediator of acute LPS developmental toxicity, then pregnant MT null mice would be expected to be less sensitive to LPS embryo toxicity than WT mice. However, we observed no difference in sensitivity to LPS between the MTKO and WT strains. Together with the Zn supplementation results, these data support the conclusion that maternal MT-induced embryonal Zn deficiency (a maternally mediated mechanism of developmental toxicity) is not a key mechanism in acute LPS-induced embryolethality in mice. While the mechanisms underlying LPS-induced embryolethality remain to be elucidated, we have previously demonstrated that LPS embryotoxicity is a maternally-mediated event. Direct exposure of embryos to LPS in whole embryo culture was not embryotoxic (Leazer et al., 2002).
From previous work, we know that TNF- is released following LPS exposure (Gendron et al., 1990
; Leazer et al., 2002
). However, we have investigated TNF-
as a mediator of LPS embryotoxicity and found that TNF-
does not singularly mediate LPS-induced embryo resorption. IL-1 and IL-6 are also involved in the initiation and maintenance of the APR. IL-1 functions similarly to TNF-
. These cytokines may work in concert, or one may compensate for the other in response to LPS (Chaplin and Hogquist, 1992
; Neta et al., 1992
). In addition, IL-6 acts directly on hepatocytes to produce the synthesis of MT (De et al., 1990
; Lee et al., 1999
; Schroeder and Cousins, 1990
). Clearly, we have shown that MT induction and Zn redistribution can occur in response to acute LPS treatment; however, other maternal physiological alterations subsequent to LPS exposure are probably more closely tied to the observed embryolethality. It is critical to note that we did not observe hypozincemia with acute LPS treatment, so the lack of protection from supplemental Zn might be predicted. With chronic LPS exposure, the situation could be different, given that hypozincemia is typical with a persistent APR. Collectively, the current data and past studies suggest that, whereas Zn supplementation may reduce the teratogenicity of some APR inducers, this effect of Zn may be limited to situations where the APR is relatively mild and prolonged.
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ACKNOWLEDGMENTS |
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NOTES |
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1 To whom correspondence should be addressed at University of Kansas Medical Center, Department of Pharmacology, Toxicology and Therapeutics, 3901 Rainbow Blvd., Kansas City, KS 66160. Fax: 913-588-7501. E-mail: tleazer{at}kumc.edu.
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