Jeanne M. Manson

The heart and diaphragm: target organs in the neonatal death induced by nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether).

This paper examines the effects of in utero exposure to 2,4-dichlorophenyl-p-nitrophenyl ether (nitrofen) on the viability of neonatal Long-Evans rats. Oral administration of this herbicide on days 8-18 of gestation reduced neonatal survival and birth weight. Day 11 of gestation was the most sensitive day for induction of neonatal mortality; 116 mg/kg to the dam on this day was the LD50 for the neonate. An increased incidence of hydronephrosis was observed in 35-day survivors. This increase was dose-related in animals exposed on day 11 of gestation. Fetuses exposed on day 11 and examined at term had reduced weights, delayed skeletal ossification, and an increased frequency of hydronephrosis and diaphragmatic hernias. While nitrofen did cause a high incidence of hydronephrosis, BUN or creatinine levels in 4-h neonates were not elevated. Detailed examination of the hearts of term fetuses revealed cardiac malformations classified as ventricular septal defect, double outlet right ventricle, and transposition of the great vessels. We conclude from these studies that the heart and the diaphragm are the target organs in nitrofen-induced neonatal death.

Evaluation of teratogenicity and behavioral toxicity with inhalation exposure of maternal rats to trichloroethylene

Female rats were exposed by inhalation to trichloroethylene (TCE) vapors at a concentration of 1800 +/- 200 ppm to determine whether exposure before mating and during pregnancy is more detrimental to reproductive outcome than exposure either before mating alone or during pregnancy alone. Four treatment groups were utilized in a two by two factorial design: exposure to TCE for 2 weeks before mating and during the first 20 days of pregnancy; TCE before mating and filtered air during pregnancy; filtered air before mating and TCE during pregnancy; and filtered air before and during pregnancy. Significant elevations in skeletal and soft tissue anomalies, indicative of developmental delay in maturation rather than teratogenesis, were observed in the group exposed during pregnancy alone. The mixed function oxidase enzymes, ethoxycoumarin and ethoxyresorufin, indicative of cytochrome P-450 and P-448 activities, respectively, were measured in maternal and fetal livers, as well as livers of non-pregnant females, and showed variable levels of activity not uniformly related to treatment or pregnancy. Behavioral evaluation of offspring indicated a lack of treatment effect in tests of general activity levels at 10, 20 and 100 days of age. However, a reduction in postnatal body weights was seen in offspring from mothers with pregestational exposure. No results indicative of treatment-related maternal toxicity, embryotoxicity, severe teratogenicity or significant behavioral deficits were obtained in any of the treatment groups.

Effects of the herbicide 2,4-dichlorophenyl-p-nitrophenyl ether (nitrofen) on fetal lung developments in rats

Abstract We have examined whether the neonatal mortality accompanied by lung hypoplasia and atelectasis induced by prenatal exposure to the diphenyl ether herbicide 2,4-dichlorophenyl-p-nitrophenyl ether (nitrofen) could be due to alterations in pulmonary surfactant synthesis. The specific hypothesis we tested was whether in utero exposure to nitrofen accelerated the catabolic metabolsim of glucocorticoids by induction of mixed function oxidase enzymes, leading to a depression in glucocorticoid levels and delay in fetal lung surfactant synthesis. Hepatic aminopyrine-n-demethylase and aniline hydroxylase activities in the 9000 g supernatant were not elevated in rat dams exposed to 50 mg/kg/day on days 8–18 of pregnancy. Cortisone and corticosterone levels in fetal plasma were not altered by nitrofen exposure. Fetal lung surfactant synthesis was also not affected, as indicated by measurements of total lung phospholipids, [14C]choline uptake into lung lipids, and cholinephosphotransferase (CPT) activity. Surface tension values from saline extracts of fetal lungs and fetal plasma corticosterone levels were monotored after co-administration of nitrofen and dexamethasone. Nitrofen exposure alone had no influence on these parameters, while dexamethasone, with or without nitrofen, depressed corticosterone levels and minimum surface tension measurements. These results suggest that the level of glucocorticoids and capacity of the fetal lung to respond to glucocorticoids by synthesizing and releasing surfactant are not affected by prenatal exposure to nitrofen.

Absence of dichloromethane teratogenicity with inhalation exposure in rats

Abstract Female rats were exposed by inhalation to dichloromethane (DCM) at a concentration of 4500 ppm to determine whether exposure before and during gestation is more detrimental to reproductive outcome than exposure either before or during gestation alone. Four treatment groups were utilized in a two by two factorial design: exposure to DCM for 3 weeks before and during the first 17 days of gestation; DCM before and filtered air during gestation; filtered air before and DCM during gestation; and filtered air before and during gestation. Maternal liver weights were increased and fetal body weights were decreased in the two groups exposed to DCM during gestation compared to those exposed to filtered air during gestation. The incidence of gross external, skeletal, or soft-tissue anomalies was not significantly increased in fetuses in any group. Exposure to DCM both before and during gestation resulted in the same low degree of maternal and embryotoxicity as exposure during gestation alone.

Behavioral toxicity in the offspring of rats following maternal exposure to dichloromethane

Abstract Rats divided in four treatment groups were exposed to dichloromethane (DCM) (4500 ppm) or filtered air before and/or during gestation in order to assess the occurrence and extent of toxic effects on developing offspring. The progeny of dams exposed to DCM either prior to and/or during gestation exhibited altered rates of behavioral habituation to novel environments. No simple relationship between exposure period and behavioral outcome was observed. Each of the treatment groups showed effects as a function of age at testing and the behavioral task used. Treatment effects were detectable in offspring as early as 10 days of age and were still demonstrable in 150-day-old male rats. Treatment effects were observed in rats of both sexes in preweaning tests but were not seen in adult female rats. No effects of subacute DCM exposure were evident in growth rate, long-term food and water consumption, wheel running activity, or avoidance learning. This study, which should be viewed as preliminary, is cf interest since altered rates of habituation to novel environments were observed in the absence of overt maternal toxicity, or teratogenicity. The effects cannot be definitely attributed to a direct effect of DCM since elevated maternal carboxyhemoglobin (COHb)- or DCM-induced changes in maternal-litter interactions could have been contributing factors. The findings do suggest that the functional development of progency of DCM-exposed dams should be further investigated.

Evaluation of teratogenicity and neurotoxicity with maternal inhalation exposure to methyl chloroform

Female Long‐Evans rats were exposed by inhalation of 2100 ±. 200 ppm methyl chlorofrom (MC) to determine whether exposure before mating and during pregnancy was more detrimental to the offspring than exposure either before mating or during pregnancy alone. Four groups were exposed for 2 wk before mating and through d 20 of gestation in a 2 X 2 factorial design: (1) MC exposure before and during pregnancy, (2) MC before mating alone, (3) MC during pregnancy alone, and (4) filtered air before and during pregnancy. A t term, half of each group were sacrificed and assessed for maternal toxicity, embryotoxicity, and teratogenicity; the other half delivered their young for later behavioral evaluation and examination for gross lesions. No significant differences were found in measurements of maternal toxicity or embryotoxicity except for a decrease in fetal body weight when dams were exposed during pregnancy alone. Significantly increased incidences of skeletal and soft tissue variations were seen in fetuses fro...

Studies of DNA damage and cell death in embryonic limb buds induced by teratogenic exposure to cyclophosphamide.

Many teratologic investigations have shown that certain types of chemical insults to the embryo (those altering replication, transcription, and translation) can cause excessive cell death in tissues destined to become malformed. Chemical carcinogens also induce cell death in target tissues, but the critical event is believed to be heritable alteration in the DNA of surviving cells. In the present study, an attempt was made to study the interaction between cell death and DNA damage in the initiation of birth defects. The pattern of DNA damage induced by cyclophosphamide was examined at time intervals before, during, and after the necrotic episode in mouse embryo limb buds. The alkaline elution assay was used to measure alkali-labile sites in single-strand DNA due to its adaptability to small tissue samples. An ip dose of 20 mg/kg of cyclophosphamide induced forelimb malformations in 85% of surviving mouse fetuses and 30% embryolethality when administered at 9 am on Day 11. As early as 5 hr after exposure, a slight excess of necrosis was observed in treated limbs by light microscopy, while at 24 hr, massive necrosis was evident. By 48 and 72 hr, excess necrosis was not observed in treated limbs. When alkaline elution analysis was conducted at prenecrotic (1-, and 5-hr), necrotic (24-hr), and postnecrotic (48-, and 72-hr) intervals, a trend toward increasing DNA damage in treated limbs with time was observed. The greatest differences in elution values occurred during the postnecrotic period. Although mean retention values were not significantly different, significantly increased variance was obtained in retention values of treated limbs at all time intervals other than 1 hr. This may reflect the actual in vivo situation where relatively few cells within a heterogeneous population of cells carry sublethal DNA damage into the postnecrotic period. These results suggest that not all limb bud cells affected by teratogenic exposure to cyclophosphamide die, but that some persist to the postnecrotic period carrying heritable alterations in their DNA.

Neonatal toxicity in mice associated with the Ahb allele following transplacental exposure to 3-methylcholanthrene

This study was conducted to determine if the Ah genotype in mice could influence the incidence of neonatal mortality following maternal exposure to 3-methylcholanthrene (3-MC). Male F1 hybrids (Ahb/Ahd) produced from a cross of C57BL/6J and DBA/2J were backcrossed to DBA/2J females. This backcross mating resulted in noninducible pregnant mice (Ahd/Ahd) containing litters with a 1:1 ratio of AHH-inducible (Ahb/Ahd) and noninducible (Ahd/Ahd) fetuses. Dams were exposed by the oral route to 3-MC in corn oil at doses of 7, 21, or 63 mg/kg/day on Days 15, 16, and 17 of pregnancy, or to the positive control agent urethane at 1 mg/g ip on Day 17. The phenotype of surviving offspring was determined by zoxazolamine paralysis time and hepatic aryl hydrocarbon hydroxylase (AHH) activity measurements. Dose-related responses in all 3-MC treatment groups were obtained in measures of neonatal toxicity, i.e., number of litters surviving to term, litter size at birth, survival to weaning, and weight gain to 13 weeks of age. Correlation of the Ah phenotype with the neonatal toxicity data indicated that genetically responsive offspring had higher levels of neonatal toxicity than nonresponsive offspring within the same exposure groups. Thus, when fetuses are exposed in the same maternal environment to 3-MC, genetic differences in Ah genotype may influence the susceptibility to neonatal toxicity.

Lung tumorigenesis and hyperplasia in offspring associated with the Ahd allele following in utero exposure to 3-methylcholanthrene

This study was conducted to determine if the Ah genotype could influence the incidence of tumorigenesis in offspring exposed to 3-methylcholanthrene (3-MC) during the fetal period. Male F1 hybrids (Ahb/Ahd) were backcrossed to Ahd/Ahd females, resulting in pregnant mice containing litters with a 1:1 ratio of Ahb/Ahd (AHH-inducible) and Ahd/Ahd (AHH-noninducible) fetuses. Dams were exposed by gavage to corn oil or to 3-MC at 7, 21, or 63 mg/kg on Days 15, 16, and 17 of gestation, or to the positive control urethane at 1 mg/g ip on Day 17. The phenotype of surviving offspring was determined by hepatic aryl hydrocarbon hydroxylase (AHH) activity. The lung was the major site of neoplastic involvement in adult offspring 6 months after in utero exposure to 3-MC. Dose-related responses in all 3-MC treatment groups were obtained for percent nodule-bearing animals, percentage adenoma-bearing animals, mean number of nodules per animal, mean nodular size per animal, and diffuse bronchiolar hyperplasia. Correlation of Ah phenotype with adult tumorigenesis indicated that genetically nonresponsive (Ahd/Ahd) offspring had a higher incidence of nodules, adenomas, and diffuse bronchiolar hyperplasia than responsive offspring within the same treatment group. Thus, when fetuses are exposed in the same maternal environment to 3-MC, genetic differences in Ah genotype may influence susceptibility to transplacental carcinogenesis.

Effects of cytosine arabinoside on in vivo and in vitro mouse limb development

SummaryWhen pregnant mice were exposed to 40 mg per kg of cytosine arabinoside (ara-C) on days 10 to 12 of gestation, adactylous limbs with large, distally located blisters were found when the fetuses were examined on day 18. Embryonic limbs exposed transplacentally under identical conditions and explanted to culture exhibited the same morphological abnormality as did limbs exposed directly in culture to 0.1 to 1 μg per ml of ara-C. Two noncytotoxic analogues of ara-C, uridine arabinoside (ara-U) and hypoxanthine arabinoside (ara-HX), had no influence on morphological differentiation of limbs in vitro. Ara-C alone caused a dose-related decrease in uptake of3H-thymidine and35SO4 in cultured limb buds. Production of this morphologically distinct malformation in vitro will allow detailed biochemical investigations on the effect of ara-C limb ectodermal-mesenchymal interactions.