Fetal mouse salivary glands and applications for determining teratogenic mechanisms in vitro.

Culture systems offer advantages for studying the actions of chemicals on mechanisms of development. Growth, [3H]thymidine incorporation, and 14C-amino acid incorporation were measured to determine culture conditions and the response of glands to three chemicals. Eagle's MEM supported the best growth and the best time for double isotope presence was the second 24 h of culture. Cyclamate had no effect on growth or isotope incorporation but cycloheximide disturbed 14C more than 3H. 5-Azacytidine was toxic but the incorporation of both isotopes exhibited the same pattern.

Metabolic activation in the fetal mouse salivary gland culture system with rat hepatocytes, rat S-9, and human S-9.

The usefulness of an in vitro assay for embryotoxicity may depend on the availability of metabolic activation systems that will function in the culture system. The fetal mouse salivary gland has been investigated as an in vitro assay system. To see if the glands would grow in the presence of metabolic activators and if the glands would react to metabolites known to be embryotoxic, the glands were grown in the presence of cyclophosphamide (CP) and several activation systems. These included isolated rat hepatocytes, uninduced rat S-9, rat S-9 induced with 3-methylcholanthrene (3-MC), rat S-9 induced with Aroclor 1254, and human S-9. Twenty salivary glands were isolated from 13 day embryos (plug day = 0) and were grown in each treatment for 48 h. One control had no activation system of CP, one had an activation system but no CP, and three treatments had the activation system and 25, 75, or 150 micrograms/ml CP. The S-9 with cofactors and the appropriate amount of CP was contained in dialysis bags. The greatest suppression of salivary gland growth occurred in co-culture with hepatocytes activating CP. The S-9 induced by Aroclor 1254 was nearly as effective as the hepatocytes. The next most effective was a group with similar activity consisting of the uninduced rat S-9 and the three samples of human S-9. The 3-MC-induced S-9 was the least effective in suppressing growth of salivary glands. All the activation systems tested can be used with the salivary gland culture system.

Use of fetal mouse salivary glands in culture to detect embryotoxicity: evaluation of eight additional chemicals.

Several developmental processes interact to convert an epithelial bud into a gland with many lobes. For each chemical tested, 20 glands were placed into a control and each of 3 concentrations of the chemical. From dose-response curves, the concentration that reduced gland growth by 50% was determined and divided into the LD50 for mice. These ratios were used to compare the toxicity of the chemicals. The ratios of cyclamate, diphenhydramine, allopurinol, nitrofen and urethane would indicate that embryotoxicity would not be expected without maternal toxicity. Promethazine, diethyldithiocarbamate, and 5-fluorouracil would be expected to show embryotoxicity without maternal toxicity.

Test of six chemicals for embryotoxicity using fetal mouse salivary glands in culture.

Many new chemicals come into use each year, and the need for rapid and cost-effective methods for testing developmental toxicity is apparent. Establishing reliable in vitro techniques is important to a tier approach to testing for developmental toxicity. The fetal mouse salivary gland was selected as a possible test system because several interacting developmental processes occur in gland growth, and development is quantifiable by counting lobes. For each chemical tested, 20 glands from 13-day embryos were treated in a control media and in three concentrations of the test chemicals. The number of lobes present after 48 hours is dependent on the number of lobes at explantation. Glands with different numbers of lobes at explantation were compared by dividing the number of lobes present after 48 hours by the number present at explantation to determine a growth ratio. Mean growth ratios were used to construct dose-response curves, and from these curves the concentration that reduced growth by 50% (TP50) was determined. Comparisons with in vivo data were made by calculating three ratios; the TP50 was divided into the lowest teratogenic dose, the lowest maternal toxic dose, and the dose that was lethal to 50%. Four in vivo teratogens, 6-aminonicotinamide, cytochalasin B, hydroxyurea, and 3-acetylpyridine, all had ratios much higher than 1, indicating a very sensitive response by the glands. One in vivo teratogen, dexamethasone, had much lower ratios, indicating less sensitivity. Acetaminophen, a nonteratogen in vivo, actually stimulated growth of the glands at 10(-5) M and had very low ratios indicating a minimal response by the glands.