Bernard A. Becker

Teratogenicity, fetal toxicity, and placental transfer of lead nitrate in rats☆

Abstract Single doses of 25–70 mg/kg of lead nitrate were administered iv to pregnant rats on Days 8–17 of gestation (Day 1 = sperm found). Malformations were produced with lead when administered on Day 9 of gestation, producing a urorectocaudal syndrome of malformations. Lead nitrate was increasingly embryo- and fetotoxic when administered on later days of gestation (Days 10–15) but not teratogenic. Hydrocephalus and hemorrhage of the central nervous system were produced on Day 16 of gestation. On Day 16 and therafter, fetal toxicity (resorptions) sharply declined. Postnatal survival of newborn exposed in utero to lead on Day 9 of gestation was very poor. Significant quantities of lead were transferred into the fetus; however, the placenta appeared to greatly limit the passage of lead since large maternal-fetal concentration gradients existed.

Diphenylhydantoin teratogenicity in rats

Diphenylhydantoin (DPH) was found to be teratogenic in Sprague-Dawley (Simonsen) rats. The rat is less susceptible than the mouse to the teratogenic actions of DPH, but DPH-induced in utero deaths and inhibition of fetal growth are similar in both species. Single ip injections of DPH into time-dated (mating equals day 0) pregnant rats produced statistically significant (P < 0.05) increases in in utero deaths. A dose-effect relationship was seen following treatment on gestational days 8, 9, 11 and 12. The lowest degree of in utero mortality was seen after treatment on days 7, 10 and 13 resulting in a biphasic mortality curve. Fetal rat body weights were significantly reduced in a dose-related manner by treatment on days 7 throught 15. Single injections of DPH produced significant incidences of hydrocephalus and hydronephrosis. A significant incidence of cleft palate was produced by administration of 150 mg/kg of DPH on gestational days 10 throught 15. Other anomalies were hydrocephalus, hydronephrosis, peritoneal hemorrhage and renal hemorrhage. The effect of DPH on fetal mouse and rat long bone length was similar; fetal long bones of the appendicular skeleton were shortened in a day- and dose-dependent fashion.

Teratogenic effects of diphenylhydantoin in swiss-webster and a/j mice.

Summary Diphenylhydantoin, 50 mg/kg, s.c., was administered to primigravid Swiss-Webster and A/J mice during early (days 7, 8, 9) and late (days 11, 12, 13) embryogenesis. Litters were delivered by caesarean section on day 18 of gestation, weighed, examined, and measured. Resorption rates were not altered in either strain treated during early embryogenesis. The A/J, but not Swiss-Webster, mice treated during late embryogenesis had a significant (p<0.05) increase in resorption. Diphenylhydantoin resulted in reduced fetal weight in both strains when given during late embryogenesis. Fetal long bone length, measured in alizarin red stained fetuses, was shortened only in late treated Swiss-Webster mice. The incidence of cleft palate was significantly elevated in late, but not early, treated progeny of both strains. Cleft palate with cleft lip occurred only in A/J mice; the rate was not different from vehicle treated controls. No other soft tissue malformations were found in either strain following Bouins fixation and freehand cross-section examination.

Teratogenicity of oral diazepam and diphenylhydantoin in mice

Abstract Diazepam (DAP) and diphenylhydantoin (DPH) have anticonvulsant activity and structural similarities. Diphenylhydantoin is teratogenic in the mouse. The effects upon the offspring of mice treated orally with DAP or DPH during pregnancy were studied. DPH, 45, 87.5, or 125 mg/kg, and equimolar doses of DAP, 50, 100, or 140 mg/kg and DAP, 500 mg/kg, were administered orally once daily for 3 days on gestation days 8–10 or days 11–13; or for 1 day only between days 8 and 15 (vaginal plug = day 1). Diazepam, 280 or 400 mg/kg, were administered once only between days 11 and 14. All drugs were suspended in sodium carboxymethylcellulose (CMC 0.5%). The animals were sacrificed on day 19 and the fetuses were weighed and examined. Significant (p ≤0.05) increases of cleft palates over CMC controls were found in offspring following 3-day maternal administration of DAP, 140 mg/kg and DPH, 87.5 or 125 mg/kg on days 11 to 13; and single-day administration of DAP 400 mg/kg on days 11 to 14; and of DAP, 500 mg/kg and DPH, 125 mg/kg on days 11 to 15. There was a greater incidence of significant increases of resorptions and significantly depressed fetal body weights following single-day administration of DAP, 500 mg/kg than after all other treatments. In conclusion, oral DPH and oral DAP, at a higher dosage level, produce a similar teratogenic effect, cleft palate, in the Swiss-Webster mouse.

Effects of organolead compounds on rat embryonic and fetal development

Abstract Tetraethyl lead (TEL) and tetramethyl lead (TML) and trimethyl lead chloride (TriML), were found to be essentially nonteratogenic in Sprague-Dawley rats. Oral doses of TEL (7.5, 15, or 30 mg/kg), TML (40, 80, 112, or 160 mg/kg) and TriML (15, 30, or 38 mg/kg) were administered as 3 divided doses during early organogenesis (days 9, 10, and 11) or late organogenesis (days 12, 13, and 14), the day of positive sperm being day 1. In addition, TriML was administered iv at doses of 20, 28, 33, or 40 mg/kg on individual gestation days 8 through 15 inclusive. The highest dose of each compound in each experiment was lethal to the maternal animal. Lower dose levels produced typical slight to severe maternal organolead toxicity, dependent upon dose. Embryo or fetal toxicity was observed to accompany the administration of the organolead compounds and was characterized by growth retardation and delayed ossification of bone. Marked fetal effects were observed only in maternal animals that exhibited severe organolead toxicity and were, therefore, severely debilitated. Infusion studies with TriML revealed that the rate of placental transfer was minimal when blood concentrations were below an apparent saturation of binding sites on maternal erythrocytes and was greatly increased at maternal blood concentrations above this saturation point. When the maternal animal was by-passed by direct intra-amniotic injections of TriML (10–100 μg/fetus) dose-related fetal lethality was observed.

Quantitative and temporal delineation of various parameters of liver dysfunction due to α-naphthylisothiocyanate

Abstract α-Naphthylisothiocyanate (ANIT) has been found to rapidly produce liver dysfunction, viz., elevation in plasma bilirubin, sulfobromophthalein (BSP) retention, and prolongation of pentobarbital-induced loss of the righting reflex, within a relatively short time following a single oral administration of this hepatotoxic agent to mice. The 24-hour LD50 was 245 mg/kg. The ED50 for hyperbilirubinemia was 40 mg/kg; the ET50 was 21 minutes. The increase in plasma bilirubin was mainly of conjugated bilirubin. The ED50 for BSP retention was 38 mg/kg; the ET50 was 102 minutes. The metabolized form of BSP was present in the blood stream. The ED50 of ANIT for the prolongation of pentobarbital-induced loss of the righting reflex was 43 mg/kg, 2 hours after ANIT. Single doses of ANIT effected persistent liver dysfunction. Hyperbilirubinemia did not subside until 4 days after treatment. BSP retention was significant for 7 days and, in occasional animals, for 26 days. Prolongation of pentobarbital hypnosis was seen for 5–12 days after ANIT. The cholestatic property of ANIT was demonstrated by a direct and an indirect method. The ET50 was 16 hours by either method. On the basis of these studies, ANIT is believed to effect liver dysfunction by a change in hepatocyte activity as well as the previously reported delayed action on bile ductules.

The nature of α-naphthylisothiocyanate-induced cholestasis

Abstract Cholestasis was established in mice by the administration of a single oral dosage of 150 mg of α-naphthylisothiocyanate per kilogram and compared to extrahepatic cholestasis effected by surgical occlusion of the common bile duct. Plasma levels of conjugated and unconjugated bilirubin, and of intravenously injected sulfobromophthalein and its metabolic product, were measured as indicators of the functional integrity of the liver of the two preparations. Equal degrees of hyperbilirubinemia and sulfobromophthalein retention were achieved by the two treatments. However, the plasma of α-naphthylisothiocyanate-treated mice contained increased levels of the metabolic products of bilirubin and sulfobromophthalein. The unconjugated form of bilirubin was elevated in animals with surgically occluded bile ducts. The onset of hyperbilirubinemia and bilirubinuria was more rapid following the drug treatment. Therefore, α-naphthylisothiocyanate-induced cholestasis differs from extrahepatic cholestasis in the following ways: (1) the onset of stasis as judged by the appearance of hyperbilirubinemia is more rapid; (2) the increase in plasma bilirubin represents an increase solely in conjugated bilirubin; (3) a higher proportion of intravenously injected sulfobromophthalein appears in the altered form; and (4) plasma levels of intravenously injected sulfobromophthalein decrease more rapidly.

Effects of phenobarbital or SKF 525A pretreatment on diphenylhydantoin disposition in pregnant mice

Abstract Teratogenicity of diphenylhydantoin (DPH) in Swiss-Webster mice and alteration of such teratogenicity by pretreatment of mice with phenobarbital or SKF 525A have been demonstrated. The present work reports the effects of phenobarbital (60 mg/kg/day for 3 days) or SKF 525A (40 mg/kg, twice on day prior and once 1 hr before DPH) on DPH metabolism, distribution, and excretion in pregnant Swiss-Webster mice and concentrations in fetuses. Neither pretreatment affected uptake of DPH-4-14C, 100 mg/kg, 20 μCi/kg, from ip injection sites. Phenobarbital pretreatment enhanced, while SKF 525A decreased, DPH metabolism, excretion, and plasma disappearance. Phenobarbital increased DPH hepatic uptake during the absorption period but subsequently decreased the hepatic storage; SKF 525A increased hepatic storage of DPH. In general, phenobarbital decreased, while SKF 525A increased, DPH concentrations in maternal fat, brain, and muscle. SKF 525A increased, and phenobarbital decreased, DPH concentrations in placenta. In the fetus, phenobarbital pretreatment resulted in lower concentrations and faster removal of DPH; SKF 525A effected high concentrations and more prolonged removal. The DPH found in the fetus after phenobarbital was mainly unbound, unmetabolized DPH (77%), but unbound, metabolized DPH (4%) was significantly greater than control (2%). SKF 525A pretreatment did not significantly alter the portion of total, metabolized, or unmetabolized DPH in the fetus.

Placental transfer, embryotoxicity, and teratogenicity of thallium sulfate in normal and potassium-deficient rats.

Abstract Thallium sulfate, a known teratogen in chicks, was administered ip to groups of pregnant Sprague-Dawley rats on days, 8,9,10 (2.5 mg/kg) or on days 12, 13, 14 (2.5 or 10.0 mg/kg) of gestation. Rats were fed either normal diet, 1 or low-potassium diet 2 from the beginning of gestation (day 0). Fetuses were delivered by cesarean section on day 21, weighed, examined, and measured. One half of each litter was examined for soft tissue anomalies, and the remainder was examined for skeletal anomalies. Low potassium diet alone significantly ( P 2 SO 4 ) at each dose and stage of gestation studied significantly reduced fetal body weight, produced hydronephrosis and nonossification of vertebral bodies, but did not increase resorption rate. Combined Tl 2 SO 4 and low-potassium treatment did not result in more severe teratogenicity than either alone. Other groups of pregnant rats fed as above were anesthetized with pentobarbital on day 20 of gestation, and the femoral vein and artery were cannulated for infusion of Tl 2 SO 4 and blood sampling. 204 Tl 2 SO 4 was infused at doses of 0.2, 0.4, 0.8, 1.6, 3.2, or 6.4 mg/min/kg, and maternal erythrocytes, plasma, and whole fetuses were sampled at 2, 4, 8, 16, 32, and 64 min after infusion was begun. Placental transfer of Tl 2 SO 4 in day 20 pregnant rats was restricted in both normal-fed and low potassium-fed rats; 32 min after beginning Tl 2 SO 4 infusion maternal blood levels were 15 times higher than fetal levels on a weight basis for the 0.2 mg/min/kg dose to 30 times higher for the 6.4 mg/min/kg dose. Maternal erythrocyte sequestering did not account for the limited placental passage of thallium. Limited placental passage of thallium may account for the lower degree of teratogenicity in rats than chicks.

Hepatotoxicity of α-naphthylisothiocyanate congeners with particular emphasis on phenylisothiocyanate☆

Abstract A structure-action study of compounds related to the hepatotoxic agent, α-naphthylisothiocyanate indicates that an aryl compound of planar configuration with a substituent radical containing N, C, and S are the probable necessary conditions for production of hyperbilirubinemia. Of sixteen congeners, only one, phenylisothiocyanate, was found to exhibit this action. Phenylisothiocyanate qualitatively and quantitatively affects liver function in mice in the same manner as α-naphthylisothiocyanate, as shown by increases in plasma bilirubin and sulfobromophthalein retention. The effects of a single oral treatment with phenylisothiocyanate persist for about 10 days. This action is similar to that of α-naphthylisothiocyanate. Effects due to phenylisothiocyanate treatment develop more slowly than effects due to α-naphthylisothiocyanate treatment.