Teratogenic effects of nigericin, a carboxylic ionophore

Nigericin (Na+ salt) was given intraperitoneally at doses of 5.0 or 7.0 mg/kg on one of gestation days 7-12 to pregnant CD-1 mice. Additional mice were injected ip with 2.5 mg/kg on day 11 or 12 only. Injections on single gestation days reduced fetal growth and increased prenatal deaths. Additional signs of toxicity to the conceptus included treatment-related extra ribs and delayed ossification. Treatment was also associated with gross and skeletal malformations, such as median facial cleft, exencephaly, encephalocele, fused ribs, and anomalous vertebrae and exoccipitals. With the possible exception of the 5.0 mg/kg dose given on gestation day 8, nigericin doses associated with gross or skeletal malformations also resulted in observable maternal toxicity.     

The effect of the time of administration of chromium trioxide on the embryotoxic response in hamsters.

Prior work has demonstrated that chromium trioxide is embryotoxic in hamsters if administered to pregnant animals early on the eighth gestation day. The major manifestations are cleft palate and an increased frequency of resorptions. In the present study a single iv dose (8 mg/kg) of chromium trioxide was injected into pregnant hamsters at 8 A.M. on either day 7, 8, 9, 10 or 11 of gestation in order to determine the effect of altering the time of treatment on embryotoxicity. Fetuses from females treated with chromium or demineralized-distilled water were collected on day 15 of gestation and were examined for the types and frequency of external and internal malformations. The number of resorption sites was recorded. Cleft palate, the major malformation detected, was produced only when chromium was administered on days 7, 8, or 9 of gestation. Since the frequency of resorptions and the incidence of cleft palate varied with the time of treatment it is concluded that the time at which chromium trioxide is injected into the pregnant hamster does influence embryotoxicity. The results suggest that an interference with embryonic growth may be an important factor in chromium-induced cleft palate in hamsters.     

Teratogenic effects of a single oral administration of methylmercuric chloride in mice.

The teratogenic effects of methylmercuric chloride (MMC) given orally as a single dose to pregnant ICR mice on day 10 of gestation were examined. The doses tested were 25, 20, 15 and 10 mg/kg. Controls received distilled water orally. Each group consisted of 20 females. Fetuses were taken on day 18 of gestation for teratological study. The number of resorbed or dead embryos was moderately increased in the 25 mg/kg group. Fetuses from dams given 25, 20 and 15 mg/kg MMC weighed significantly less than those in the control group. Many fetuses with malformations were observed in the treated groups; cleft palate occurred in 100, 58.6 and 28.0% of fetuses from dams given 25, 20 and 15 mg/kg MMC, respectively (statistically significant). Hydronephrosis appeared in 23.8 and 18.5% of fetuses from dams given 25 and 20 mg/kg MMC, respectively (statistically significant). Skeletal variations, incomplete ossification of sternebrae, for example, were also observed in the treated groups. These results indicate that MMC is teratogenic so far as cleft palate is concerned and embryotoxic in ICR mice.     

Teratogenic effects of nickel chloride on embryonic mice and its transfer to embryonic mice.

Administration of nickel chloride to the pregnant mice on the seventh to eleventh day of their gestational period, resulted in significant embryotoxic effects in terms of an increased resorption rate, a decreased fetal weight, delay in skeletal ossification and high incidence of malformation. Among the cases of fetal malformation, the following malformations were observed to occur at a higher rate of incidence: acephalia, exencephaly, cerebral hernia, open eyelid, cleft palate, micromelia, ankylosis of the extremity, club foot and skeletal anomalies. Most skeletal anomalies were in the form of vertebral and/or rib fusions and were found mostly at thoracic and lumbar levels. The concentration of nickel retained in embryonic tissues was 800 times higher in the exposed compared to control groups and indicated that increased tissue levels of nickel chloride had a toxic influence on the developing embryo.     

Mechanism of dimethylsulfoxide protection against the teratogenicity of secalonic acid D in mice

Dimethylsulfoxide (DMSO) is known to antagonize the teratogenic effects of secalonic acid D (SAD) in mice. To establish the optimum protective dose of DMSO, pregnant CD-1 mice were treated, i.p., with 30 mg/kg of SAD in 5% (w/v) NaHCO3, containing 0, 10, 20, or 30% (v/v) DMSO on day 11 of gestation. Data indicate that at 10% and 20% levels, DMSO affords an apparent dose-related protection against SAD-induced cleft palate, whereas 30% DMSO enhanced fetal resorption with no reduction in the incidence of cleft palate. Ultraviolet spectra and TLC mobility indicated that DMSO at 20% did not directly interact with SAD. Distribution and elimination of 14C-SAD was studied in fetal and maternal tissues from pregnant mice at 24 and 48 hr after exposure to 30 mg/kg of 14C-SAD, i.p., in NaHCO3 (control) or in 20% DMSO. Compared with those not receiving DMSO, maternal exposure to DMSO: 1) significantly reduced (42-75%) radioactivity in fetal heads and bodies, placenta, and maternal tissues other than liver; 2) significantly increased (up to 222%) the radioactivity in maternal liver; and 3) significantly reduced (44-58%) fecal and urinary elimination of SAD-derived radioactivity. These results suggest that the antiteratogenic effect of DMSO against SAD may be at least partly mediated by increased SAD (or its metabolites) retention by maternal liver leading to reduced SAD uptake by the fetus.     

Effect of route of administration on phenytoin teratogenicity in A/J mice

Acute administration of the anticonvulsant drug, phenytoin (PHT) has been shown to result in embryotoxicity and teratogenicity in several strains of mice. The A/J strain is reported to be most susceptible to the effects of the drug including an increased incidence of resorptions and orofacial clefts in treated animals. When administered chronically, the drug has been shown to be teratogenic in the absence of maternal toxicity and embryolethality in Swiss Webster mice [Hansen and Billings, 1985]. In this paper, we have compared the embryopathic effects of chronic and acute administrations of PHT to A/J mice. PHT was administered to pregnant females by intraperitoneal (i.p.) injection on day 10 of gestation at a dose of either 60 or 75 mg/kg body weight. Alternatively, PHT was added to ground chow and fed to animals prior to and throughout gestation; animals received a daily dose of either 60 or 75 mg/kg body weight. Pregnant animals were sacrificed on day 18 or 19 of gestation, and fetuses were examined for the presence of orofacial clefts and other anomalies. There was a significant increase in the frequency of cleft lip and palate in animals receiving the drug by i.p. administration, but there was no increase in the incidence of clefts if the drug were added to the diet. The results of this study reiterate the importance of the route of administration of a drug in determining its embryopathic effect.     

The relation between maternal restraint and food deprivation, plasma corticosterone, and induction of cleft palate in the offspring of mice.

The blood level of corticosterone was measured in mice following the injection on day 14 of pregnancy of a dose of corticosterone sufficient to cause a low frequency of cleft palate in the fetuses. This was compared with the blood levels present during maternal restraint and food deprivation that produced a similar frequency of cleft palate. The mean blood level over the 24 h following injection of corticosterone was 660 mug/100 ml, and during a similar period of restraint was 485 mug/100 ml. Other mice were subjected either to restraint or food deprivation for 24 h beginning day 14 of pregnancy, the plasma corticosterone levels measured during that time, and the frequency of cleft palate in late fetuses compared with the individual plasma corticosterone levels during treatment. There was a significant (P less than 0.025) correlation between high maternal corticosteroid levels and the frequency of cleft palate in the offspring of the restrained mice but not in the food-deprived animals. It is suggested that in some stressed mice endogenous plasma corticosterone can reach levels sufficient to account for the development of cleft palate.     

Inhibition of isotretinoin teratogenicity by acetylsalicylic acid pretreatment in mice.

Although isotretinoin (ITR) has been suggested to cause malformations via cytopathic effects on embryonic cells, the molecular mechanisms of ITR cytotoxicity in teratogenesis are not clear. Since ITR undergoes metabolism by prostaglandin synthase to a potentially cytotoxic peroxyl free radical, the possible role of prostaglandin synthase metabolism as a modulator of ITR teratogenicity was evaluated. Craniofacial and limb abnormalities were noted in fetuses on day 18.5 of gestation following administration of ITR to pregnant CD-1 mice in a three dose regimen of 100 mg/kg at 4 hr intervals on day 10.5 of gestation (plug day = day 0.5 of gestation). Mice were also treated with acetylsalicylic acid (ASA), an irreversible inhibitor of the cyclooxygenase component of prostaglandin synthase, at doses of 20 and 60 mg/kg body weight 2 hr prior to each ITR dose. ASA pretreatment of mice receiving ITR treatment showed a dose-dependent decrease in the overall incidence of malformations, number of defects per fetus, and the incidence of specific craniofacial and limb defects. Equivalent doses of ASA given to control mice did not cause malformations or alter the incidence of resorptions. These results demonstrate that ASA is able to ameliorate the teratogenic effects of ITR observed in fetal mice near term and indicate that prostaglandin metabolism could play a mechanistic role in ITR teratogenicity.     

Teratogenic effect of trifluoperazine in rats and mice

Trifluoperazine was administered to pregnant mice and rats by gastric intubation during the period of organogenesis. Dams were treated at doses of 0.5, 5, 50 (mice and rats) and 100 (only rats) mg/kg/day. The drug affected the pregnant weight increment in a dose-related manner in rats but only the 50 mg/kg/day dose level affected the weight gain of mice. The foetal weight was not markedly affected in either species. The drug induced cleft palate and micrognathia in rats but did not produce a teratogenic effect in mice. Trifluoperazine caused abortions in mice and there were significant increases in the number of resorptions with the top dose levels in both species.     

Pharmacological studies on the potentiation of phenytoin teratogenicity by acetaminophen

An in vivo murine model was developed to measure maternal phenytoin biotransformation along with the covalent binding of phenytoin to fetal tissues in the same fetuses which were assessed for fetal anomalies. Acetaminophen was administered to pregnant CD-1 mice 1 hour prior to phenytoin, both given i.p. at varying doses and gestational times between days 11 and 13. Dams were killed between days 12 and 19. Metabolites reflecting the enzymatic bioactivation of phenytoin were quantified in maternal plasma and urine with high-performance liquid chromatography (HPLC). Acetaminophen pretreatment caused a threefold increase in phenytoin-induced fetal cleft palates without increasing resorptions. The covalent binding of radiolabeled phenytoin to fetal and placental tissues measured on day 13 was increased twofold and threefold, respectively, by acetaminophen pretreatment. Phenytoin covalent binding measured on day 16 was significantly increased in the livers of fetuses with cleft palates, but not in the livers of dams with fetuses having cleft palates. Binding to fetal brain on day 16 was over fourfold higher than that in maternal brain. Acetaminophen pretreatment differentiated dams into poor and extensive metabolisers of phenytoin, with only the latter group carrying fetuses with cleft palates. The incidence of fetal cleft palates correlated positively with maternal urinary levels of phenytoin (r = +.81, P less than .01) and its dihydrodiol metabolite (r = +.61, 0.05 less than P less than .1), and negatively with levels of para-hydroxylated phenytoin (r = -.85, P less than .01). These findings related both to the mechanism of phenytoin teratogenicity and its potentiation by acetaminophen.     

Reduced weight in mice offspring after in utero exposure to 2450-MHz (CW) microwaves

Time-bred CD-1 mice (100) were sham-irradiated or irradiated with 2450-MHz (CW) microwaves at 28 mW/cm² for 100 minutes daily from the 6th through 17th day of gestation. The offspring were examined either as fetuses after hysterotomy on the 18th day of gestation or as naturally born neonates on the 1st and 7th day of age. Fetuses of half of the dams were examined on the 18th day of gestation. The incidence of pregnancy and the numbers of live, dead, resorbed, and total fetuses were similar in both groups. The mean weight was significantly lower (10%) in live microwave-irradiated fetuses, and ossification of sternal centers was significantly delayed. In the offspring that were born naturally, the mean weight of microwave-irradiated 7-day-old suckling mice was significantly lower (10%) than that of the sham-irradiated group. Survival rates of neonates in these two groups were not different. These data demonstrate that the decreased fetal weight seen in microwave-irradiated mice is retained at least 7 days after birth. Evidence from other published studies is presented to show that the retarded growth is persistent and might be interpreted as permanent stunting.     

Both Maternal Over- and Undernutrition During Gestation Increase the Adiposity of Young Adult Progeny in Rats

FIOROTTO, MARTA L, TERESA A DAVIS, PATRICIA SCHOKNECHT, HARRY J MERSMANN AND WILSON G POND. Both maternal over- and undernutrition during gestation increase the adiposity of young adult progeny in rats. ObesRes. 1995;3:131–141. We examined the influence of maternal diet during gestation on the growth and body composition of the progeny. On day 1 of gestation, rat dams were assigned to one of four feeding regimens: free access to standard rodent chow throughout gestation (AL); 20 g feed/day (prebreeding intake) throughout gestation (PB); 10 g feed/day from day 1 to day 14, then ad libitum from day 15 to parturition (RAL); 10 g feed/day from day 1 to 14, then 20 g/day to parturition (RPB). Progeny were fed ad libitum on standard chow diet from 3 to 12 weeks of age; food intake and weight gain were measured over this time. Body composition was measured at 12 weeks. The PB regimen restricted maternal food intake during the third trimester only; the RAL regimen restricted intake by 50% for two trimesters and produced hyperphagia in the third; the RPB regimen restricted intake by 50% for two trimesters, then intake (per unit body weight) was similar to that of AL dams during the third trimester. Litter size and progeny birth, weaning, and 12-week body weights were similar among the four groups. At 12 weeks of age, PB progeny had the highest body fat (per kg fat-free mass), despite similar feed intake during the 9-week postweaning period. The increased fat was proportionally distributed among intra-abdominal and subcutaneous depots. Progeny of RAL, AL, and RPB dams had similar amounts of body fat, but in RAL progeny more fat was present in intra-abdominal depots. The weights of fat-free mass, gastrointestinal tract and hindlimb skeletal muscles were unaffected by maternal diet Restriction of maternal feed intake during the third week of gestation had subtle effects on the body composition of young adult progeny that could not be explained on the basis of differences in postweaning voluntary feed intake.     

The effects of continuous exposure to 20-kHz sawtooth magnetic fields on the litters of CD-1 mice.

Mated CD-1 mice were exposed to 20-kHz sawtooth magnetic fields similar to those associated with video display terminals (VDT). Four groups of animals were continuously exposed from day 1 to day 18 of pregnancy to field strengths of 0, 3.6, 17, or 200 microT. There were no less than 185 mated dams in each exposure group. On day 18, the dams were sacrificed and assessed for weight gain and pregnancy. The litters were evaluated for numbers of implantations, fetal deaths and resorptions, gross external, visceral and skeletal malformations, and fetal weights. There were no less than 140 pregnant females in each group, and there were no significant differences between any of the exposure groups and the sham group (0 microT) for any of the end points. The results of this study do not support the hypothesis that the 20-kHz VLF magnetic fields associated with video display terminals are teratogenic in mammals.     

Alcohol and smokeless tobacco effects on the CD-1 mouse fetus.

Tobacco products and alcohol are commonly used as nonmedicinal drugs by pregnant women, and both are known to cause various effects on the fetus and the newborn. The objective of this study was to examine the fetal effects of both drugs when administered individually and simultaneously to pregnant CD-1 mice at moderate dosages. Specifically, we wanted to determine whether or not the effect on the fetus of these two biologically active substances was additive, ameliorative, or synergistic. A total of 65 CD-1 dams were divided among four groups receiving either ST equivalent to 8 mg/kg nicotine, ethanol (ETOH) 1.8 g/kg, a combination of ST+ETOH in the same dosages, or D-glucose (controls and ST alone) to supply calories equivalent to the dose of ethanol. Mice were dosed three times per day on gestational days 6-15. On gestational day 17 all dams were killed, fetal and placental weights recorded, and the number of resorbed, dead, and malformed fetuses noted. The mean maternal plasma drug levels were: nicotine-321 ng/ml and ethanol-0.105 g%. No significant differences were observed in maternal weight gain, litter size, or in the incidence of resorptions, deaths and/or malformations. Fetal weights were reduced in all three treatment groups (P less than 0.05), with the greatest reduction (13% decrease) recorded in the ST group, followed by a 9% decrease in the ETOH group, and a 7% decrease in the ST+ETOH group. Placentas of the ST group weighed significantly less (P less than 0.05) than controls. Ossification of the fetal skeleton, observed in ten sites, was affected to the greatest extent in the ST group, followed by the ETOH and ST+ETOH groups. Craniofacial measurements were significantly affected (P less than 0.05) in all three treatment groups, compared to controls. We conclude that under these experimental conditions, in terms of fetal growth and ossification, ST had the greatest effect, followed by ETOH and ST+ETOH. The interaction of ST+ETOH was neither additive, synergistic, nor ameliorative.     

Effects of preconceptional and gestational exposure to Tordon 202c on fetal growth and development in CD-1 mice

Female CD-1 mice were exposed to Tordon 202c (a picloram and 2,4-D combination herbicide) in the drinking water at concentrations of 0.21, 0.42, and 0.84% for 60 days prior to mating with untreated males. One-half of the pregnant females subsequently continued treatment throughout gestation while the remaining females were maintained on distilled water. Fetal weight, crown-rump length, placental weight, and maternal gestational weight gain were reduced in a dose-dependent manner following combined preconceptional and gestational exposure. The incidence of malformed fetuses (cleft palate, renal agenesis, hydronephrosis, unilateral testicular agenesis, and umbilical hernia) and fetuses with variants (especially incomplete ossification of the skeleton) were increased in a dose-dependent manner following combined exposure. Increased maternal mortality and decreased preconception weight gain were observed in the highest-dosage group. Relative maternal liver weight was increased in a dose-dependent manner. The results suggest that combined preconceptional and gestational exposure to Tordon 202c is required for teratogenesis and fetal growth depression. Preconceptional exposure alone is not effective in increasing the risk for embryotoxicity.     

Diazepam-induced cleft palate in the mouse and lack of correlation with the H-2 locus

Cleft palate frequencies were studied in AJ and SW mice following either 1- or 2-day dosing schedules with the anxiolytic drug diazepam (DAZ). In all cases, mice were food and water deprived for 24 and 48 hours in the 1- and 2-day dosing schedules, respectively. High cleft palate frequencies in control mice of both strains resulting from 48-hour food and water deprivation (on days 13.5 and 14.5 of gestation) were reduced in mice deprived for 24 hours, indicating a stress related effect. Two-day dosing with DAZ (400 mg/kg) produced a net increase in cleft palate frequency in SW (33%) and AJ (18%) mice. Mice treated only on day 13.5 had reduced control and DAZ cleft palate frequencies, neither of which were significant. Clefting was significant but reduced following 1-day dosing on day 13/20 of gestation (13 days 20 hours) in SW mice (18%), whereas no clefting was seen in the AJ strain. This strain difference was shown not to be related to differences in developmental timing. Production of cleft palate seen in AJ mice after 2 days of dosing may be indicative of an interaction of DAZ with the stresses resulting from food and water deprivation. Genes of the major histocompatibility locus, H-2, have been shown to regulate cleft palate formation following glucocorticoid and phenytoin administration to mice. Despite pharmacological similarities between DAZ and phenytoin, comparison of cleft palate frequencies following administration of DAZ to various strains of mice of different H-2 haplotypes indicated that genes associated with the H-2 locus do not regulate DAZ-induced cleft palate in these strains.     

Teratogenic effects of the plant hormone indole-3-acetic acid in mice and rats.

These studies evaluated the teratogenic potential of indole-3-acetic acid (IAA), a naturally occurring plant hormone, in CF-1 mice and Sprague-Dawley rats. Mice were given 5, 50, 200, or 500 mg IAA/kg/day by gavage on days 7 through 15 of gestation. Rats were given 50, 200, or 500 mg IAA/kg/day by gavage on days 7 through 15 of gestation. IAA was teratogenic in mice and rats at 500 mg/kg/day; cleft palate was induced in both species at this dose level. In mice, other malformations including exencephaly, ablepharia, dilated cerebral ventricles, and crooked tail were also observed. Mice given 500 mg/kg of IAA gained less than control mice during gestation; no evidence of maternal toxicity was observed in rats. IAA did not cause fetal resorptions in either species and was not teratogenic at dose levels below 500 mg/kg.     

Effects of tert-butyl acetate on maternal toxicity and embryo-fetal development in Sprague-Dawley rats

This study investigated the potential adverse effects of tert-butyl acetate (TBAc) on maternal toxicity and embryo-fetal development after maternal exposure of pregnant rats from gestational days 6 through 19. TBAc was administered to pregnant rats by gavage at 0, 400, 800, and 1,600 mg/kg/day. All dams were subjected to a Caesarean section on day 20 of gestation, and their fetuses were examined for any morphological abnormalities. At 1,600 mg/kg, maternal toxicity manifested as increases in the incidence of clinical signs and death, lower body weight gain and food intake, increases in the weights of adrenal glands and liver, and a decrease in thymus weight. Developmental toxicity included a decrease in fetal weight, an increase in the incidence of skeletal variation, and a delay in fetal ossification. At 800 mg/kg, only a minimal developmental toxicity, including an increase in the incidence of skeletal variation and a delay in fetal ossification, were observed. In contrast, no adverse maternal or developmental effects were observed at 400 mg/kg. These results show that a 14-day repeated oral dose of TBAc is embryotoxic at a maternally toxic dose (i.e., 1,600 mg/kg/day) and is minimally embryotoxic at a nonmaternally toxic dose (i.e., 800 mg/kg/day) in rats. However, no evidence for the teratogenicity of TBAc was noted in rats. It is concluded that the developmental findings observed in the present study are secondary effects to maternal toxicity. Under these experimental conditions, the no-observed-adverse-effect level of TBAc is considered to be 800 mg/kg/day for dams and 400 mg/kg/day for embryo-fetal development.     

The role of RXR-alpha in retinoic acid-induced cleft palate as assessed with the RXR-alpha knockout mouse

Treatment of pregnant mice with retinoic acid (RA) in mid-gestation produces cleft palate and limb defects in the fetuses. RXR-alpha has been previously shown to mediate the teratogenic effects of RA in the limb. In this study, we show that RXR-alpha is also involved in retinoid-induced palatal clefting. Treatment of RXR-alpha knockout mice with a teratogenic dose of RA on gestation day 11 or 12 induces cleft palate at a lower frequency than that seen in wild-type animals.