Cardiac abnormalities induced by zinc deficiency are associated with alterations in the expression of genes regulated by the zinc-finger transcription factor GATA-4.

Zinc (Zn) deficiency during pregnancy results in a wide variety of developmental abnormalities. The objective of this study was to determine if expression of cardiac developmental genes regulated by Zn-finger transcription factors could be modulated during dietary Zn deficiency. Rats were fed 0.5 (low Zn) or 90 (controls) microg Zn/g diet throughout pregnancy. Fetal development was examined and RNA isolated at gestation day (GD) 13 and 20. Cardiac abnormalities were detected at GD 20 in 82% of fetuses from dams fed low Zn diets compared with only 2% in controls. Cardiac developmental gene expression regulated by the Zn-finger transcription factor, GATA-4, was measured by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). In GD 13 and 20 hearts, two genes critical for heart development, alpha-myosin heavy chain (alpha-MHC) and cardiac troponin I (cTnI), were down-regulated in Zn-deficient fetuses. Expression of alpha-MHC was 66 and 40% lower at GD 13 and 20, respectively, in fetuses from dams fed low Zn diets compared with fetuses from control dams (p<0.05). Fetal cardiac TnI RNA levels were reduced 40 and 45% at GD 13 and 20 in the Zn-deficient group compared with controls (p<0.05). Fetal cardiac transcript levels of GATA-4 and MHox, a gene regulated by a helix-loop-helix transcription factor, whose expressions are not Zn-dependent, were unaffected by diet. These data indicated that alterations in gene regulation might be an underlying mechanism of cardiac abnormalities. Dysfunction of other Zn-dependent transcription factors may be an integral part of the extensive teratogenesis associated with Zn deficiency.

Repeated administration of alpha-hederin results in alterations in maternal zinc status and adverse developmental outcome in the rat.

The administration of alpha-hederin, an inducer of metallothionein, results in a secondary zinc deficiency that may be an important maternally mediated mechanism of developmental toxicity. Previous studies have shown adverse developmental outcome with a single administration of alpha-hederin to rats on gestation day (GD) 8 or 11. The objective of this study was to determine whether dosing of alpha-hederin throughout organogenesis would result in a sustained elevation of maternal hepatic metallothionein and subsequent developmental abnormalities. Rats were administered dosage levels of 0 (vehicle only), 20, or 30 mumol/kg from GD 6-15. Maternal hepatic metallothionein levels were 10-fold higher on GD 16 in the treatment groups than the controls. Consequently, liver zinc concentrations increased 60% and 54%, whereas plasma levels decreased 23% and 33% in the 20 and 30 mumol/kg treatment groups, respectively. At GD 20, mean fetal weights of the treatment litters were 11% less than control litters. The administration of alpha-hederin resulted in a threefold increase in the number of offspring that exhibited developmental abnormalities, including visceral and skeletal malformations. Following an oral pulse of 65Zn subsequent to treatment with 0 or 20 mumol/kg of alpha-hederin, the distribution of 65Zn to the liver of treated dams was twice that of controls, whereas the radiolabeled zinc apportioned to the decidua and uterus decreased by 44%. Furthermore, the 65Zn detected in the embryos from treated dams was 70% lower than in embryos from control dams. In conclusion, low doses of a metallothionein inducer administered to the dam from GD 6-15 resulted in a sustained elevation of hepatic metallothionein and a subsequent redistribution of zinc leading to a decrease in the zinc available to the embryo and ultimately to adverse development of the offspring. Repeated dosing throughout organogenesis, as required in regulated safety assessment testing, increased the severity of the effects previously observed with single large dosages of the toxicant administered during midgestation.

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)

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.

Association and dissociation of Escherichia coli heat-stable enterotoxin from rat brush border membrane receptors.

Escherichia coli heat-stable enterotoxin (ST) binds to receptors on rat intestinal cells and brush border membranes (BBM). We devised experiments to examine the reversibility of ST binding. We found that both 125I-labeled ST and native ST were spontaneously dissociable from the BBM receptor. Radiolabeled ST bound to BBM was also dissociated by the addition of avid goat anti-ST antiserum. Furthermore, using a computer program for analysis of ligand binding, we calculated an apparent Ka of 10(8) liters/mol from competitive inhibition and saturation-binding data. This is significantly lower than the value previously reported by others. Our findings, of a lower Ka and a reversible ST-binding process, suggest that a therapeutic strategy of removing bound ST from its receptor or competing with the enterocyte receptor for unbound ST might be successful in terminating ST-induced secretion.