Zinc alters actin filaments in Madin-Darby canine kidney cells.

Exposure of semiconfluent cultures of Madin-Darby canine kidney cells to 10 microM zinc leads to a change in the organization of the actin filament system. Most of the stress fibers at the basal end of the cell are lost and the actin associated with the lateral membrane and junctional regions appears to retract into the cytoplasm. In addition, at the base of the cell in regions of cell-substratum contact, dense, actin-rich plaques appear. These alterations in actin filaments are associated with a change in cell shape. Microtubules were unaffected by exposure to 10 microM zinc. At zinc concentrations greater than or equal to 50 microM the microtubules depolymerized. Exposure to cadmium alters the actin filaments as well but the effect is different from the change seen with zinc. When the cells are exposed simultaneously to zinc and cadmium the cells appear the same as they would if exposed to zinc alone. Exposure of MDCK cells to either metal, individually or in combination, results in a significant and similar increase in F-actin content as determined spectrofluorometrically. The changes in organization and amount of F-actin are associated with a reduction in the ability of the cells to remain attached to the substrate, a toxic effect of these metals with regard to epithelial function. The results indicate that zinc, an essential metal, and cadmium, a highly toxic metal, interact with the actin cytoskeleton in intact cells.

Embryotoxic effects of sodium azide infusions in the Syrian hamster.

Pairs of osmotic minipumps containing 400 mg/ml (6.15 M) sodium azide in distilled water were subcutaneously implanted in timed pregnancy Syrian golden hamsters. The total delivered dose was calculated as 6 X 10(-2) mmol kg-1 hr-1 at the maximal pumping rate. Most dams exhibited obvious signs of toxicity during the period of pump implantation which was Days 7 through 9 of gestation. After removal of the pumps the dams were euthanized on Day 13 of gestation, and the uteri were removed for counting of the number of living, malformed, and resorbed fetuses. This dose rate resulted in a significantly increased incidence of resorptions of embryos over that in a control group implanted with pumps delivering only distilled water. The incidence of gross malformations exclusively in the form of encephaloceles was not different between control and azide-infused groups. The extent of nitrosylation of circulating hemoglobin was followed with time and found to involve only about 0.1% of the total blood pigment. Thus, this commercially important and widely distributed chemical with high acute toxicity is not considered to be teratogenic in hamsters, and it produces embryotoxicity only at dose rates that result in toxic signs in the dams.

Route-dependent pharmacokinetics, distribution, and placental permeability of organic and inorganic selenium in hamsters.

Inorganic selenium (Se) salts (selenite and selenate oxyanions) and the organic selenoamino acids (selenomethionine and seleniferous grains) are teratogenic and embryolethal in domestic and wild birds. Selenium bioaccumulation has been held responsible for reproductive failure among waterfowl at the Kesterson Reservoir (California), the Ouray and Stewart Lake Wildlife Refuges (Utah), and the Carson Sink (Nevada). Anecdotal field and controlled laboratory reports have implicated Se exposure in mammalian embryotoxicity (including human), but developmental toxicity studies in hamsters failed to demonstrate an adverse response, except at maternally toxic doses (Ferm et al., Reprod. Toxicol., in press). Uptake, distribution, and elimination of Se after a single bolus equimolar dose (60 mumol/kg) of selenate or selenomethionine by oral or intravenous administration were compared using day 8 pregnant hamsters. Intravenous selenate was eliminated ten times more rapidly from maternal plasma than oral selenate, but concentrated in liver, kidney, and placenta to the same degree. Intravenous (iv) L-selenomethionine achieved lower maximum circulating total [Se], but it was eliminated more slowly than iv selenate. Larger areas under the plasma and peripheral tissue [Se]:time curve (AUC) after oral or parenteral selenomethionine than after equimolar selenate were consistent with previous studies in rodents and in humans. Embryonic [Se] plateaued at 3 nmol/g after selenate, but embryonic [Se] after selenomethionine continued to accumulate (80 nmol/g) as gestation progressed. The lack of a teratogenic response in hamsters at doses of either selenate or selenomethionine less than those associated with maternal intoxication cannot be attributed to lack of Se accumulation in early embryonic and placental tissue.

Embryotoxicity and dose-response relationships of selenium in hamsters.

Pregnant hamsters were treated with selenite, selenate, and selenomethionine during the critical stages of embryogenesis. The dosing regimens were oral, intravenous, and osmotic minipump infusion. Malformations, mainly encephaloceles, were noted with oral and intravenous selenite and selenate but were associated with maternal toxicity manifested by inanition and weight loss. Fetal body weights and lengths were reduced in a dose-dependent manner with the inorganic forms. Single oral doses of selenomethionine above 77 mumol/kg induced similar malformations but not when the dose was delivered orally over four days nor by minipump over several days. Fetal body weights and lengths were decreased by selenomethionine in a dose-dependent manner. Maternal toxicity was pronounced with the higher doses of selenomethionine. Assigning a specific teratogenic effect to selenium is confounded by maternal toxicity.

Effect of cadmium on F-actin and microtubules of Madin-Darby canine kidney cells.

The effect of cadmium on F-actin and microtubules of Madin-Darby Canine Kidney cells was studied by cytochemical methods. A 6-hr exposure to cadmium (10 microns) in a buffered salt solution resulted in the breakdown of actin filaments, particularly those associated with both the stress fibers and the lateral membranes in areas of intercellular contact. Microtubules were not dramatically altered during this exposure period and cell viability, determined by trypan blue exclusion, was similar to controls. The effect of cadmium on actin was reversible if the cells were returned to culture medium. The results indicate that one possible mechanism of cadmium toxicity is via an effect on the organization of actin filaments.

Cadmium effects and biochemical status in hamsters following acute exposure in late gestation.

A normally teratogenic dose of cadmium ions administered to hamsters late in gestation does not cross the placental barrier nor does it result in placental pathology. Our finding, which differs from data for other rodents, may be due to differences in the chemical status of cadmium in the placental cytosols.

Cadmium disposition following repeated exposure in the pregnant hamster.

Pregnant Syrian hamsters were given a dose of 3.9 micromoles cadmium ion/kg (radiolabelled with 109cadmium) on days 7, 8, 9, and 11 of gestation. Animals were sacrificed on day 13 of gestation and maternal blood, liver, kidney, placentas, and fetuses collected. No pathology was observed in placentas or fetuses. Maternal liver accounted for 61% of the total cadmium injected (15.6 micromoles/kg dam), maternal kidney 4%, and placentas 0.24%. Maternal blood and fetuses contributed negligibly to the total body burden of cadmium. Gel filtration experiments indicated two major cadmium fractions in maternal liver, maternal kidney, and placental cytosols, one at the excluded volume and another nearly twice the excluded volume. Further purification and characterization of the cadmium fraction eluting at twice the excluded volume in the gel filtration experiments showed it to be cadmium metallothionein. Ion exchange chromatography on DEAE Sephacel revealed two forms of cadmium metallothionein in the cytosols of maternal liver and placentas. Maternal kidney cytosols contained one cadmium metallothionein. Amino acid analyses suggest that the thionein components of placental cytosols are synthesized in situ.

The concentration and chemical status of arsenic in the early placentas of arsenate-dosed hamsters.

We determined the concentration and chemical status of arsenic in the placentas of hamsters following continuous exposure via the osmotic minipump to minimally and frankly teratogenic doses of arsenate. Close to 70% of the placental arsenic is bound to macromolecules, two-thirds of which is dialyzable. The remaining 30% of arsenic consists of low molecular weight species, predominantly inorganic arsenic. This mix is the same for minimally teratogenic and frankly teratogenic doses of arsenate.

Comparative teratogenic activity of cancer chemopreventive retinoidal benzoic acid congeners (arotinoids).

The benzoic acid derivatives of retinoic acid, often referred to as arotinoids, are synthetic retinoids that possess some of the properties of vitamin A. In general, these retinoids have more favorable therapeutic ratios, based on acute toxicity in adults, than all-trans-retinoic acid in cancer chemoprevention. In the present study, a single dose of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]benzoic acid (Ro 13-7410; arotinoic acid), ethyl-(E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8- tetramethyl-2-naphthalenyl)-1-propen-1-yl]benzoate (Ro 13-6298; arotinoid ethyl ester), (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8, 8-tetramethyl-2-naphthalenyl)-1-propen-1-yl]phenylmethanol (Ro 13-8320; arotinoic methanol), or (E)-1,2,3,4-tetrahydro-1,1,4, 4-tetramethyl-6-[1-(4-methylphenyl)-1-propen-2-yl]naphthalene (Ro 13-9272; methyl arotinoid) was administered to pregnant Syrian golden hamsters during the early primitive streak stage of gestation. A significant increase in the numbers of litters containing one or more malformed offspring occurred at all doses of each retinoid studied. The types of malformations induced by oral arotinoid treatment were essentially identical to those found after maternal treatment with all-trans-retinoic acid or other teratogenic retinoids during the same gestational age. The results indicate that the alcohol congener was approximately 400 times more potent on a milligram per kilogram basis than all-trans-retinoic acid as a teratogen, and it was 70 times as embryolethal as all-trans-retinoic acid. The ethyl ester congener was 132 times, and the free acid 123 times, as embryolethal as all-trans-retinoic acid. On a molar basis, the arotinoic acid was at least 375 times as teratogenic as all-trans-retinoic acid and at least 140 times as teratogenic as etretinate in hamsters. Because the dose-response curves for arotinoids were significantly parallel to that for all-trans-retinoic acid, and because the spectrum of congenital defects induced by arotinoids was identical to that induced by all-trans-retinoic acid and other teratogenic retinoids, the mechanism of embryopathic action of these conformationally restricted retinoids was concluded to be similar to that of all-trans-retinoic acid.

Inhibition of cadmium teratogenesis by a mercaptoacrylic acid (MFA).

The teratogenic effect of cadmium can be diminished by a number of mechanisms including zinc and pretreatment with cadmium and mercury. In this study, the oral administration of alpha-mercapto-beta-(2-furyl)-acrylic acid (MFA) protects against cadmium-induced malformations and embryonic death. This protection is probably mediated by the chelation of the cadmium ion rather than metallothionein (MT) synthesis.

Teratogen concentration changes as the basis of the heat stress enhancement of arsenate teratogenesis in hamsters.

Hamster dams dosed continuously with arsenate and exposed to short-term hyperthermia produced a greater percentage of malformed offspring than did hamster dams dosed with arsenate alone. Hamsters receiving both treatments possessed elevated arsenic concentrations in the maternal blood and placentas immediately after cessation of the hyperthermic insult. Blood levels of arsenic were the same as those of animals not receiving the heat treatment within several hours post-hyperthermia; however, arsenic concentrations remained elevated in placentas, the duration being dependent on the dose of arsenate. We suggest that the rise in placental arsenic concentrations is the basis of the increase in the production of fetal malformations for hamsters treated continuously with arsenate and heat stressed during critical organogenesis.

Arsenate-induced neural tube defects not influenced by constant rate administration of folic acid.

Serious suggestions have been made that dietary supplementation with folic acid (FA) and perhaps other vitamins during pregnancy may reduce the incidence of neural tube defect (NTD) in human newborns. The purpose of these experiments was to evaluate the effect of continuous infusion of FA on the incidence of NTDs induced by arsenate. This teratogen induces NTDs in up to 90% of golden hamster fetuses when administered acutely during critical stages of embryogenesis. FA was administered by subcutaneously implanted osmotic minipumps beginning on the 6th day of gestation, 48 h before an acutely administered dose of sodium arsenate. The protective effect of FA was examined at three teratogenic dose levels of arsenate: optimal, with 905 NTDs, intermediate, with 38% NTDs, and low, with 20% NTDs. Fetuses were recovered at day 13 of gestation and examined for NTDs and other malformations. Maternal red cell folate levels were determined on day 8, 48 h after implantation of the pumps. The results show that the maternal red blood cell level of FA can be significantly increased within 48 h by chronic infusion to levels which are almost two times (550 ng/ml) control levels. There was no significant protection against arsenate-induced NTDs following FA supplementation at any of three levels of this teratogen.

Concentration and chemical status of arsenic in the blood of pregnant hamsters during critical embryogenesis. 1. Subchronic exposure to arsenate utilizing constant rate administration.

We have determined the concentration, availability, and chemical status of radiolabeled arsenic in the blood of pregnant hamsters at the beginning (morning of Day 8) and the end (morning of Day 9) of the critical period of embryogenesis. Hamster dams were exposed to teratogenic doses of arsenate by means of osmotic minipumps implanted on the morning of Day 6 of the gestation period. Whole blood arsenic concentrations were the same for 48 and 72 hr postimplant. The arsenic concentration of plasma equaled that of red cells. Plasma arsenic was not bound to macromolecules and had the same chemical status 48 and 72 hr postimplant. Arsenate was the dominant form (67% of the total). However, the presence of dimethylarsinic acid and arsenite indicates that the pentavalent species was metabolized. Red cell arsenic was bound to macromolecules in the cell sap. Seventy percent of red cell sap arsenic was dialyzable 48 hr postimplant, but only 56% 72 hr postimplant. Arsenate was the dominant dialyzable red cell species on Day 8 and arsenite was the major dialyzable form on Day 9. Our findings demonstrate a relationship between the maternal blood concentration and chemical status of arsenic and the presence of malformations resulting from a constant rate exposure of pregnant hamsters to arsenate via the osmotic minipump.

Concentration and chemical status of arsenic in the blood of pregnant hamsters during critical embryogenesis. 2. Acute exposure.

We have determined the concentration and chemical composition of arsenic in the blood of pregnant hamsters between 0.2 and 6 hr after an intraperitoneal injection of a teratogenic dose of radiolabeled sodium arsenate on the morning of the eighth day of gestation. Arsenic was present in plasma and red cells 0.20 hr postinjection. The plasma arsenic concentration reached a maximum of 220 mumole/kg blood near 0.5 hr postinjection. Plasma arsenic existed entirely as low-molecular-weight species. Both arsenite and dimethylarsinate (DMA) were present in plasma 0.20 hr postinjection, indicating that arsenate reduction and methylation of arsenic are rapidly initiated. However, the arsenite contribution remained small while the DMA contribution increased with time. Red cell arsenic included macromolecular arsenic (AsP) as well as three low-molecular-weight forms. The contribution of DMA remained small, but arsenite and AsP contributions increased with time. These findings identify the maternal blood concentration and chemical status of arsenic following the administration of a teratogenic dose of arsenate during the period of organogenesis. They could prove useful for predicting the likelihood of a teratogenic outcome in other mammalian species.

Constant rate exposure of pregnant hamsters to arsenate during early gestation.

We have examined the teratogenic and embryotoxic effects of constant-rate exposure of pregnant hamsters to arsenate by means of subcutaneous implants of osmotic minipumps. Different total exposure regimens were established by varying the duration of minipump implants and by varying the concentration of arsenate in the minipumps. Dams were killed on Day 13 of pregnancy, 5 days after the critical stage of organogenesis. Numbers of resorptions, dead fetuses, and living fetuses were obtained. Fetal weights, crown-rump lengths, and the incidence of malformations were recorded. Control animals were treated identically with minipumps containing demineralized water. The percentage of malformations per litter, a direct measure of teratogenesis, was dependent only upon the concentration of arsenate in the minipumps. The minimum teratogenic response was achieved with a dose of 70 mumol/kg dam/24 hr during the critical stages of organogenesis. The embryotoxic (fetotoxic) indicators, fetal weight and crown-rump length, decreased with increases in exposure time and with increased concentrations of arsenate. The resorption rate also depended directly upon duration of exposure and concentration of arsenate in the minipump.

Heat- and alcohol-induced neural tube defects: interactions with folate in a golden hamster model.

Maternal hyperthermia or ethanol each can induce fetal neural tube defects (NTD) following exposures on the 8th day of gestation in golden hamsters. To explore the relationship between NTD and varying doses of either heat or ethanol, timed pregnant golden hamsters were exposed to various doses of either 25% ethanol, or heat in an incubator at 39.5 degrees C on the morning of the 8th day of gestation. Two doses of 0.015 ml/g body weight of 25% ethanol 4 h apart resulted in a 44% incidence of NTD when fetuses were examined on day 13. Single doses of 25% ethanol (either 0.015 or 0.0075 ml/g) resulted in very low incidences of NTD that were not significantly different from zero. A 50-min exposure to heat resulted in a 35% incidence of NTD. A shorter exposure (44 min) resulted in a 23% incidence, and a longer exposure (56 min) resulted in a 68% incidence of NTD. A 0.0075 ml/g dose of 25% ethanol, followed by these same durations of heat, resulted in incidences of NTD that were not significantly different from heat alone. In order to determine what effect folate supplementation might have on ethanol- or heat-induced NTD, osmotic pumps filled with either folate or saline were placed subcutaneously in pregnant hamsters on the 6th day of gestation. Animals were then exposed to ethanol or heat on the morning of day 8. No significant protection from NTD was evident among fetuses from mothers supplemented with folate despite significant elevations in their red cell folate levels on day 8 of gestation.

Teratogenic dose-response relationships of etretinate in the golden hamster.

Etretinate (Ro 10-9359; Tigason; 4-methoxy-2,3,6-trimethylphenyl analog of retinoic acid, ethyl ester) was evaluated for teratogenic activity in the Syrian golden hamster. Groups of pregnant hamsters were given a single oral dose of 2.8-88 mg/kg etretinate during the early primitive streak stage of gestation. No signs of maternal intoxication were observed in any of the hamsters given the retinoid and maternal body weight changes throughout gestation were not significantly different from those of the vehicle-treated group. Etretinate administration was associated with a dose-dependent increase in the incidence and severity of malformations. The average fetal body weight was significantly less in litters recovered from dams given 44 or 88 mg/kg of etretinate when compared to the average body weight of fetuses recovered from dams given an equivalent volume of the vehicle. The average crown-rump lengths also were significantly shorter in fetuses taken from the dams given 44 or 88 mg/kg etretinate as compared to the control group. The malformations induced by etretinate administration were similar to those noted following an oral dose of all-trans-retinoic acid (Willhite and Shealy, 1984). A comparison of the dose-response curves for induction of terata following treatment with etretinate or all-trans-retinoic acid revealed that etretinate was twice as potent as a teratogen in the hamster as all-trans-retinoic acid. Teratogenic activity of etretinate in the hamster was achieved at doses (mg/kg body wt) used in patients at current clinical therapeutic levels.

Prenatal and developmental toxicology of arsenicals.

A variety of species, including the human, have been shown to be susceptible to the embryotoxic effects of inorganic arsenic. Malformations of the axial skeleton, neurocranium, viscerocranium, eyes, and genitourinary systems as well as prenatal death followed a bolus dose of trivalent or pentavalent inorganic arsenic. Trivalent arsenic was more teratogenic than pentavalent arsenic; in contrast, the methylated metabolites of arsenic possessed only limited teratogenic activity. Administration of inorganic arsenic to mammals results in concentration of arsenic within the placenta and small amounts are deposited within the embryo. Studies concerning the pathogenesis of arsenic-induced axial skeletal lesions revealed early failure of neural fold elevation and a subsequent, persistent failure of closure of the neural tube. Physical factors, drugs and heavy metals may modify the response to a teratogenic dose of inorganic arsenic. Medical problems associated with industrial or agricultural arsenicalism are most often typified by chronic exposure; future studies should emphasize those routes of administration and types of exposure that are characteristic of arsenic intoxication.