M. Kate Smith

Dominant lethal effects of subchronic acrylamide administration in the male Long-Evans rat

Acrylamide, a widely used vinyl monomer, is well known as a neurotoxin but inactive as a mutagen in bacterial test systems. The experiments reported demonstrate that after subchronic oral dosing in the male rat, acrylamide induced significant elevations in both pre- and post-implantation loss following dominant lethal testing. These effects were seen at doses which failed to produce clinical or pathological evidence of neurotoxicity. In an accompanying cytogenetic study, no increase in chromosome aberrations was observed in spermatogonia or spermatocytes of treated animals. When spermatocytes from treated spermatogonial stem cells were analyzed, reciprocal translocations (4) were observed in the treated animals and not in the control, but the significance of this result still needs to be established.

A comparison of the in vivo and in vitro response of mammalian embryos to a teratogenic insult

This study serves to further define the capabilities of the whole embryo culture system using the known teratogen, hydroxyurea (HU). An initial in vivo study was performed whereby day 9 pregnant mothers were injected i.p. with 300 mg/kg HU. Dams were sacrificed 2 days later and embryos were analyzed for malformations and total embryonic protein. In addition, the peak plasma value from injected dams was found to be approximately 300 micrograms/ml with a plasma half-life of 30 min. These values were then reproduced in the culture system with results noted in cultured embryos with respect to the types of malformations found. Additional in vitro experiments were performed varying both exposure time and drug level concentrations. Results indicate that both of these parameters are important considerations when designing in vitro experiments.

The role of pharmacokinetics in determining the response of rodent embryos to teratogens in whole-embryo culture.

The rodent embryos response to teratogenic insult in whole-embryo culture during the period of neurulation was characterized by determining the role of pharmacokinetics and embryonic recovery in producing abnormal growth and development. Five known teratogens, hydroxyurea, cyclophosphamide, cadmium, diphenylhydantoin, and the ketone body beta-hydroxybutyrate were employed. The dose and exposure times in vitro were designed to reproduce the peak serum concentrations and half-life of the compounds present following the administration of a teratogenic or maximum maternally tolerated dose of the agents in vivo. The results showed: first, that both the serum concentration and duration of exposure to an agent play a role in determining the embryonic response in vitro; secondly, that compounds that do not produce effects during the period of neurulation or that require maternal metabolic activation are not teratogenic in culture; and thirdly, that embryos have the capacity to recover from certain teratogenic insults in vitro. Thus, both pharmacokinetics and the potential for embryonic recovery should be considered when applying the whole-embryo culture technique to studies in teratology and toxicology.

Animal virus screens for potential teratogens. I. Poxvirus morphogenesis.

The growth of poxvirions in cell culture is considered a teratogen screening test, since this virus has a rapid, simple morphogenetic pathway that is dependent upon cell proliferation. Vaccinia WR-infected BSC 40 monolayers were exposed to 42 known teratogens and 9 nonteratogens at dosages from 1 microM to 100 mM. After 24 h of infection, the number of functional virions was determined by plaque assay. Thirty-three of the 42 teratogens inhibited the virus, 3 teratogens stimulated the virus, and 6 teratogens were false-negatives. Eight of the 9 nonteratogens had no effect on virus proliferation at dosages as high as 600 times the lowest reported teratogenic dosage. The number of new virions could be directly related to the concentration of the teratogen in vitro, thus allowing each compound to be characterized by an RD50. The RD50 dosage in milligrams per liter was 98% correlated with the lowest reported teratogenic dose in vivo in milligrams per kilogram. In sum, vaccinia-infected cells have an easily identifiable endpoint, plaque-forming units, which may be an accurate prognosticator of teratogenesis.

The developmental toxicity of bromochloroacetonitrile in pregnant long‐evans rats

Bromochloroacetonitrile (BCAN) is a by‐product of the chlorine disinfection of water containing natural organic material. Adverse effects of BCAN in an in vivo teratology screen (i.e. neonatal survival assay) gave reason for further investigation into the developmental toxicity of this compound. BCAN was administered orally to pregnant Long‐Evans rats on gestation days 6–18 (vaginal plug = day 0). Four groups of approximately 20 females received BCAN at 5, 25, 45 or 65 mg/kg/day in a tricaprylin vehicle. Endpoints assessed at necropsy (day 20) included maternal organ weights, number of corpora lutea and uterine contents (number of implants and fetuses); live fetuses were weighed, measured and subsequently examined for external, skeletal and soft tissue malformations. Gestational maternal weight gain was reduced at 45 and 65 mg kg‐1. Maternal toxicity, manifested as increased organ weights and deaths, occurred at 65 mg kg‐1. Postimplantation loss was elevated at 45 mg kg‐1 while total litter loss was obser...

Determination of Genetic Diversity and Paternity in the Gray-Tailed Vole (Microtus canicaudus) by RAPD-PCR

Genetic relatedness of gray-tailed voles ( Microtus canicaudus ) was determined by random amplified polymorphic DNA (RAPD). This work is the first reported use of the RAPD method for pedigree analysis of M. canicaudus and demonstrates the feasibility of RAPD for assessing paternity and genetic similarity between inbred and outbred individuals. Amplified polymorphisms and two statistical approaches were used to evaluate genetic similarities between individual voles. DNA profiles of wild-caught (outbred) voles were significantly less similar than were inbred voles, using two different primers ( P ≤ 0.03 and P ≤ 0.003). In pedigree analyses using three unrelated males in a discrimination test, indication of paternity was based upon bands shared by the progeny and the male in question. Non-sire males produced bands that were not seen in the probable father or offspring and, conversely, bands were found in the progeny that were apparently inherited only from the probable sire. The RAPD procedure may have wide applications in identifying genetic relationships among individuals to estimate genetic heterogeneity, determine parentage, and address questions concerning gene flow and kinship in feral vole populations.

Evaluation of the Developmental Toxicity of Sodium Nitrite in Long-Evans Rats,

Sodium nitrite administered in the drinking water to Long-Evans rats during pregnancy and lactation severely affected erythropoietic development, growth, and mortality in their offspring. Pregnant rats were maintained throughout gestation on 0.5, 1, 2, or 3 g NaNO2/liter. There were no significant differences between treated and control litters at birth. Thereafter, pups of treated dams on 2 and 3 g NaNO2/liter gained less weight, progressively became severely anemic, and began to die by the third week postpartum. By the second week postpartum, hemoglobin levels, RBC counts, and mean corpuscular volumes of these pups were all drastically reduced compared to controls. Blood smears showed marked anisocytosis and hypochromasia. Gross chylous serum lipemia and fatty liver degeneration were noted. Histopathology demonstrated cytoplasmic vacuolization of centrilobular hepatocytes and decreased hematopoiesis in bone marrow and spleen. Administration of 1 g NaNO2/liter resulted in hematological effects but did not affect growth or mortality. NaNO2 (0.5 g/liter) was at or near the no observed effect level. Cross-fostering indicated that treatment during the lactational period was more instrumental in producing lesions than treatment during the gestational period. The data presented are consistent with the lactational induction of severe iron deficiency in the neonate.

Nitrite-induced iron deficiency in the neonatal rat

Neonatal rats from dams receiving 2 or 3 g NaNO2/liter in the drinking water through -gestation and lactation suffered severe microcytic anemia as well as growth retardation and high mortality. Lipemia, fatty liver damage, decreased erythropoiesis of spleen and bone marrow, and reduced plasma and tissue iron levels were noted in affected pups. These effects were all consistent with and characteristic of iron deficiency. Experiments presented here were designed to show that the maternally mediated toxicity of nitrite is actually an iron deficiency syndrome in the pups caused by inadequate iron transfer from dam to pup. It was found that administration of exogenous iron supplement to pups of treated mothers reversed the anemia and other effects of nitrite toxicity noted both in previous studies and in unsupplemented littermates. Mothers of affected pups were themselves anemic. Finally, we fully documented severe iron deficiency in pups of nitrite-treated mothers and showed that these mothers produced milk of reduced iron content. It appears then that nitrite-consuming dams have a reduced capacity to transfer iron to their pups. The nitrite-associated toxicities in the pups are actually a result of an iron deficiency.