The Embryolethality of Lipopolysaccharide in CD-1 and Metallothionein I–II Null Mice: Lack of a Role for Induced Zinc Deficiency or Metallothionein Induction

Tyra M. Leazer*,{dagger},1, George P. Daston{ddagger}, Carl L. Keen§ and John M. Rogers*,{dagger}

* Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina 27599; {dagger} Developmental Biology Branch, Reproductive Toxicology Division, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711; {ddagger} 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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Lipopolysaccharide (LPS) is embryolethal in CD-1 mice. LPS induces metallothionein (MT) via cytokines, including TNF-{alpha}, IL-1, and IL-6, which initiate and maintain the acute phase response. Maternal hepatic MT induction in pregnant rats, by diverse toxicants, can result in maternal hypozincemia and subsequent embryonal zinc (Zn) deficiency. We examined the hypothesis that LPS causes embryo toxicity in CD-1 mice via MT induction and subsequent embryo Zn deficiency by (1) determining whether LPS induces maternal hepatic MT and causes Zn redistribution, (2) assessing the effects of maternal Zn supplementation on LPS developmental toxicity, and (3) assessing the role of MT with MT I-II null mice (MTKO). Timed pregnant CD-1 mice were dosed i.p. with LPS (S. typhimurium) (0.05 mg/kg) on gestation day (gd) 9. Zn supplementation was administered on gd 8 (10 mg/kg, pretreatment) or on gd 9 as a cotreatment (5 or 10 mg/kg). MTKO and wild type (WT) mice were dosed with LPS (0.05 or 0.1 mg/kg) on gd 9, and maternal liver MT and Zn and plasma Zn were measured. In CD-1 mice, maternal hepatic MT was elevated 24 h after LPS treatment, and cotreatment with Zn caused further elevation of MT. Maternal hepatic Zn concentrations paralleled hepatic MT concentrations. Maternal plasma Zn on gd 10 showed no consistent effect of LPS treatment or Zn cotreatment on gd 9. Zn pretreatment (10 mg/kg) on gd 8 did not ameliorate LPS embryolethality, while Zn cotreatment (5 or 10 mg/kg) on gd 9 exacerbated the toxicity of LPS. LPS produced a similar incidence of embryolethality in MTKO and WT strains on gd10. Plasma Zn concentrations were similar in both strains, while hepatic Zn concentrations were significantly higher in WT than in the MTKO strain. In conclusion, while LPS can induce maternal hepatic MT and Zn redistribution in CD-1 mice, this does not appear to be a key mechanism leading to LPS embryotoxicity.

Key Words: lipopolysaccharide; embryolethality; metallothionein; metallothionein null mice; zinc deficiency.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The acute phase response (APR) is the body’s first defense against the stress of infection, toxicity, or trauma, and it functions to restore metabolic homeostasis following insult. Lipopolysaccharide (LPS), the endotoxic component of gram negative bacteria and a prototypical inducer of the APR (Burd et al., 1992Go), produces embryo death when administered to pregnant CD-1 mice during organogenesis (Leazer et al., 2002Go). Exposure to LPS initiates a cytokine cascade including tumor necrosis factor alpha (TNF-{alpha}), interleukin-1 (IL-1), and interleukin-6 (IL-6), and increased synthesis of acute phase proteins including metallothionein (MT). De and coworkers (1990)Go reported that rapid induction of MT by LPS in the CD-1 mouse could be mimicked by the cytokines TNF-{alpha} or IL-1, but MT induction was attenuated in LPS- resistant C3HeJ mice. Liu et al. (1991)Go demonstrated that LPS induces MT via cytokines by comparing an LPS-sensitive (normal) strain of mouse to an LPS-insensitive (low-cytokine-producing) strain. The low-cytokine-producing mice were much less responsive to the induction of MT by LPS than was the normal (sensitive) strain, but were equally responsive to the induction of MT by IL-1, IL-6, and TNF-{alpha}, supporting the hypothesis that these cytokines induced MT synthesis.

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, 1991Go). 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, 1989Go), and cytokines (Dunn et al., 1987Go). 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., 1991Go, 1994Go; Taubeneck et al., 1994bGo). 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, 1992Go; Keen and Hurley, 1989Go).

Daston and coworkers (1994)Go 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-{alpha} in pregnant CD-1 mice (Leazer et al., 2002Go). Taubeneck et al. (1994a)Go examined the effects of TNF-{alpha} on maternal and embryonic Zn metabolism in C3HeB/FeJ mice (chosen for their well-characterized cytokine production and endotoxin sensitivity) and found TNF-{alpha} to be developmentally toxic. These researchers postulated that the developmental toxicity of TNF-{alpha} in this mouse strain was, in part, modulated by maternal Zn status. This report stated that (1) maternal TNF-{alpha} treatment in the mouse was teratogenic; (2) TNF-{alpha} induced maternal hepatic MT and resulted in increased maternal, and decreased embryonic, Zn concentrations; (3) maternal Zn status modulated the teratogenicity of TNF-{alpha}; and (4) the teratogenicity to embryos cultured in serum from TNF-{alpha}-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-{alpha}, 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animals and treatment.
Timed pregnant CD-1 mice were obtained from Charles River Laboratories (Raleigh, NC). Metallothionein I-II null (MTKO) mice and 129SvCPJ wild type (WT) mice were obtained from Jackson Laboratories (Bar Harbor, ME). The MTKO mice were from nullizygous knockout mice (Masters et al., 1994Go) and cross-bred to the 129SvCPJ background strain. Gestation day (gd) 0 was established upon detection of a copulatory plug after overnight mating. Animals were housed 5–10 per cage, provided Prolab RMH 3000 diet and water ad libitum, and maintained on a 12 h light/12 h dark cycle. All animals were housed in accordance with the American Association for the Accreditation of Laboratory Animal Care, and all procedures involving the use of laboratory animals were in accordance with the National Institutes of Health. Dams were administered 0.05 mg/kg LPS (S. typhimurium; Sigma, St. Louis) in saline by single i.p. injection on gd 9 in a dosing volume of 0.2 ml. MTKO and WT mice were injected i.p. with LPS at 0.05 or 0.1 mg/kg dosages on gd 9. Control animals received saline vehicle. Dams were killed on gd 10 (24 h after LPS) by cervical dislocation. Laparotomies were performed, gravid uteri were excised and weighed, and the numbers of implantation sites, resorptions, and live and dead embryos were determined. Maternal liver and blood (plasma) were harvested on gd 10 for MT and Zn assessments. CD-1 mice were pretreated or cotreated by i.p. injection with 5 mg/kg Zn acetate on gd 8 or with 5 or 10 mg/kg Zn acetate on gd 9. Each of these adjunct treatments was combined with LPS treatment (0.05 mg/kg on gd 9). Control animals received saline vehicle. Doses were administered in a 0.2 ml volume.

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, 1982Go).

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 98–102% (Clegg et al., 1981Go).

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, 1990Go) was used to determine differences among all dose groups. Pairwise t-tests with the least-squares means procedure of SAS (Cary, NC) (SAS, 1990Go) 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
LPS Embryolethality in CD-1 Mice
LPS (0.05 mg/kg) on gd 9 caused approximately 50% embryo resorption per litter by gd 10 (Fig. 1Go). Zn pretreatment (10 mg/kg) on gd 8 did not significantly reduce the embryolethality of 0.05 mg/kg LPS administered on gd 9. When compared to LPS alone, the Zn cotreatments (5 or 10 mg Zn acetate/kg concomitant with 0.05 mg/kg LPS on gd 9) were associated with about a 40% increase in embryo resorption. None of the Zn treatments alone affected the incidence of resorptions on gd 10.



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FIG. 1. Effects of gd 9 LPS treatment on the incidence of resorptions at gd 10. Dams were treated with 0 or 0.05 mg/kg LPS by i.p. injection on gd 9. The effects of Zn supplementation were tested by pretreating dams with 10 mg/kg Zn acetate on gd 8 (10 Zn PTX) or 5 or 10 mg/kg Zn acetate on gd 9 (5 Zn CoTX and 10 Zn CoTX, respectively) and comparing them to dams receiving no Zn treatment (No Zn TX). *different from 0 mg/kg LPS, No ZnTX by p <= 0.05; ** different from 0.05 LPS, No Zn TX by p <= 0.05. (n = 32, No Zn TX group; n = 23, 10Zn PTX group; n = 16, 5Zn CoTX group; n = 22, 10Zn CoTX group).

 
LPS Induced MT Synthesis and Zn Redistribution in CD-1 Mice
LPS (0.05 mg/kg on gd 9) exposure alone or in concert with Zn pretreatment (10 mg Zn/kg on gd 8; 10 Zn PTX) or Zn cotreatment (5 or 10 mg Zn/kg on gd 9; Zn CoTX) resulted in induction of maternal hepatic MT (Fig. 2Go). LPS treatment on gd 9 alone resulted in a significant increase in maternal hepatic MT on gd 10. LPS-induced MT concentrations were 677 ± 87 µg MT/g liver compared to basal concentrations of 417 ± 86 µg MT/g liver (reported here as mean ± SEM). Zn pretreatment on gd 8, followed by LPS treatment on gd 9, resulted in maternal hepatic MT concentrations similar to LPS treatment alone. Control animals in the Zn pretreatment group had hepatic MT concentrations similar to baseline concentrations. Both Zn cotreatment groups had increased hepatic MT concentrations on gd 10 compared to their saline control groups, and 10 mg/kg Zn coadministered with LPS significantly increased MT (1137 ± 88) concentrations compared to LPS alone (677 ± 87 µgMT/g liver).



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FIG. 2. Effects of gd 9 LPS treatment on maternal hepatic metallothionein at gd 10. Dams were treated with 0 or 0.05 mg/kg LPS by i.p. injection on gd 9. The effects of Zn supplementation were tested by pretreating dams with 10 mg/kg Zn acetate on gd 8 (10 Zn PTX) or 5 or 10 mg/kg Zn acetate on gd 9 (5 Zn CoTX and 10 Zn CoTX, respectively) and comparing them to dams receiving no Zn treatment (No Zn TX). *different from 0 mg/kg LPS, No ZnTX by p <= 0.05; ** different from 0.05 LPS, No Zn TX by p <= 0.05. (n = 32, No ZnTX group; n = 23, 10Zn PTX group; n = 16, 5Zn CoTX group; n = 22, 10Zn CoTX group).

 
Maternal Hepatic and Plasma Zn Concentrations in CD-Mice
Maternal hepatic Zn concentrations were increased following all LPS treatments compared to their saline control groups. Significant differences were observed for both Zn cotreatments (Fig. 3Go). The Zn cotreatment groups produced a greater increase in liver Zn concentrations compared to LPS alone. As would be expected, maternal hepatic Zn concentration paralleled hepatic MT concentrations. Plasma Zn was measured in samples taken 18 h (maximum MT induction) following LPS exposure. Plasma Zn concentrations at this time point were not significantly affected by LPS exposure or Zn treatments (Fig. 4Go).



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FIG. 3. Effects of gd 9 LPS treatment on maternal hepatic Zn at gd 10. Dams were treated with 0 or 0.05 mg/kg LPS by i.p. injection on gd 9. The effects of Zn supplementation were tested by pretreating dams with 10 mg/kg Zn acetate on gd 8 (10 Zn PTX) or 5 or 10 mg/kg Zn acetate on gd 9 (5 Zn CoTX and 10 Zn CoTX, respectively) and comparing them to dams receiving no Zn treatment (No Zn TX). *different from 0 LPS, No Zn TX by p <= 0.05. ** different from 0.05 LPS, No Zn TX by p <= 0.05. (n = 32, No ZnTX group; n = 23, 10Zn PTX group; n = 16, 5Zn CoTX group; n = 22, 10Zn CoTX group).

 


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FIG. 4. Effects of gd 9 LPS treatment on maternal plasma Zn at gd 10. Dams were treated with 0 or 0.05 mg/kg LPS by i.p. injection on gd 9. The effects of Zn supplementation were tested by pretreating dams with 10 mg/kg Zn acetate on gd 8 (10 Zn PTX) or 5 or 10 mg/kg Zn acetate on gd 9 (5 Zn CoTX and 10 Zn CoTX, respectively) and comparing them to dams receiving no Zn treatment (No Zn TX). *different from 0 LPS by p <= 0.05. ** different from 0.05 LPS by p <= 0.05. (n = 32, No Zn TX group; n = 23, 10Zn PTX group; n = 16, 5Zn CoTX group; n =, 10Zn CoTX group).

 
LPS Embryolethality in MT I/II Null Mice
We hypothesized that if maternal hepatic MT induction played a significant role in LPS developmental toxicity, MTKO mice would be less sensitive to LPS developmental toxicity. However, a similar incidence of resorption was observed in MTKO and WT mice on gd 10 (Fig. 5Go). Maternal liver and plasma Zn concentrations were measured in both the MTKO and WT strains (Fig. 6Go). Liver Zn concentrations in the MTKO mice were at a baseline mean of 400 ± 13.8 nmol/g liver tissue. The WT liver Zn concentrations were significantly higher than the MTKO mice. LPS treatment resulted in increased hepatic Zn in WT mice (no data for 0.05 group) but did not affect hepatic Zn concentration in the MTKO mice. The increase of hepatic Zn by LPS in WT mice was not statistically significant. Plasma Zn concentrations were similar for both groups and were not affected by LPS treatment.



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FIG. 5. Effects of gd 9 LPS treatment on incidence of embryo resorption at gd 10 in MTKO mice and WT controls. MTKO and WT mice were injected i.p. with LPS at 0.05 or 0.1 mg/kg dosages on gd9. Both dosages of LPS induced significant embryolethality in both strains of mice (p < 0.001), but there was no difference in response between the strains. (n = 2 MTKO, control and 0.05 treatment; n = 6 MTKO, 0.1 treatment; n = 2 WT, control; n = 2 MTKO, 0.05 treatment; n = 8 WT, 0.1 treatment).

 


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FIG. 6. Effects of gd 9 LPS treatment on maternal hepatic plasma concentrations at gd 10 in MTKO mice and WT controls. MTKO and WT mice were injected i.p. with LPS at 0.05 or 0.1 mg/kg dosages on gd 9. Maternal hepatic Zn concentrations were significantly elevated by LPS treatment in the WT, but not the MTKO mice. Plasma Zn concentrations were not affected by maternal LPS treatment. (n = 2 MTKO, control and 0.05 treatment; n = 6 MTKO, 0.1 treatment; n = 2 WT, control; n = 8 WT, 0.1 treatment).

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have demonstrated for a variety of developmental toxicants that a single maternal dose can alter Zn distribution due to increased MT synthesis (Daston et al., 1991Go, 1994Go; Taubeneck et al., 1994bGo). LPS is a known APR and MT inducer, and we predicted that it would produce an APR, including MT induction and subsequent maternal plasma Zn redistribution. While we did observe maternal hepatic MT induction and altered hepatic Zn concentrations following acute LPS treatment, our data indicate that these changes were not key mediators of acute LPS-induced embryolethality.

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)Go. 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)Go.

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., 2002Go).

From previous work, we know that TNF-{alpha} is released following LPS exposure (Gendron et al., 1990Go; Leazer et al., 2002Go). However, we have investigated TNF-{alpha} as a mediator of LPS embryotoxicity and found that TNF-{alpha} 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-{alpha}. These cytokines may work in concert, or one may compensate for the other in response to LPS (Chaplin and Hogquist, 1992Go; Neta et al., 1992Go). In addition, IL-6 acts directly on hepatocytes to produce the synthesis of MT (De et al., 1990Go; Lee et al., 1999Go; Schroeder and Cousins, 1990Go). 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.


    ACKNOWLEDGMENTS
 
This research was funded by the EPA/UNC Toxicology Research Program, Training Agreement T901915, with the Curriculum in Toxicology, University of North Carolina at Chapel Hill, and supported by HD01743 (C.L.K.).


    NOTES
 
This manuscript has been reviewed in accordance with the policy of the National Health and Environmental Effects Research Laboratory, U.S. Environmental Protection Agency, and approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.

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. Back


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