Embryotoxicity of phenytoin in adrenalectomized CD-1 mice.

It has been proposed that the anticonvulsant drug phenytoin (PHT) and glucocorticoids induce orofacial clefting by the same mechanism. Previous work had demonstrated that PHT treatment significantly increased endogenous maternal corticosterone concentrations for approximately 48 hr after dosing in A/J mice. The purpose of the present investigation was to determine whether PHT is embryotoxic in the absence of endogenous maternal glucocorticoids. Maternal adrenal glands were removed on Day 7 of gestation, and the incidence of clefting after PHT treatment was determined. There was a high level of maternal toxicity following adrenalectomy (ADX) and PHT treatment at either 60 or 75 mg/kg. This increased toxicity did not appear to be due to altered maternal drug levels in ADX mice. There was a significant increase in the clefting incidence among offspring of ADX dams treated with PHT at 60 mg/kg. This dose of PHT did not elevate maternal corticosterone levels in ADX dams. These data suggest that PHT is capable of producing clefts in the absence of endogenous maternal corticosterone.     

Effect of 6-azauridine on calcium metabolism in rats.

After 12 days of intramuscular administration of 6-azauridine (500 mg/kg b.w.) rats displayed a significant decrease in plasma calcium, inorganic phosphorus, and total hydroxyproline levels and alkaline and acid phosphatase activity. The biological method employed revealed no changes in calcitonin activity. 6-Azauridine reduced the citric acid concentration in the kidneys, liver, heart and bones. Alkaline phosphatase activity in the kidneys, heart and liver was unaffected. The results indicate that 6-azauridine inhibits calcium resorption from the bones and interferes with collagen synthesis. It cannot be ruled out that the described changes are elicited by the antimetabolic effect of this cytostatic drug.     

The 6-azauridine analogues possessing sedative and hypnotic effects.

Pharmacological effects of 6-azauridine (4) analogues were evaluated using hypnotic activity, pentobarbital (PB)-induced sleep prolongation and locomotor activity as indices. Compound 4, N3-benzyl- (10), N3-o-xylyl- (11), N3-m-xylyl- (12), N3-p-xylyl- (13), N3-alpha-phenylethyl-substituted 6-azauridine (14) exhibited hypnotic activity and PB-induced sleep prolongation, whereas N3-alkyl substituted analogues (methyl-, ethyl-, n-propyl-, n-butyl- and allyl-substitution) did not. Compound 4 and xylyl analogues (11-13) significantly decreased locomotor activity of mice by i.c.v. injection and produced motor incoordination. The results indicate that 4 and its benzyl related analogues, but not alkyl analogues have depressant effects on the central nervous system (CNS).     

In vivo synthesis of 6-azauridine 5'-triphosphate and incorporation of 6-azauridine into RNA of germinating wheat embryonic axes

The cytostatic effect of 6-azauridine on cell growth is generally regarded to be a consequence of the inhibition of de novo pyrimidine biosynthesis by the metabolite, 6-azauridine 5'-monophosphate. We show here that wheat embryonic axes further metabolize 6-azauridine to the 5'-triphosphate and incorporate the analogue into RNA, thus offering an alternative mechanism for growth inhibition. At a level of 6-azauridine required to maximally inhibit UTP biosynthesis, the ratio of 6-azaUTP to UTP is about 2:1 and substitution of 6-azauridine for uridine in new RNA is on the order of 1 in 18. The new metabolites of 6-azauridine are identified by high pressure and thin layer chromatography coupled with enzyme treatments.     

Leydig cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol

Pregnant female mice were given ethinyl estradiol on days 11 through 17 of gestation. On day 18 the dams were killed and the male fetuses were examined for testicular differentiation. Three of 12 males from dams treated with the highest dose of ethinyl estradiol showed cryptorchid testes with uterine tubes. Light and electron microscopic evaluation of the testes, both cryptorchid and normal, demonstrated foci of hyperplasia of Leydig cells showing cytoplasmic and nuclear pleomorphism, increase in lipid droplets, and decrease in smooth endoplasmic reticulum and ribosomes when compared to testes from control fetal mice. Morphometric determinations of the testes indicated that the number of Leydig cells in a unit area (mm2) in the interstitial tissue showed a dose-response relationship to ethinyl estradiol in the normal testes. The number of Leydig cells in the testes exposed to the highest dose of estrogen showed a significant difference between cryptorchid and normal testes: the former had fewer Leydig cells than the latter. These morphological observations indicate that hyperplasia of Leydig cells of fetal mouse testis at term can be induced by transplacental treatment with ethinyl estradiol and suggest that a malignant transformation into a Leydig cell tumor is possible.     

Embryotoxicity and teratogenicity of uranium in mice following subcutaneous administration of uranyl acetate

The effects of multiple maternal subcutaneous injections of uranyl acetate dihydrate (0.5, 1, and 2 mg/kg/d) from d 6 to d 15 of gestation were evaluated in Swiss mice. External, internal soft-tissue and skeletal examinations of fetuses were performed on gestation d 18. Maternal toxicity occurred in all uranium-treated groups as evidenced primarily by deaths as well as significant decreases in weight gain and in body weight at termination. Although it was not dose-related, embryotoxicity also occurred in all uranium-treated groups (significant increases in the number of nonviable implantations and in the percentage of postimplantation loss). Fetal body weight was significantly decreased at 1 and 2 mg/kg/d, whereas the number of total internal and total skeletal defects showed dose-dependent increases at 0.5, 1, and 2 mg/kg/d. Most morphological defects were developmental variations, whereas malformations were only detected at 1 and 2 mg/kg/d. On the basis of these data, both the maternal no-observable-adverse-effect level (NOAEL) and the NOAEL for embryotoxicity of uranyl acetate dihydrate were below 0.5 mg/kg/d, whereas the NOAEL for teratogenicity was 0.5 mg/kg/d.     

Concurrent development of resistance to 6-azauridine and adenosine in a mouse cell line

A variant of the hypoxanthine-guanine phosphoribosyltransferase deficient, and adenine phosphoribosyltransferase deficient mouse A9 cell line has been obtained by selecting cells which are resistant to 6-azauridine. These cells are not only resistant to 6-azauridine (5 × 10⁻⁴ M), but also to adenosine (10⁻³ M). Resistance persists indefinitely even in the absence of both compounds. The resistant cells are killed by 5-fluorouridine (10⁻⁶ M), indicating that the part of the salvage pathway for pyrimidine ribonucleotide biosynthesis which is relevant to the action of 6-azauridine is intact. The heritable change producing concurrent resistance to 6-azauridine and adenosine probably involves the de novo pyrimidine biosynthetic pathway.     

Ovarian follicular cell hyperplasia in fetal mice treated transplacentally with ethinyl estradiol

The occurrence of follicular cell hyperplasia was studied by light and electron microscopy in fetal mouse ovaries exposed to ethinyl estradiol (EE) from day 11 through day 17 of pregnancy. Pregnant mice were given EE in olive oil (0.02, or 0.2 mg/kg of body weight) and were sacrificed on day 18. The female fetuses were examined for ovarian histogenesis. Follicular cell hyperplasia was detected in both of the experimental groups, but the incidence was statistically significant only in fetuses exposed to 0.2 mg/kg of EE. Light and electron microscopic observations of the ovaries showed that the hyperplasia was located in the medullary region, and the follicular cells showed pleomorphism. Accumulation of abundant lipid droplets, enlarged rough endoplasmic reticulum with granular material, dense bodies, and vague masses of fibrous structures were seen in the cytoplasm. These morphological observations indicate that hyperplasia of follicular cells in fetal mouse ovaries at term can be induced by prenatal treatment with EE.     

Acetylation and cleavage of purine nucleosides. Synthesis of 6-azauridine, 5-fluorouridine, and 5-methyluridine.

Inosine (I) when acetylated with acetic anhydride in the presence of acetyl chloride in acetic acid solution (the so called "acid acetylation"), affords an acetylated nucleoside III (75%) along with cleavage products of the nucleoside (hypoxanthine, 19%). The reaction of I with acetyl chloride (7 days) results in the formation of hypoxanthine (95%) and triacetylribofuranosyl chloride (IV) isolated in the form of tetraacetylribofuranose (47%). The acetylated purine nucleoside affords a similar result by reaction with acetyl chloride or acetyl bromide. 2'-Deoxyuridine gives a diacetyl derivative (80%) by reaction with acetyl bromide. On treatment with acetyl bromide, the nucleoside bond of purine nucleosides is quantitatively cleavaged (4 h, 20 degrees C) with the formation of tri-O-acetyl-D-ribofuranosyl bromide (X). The halogenose X affords pure beta-anomers, namely, 1,2,3,5-tetra-O-acetyl-beta-D-ribofuranose (75%), the triacetyl derivatives of 5-methyluridine (XVIIa; 75%, referred to guanosine), 6-azauridine (XVIII; 71%), and 5-fluorouridine (XIXa; 75%).     

Drugs elevating extracellular adenosine administered in vivo induce serum colony-stimulating activity and interleukin-6 in mice

Our previous studies have shown that the combined administration of drugs elevating extracellular adenosine, i.e. dipyridamole (DP) and adenosine monophosphate (AMP), enhances murine hematopoiesis and potentiates the action of granulocyte colony-stimulating factor (G-CSF). In this study, colony-stimulating activity (CSA) of blood serum of mice treated with DP+AMP, G-CSF or all these drugs in combination, i.e. the ability of the sera to stimulate the growth of GM-CFC colonies, was assayed in vitro. Furthermore, the concentration of GM-CSF and IL-6 in the sera was determined. Administration of DP+AMP was found to enhance significantly serum CSA at all time intervals of serum sampling including 24 h after the last injection of the tested drugs. Additive effects of DP+AMP and G-CSF on serum CSA were noted at early intervals after administration of the drugs. Furthermore, IL-6 levels were significantly elevated in the sera of mice which were administered DP+AMP either alone or in combination with G-CSF. Our results show that the effects of DP+AMP are indirect, mediated through the induction of some cytokine(s) and/or growth factor(s) and that extracellular adenosine can act in cooperation with G-CSF. These findings contribute to the further elucidation of the role of adenosine in hematopoiesis.     

A novel effect of 6-azauridine on growth and protein synthesis in wheat embryonic axes

Growth and the rate of protein synthesis in germinating wheat embryonic axes are inhibited by the analog 6-azauridine via a mechanism which is independent of the usual effect of this compound as an inhibitor of de novo synthesis of UTP. The effects on growth and protein synthesis can be separated from that on UTP biosynthesis by analyses of the kinetics by which each effect is maximized following a 1.5-h pulse with 6-azauridine, and by saturation of the responses at different doses of the analog. The inhibitions of growth and protein synthesis are apparently not mediated through the rate of poly A(+) RNA synthesis (reduced as little as 8%), but rather by an effect on translation. Since cordycepin reduces the azauridine inhibitions of growth and protein synthesis, it is suggested that these latter effects of 6-azauridine may depend upon the synthesis of an inhibitory azauridyl-RNA.