Altered zinc metabolism contributes to the developmental toxicity of 2-ethylhexanoic acid, 2-ethylhexanol and valproic acid.

It has been hypothesized that the developmental toxicity of certain compounds is, in part, due to maternal toxicity resulting in alterations in zinc (Zn) metabolism that affects the developing conceptus. In the present work the effects of developmentally toxic doses of 2-ethylhexanoic acid (EHXA), 2-ethylhexanol (EHXO), and valproic acid (VPA) on Zn metabolism were investigated in the pregnant rat. In experiment 1, dams were intubated with EHXA (3.13, 6.25, 9.38 or 12.5 mmol/kg), EHXO (6.25, 9.38 or 12.5 mmol/kg), VPA (1.56, 3.13, 6.25 or 9.38 mmol/kg), or corn oil (control; 1.0 ml/kg) at 14:00 h on gestation day (GD) 11.5, intubated with 32 microCi 65Zn at 22:00 h, and then killed at 08:00 h on GD 12.5. At the higher dose levels of EHXA and EHXO, and at all dosages of VPA, the percentage of 65Zn retained in maternal liver was higher, while that in the embryos was lower, than in controls. Chemical-associated changes in 65Zn distribution were associated with increased maternal liver metallothionein (MT) concentrations. In experiment 2, dams were fed diets containing 1, 25 or 97 microg Zn/g from GD 0-16 and intubated with 3.5 mmol EHXA or 1.0 ml corn oil/kg/d from GD 8-15. Dams were killed on GD 16 or 19. High incidences of encephalocele and tail defects were noted in the GD 16 fetuses of EHXA-treated dams fed either the low or adequate Zn diet, the highest incidences being in the low Zn group. On GD 19 the incidence of tail defects tended to be higher in the EHXA groups than in oil-treated controls, the highest incidence occurring in the low Zn EHXA group. Encephalocele was only observed in the low Zn EHXA-treated group. Fetal weight and crown-rump lengths were decreased by EHXA treatment and low dietary Zn. The incidence of rib anomalies was higher in the EHXA-exposed groups than in their respective oil controls. In experiment 3, GD 10.5 embryos collected from control dams were cultured for 48 h in serum from control or EHXA-treated male rats fed 4.5 or 25.0 microg Zn/g diets. Embryos cultured in either EHXA or low Zn sera exhibited delayed development; the addition of Zn to these sera eliminated their developmental toxicity. These results support the hypothesis that certain chemicals which induce maternal toxicity act, in part, to influence embryonic Zn metabolism and trigger abnormal development. Importantly, the teratogenic effects of these chemicals can be modulated by dietary Zn intake.

Assessing the effects of low boron diets on embryonic and fetal development in rodents using in vitro and in vivo model systems.

To date, boron (B) essentiality has not been conclusively shown in mammals. This article summarizes the results of a series of in vitro and in vivo experiments designed to investigate the role of B in mammalian reproduction. In the first study, rat dams were fed either a low (0.04 microg B/g) or an adequate (2.00 microg B/g) B diet for 6 wk before breeding and through pregnancy; reproductive outcome was monitored on gestation day 20. Although low dietary B significantly lowered maternal blood, liver, and bone B concentrations, it had no marked effects on fetal growth or development. The goal of the second study was to assess the effects of B on the in vitro development of rat postimplantation embryos. Day 10 embryos collected from dams fed either the low or adequate B diets for at least 12 wk were cultured in serum collected from male rats exposed to one of the two dietary B treatments. Dams fed the low B diet had a significantly reduced number of implantation sites compared to dams fed the B-adequate diet. However, embryonic growth in vitro was not affected by B treatment. The aim of study 3 was to define the limits of boric acid (BA) toxicity on mouse preimplantation development in vitro. Two-cell mouse embryos were cultured in media containing graded levels of BA (from 6 to 10,000 microM). Impaired embryonic differentiation and proliferation were observed only when embryos were exposed to high levels of BA (>2000 microM), reflecting a very low level of toxicity of BA on early mouse embryonic development. Study 4 tested the effects of low (0.04 microg B/g) and adequate (2.00 microg B/g) dietary B on the in vitro development of mouse preimplantation embryos. Two-cell embryos obtained from the dams were cultured in vitro for 72 h. Maternal exposure to the low B diet for 10, 12, and 16 wk was associated with a reduction in blastocyst formation, a reduction in blastocyst cell number, and an increased number of degenerates. Collectively, these studies support the concept that B deficiency impairs early embryonic development in rodents.

Di(2-ethylhexyl) phthalate induces a functional zinc deficiency during pregnancy and teratogenesis that is independent of peroxisome proliferator-activated receptor-alpha.

Di(2-ethylhexyl) phthalate (DEHP) is a peroxisome proliferator whose administration to rodents induces a pleiotropic response mediated by the peroxisome proliferator-activated receptor-alpha (PPAR alpha). The mechanisms underlying DEHP-induced reproductive toxicity and teratogenicity are not well understood but could be the result of an alteration in gene expression by PPAR alpha. Additionally, phthalate exposure is known to impair fetal zinc (Zn) levels during pregnancy. In this work, we investigated whether the reproductive toxicity and teratogenicity of DEHP are mediated by PPAR alpha and whether the receptor influences maternal and/or embryonic Zn metabolism. Pregnant female mice, homozygous wild-type (+/+) or PPAR alpha -null (-/-), were intubated with either vehicle alone or 1,000 mg DEHP/kg body weight on gestation day (GD) 8 and 9. Pregnancy outcome was evaluated on GD10 and GD18 in two cohorts of animals. Compared to controls, DEHP administration resulted in maternal toxicity, embryo/ fetal toxicity, and teratogenicity in both (+/+) and (-/-) mice. Maternal liver mRNA for cytochrome P-450 4A1 (CYP4A1) was higher in DEHP-treated (+/+) mice but not in DEHP-treated (-/-) mice on GD10, consistent with their respective phenotype. Maternal liver MT and Zn levels were significantly higher than in controls on GD10. In addition, embryonic Zn content was significantly lower in both genotypes treated with DEHP compared to controls. Results from this work show that DEHP-induced reproductive toxicity, teratogenicity, and altered Zn metabolism are not mediated through PPAR alpha-dependent mechanisms. In addition, this work suggests that DEHP-induced alterations in Zn metabolism contribute to the mechanisms underlying DEHP-induced reproductive toxicity and teratogenicity.

Toxicant exposure and trace element metabolism in pregnancy.

A review of the literature provides support for the concept that maternal nutritional status has a significant influence on embryonic and fetal development. The consumption of `poor' diets has been shown to be a risk factor for poor pregnancy outcome, while the provision of selected nutritional supplements prior to and during pregnancy has been associated with improved pregnancy outcome. Despite the above, it has been difficult to identify specific nutrient deficiencies as causative factors of abnormal development. One explanation for this failure is that embryo/fetal nutritional deficiencies can arise through a number of mechanisms in addition to a low maternal intake of a nutrient(s). Evidence is presented for the hypothesis that the developmental toxicity of a number of teratogens can be ascribed, in part, to their ability to induce alterations in the partitioning of essential trace elements between the maternal and fetal unit. An implication of the above hypothesis is that maternal diet can be an important modulator of the developmental toxicity of several agents.

Zinc deficiency causes apoptosis but not cell cycle alterations in organogenesis-stage rat embryos: effect of varying duration of deficiency.

Zinc deficiency is teratogenic in all species in which it has been examined. Zinc is an essential component of enzymes involved in DNA synthesis and cell proliferation, and may play an as yet undetermined role in apoptosis. To further our understanding of the role of zinc in normal development, we examined cell death and cell cycle parameters in embryos of pregnant rats fed a zinc-deficient diet for 2 to 10 days (0.5 microgram zinc/g diet; zinc-adequate diet was 25 micrograms zinc/g). To elucidate sensitive periods of development and susceptible cell populations, dams were fed the zinc-deficient diet from gestation day 1, 3, 7, or 9 and killed on day 11. Embryos were examined for morphology and developmental stage. From each litter, 2-3 embryos were stained with Nile blue sulfate (NBS) to visualize cell death, 3 embryos were frozen for flow cytometric cell cycle analysis and cell counts, and selected embryos were preserved for histological examination. Dams fed the zinc-deficient diet for more than 3 days reduced their food intake through gestation day 8 but increased food intake on day 9. Maternal plasma zinc dropped to 10-25% of control levels in the zinc-deficient groups. Zinc deficiency from gestation day 1 or 3 resulted in two categories of affected litters on day 11. One category had embryos which were morphologically normal but displayed extensive NBS staining in the visceral arches, neural tube, and somites. The second category had developmentally retarded or maldeveloped embryos which showed little NBS staining. Zinc deficiency from gestation day 7 produced cell death in the posterior dorsal midline in the area of premigratory neural crest cells, which was confirmed by histological examination. Zinc deficiency from gestation day 9 did not affect morphology or NBS staining. Percentages of cells in the G0/G1, S, and G2M phases of the cell cycle on gestation day 11, determined by flow cytometry, were similar to controls in all groups. This study shows that as few as 4 days of maternal zinc deficiency can produce excess embryonal cell death, and that neural crest cells may be particularly sensitive.

Tumor necrosis factor-alpha alters maternal and embryonic zinc metabolism and is developmentally toxic in mice.

We tested the hypothesis that tumor necrosis factor-alpha (TNF-alpha) would be teratogenic in mice due in part to its effects on zinc metabolism. In Experiment 1, nonpregnant mice were injected with a single dose of TNF-alpha (40,000 U) or PBS and then received a 65Zn-labeled meal. Mice killed 10 h after TNF-alpha treatment had high liver 65Zn and low plasma 65Zn, compared with controls. In Experiment 2, gestation day 8 (GD 8) mice were injected with PBS or TNF-alpha and then received a 65Zn-labeled meal. Dams killed 10 h after TNF-alpha treatment had higher liver and kidney 65Zn and lower plasma and embryonic 65Zn accumulation than controls. In Experiment 3, TNF-alpha dosing from GD 7-12 was associated with high maternal liver Zn and metallothionein concentrations on GD 13 and a high frequency of exencephaly on GD 18. In Experiment 4, dams fed diets containing 4.5, 12.5 or 50.0 micrograms Zn/g were given PBS or TNF-alpha on GD 7-12. Gross fetal defects were not observed in the PBS-treated litters evaluated on GD 18. In contrast, TNF-alpha-treated litters were characterized by multiple defects, with the incidence and severity being highest in the low Zn diet group. In Experiment 5, embryos cultured in serum from TNF-alpha-treated animals exhibited a high frequency of defects; the developmental toxicity of this serum was ameliorated when it was supplemented with Zn. Thus, the developmental toxicity of TNF-alpha is due in part to its influence on Zn metabolism.

Altered maternal zinc metabolism following exposure to diverse developmental toxicants.

It has been hypothesized that one mechanism contributing to the developmental toxicity of some xenobiotics is an embryonic/fetal zinc (Zn) deficiency that occurs secondary to toxicant-induced changes in maternal Zn metabolism. We studied the influence of diverse toxicants (urethane, ethanol, melphalan, arsenic, and alpha-hederin) on maternal-embryonic Zn metabolism and maternal liver metallothionein (MT) induction in Sprague-Dawley rats given a 65Zn-labelled meal by gavage 8 h after toxicant exposure and killed 10 h later on gestation day 12.5. Exposure to the toxicants resulted in increases in maternal hepatic MT concentrations that generally exceeded that which could be accounted for by reductions in food intake. 65Zinc retention was higher in maternal liver and lower in the products of conception in the toxicant-exposed groups. Strong linear relationships were found; as maternal liver MT concentrations increased, 65Zn retention in maternal liver was increased and 65Zn distribution to the conceptuses was decreased. These results support the hypothesis that diverse insults can produce developmental toxicity, in part, by altering maternal and embryonic Zn metabolism.

Altered Zn status by alpha-hederin in the pregnant rat and its relationship to adverse developmental outcome.

The hypothesis that an acute-phase reaction in the pregnant animal causes a systemic redistribution of Zn, resulting in a transient but developmentally adverse Zn deficiency in the embryo, was tested by treating pregnant rats during organogenesis with alpha-hederin, an agent reported to induce substantial metallothionein (MT) synthesis in rat liver, and determining hepatic MT concentration, hepatic and plasma Zn concentration, and systemic distribution of a pulse of 65Zn after treatment. Developmental toxicity was assessed by evaluating morphologic development in term fetuses. A single dose of alpha-hederin, injected sc at dosages of 3 to 300 mumol/kg, caused an acute phase response, indicated by decreased Fe and Zn, and increased Cu, alpha 1-acid glycoprotein, and ceruloplasmin concentration in plasma, along with a dosage-related increase in maternal hepatic MT concentration. The maximum induction of MT was 11 to 15-fold greater than control and occurred at dosages of 30 mumol/kg and higher, and MT concentration reached its peak 12 to 24 h after treatment. Zn concentration in liver and liver cytosol increased along with MT, reaching a maximum level at dosages of 30 mumol/kg and higher. Plasma Zn concentration decreased after alpha-hederin treatment to a level approximately 75% of control at a dosage of 30 mumol/kg and 50% of control at 300 mumol/kg. Therefore, hepatic MT induction was associated with most, but not all, of the decrease in plasma Zn concentration. Zn distribution was evaluated by giving an oral pulse of 65Zn 8 h after treatment with 0, 30, or 300 mumol/kg alpha-hederin on gestation day 11, and measuring 65Zn levels 18 h after treatment. The fraction of 65Zn distributed to the liver of treated rats (either dosage) was twice that of control, but distribution of 65Zn to other maternal tissues was decreased. 65Zn accumulation by conceptuses was significantly decreased, attributable to decreased accumulation in decidua, but not in visceral yolk sacs or embryos; however, at this stage of development the decidua accounts for a greater quantity of Zn than either of the other products of conception and may serve as the Zn-storing tissue for the conceptus. Both 30 and 300 mumol/kg increased resorption incidence, and 300 mumol/kg also decreased fetal weight and increased the incidence of abnormal fetuses. Serum collected from rats two hours after alpha-hederin treatment (i.e., before the onset of MT synthesis) supported rat embryo development in vitro, whereas serum collected 18 h after treatment did not. Adding Zn to this serum restored normal embryonic development.(ABSTRACT TRUNCATED AT 400 WORDS)

Meso-2,3-dimercaptosuccinic acid (DMSA) affects maternal and fetal copper metabolism in Swiss mice.

Meso-2,3-dimercaptosuccinic acid (DMSA) is a chelating agent used to treat heavy metal intoxication. DMSA has been reported to be teratogenic in the mouse, and it has been suggested that this teratogenicity may be secondary to DMSA-induced alterations in Zn metabolism. In the present study, 0, 400 or 800 mg DMSA/kg body weight were administered on gestation days 6-15 to pregnant Swiss mice by gavage (PO) or subcutaneous injection (SC). Mice were fed a diet containing 14 micrograms Zn, 10 micrograms Cu, 120 micrograms Fe, 1175 micrograms Mg and 6.8 mg Ca/g diet. A sub-group of mice in the 800 mg DMSA/kg SC group was fed a diet containing 250 micrograms Zn/g. DMSA administration did not result in overt maternal toxicity. There was no effect of the drug on fetal or placental weight, or on crown-rump length. However, some fetuses from DMSA-treated dams were characterized by skeletal abnormalities including supernumerary ribs, unossified anterior phalanges and malformed sternebrae. Drug exposure was not associated with consistent changes in tissue Zn, Fe, Ca or Mg levels. Supplemental Zn had no marked effects on the fetus. Fetal liver Cu concentrations exhibited dose-dependent decreases with increasing DMSA dose. This finding suggests that the developmental toxicity of DMSA may be mediated through disturbed maternal/fetal copper metabolism.

The role of metallothionein induction and altered zinc status in maternally mediated developmental toxicity: comparison of the effects of urethane and styrene in rats.

We hypothesize that maternal metallothionein (MT) induction by toxic dosages of chemicals may contribute to or cause developmental toxicity by a chain of events leading to a transient but developmentally adverse decrease in Zn availability to the embryo. This hypothesis was tested by evaluating hepatic MT induction, maternal and embryonic Zn status, and developmental toxicity after exposure to urethane, a developmental toxicant, or styrene, which is not a developmental toxicant. Pregnant Sprague-Dawley rats were given 0 or 1 g/kg urethane ip, or 0 or 300 mg/kg styrene in corn oil po, on Gestation Day 11 (sperm positive = Gestation Day 0). These were maternally toxic dosages. As both treatments decreased food consumption, separate pair-fed control groups were also evaluated for effects on MT and Zn status and development. In addition, Gestation Day 11 rat embryos were exposed to urethane in vitro in order to determine whether urethane has the potential to be directly embryotoxic. Urethane treatment induced hepatic MT 14-fold over control; styrene treatment induced MT 2.5-fold. The MT induction by styrene could be attributed to decreased food intake, as a similar level of induction was observed in a pair-fed untreated control group. However, the level of MT induction by urethane was much greater than that produced by decreased food intake alone. Hepatic Zn concentration, particularly in the cytosol, was increased in the presence of increased hepatic MT concentration. Plasma Zn concentration was significantly decreased (approximately 30%) by urethane treatment, but not by styrene or food restriction (pair-feeding). Distribution of 65Zn to the liver of urethane-treated dams was significantly greater (by 30%), while distribution to embryonic tissues was significantly lower (by at least 50%) than in pair-fed or ad lib.-fed controls. Styrene treatment had no effect on 65Zn distribution. Urethane was developmentally toxic, causing an 18% decrease in fetal weight and a significant delay in skeletal ossification, but was not toxic to rat embryos in vitro. Styrene was not developmentally toxic. The changes observed after urethane treatment, namely substantial hepatic MT induction and altered maternal and embryonic Zn status, along with the lack of direct embryotoxicity of urethane in vitro, support the hypothesis that these maternal effects contribute to developmental toxicity. The lack of similar changes in styrene-intoxicated dams provides one explanation for its low developmental toxicity at maternally toxic dosages.