Induction of postaxial forelimb ectrodactyly with anticonvulsant agents in A/J mice.

Exposure of A/J mice on day 9.5 of gestation to the derivatives of three acidic anticonvulsant agents, namely dimethadione, sodium valproate, and sodium diphenylhydantoin, each induced postaxial forelimb ectrodactyly predominantly of the right side. This specific malformation has previously been associated with the administration of acetazolamide to rodents; however, several agents can induce this same defect including other carbonic anhydrase inhibitors, carbon dioxide, cadmium, ethanol, ammonium chloride, and 13-cis retinoic acid. The relative potency of the three agents indicates no direct relationship to the pKa of the acid. Other than ectrodactyly, each of the anticonvulsant agents induced a compound-specific spectrum of malformations despite the uniform administration time. This finding suggests that these agents are capable of acting via different mechanisms or by the differential spatial and temporal dynamics of a common mechanism.

Teratologic potential of 2-methoxyethanol and transplacental distribution of its metabolite, 2-methoxyacetic acid, in non-human primates.

The embryotoxic effects of 2-methoxyethanol (2-ME) were studied in non-human primates to better assess the risk for women of child-bearing age exposed to this agent. Macaca fascicularis females were treated daily throughout the organogenetic phase of pregnancy (days 20-45) by gavage and the fetuses collected at day 100 by Caesarean section. At the highest dose (0.47 mmole/kg), all eight pregnancies ended in death of the embryo. One of these dead embryos was abnormal, missing a digit on each forelimb. At the middle dose (0.32 mmole/kg), three of 10 pregnancies ended in embryonic death, presumably due to 2-ME exposure and three of 13 pregnancies met a similar fate at the low dose (0.16 mmole/kg). In each of these two groups, an additional pregnancy was lost to abortion, but both were thought to be spontaneous, which usually occurs in 10-20% of untreated macaque pregnancies. These results indicate that 2-ME is a potent toxin to the developing primate embryo and thereby furthers the concern about exposure of pregnant women to this agent, although maternal toxicity was evident in nearly all treated pregnancies and was especially severe in the high-dosage animals. Distribution of the major metabolite of 2-ME, 2-methoxyacetic acid (2-MAA), indicated a long half-life (ca. 20 h), resulting in accumulation of metabolite in maternal serum after repeated daily dosing. Transplacental studies revealed uniform distribution in the embryo and extraembryonic fluids at a concentration similar to that in maternal serum. The yolk sac, on the other hand, accumulated a very high concentration of 2-MAA, but the embryotoxic significance of this observation is unknown.

Attenuation of the virulence of the M strain of Plasmodium cynomolgi during prolonged multiplication in splenectomized rhesus monkeys.

The primary data in this report showed that prolonged multiplication of the M strain of Plasmodium cynomolgi in splenectomized rhesus monkeys led frequently to emergence of parasites whose virulence for monkeys with intact spleens was markedly attenuated while that for splenectomized monkeys was unimpaired. Expressions of attenuation regularly included reductions in the height of the peak parasitemia and in some instances failure to induce infection with inocula 1,000-fold those infective for splenectomized monkeys. Attenuation of virulence, once established, was maintained through as many as 17 serial passages in intact monkeys and more than 100 such transfers in splenectomized monkeys. Not only asexual blood stages, but also sexual stages (gametocytes) carried the attenuated characteristic, as indicated by its ready passage through mosquitoes. Studies in monkeys with sporozoite-induced infections showed that the persisting exoerythrocytic stages did not participate in the attenuation phenomenon; for when erythrocytic parasites of reduced virulence were cleared from the blood by either immune processes or chemotherapy, they were replaced upon relapse with parasites of unimpaired virulence. A major effort to determine whether splenectomized monkeys carried plasma or erythrocyte factors responsible for displays of attenuated virulence was nonproductive. An intact spleen was the essential element in these displays, for they disappeared immediately upon removal of this organ. The attenuation phenomenon probably reflects selection of spontaneously occurring mutants with limited capacity to escape normal splenic clearance mechanisms and unimpaired or even enhanced capacity to multiply in splenectomized monkeys.

Antimalarial activities and subacute toxicity of RC-12, a 4-amino-substituted pyrocatechol.

RC-12 [1,2-dimethoxy-4-(bis-diethylaminoethyl)-amino-5-bromobenzene] was evaluated for prophylactic, radical curative, and suppressive activities against infections with Plasmodium cynomolgi and subacute toxicity in rhesus monkeys. Applied as a prophylactic agent, RC-12, administered in doses of 6.25 to 25.0 mg/kg daily throughout the incubation period, provided near-complete to complete protection against 10(5) to 10(6) times the minimum infective dose of sporozoites. Applied as a suppressive agent, daily doses of 100.0 mg of RC-12 per kg did not eradicate blood schizonts regularly; hence, the need for concomitant administration of a blood schizonticide, such as chloroquine, in assessments of radical curative activity. In such appraisals, daily doses of 6.25 to 25.0 mg of RC-12 per kg for 14 days, in combination with 2.5 mg of chloroquine per kg daily for 7 days, effected cure of 69 and 93% of established infections, respectively. The curative activity of RC-12 was related to the total dose and could be achieved with a regimen as brief as 4 days. With respect to outward expressions of toxicity, daily doses of 50.0 mg/kg or lower for 15 to 225 days evoked no reactions. Doses of 100.0 or 200.0 mg/kg, scheduled for 15 days, evoked convulsions and depression and were, respectively, lethal to 4 of 17 and 7 of 7 recipients. Doses of 25.0 mg/kg or lower evoked no discrete reactions. Doses of 50.0 mg/kg and higher evoked hepatomegaly, vacuolation of hepatocytes, and elevations of glutamic oxalacetic and glutamic pyruvic transferase activities in serum, reactions related in intensity to dose but not duration of dosage.

The effects of prenatal ethanol in cynomolgus monkeys.

Ethanol was administered to pregnant cynomolgus monkeys (Macaca fasicularis) from day 20 to 150 of gestation. The ethanol was delivered by intragastric intubation twice a day at 8 AM and 5 PM. Dosage was begun at a relatively low level, 2 g/kg/day and increased gradually to attain a final dosage of 4 or 5 g/kg/day. The offspring were allowed to be delivered naturally and remained with their mother until 23 days of age when they were sacrificed. Maternal blood ethanol and acetaldehyde levels were monitored at weekly intervals throughout pregnancy. Pregnancy wastage in the form of abortions and stillbirths was increased by ethanol treatment especially at the high dose. Birthweight was significantly lowered in offspring of mothers receiving 5 g/kg/day of ethanol. However, no structural malformations or facial changes suggestive of those seen in the human fetal alcohol syndrome were found in any of the offspring. We conclude that the conditions of our study did not produce a satisfactory model of human fetal alcohol syndrome.

The course of untreated Plasmodium inui infections in the rhesus monkey (Macaca mulatta).

This report deals with the major features of untreated infections with Plasmodium inui as exhibited in a group of 31 rhesus monkeys (27 inoculated with trophozoites, four with sporozoites). Infections in nine monkeys were followed to self-cure. In 22 subjects they were interrupted prematurely; in 17 by chemotherapy 454 to 3,931 days after onset of patency; in 5 by death 491 to 1,025 days after onset of patency. Substantial monkey-to-monkey variations were encountered in the intensity of the parasitemia and its cyclic undulations, in the morbid features of the disease, and in the duration of infection. Thus initial peak parasitemias varied by as much as 15-fold. Some post-peak parasitemias were sustained at relatively constant and moderately high levels for many months; others fluctuated cyclically between barely detectable and readily countable numbers. In most subjects, there was no evidence of morbidity attributable to malarial infection; in two, reactions were severe enough to lead to death. The duration of untreated infections could be as brief as 14 months or could approach 14 years. With due allowance for premature interruptions, the majority of infections could be expected to persist for 4--6 years.

Non-confirmation of thalidomide induced teratogenesis in rats and mice.

It has been reported that thalidomide, dissolved in a 1:3 mixture of Tween 20 and physiological saline and administered intraperitoneally to pregnant mice and rats induces a variety of malformations, including limb deformaties, characteristic of the primate syndrome. The studies reported herein attempted to confirm these findings without success although the rodent strains used were not the same. A low level of non-specific malformations was observed in the fetuses of both species at dose levels reported to cause a 47 percent and 92 percent rate of malformation in mice and rats respectively. One possible source of difference was Tween 20 which was toxic to the point of lethality in these studies at dose levels reported to be non-toxic in the earlier studies.

Differences in the virulence of Plasmodium knowlesi for Macaca irus (fascicularis) of Philippine and Malayan origins.

This report summarizes the results of a comparative study of the virulence of the "S-M," H, and C strains of P. knowlesi for Indian rhesus monkeys (Macaca mulatta) and cynomolgus monkeys [M. irus (fascicularis)] of Malayan (West Malaysia) and Philippine origins. Each of the above strains produced fulminating, uniformly fatal infections in the rhesus monkey and mild, chronic infections, characterized by relatively low level parasitemias in cynomolgus monkeys of Philippine origin. In striking contrast, the H and C strains produced infections in cynomolgus monkeys of Malayan origin which were indistinguishable in severity from infections produced in M. mulatta. The circumstances of the study precluded evaluation of the virulence of the "S-M" strain for M. irus of Malayan origin. Even so, the available data make it necessary to qualify the long-held belief that infections with P. knowlesi in M. irus invariably follow a benign course.

Comparative distribution and embryotoxicity of hydroxyurea in pregnant rats and rhesus monkeys.

Hydroxyurea was given to pregnant rhesus monkeys and pregnant rats in regimens adjusted to produce similar degrees of teratogenicity, for the purpose of comparing the distribution of the drug in the females and their embryos. According, in rats 137 mg/kg/day ip on days 9-12 resulted in a drug half-life in maternal plasma of about 15 min and in embryos about 85 min, after the last injection; and in monkeys 100 mg/kg/days iv on days 23-32 resulted in drug half-life in maternal plasma estimated to be 120 min and in embryos 265 min, after the last injection. Using as a baseline of biological effects the minimal concentration known to inhibit DNA synthesis in rat embryos and cancer cells, namely 10(-4) M, it was calculated that the rat embryos in the present study were exposed to this level or more for approximately 12 h whereas the monkey embryos were exposed for approximately 100 h. Although the teratogenic effects were not identical in the two species, these data are interpreted to mean that rat embryos are teratogenically much more sensitive to hydroxyurea than monkey embryos. These observations have important implications in the selection of appropriate species for tests to estimate human teratogenic risks. The rat, which is currently the most widely used animal for such tests, displays sizeable differences from rhesue monkeys, which is one of the animals thought to be most like man in teratogenic susceptibility.