In vitro teratogenicity of ethylenethiourea in the rat.

We have demonstrated that ethylenethiourea (ETU) is a potent teratogen to the rat embryo developing in vitro. Sprague Dawley rat embryos were explanted on gestation day 10 and cultured for 48 hours in the presence of 40-200 micrograms/ml ETU. This resulted in a dose-related inhibition of growth and differentiation as assessed by crown-rump length, protein and DNA content, and somite number and in an increase in the frequency of abnormalities. A variety of anomalies was produced, including fluid accumulation in the brain (hydrocephalus), decreased mandibular size, decreased telencephalon size, abnormal dorsiflexion, as well as subectodermal blisters on the tail and limb buds and maxilla. Frank malformations have been observed at these same sites--hydrocephalus, brachygnathia, kyphosis, limb and tail defects, cleft palate--in the term fetus in vivo. The presence of abnormal fluid accumulation in the embryos--distended neural tube and subectodermal blisters--suggesting that the osmotic environment of the embryo had been altered by ETU exposure. Osmolality of the exocoelomic fluid (ECF) surrounding the embryo was measured after 48 hours of exposure to a concentration of ETU that caused nearly a 100% incidence of subectodermal blisters. ECF osmolality was found to be significantly lower than that of control embryos. Lowered osmolality would cause water to move out of the ECF, presumably causing the observed fluid accumulation in the embryo. It is speculated that altered osmotic balance and localized edema in the embryo are contributory steps in the formation of defects after ETU exposure.

Co-culture of rat embryos and hepatocytes: in vitro detection of a proteratogen.

The technique of whole embryo culture developed by New [Environ Health Perspect 18:105-110, 1976] provides a sensitive assay to evaluate the effects of a test chemical on embryo development independent of maternal influences. To detect proteratogens, this assay must be coupled with an exogenous metabolic activation system. We have developed methods for the co-cultivation of rat embryos with primary hepatocytes, which offers several advantages over subcellular fractions when providing metabolic activation for in vitro assays. In the present study, rat embryos removed from the dam on day 10 of pregnancy were co-cultivated in vitro with primary cultures of rat, rabbit, or hamster hepatocytes. Embryos co-cultivated with hepatocytes developed normally, as did embryos exposed to a test chemical, cyclophosphamide (CP) in the absence of hepatocytes. When embryos were co-cultivated with hepatocytes and exposed to CP, a dose-related embryotoxicity was observed, indicating metabolic activation of the proteratogen. Using hepatocytes isolated from rats pretreated in vivo with phenobarbital, we observed an increase in CP-induced malformations and embryotoxicity compared to those of embryos exposed to CP in the presence of uninduced hepatocytes. The teratogenic bioactivation of CP was inhibited in vitro by the addition of metyrapone. When similar numbers of hepatocytes were used for metabolic activation of CP the induced embryotoxicity was greater in the presence of rabbit and hamster hepatocytes than with rat hepatocytes. Development of procedures for the culture of rat embryos with hepatocytes from other species suggests the utility of this in vitro system for the investigation of species differences in sensitivity to chemical teratogens.

Studies on the mechanism of trypan blue teratogenicity in the rat developing in vivo and in vitro.

Trypan blue is a potent teratogen in vivo and in vitro in the rat. Many of the abnormalities produced by trypan blue--including swollen neural tube and pericardium, subectodermal blisters, hematomas, and generalized edema--may result from altered fluid balance in and around the embryo. The present study demonstrates relationships between changes in the fluid environment around the embryo and appearance of anomalies. Rat embryos were exposed in utero or in vitro to trypan blue during the early period of organogenesis. Both exposures resulted in defects that are typical of trypan blue treatment. Osmolality of exocoelomic fluid (ECF) was measured on gestation day 10 in vivo and day 12 in vitro, both after 48 hr of exposure to trypan blue. In both cases ECF osmolality was significantly lower than controls. This was correlated with the presence of edema-related anomalies in the embryo. On gestation day 11 in vivo, three days after maternal injection of trypan blue, ECF osmolalities were significantly higher than controls; however, there was tremendous variability in this parameter in day 11 treated embryos, and some had ECF osmolalities below the control range. Increased frequency of abnormalities was correlated with abnormal ECF osmolality, below and above the control range. Trypan blue probably exerts its teratogenic effects by disturbing the function of the visceral yolk sac. The movements of an amino acid and a monosaccharide across the visceral yolk sac were measured on gestation day 12 embryos in vitro. This aspect of yolk sac function was not altered by trypan blue exposure. Ultrastructure of the visceral yolk sac was observed after trypan blue exposure in vivo and in vitro. Endodermal cells in trypan blue-treated yolk sacs contained fewer large, electron dense lysosomes than controls. These were replaced by numerous small vacuoles, which may contain trypan blue. Trypan blue causes osmotic changes in the rat embryo in vivo and in vitro. These changes are correlated with embryonic malformations. Alterations in yolk sac ultrastructure indicate that trypan blue affects the function of this membrane.

Postnatal effects of hexachlorobenzene (HCB) on cardiac lactic dehydrogenase (LDH) and creatine kinase (CK) isozymes in CD-1 mice.

Pregnant CD-1 mice were treated with hexachlorobenzene (HCB) by gavage at doses of 0, 1, 10 and 50 mg HCB/kg body weight on days 6-17 of gestation and studied on day 1 or 21 postpartum (pp). Hearts of the dams and pups were assayed for lactic dehydrogenase (LDH) and creatine kinase (CK) activities and isozyme profiles. LDH and CK activities and CK isozyme profiles were not affected in the dams by HCB treatment, but isozyme LDH-5 was significantly depressed and isozyme LDH-3 significantly increased at the 50 mg/kg dose of HCB on day 1 pp. Hearts of the pups were studied on days 1, 8, 10, 15 and 21 pp. The 50 mg/kg dose level resulted in a statistically significantly increased mortality in the pups. The mortality at the other doses showed a dose-related effect. LDH and CK activities and CK isozyme profiles were not affected by HCB exposure in utero. The only isozyme affected was LDH-5 with a statistically significant increase in the 50 mg/kg dose group on day 21 pp.

Further development of rodent whole embryo culture: solvent toxicity and water insoluble compound delivery system.

In order to study the in vitro embryotoxicity and dysmorphogenesis of water insoluble compounds, solvents or chemical delivery systems of low toxicity and teratogenicity to the developing embryo must be found. Therefore, day 10.5 rat embryos were cultured for 2 days in whole rat serum containing 0.1, 0.5 and 2.5 vol.% of ethyl alcohol, dimethylsulfoxide, acetone, Tween 80, corn oil and 10% acetone/90% corn oil. No adverse effects occurred with any of the solvents at the 0.1% concentration level. At 0.5% ethyl alcohol and Tween 80 significantly reduced embryonic growth and increased the incidence of embryonic abnormalities. With the exception of corn oil and acetone/corn oil, embryos cultured in media containing 2.5% of various solvents failed to grow, did not differentiate, and died during the culture period. Corn oil suspended in rat serum by use of ultrasound was non-toxic even at concentrations of 2.5% and 10%. Growth parameters of embryos cultured in serum containing corn oil were indistinguishable from controls and overall morphogenesis was good (particularly at 2.5%). The order of increasing embryotoxicity and dysmorphogenesis of the studied liquids was corn oil less than acetone/corn oil less than dimethylsulfoxide less than ethyl alcohol, acetone less than Tween 80. Any of the 4 water miscible solvents (at 0.1%) or a sonicated suspension of corn oil in serum (up to 2.5%) met the criteria of a non-toxic and non-teratogenic water insoluble compound delivery system for in vitro embryo culture.

In vitro study of embryotoxic and dysmorphogenic effects of mercuric chloride and methylmercury chloride in the rat.

Rat-embryo explants removed on day 10.5 of gestation were cultured for 48 hr in various concentrations of HgCl2, CH3HgCl or glutathione. Dose-related dysmorphogenesis and growth retardation occurred with increasing concentrations of HgCl2 (1-10 microM). Increasing concentrations of CH3HgCl (3-100 microM) produced a similar pattern of embryonic effects. The rat-serum incubation medium had no detectable level of reduced glutathione (less than 0.02 mM) and only 0.12 mM total sulphydryl groups. Reduced glutathione (60-300 microM) added to the incubation medium was relatively non-toxic. The addition of exogenous glutathione to culture medium containing HgCl2 partially antagonized the embryonic growth retardation and prevented most of the embryolethality observed in cultures to which only HgCl2 had been added.

Combined use of a water-insoluble chemical delivery system and a metabolic activation system in whole embryo culture.

An integrated water-insoluble chemical delivery/metabolic activation/rat embryo culture system is described. Increasing concentrations of diallate dissolved in corn oil caused dose-related embryonic growth retardation in vitro. The presence of a metabolic activating system did not potentiate the effects of diallate in corn oil. The rat hepatic microsomal monooxygenase system was enzymatically active in the presence of a high concentration of corn oil in the rat serum incubation media. Thus, the failure of the metabolic activation system to potentiate the toxicity of diallate in corn oil is not due to an enzymatic deficiency of the hepatic metabolic activation system. As high concentrations of some organic solvents are known to act directly as embryonic poisons in vitro, rat embryos were cultured in incubation media that contained chloroform (0.5 or 2.5%) dissolved in corn oil. This treatment was found to be severely toxic to embryonic growth and differentiation in vitro. Thus, unlike diallate, the embryonic toxicity of chloroform was fully evident when dissolved in the solvent corn oil. Diallate dissolved in acetone retarded embryo growth and caused dysmorphogenesis. When a metabolic activation system and either corn oil or acetone were used together, the incidence of embryolethality and abnormalities were so high that compound treatment effects were difficult to evaluate.

Studies of saccharin and cyclohexylamine in a coupled microsomal activating/embryo culture system.

Rat embryos undergoing organogenesis (day 10.5 of pregnancy) were exposed to 1.0 mM-saccharin or 0.1, 0.3 or 1.0 mM-cyclohexylamine in an in vitro metabolic activating/embryo culture system. Either in the absence or presence of a metabolic activating system, 1.0 mM-saccharin had few adverse effects on yolk-sac growth or embryo growth or differentiation. However, rat embryos cultured in vitro with cyclohexylamine and an adult rat hepatic microsomal activating system showed more growth retardation and dysmorphogenicity than embryos exposed to the same concentrations of cyclohexylamine alone. A concentration of 1.0 mM-cyclohexylamine alone reduced yolk-sac DNA content (by 51%), embryo DNA content (69%) and crown-rump length (26%). In the presence of a hepatic microsomal activating system this same dose of cyclohexylamine decreased yolk-sac DNA (60%), embryo DNA (84%) and crown-rump length (49%). The effects of the metabolic activating system and of cyclohexylamine were additive. Other than decreasing final embryo DNA content, exposure to 0.1 and 0.3 mM-cyclohexylamine did not produce any deleterious effects on in vitro rat embryo growth or differentiation. Thus a substantial concentration of saccharin (1.0 mM) or cyclohexylamine (0.3 mM) in vitro did not elicit markedly deleterious effects on rat organogenesis, a result in keeping with published in vivo findings.

Maternal hepatic effects of 1,2,4,5-tetrachlorobenzene in the rat.

1,2,4,5-Tetrachlorobenzene (TCB) is an industrial intermediate used in the production of 2,4,5-trichlorophenoxyacetic acid. This herbicide contains trace quantities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Because of possible maternal hepatic or reproductive effects of this uncharged, low-molecular weight, lipophilic chlorinated benzene 0, 30, 100, 300, and 1000 mg/kg/day of TCB was orally administered to rats on Days 9, 10, 11, 12, and 13 of gestation and the animals were sacrificed on Day 14 of pregnancy. No maternal deaths were recorded and body weight gain was significantly decreased only in the 1000 mg/kg/day group. Maternal liver weight, liver to body weight ratio, and hepatic microsomal protein content were unaffected by TCB treatment. Although Day 14 NADPH-cytochrome c reductase activity was not affected, the maternal hepatic microsomal cytochrome P-450 content was significantly increased by administration of 1000 mg/kg/day of TCB. Microsomal N-demethylation of aminopyrine was slightly increased from 2.4 to 3.4 and 3.5 nmole/mg protein/min at doses of 300 and 1000 mg/kg TCB. However, maternal hepatic microsomal ethoxyresorufin O-deethylase activity was greatly increased from 14 to 30, 40, 50, and 49 pmole/mg protein/min in pregnant rats administered 0, 30, 100, 300, and 1000 mg/kg/day TCB. The microsomal rates of p-nitrophenol and phenolphthalein glucuronidation in vitro were not increased by TCB administration. The maternal hepatic microsomal enzyme induction observed after TCB administration to pregnant rats suggests the presence both cytochrome P-450 and P-448 inducers in the sample of 1,2,4,5-tetrachlorobenzene used.

Maternal hepatic and embryonic effects of 1,2,4-trichlorobenzene in the rat.

The possible maternal hepatic and reproductive effects of 1,2,4-trichlorobenzene (TCB) were assessed in rats given 0, 36, 120, 360, and 1200 mg/kg/day of TCB on Days 9-13 of gestation. The animals were sacrificed on Day 14 of pregnancy. Maternal deaths (2/9 rats, 6/6 rats) were recorded in the 360 and 1200 mg/kg/day treatment groups and body weight gain was significantly decreased in the 360 mg/kg/day TCB group. Maternal liver weight, liver/body weight ratio, and hepatic microsomal protein content were unaffected by TCB treatment. Although Day 14 NADPH-cytochrome c reductase activity was affected only at 360 mg/kg/day TCB, the maternal hepatic microsomal cytochrome P-450 content was significantly increased by administration of both 120 and 360 mg/kg/day of TCB. Hepatic microsomal aminopyrine N-demethylase, ethoxyresorufin O-deethylase, and UDP-glucuronyl transferase activity towards p-nitrophenol were also increased at 120 and 360 mg/kg TCB. Glutathione S-transferase activity to 1-chloro-2,4-dinitrobenzene and 1,2 dichloro-4-nitrobenzene were both increased by pretreatment with TCB. Although pretreatment with 360 mg/kg/day TCB did not increase resorptions, embryolethality, or teratogenicity, embryonic development was significantly retarded by all four growth criteria used (head length, crown-rump length, somite number, and protein content).

Maternal hepatic and embryonic effects of 1,2,3,4-tetrachlorobenzene in the rat.

To assess possible maternal hepatic and reproductive effects of this uncharged, low molecular weight, lipophilic chlorinated benzene 0, 100, 300 and 1000 mg/kg/day of 1,2,3,4-tetrachlorobenzene (TCB) was orally administered to pregnant rats on days 9-13 of gestation and the animals were killed on day 14 of pregnancy. Phenobarbital and beta-naphthoflavone were administered to other pregnant rats as positive hepatic controls. Maternal mortality (7/19 rats) was increased and body weight gain was greatly decreased in the 1000 mg/kg/day TCB group. Liver to body weight ratio and hepatic microsomal protein content were unaffected by any TCB treatment. On day 14 maternal NADPH-cytochrome c reductase activity was increased at 1000 mg/kg/day, while the maternal hepatic microsomal cytochrome P-450 content was significantly induced by both 300 and 1000 mg/kg/day of TCB. Microsomal N-demethylation of aminopyrine was increased from 2.6 to 4.0 and 4.5 nmol/mg protein/min at doses of 300 and 1000 mg/kg TCB, respectively. However, maternal hepatic microsomal ethoxyresorufin O-deethylase activity was not consistently increased by TCB. Hepatic glutathione S-transferase activity towards 1,2-dichloro-4-nitrobenzene was increased only by the 1000 mg/kg/day TCB treatment. The rate of microsomal p-nitrophenol and phenolphthalein glucuronidation was increased by TCB administration. Embryonic growth was adversely affected by TCB treatment. Yolk sac diameter, embryonic crown-rump length, and head length were all decreased by treatment with 300 mg/kg/day TCB. This TCB treatment did not significantly elevate the number of dead or abnormal embryos.

2,4,5-T effects on cardiac and serum lactic dehydrogenase (LDH) and creatine kinase (CK) isozymes. II. Neonatal enzyme activities and isozyme profiles.

Neonates from CD-1 mice, which were treated during gestation with 100 mg/kg of 2,4,5-T, were studied from day 1 to 30 postpartum (pp) for effects on cardiac development by determining total cardiac activities and isozyme profiles of LDH and CK. On day 1pp, the total activities and isozyme profiles of LDH of the neonatal hearts were normal. During the developmental period of day 7 through 15, changes were noted in the developmental pattern of LDH isozymes some of which continued to day 30. The CK isozyme profile on day 1pp showed a significant change and changes continued throughout the lactational period. During this period, the normal developmental isozyme patterns of LDH and CK were altered by prenatal exposure to 2,4,5-T suggesting metabolic derangement or pathological changes in the neonatal heart.

2,4,5-T effects on cardiac and serum lactic dehydrogenase (LDH) and creatine kinase (CK) isozymes. I. Maternal enzyme activities and isozyme profiles.

Pregnant mice were exposed to 100 mg/kg 2,4,5-T, IG, on gestation days 6 through 17, and sacrificed on postpartum day 1 or 21. Toxicologic evaluation showed no changes in body weight, heart weight, heart to body weight ratio, or cardiac supernate protein between corn oil control and 2,4,5-T treated mice on days 1 or 21 postpartum (pp). There were no effects of 2,4,5-T treatment on the total LDH enzyme activity of cardiac supernate on day 1 or 21pp. Serum LDH total activity was depressed on day 1pp and comparable to control values on day 21pp. Cardiac CK total activity was elevated on day 1pp, but not on day 21pp. Serum CK total activity on day 1 was comparable to control values; however, on day 21, a significant decrease in activity was observed. Cardiac LDH and CK isozyme profiles were normal on days 1 and 21pp. The serum LDH isozyme profile was normal at both times. The serum CK isozyme profile on Day 1 was markedly altered by athe appearance of two aberrant isozyme bands while on day 21, there was a profile shift with an increase in the BB band and a compensatory decrease in the MM band. These changes in creatine kinase suggest metabolic or pathologic changes in the cardiac muscle.

Isozyme profiles of lactic dehydrogenase and creatine phosphokinase in neonatal mouse hearts.

Isozyme profiles of lactic dehydrogenase (LDH) and creatine phosphokinase (CPK) were determined in cardiac tissue of mice during postnatal development. LDH isozymes 1 and 5 showed a definite developmental change, achieving the adult values by 20 days of age, while the other three isozymes showed no change. During the first 2 postnatal weeks, a developmental change was seen in the activities of the 'mito' and MB isozymes of CPK, with the adult profile being established by day 20. CPK isozymes MM and BB did not change during this interval.