Constant rate of infusion--improvement of tests for teratogenicity and embryotoxicity

Sodium salicylate was used as a model substance to investigate whether the embryotoxic effects on rat fetuses varies between two modes of administration. A marked increase in fetal adverse effects was observed at analgetic doses with the once-a-day bolus regimen compared to the constant rate input. The difference was less marked at antirheumatic levels.     

Phenytoin teratogenicity and midgestational pharmacokinetics in mice.

Mice of the A/J and C57BL/6J (C57) strains were dosed with phenytoin (PHT) every 48 hr throughout pregnancy by gastric intubation to test the hypothesis that maternal plasma PHT concentration may be the significant factor in determining PHT reproductive and developmental toxicity. Serial serum samples were obtained from each mouse from gestation day (GD) 10-GD 12 for determination of individual dam PHT pharmacokinetics. Maximum PHT concentration and PHT AUC (area under-the-time-concentration curve) were regressed to laparotomy and fetal evaluation endpoints to determine whether significant association existed. Although serum PHT concentrations exceeded levels associated with teratogenicity (greater than 10 micrograms/ml), few major malformations were induced in either strain. However, in the A/J strain, there was a significant increased incidence of hydrocephaly and open eyelid. Regression of pharmacokinetic parameters with embryo and maternal endpoints indicated significant associations between gestational weight gain and maximum concentration measured (Cmax) or AUC in both strains. This association was also found for fetal weight in the C57 strain. In the A/J strain, the induction of decreased ossification of the sternebrae was also associated with maternal PHT concentration; however, linear regression of hydrocephaly and open eyelid to PHT concentration was not statistically significant. These results suggest that maternal plasma PHT concentration may be a quantifiable determinant of certain aspects of PHT developmental toxicity in the mouse.     

Inhibition of isotretinoin teratogenicity by acetylsalicylic acid pretreatment in mice.

Although isotretinoin (ITR) has been suggested to cause malformations via cytopathic effects on embryonic cells, the molecular mechanisms of ITR cytotoxicity in teratogenesis are not clear. Since ITR undergoes metabolism by prostaglandin synthase to a potentially cytotoxic peroxyl free radical, the possible role of prostaglandin synthase metabolism as a modulator of ITR teratogenicity was evaluated. Craniofacial and limb abnormalities were noted in fetuses on day 18.5 of gestation following administration of ITR to pregnant CD-1 mice in a three dose regimen of 100 mg/kg at 4 hr intervals on day 10.5 of gestation (plug day = day 0.5 of gestation). Mice were also treated with acetylsalicylic acid (ASA), an irreversible inhibitor of the cyclooxygenase component of prostaglandin synthase, at doses of 20 and 60 mg/kg body weight 2 hr prior to each ITR dose. ASA pretreatment of mice receiving ITR treatment showed a dose-dependent decrease in the overall incidence of malformations, number of defects per fetus, and the incidence of specific craniofacial and limb defects. Equivalent doses of ASA given to control mice did not cause malformations or alter the incidence of resorptions. These results demonstrate that ASA is able to ameliorate the teratogenic effects of ITR observed in fetal mice near term and indicate that prostaglandin metabolism could play a mechanistic role in ITR teratogenicity.     

Maternal protein deprivation enhances the teratogenicity of ochratoxin A in mice

Teratogenic effects of the mycotoxin ochratoxin A (OA) were investigated in protein-deprived mice. Pregnant CD-1 mice were assigned to control (26%), 16%, 8%, and 4% protein diet groups and treated by gavage with 0, 2, or 3 mg/kg OA on gestation day 8 (plug = day 1). They were killed on day 18 and subjected to teratological examination. OA treatment decreased prenatal survival, particularly at the two lowest dietary protein levels. OA at the higher dose also inhibited fetal growth in all groups, with a similar effect at the low dose in the 16% and 4% protein groups. Gross malformations associated with OA were inversely related to dietary protein levels; for example, 26% and 100% of the litters were affected in the 26% and 4% protein groups, respectively, at the high OA dose. A similar trend was also seen for skeletal malformations and variations. Thus maternal protein deprivation was seen to exacerbate adverse effects of OA in a developing mammal. Such results may have implications for areas where lack of adequate food supply may cause consumption of moldy grain by women or domestic animals already living on a protein-deficient diet.     

Lack of teratogenicity of aluminum hydroxide in mice

The embryotoxic and teratogenic potential of aluminum hydroxide, a therapeutic drug used as an antacid and phosphate binder, was investigated in Swiss mice. Mated female mice were given by gavage daily doses of 0, 66.5, 133 or 266 mg/kg of A1 (OH)3 on gestation days 6 through 15 and killed on gestation day 18. Females were evaluated for body weight gain, food consumption, appearance and behavior, survival rates, and reproduction data. No significant effects attributable to A1(OH)3 were noted in comparison of maternal body weight and food consumption values, appearance and behavior. No treatment-related changes were recorded in the number of total implants, resorptions, the number of live and dead fetuses, fetal size parameters or fetal sex distribution data. Gross external, soft tissue and skeletal examination of the A1-treated fetuses did not reveal differences at any dose in comparison with the controls. Thus, no evidence of maternal toxicity, embryo/fetal toxicity or teratogenicity was observed with A1(OH)3 in mice.     

Mitigation of caffeine-induced teratogenicity in mice by prior chronic caffeine ingestion.

Pregnant A/J female mice, which had drunk tap water or a 0.05% caffeine solution for 8-19 weeks after weaning, were each injected sc with 150 or 250 mg/kg caffeine once on day 13 of gestation. After 150 mg/kg caffeine the frequencies at term of fetal death, external malformation, and subcutaneous hematomas were significantly lower in the caffeine- than water-drinking group. After 250 mg/kg caffeine the frequency of fetal death but not of malformations and hematomas was lower in the group with caffeine pretreatment. These findings were explained by assuming that long-term ingestion of caffeine induced and increased rate of degradation of caffeine administered during pregnancy.     

Effect of cyclosporin A on lymphopoiesis. I. Absence of mature T cells in thymus and periphery of bone marrow transplanted mice treated with cyclosporin A

In this report, we investigate the effect of cyclosporin A (CsA) on lymphopoiesis, and demonstrate that CsA selectively abrogates the development of CD4+CD8- and CD4-CD8+ T cells (single positive cells) in the thymus. This developmental arrest results in the complete absence of mature T cells (assessed both by phenotypic and functional analyses) in the spleen of syngeneic bone marrow transplanted mice subsequently treated with CsA. In contrast to its remarkable effect on T cells, CsA had no detectable effect on B cells differentiation. In the thymus, the generation of CD4+CD8+ thymocytes was not affected by CsA treatment, and CD4-CD8- thymocytes of CsA-treated mice expressed surface markers characteristic of normal CD4-CD8- thymocytes, and exhibited normal functional activity when stimulated with anti-CD3 antibody. Thus, CsA appears to prevent the generation of mature, single positive T cells without affecting the development of immature T cells in the thymus. In addition to its immunosuppressive effect on immunocompetent cells, these results indicate a novel feature of CsA, which involves arrest of T cell differentiation, a finding that may be important for applications in clinical bone marrow transplantation.     

Effect of route of administration on phenytoin teratogenicity in A/J mice

Acute administration of the anticonvulsant drug, phenytoin (PHT) has been shown to result in embryotoxicity and teratogenicity in several strains of mice. The A/J strain is reported to be most susceptible to the effects of the drug including an increased incidence of resorptions and orofacial clefts in treated animals. When administered chronically, the drug has been shown to be teratogenic in the absence of maternal toxicity and embryolethality in Swiss Webster mice [Hansen and Billings, 1985]. In this paper, we have compared the embryopathic effects of chronic and acute administrations of PHT to A/J mice. PHT was administered to pregnant females by intraperitoneal (i.p.) injection on day 10 of gestation at a dose of either 60 or 75 mg/kg body weight. Alternatively, PHT was added to ground chow and fed to animals prior to and throughout gestation; animals received a daily dose of either 60 or 75 mg/kg body weight. Pregnant animals were sacrificed on day 18 or 19 of gestation, and fetuses were examined for the presence of orofacial clefts and other anomalies. There was a significant increase in the frequency of cleft lip and palate in animals receiving the drug by i.p. administration, but there was no increase in the incidence of clefts if the drug were added to the diet. The results of this study reiterate the importance of the route of administration of a drug in determining its embryopathic effect.     

Hydrocortisone-induced embryotoxicity and embryonic drug disposition in H-2 congenic mice

Congenic mouse strains C57BL/10Sn (B10) and B10.A/SgSn (B10A), genetically different only in the region of the H-2 complex, were compared for sensitivity to hydrocortisone-induced embryotoxicity and embryonic drug disposition. Pregnant B10A mice dosed intramuscularly with 0, 100, 150, and 200 mg hydrocortisone/kg body weight and B10 mice injected with 0, 200, 400, 600, and 800 mg/kg, both on gestational day (GD) 12, were evaluated on GD 18 for reproductive toxicity. The induction of cleft palate demonstrated a linear dose-response by probit analysis: The ED50s were 143.6 mg/kg and 512.0 mg/kg for B10A and B10 mice, respectively. Comparison of fetal weight revealed statistically significant intrauterine growth retardation at all doses administered to B10 mice. However, growth retardation was shown only in the high-dose group in the B10A strain. Embryonic drug concentrations were evaluated by administration of hydrocortisone to mice of both strains on GD 12, at the ED50 for cleft palate production in the B10A strain, with 3H-hydrocortisone (5 muci/mouse) added as a tracer. Maternal serum and embryos were analyzed for steroid content. Disposition and pharmacokinetics of 3H-hydrocortisone were similar in both strains, with the majority of serum radioactivity recovered as hydrocortisone and the major radioactive peak in embryos comigrating with cortisone. The results indicate that H-2 haplotype does not influence hydrocortisone-induced cleft palate sensitivity through an alteration of embryonic drug exposure.     

Effect of cyclosporin A on lymphopoiesis. III. Augmentation of the generation of natural killer cells in bone marrow transplanted mice treated with cyclosporin A

The effects of cyclosporin A (CsA) on the generation of NK cells were studied using syngeneic bone marrow transplanted mice subsequently treated with CsA (BMT/CsA mice). In contrast to a severe reduction in T cells that was reported previously, these mice exhibited a marked enhancement of splenic NK activity. The enhanced NK activity was mediated by NK1.1+, Thy-1- cells as assessed by antibody plus complement treatment, and was concomitant with an absolute increase in the numbers of NK1.1+ cells as assessed by flow cytometry. Because the depletion of host-derived, mature NK cells by injection of anti-asialo GM1 antibody before bone marrow reconstitution did not affect the enhancement of NK activity, CsA appeared to augment the generation of NK cells from bone marrow precursors. To investigate a possible relationship between the enhancement of NK activity and the maturational arrest of T cells in the thymus induced by CsA, mice were thymectomized, followed by irradiation, bone marrow reconstitution, and CsA treatment. These mice exhibited as strong enhancement of splenic NK activity as BMT/CsA mice, suggesting that the CsA-induced effect on NK cells is distinct from its effect on T cell development in the thymus. Taken together, these results are the first demonstration of the positive effect of CsA on NK cell generation and may be of importance in clinical bone marrow transplantation.     

A new model for embryotoxicity testing: teratogenicity and pharmacokinetics of valproic acid following constant-rate administration in the mouse using human therapeutic drug and metabolite concentrations

The limitations of a conventional testing procedure for embryotoxicity-multiple dosing throughout the organogenesis period of the mouse or rat-are discussed for drugs such as valproic acid (VPA) which exhibit pharmacokinetic properties in these species which are very different from those in the human. Administration of VPA to the mouse, once each day, resulted in peak plasma drug concentrations 10 fold higher than human therapeutic plasma levels, after which the drug levels rapidly decreased to insignificant levels which persisted until the time of the next dose. Dose-dependent growth retardation, a sharp increase in the resorption rate and a high incidence of exencephaly were observed with this dosing regimen. A new mouse model was developed where constant-rate application of the drug with osmotic minipumps resulted in drug levels throughout the organogenesis period which were similar to those observed during human therapy. Such therapeutic drug levels produced a slight but significant fetal growth retardation and increased resorption rates, but not exencephaly. It is suggested that maintaining plasma concentrations in the experimental animal comparable to human therapeutic drug levels should offer a more realistic model for drug-embryotoxicity testing.     

Amidic modification of valproic acid reduces skeletal teratogenicity in mice

BACKGROUND: The antiepileptic drug valproic acid (VPA) is well known to cause neural tube and skeletal defects in both humans and animals. The amidic VPA analogues valpromide (VPD) and valnoctamide (VCD) have much lower teratogenicity than VPA inducing exencephaly in mice. The objective of this study was to investigate the teratogenic effects of VPA, VPD, and VCD on the skeleton of NMRI mice. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of VPA (400 and 800 mg/kg), VPD (800 mg/kg), or VCD (800 mg/kg) on the morning of gestation day (GD) 8. Cesarean section was carried out on GD 18. Live fetuses were double‐stained for bone and cartilage and their skeletons were examined. RESULTS: Significant increases in fetal loss and exencephaly rate were observed with VPA at 800 mg/kg compared to the vehicle control. There were no significant differences between either VPD or VCD and the control groups for any parameter at cesarean section. A number of abnormalities were dose‐dependently induced at high incidences by VPA in both the cartilage and bone of vertebrae, ribs and sternum. In contrast, lower frequencies of abnormality were exhibited with VPD and VCD than VPA in all skeletons affected by VPA. CONCLUSIONS: These findings clearly indicate that VPD and VCD are distinctly less teratogenic than VPA in the induction of not only neural tube defects, but also skeletal abnormalities. A structure‐teratogenicity relationship of VPA on the skeleton is suspected. Birth Defects Res B 71:47–53, 2004. © 2004 Wiley‐Liss, Inc.     

Effects of cyclosporin A on humoral immune response and resistance against vesicular stomatitis virus in mice.

The effect of cyclosporin A (CS-A) on the antiviral humoral response was studied by using vesicular stomatitis virus (VSV); VSV provided the opportunity to simultaneously assess both T-independent and T-dependent antibody responses. The T-independent anti-VSV immunoglobulin M (IgM) response was virtually unaffected, whereas the T-dependent primary anti-VSV IgG response was suppressed by CS-A; in contrast, the secondary IgG response was highly resistant to CS-A. Moreover, once the switch from IgM to IgG had occurred, the primary response also became refractory to suppression by CS-A. We concluded that the effect of CS-A on the primary anti-VSV antibody response was mediated via impairment of a T-dependent mechanism; in contrast, memory T cells or memory B cells or both were quite resistant to the suppressive effects of CS-A. CS-A treatment rendered mice highly susceptible to VSV infection; under CS-A treatment, mortality was 100% after infection via footpads, whereas immunocompetent mice survived. Since CS-A does not impair induction of early T-independent anti-VSV IgM neutralizing antibodies, this high mortality in CS-A treated mice illustrates the crucial role of CS-A-sensitive cells in resistance against VSV.     

Polycomb homologs are involved in teratogenicity of valproic acid in mice

BACKGROUND: Valproic acid (VPA) is widely used to treat epilepsy and bipolar disorder and is also a potent teratogen, but its teratogenic mechanisms are unknown. We have attempted to describe a fundamental role of the Polycomb group (Pc-G) in VPA-induced transformations of the axial skeleton. METHODS: Pregnant NMRI mice were given a single subcutaneous injection of vehicle or VPA (800 mg/kg) on gestation day (GD) 8. The expression of genes encoding Polycomb and trithorax groups was measured by quantitative real-time RT-PCR using total RNA isolated from the embryos exposed to vehicle or VPA for 1, 3, and 6 hr. In addition, the use of two less teratogenic antiepileptic chemicals valpromide (VPD) and valnoctamide (VCD) provide reliable evidence to support the relationship between VPA teratogenicity and the Polycomb group. RESULTS: At a teratogenic level, VPA inhibits the expression of the Polycomb group genes, including Eed, Ezh2, Zfp144, Bmi1, Cbx2, Rnf2, and YY1 in the mouse embryos. In contrast, neither VPD nor VCD have significant effects on the expression of those genes affected by VPA. The trithorax group (trx-G) gene MLL, which is known to be required to maintain homeobox gene expression such as the Polycomb gene, is not affected by a teratogenic dose of VPA. CONCLUSIONS: We propose that, during embryonic development, VPA may affect the gene silencing pathway mediated by the Polycomb group complex. The epigenetic mechanism of VPA teratogenicity on anteroposterior patterning is suspected.     

Effects of protein deficiency on the teratogenicity of cytochalasins in mice

The developmental toxicity of cytochalasins B (CB) and D (CD) was evaluated in protein-deprived mice. Pregnant CD-1 mice were assigned to control (26%), 16%, 8%, or 4% dietary protein groups on gestation day 1 and dosed by gavage with 0 or 1.5 mg/kg CB or CD on gestation day 8 (plug = day 1). They were killed and subjected to teratological examination on day 18. CD, but not CB, increased prenatal mortality but failed to interact significantly with dietary protein level. Fetal weights were decreased in the 4% and 8% dietary protein groups, but cytochalasin treatment did not exacerbate this effect. Cytochalasin treatment was associated with gross fetal malformations, primarily neural tube defects. Although CB and CD did not significantly increase the percentage of grossly malformed fetuses per litter, the data was suggestive of such an effect, and the incidence of affected litters was increased by cytochalasin treatment in all but the 4% protein group. Skeletal defects, such as jaw malformations, rib or sternebrae variations, and unossified skull bones appeared to be increased by both cytochalasin treatment and dietary protein deficiency. The differences from control values were nonsignificant, however, except for some cases of cytochalasin effects on skull ossification. These results show a general lack of effect of protein deprivation on the developmental toxicity of cytochalasins.