Deborah K. Hansen

Leukoencephalomalacia and hemorrhage in the brain of rabbits gavaged with mycotoxin fumonisin B1.

Two of five pregnant rabbits gavaged with purified fumonisin B1 at 1.75 mg/kg/day died, one after 9 and one after 13 doses. Microscopic examination revealed focal small hemorrhages in cerebral white matter in both animals, with malacia and hemorrhage also present in the hippocampus of one. The lesions were bilateral. Both animals also had marked degeneration of renal tubule epithelium and of hepatocytes. Apoptosis was the dominant degenerative change in kidney and liver. Fumonisin is known to cause leukoencephalomalacia and hemorrhage in equines, but CNS changes associated with exposure to fumonisins apparently have not been reported in other species. This preliminary observation in rabbits is reported to alert other investigators of a potential model of the disease in equines, as well as for investigation of potential mechanisms of toxicity to the CNS.

Renal Effects of Fumonisin Mycotoxins in Animals

Fumonisins are mycotoxins produced worldwide by Fusarium fungi, principally F. moniliforme. The fungus is present in virtually all harvested com, but the toxins produced are variable. The toxins, especially fumonisin B1, cause mild to fatal diseases in animals, with peculiar species specificity for the dominant signs of toxicity. The mechanism of toxicity is poorly understood, but it appears to be related to interference with sphingolipid biosynthesis in multiple organs. Whereas brain, lung, and liver are well-known target organs, toxic effects on the kidney are also widespread and have only recently begun to be characterized. Increased urine volume and decreased osmolality are early changes associated with the toxin, as are increased excretions of high-and low-molecular-weight proteins. Enzymuria in vivo, reduced ion transport in vitro, and elevation of free sphinganine in renal tissue and in urine are present. An increase in serum creatinine and blood urea nitrogen and histopathologic change in renal tubules occur later and at higher doses. The morphologic change principally affects the junction of cortex and medulla and includes prominent apoptosis of epithelial cells of proximal convoluted tubules. Nephrotoxicity has been reported in several species, and in rats and rabbits, the kidney appears to be the most sensitive target organ.

Prenatal dexamethasone exposure in rats : effects of dose, age at exposure, and drug-induced hypophagia on malformations and fetal organ weights

Glucocorticoids cause stunting and cleft palate in rodents. The aim of this study is to identify fetal organs and developmental periods sensitive to stunting induced by maternal exposure to dexamethasone (DEX). DEX (0.2 or 0.4 mg/kg) or saline was given sc to pregnant CD albino rats on Gestation Days (GD) 9-14 or 14-19. On GD 20 dams were euthanized. Fetuses were weighed and examined for cleft palate. Eight fetuses/litter were randomly selected, and weights were obtained. Fetal skeletons were examined for abnormalities, and long bone measurements were taken. A dose-related decrease in maternal and fetal body weights occurred at both exposure periods. Developmental stage-specific malformations were noted in the high-dose group on GD 9-14 (cleft palate) and on GD 14-19 (wavy ribs). A dose-response in stunting occurred in all organs except cerebellum in at least one exposure period. Across both exposure periods the brain, heart, testes, and long bones were relatively resistant to DEX. Sensitive organs included thymus, spleen, adrenals, lungs, liver, and kidneys. DEX substantially reduced maternal food intake and increased water intake in some dams. Pair-feeding experiments suggested that the hypophagic effect of DEX was not responsible for the noted malformations and had little impact on growth stunting. The present findings have identified fetal organs, skeletal regions, and developmental periods sensitive to DEX exposure.

Lack of embryotoxicity of homocysteine thiolactone in mouse embryos in vitro.

Recent work from humans and chick embryos has suggested that homocysteine may play a role in producing neural tube defects (NTDs). In an effort to determine if homocysteine is able to produce NTDs in mammalian embryos, mouse embryos were explanted on GD 8 and cultured for 44 h. When either homocysteine or homocysteine thiolactone was added to the culture medium, treated embryos developed as well as controls and had closed neural tubes. Homocysteine thiolactone was also microinjected into the amniotic sac of mouse embryos. Again, development proceeded normally with no significant increase in the number of embryos with open neural tubes at the end of the culture period. HPLC analysis of embryonic thiols 24 h after microinjection revealed a significant increase in embryonic cystathionine levels. These data suggest that homocysteine does not produce NTDs in mouse embryos cultured in vitro and that early organogenesis-stage embryos are able to metabolize homocysteine.

Alterations in maternal plasma corticosterone levels following treatment with phenytoin

The mechanisms of the embryotoxic effects of the anticonvulsant drug, phenytoin (PHT), are unknown. Glucocorticoids and PHT demonstrate similar embryopathic effects and strain sensitivity in that A/J mice are very sensitive to the embryopathic effects of synthetic glucocorticoids and PHT while C57BL/6 (B6) mice are comparatively resistant to both. It is possible that teratogenic consequences of PHT are not a result of drug interaction at the target site but are mediated indirectly by glucocorticoids. In this study, PHT was administered by intraperitoneal injection at 25 (a nonteratogenic dose) or 75 mg/kg body weight (a teratogenic dose) to pregnant A/J mice on Day 10 of gestation. Mice of the B6 strain received the drug at 75 mg/kg on Day 10. Control mice received vehicle (pH 11.0 distilled water). Dams were killed at various times after the injection; plasma samples were obtained, and corticosterone levels were determined by radioimmunoassay. In control animals, maternal plasma corticosterone levels were elevated soon after dosing but gradually declined, except for an apparent circadian rhythm effect seen in samples obtained in the afternoon. Administration of a nonteratogenic dose of PHT to A/J mice caused a temporary increase in plasma corticosterone levels which decreased to the control level between 6 and 24 hr following dosing. Treatment with a teratogenic dose in A/J mice led to plasma levels that remained elevated for the entire 48-hr period examined in this study. In B6 mice, treatment with 75 mg/kg increased plasma corticosterone levels for 24 hr, after which they declined to the control value by 30 hr. The adrenal corticosteroid response of A/J mice to PHT appeared to be much more sensitive than that of B6 mice, and there appeared to be a relationship between plasma levels of PHT and corticosterone. The lengthy increase in plasma corticosterone during organogenesis may be a factor in the increased incidence of cleft lip and palate seen after administration of PHT to A/J mice.

Retinoic acid induction of stress proteins in fetal mouse limb buds

Retinoic acid (RA) is teratogenic in rodent embryos. Several teratogens have been shown to induce the synthesis of a subset of heat shock proteins (stress proteins) in Drosophila. To determine if RA induces the synthesis of these proteins in rodent embryos, pregnant ICR mice were dosed with 100 mg/kg RA on Day 11 of gestation. Forelimb buds were removed from embryos 2.5 hr post-RA-treatment and nuclei were isolated, stained, and sorted from stages of the cell cycle. Nuclear proteins were extracted and analyzed by two-dimensional polyacrylamide gel electrophoresis. Nuclear proteins with molecular weights of approximately 84 and 25 kDa were synthesized in embryos in the G0 + G1 phase after pregnant dams were treated with RA. Isoelectric points, molecular weights, immunochemical blotting, and polypeptide mapping demonstrated that these proteins are indistinguishable from stress proteins isolated under a variety of conditions from rat submaxillary glands and mouse lymphoma cells. These results suggest that treatment with RA induces the synthesis of a subset of stress proteins; the role of these proteins in the teratogenic effects of RA is not known.

Effects of fumonisin B1 in pregnant rats. Part 2

The developmental toxicity of purified fumonisin B1 (FB1), a mycotoxin from the common corn fungus Fusarium moniliforme, was examined in Charles River rats. Pregnant rats were dosed orally on gestation days 3-16 at 0, 6.25, 12.5, 25 or 50 mg FB1/kg body weight/day. FB1 was not teratogenic at the doses tested. At 50 mg/kg, maternal toxicity (inappetence, emaciation, lethargy, death, resorption of entire litters) and foetal toxicity (increased number of late deaths, decreased foetal body weight, decreased crown rump length, increased incidence of hydrocephalus, increased incidence of skeletal anomalies) were seen. The foetal toxicity observed at 50 mg/kg may be related to maternal toxicity. Histopathological evaluation of tissues from dams of control and all treated groups revealed dose-related toxic changes in kidney and liver tissues. Acute toxic tubular nephrosis was seen in kidneys from all treated groups. Hepatocellular cytoplasmic alteration and individual cellular necrosis of the liver was seen in the two high-dose groups. Sphinganine (Sa) and sphingosine (So) were measured in day-17 adult and foetal tissues. Dose related increases in Sa/So ratios were seen in maternal liver, kidney, serum and brain, but there was no effect on foetal liver, kidney and brain. These data suggest that FB1 does not cross the placenta and further suggest that the observed foetal toxicity is a secondary response to maternal toxicity.

The embryotoxicity of phenytoin : an update on possible mechanisms

Summary and Conclusions PHT has multiple effects in adult humans and animals, and there is no reason to assume that it will not have multiple effects in embryos and fetuses. Although one of the first associations between anticonvulsant therapy and an adverse development effect in humans was noted in 1964 (104), the mechanism(s) whereby these adverse effects occur has thus far eluded research efforts. In this review, I have focused on three possible mechanisms. Overall, the evidence does not appear to implicate folate deficiency in PHT-induced embryotoxicity. A role for glucocorticoids or interaction between PHT and the glucocorticoid receptor has not been ruled out. However, a significant amount of work remains to be done to examine the involvement of the arachidonic acid cascade in PHT-induced embryotoxicity in vivo. The bulk of the experimental evidence would seem to favor a role for the generation of a reactive intermediate and its subsequent binding to embryonic macromolecules. This metabolite(s) has not been identified. Additionally, the association between covalent binding of metabolites and embryotoxicity remains simply an association; a causal relationship has not been established. Much work remains to be done to determine whether any of these possibilities, some other possibility, or a combination of several mechanisms will explain the adverse developmental effects of this very important, therapeutically useful anticonvulsant. The author would like to thank Drs. Ruth Billings and Daniel M. Sheehan for their helpful discussions and critical reviews of the manuscript.

Effects of silver nanoparticles on human and rat embryonic neural stem cells

Silver nano-particles (Ag-NPs) are becoming increasingly prevalent in consumer products as antibacterial agents. The increased use of Ag NP-enhanced products will almost certainly increase environmental silver levels, resulting in increased exposures and the potential for increased adverse reactions including neurotoxic effects. In the present study, embryonic neural stem cells (NSCs) from human and rat fetuses (gestational day-16) were used to determine whether Ag-NPs are capable of causing developmental neurotoxicity. The NSCs were cultured in serum free medium supplemented with appropriate growth factors. On the eighth day in vitro (DIV 8), the cells were exposed to Ag-NPs at concentrations of 1, 5, 10, and 20 μg/ml for 24 h. The cultured cells then were characterized by NSC markers including nestin and SOX2 and a variety of assays were utilized to determine the effects of Ag-NPs on NSC proliferation and viability and the underlying mechanisms associated with these effects. The results indicate that mitochondrial viability (MTT metabolism) was substantially attenuated and LDH release was increased significantly in a dose-dependent manner. Ag-NPs-induced neurotoxicity was further confirmed by up-regulated Bax protein expression, an increased number of TUNEL-positively stained cells, and elevated reactive oxygen species (ROS). NSC proliferation was also significantly decreased by Ag-NPs. Co-administration of acetyl-L-carnitine, an antioxidant agent, effectively blocked the adverse effects associated with Ag-NP exposure.

Retinoic acid-induced stress protein synthesis in the mouse

We have previously demonstrated that stress proteins (SPs) are synthesized in tissues in which malformations are later observed following treatment with the developmental toxicant, retinoic acid (RA), on day 11 of gestation (GD 11). These proteins were not synthesized in tissues which did not present with malformations near partuition. The purpose of the present investigation was to determine if this correlation between early SP synthesis and later malformation was present at other times during gestation. CD-1 strain mice were dosed orally with corn oil or 100 mg/kg body weight RA on GD 10 or 13. Some of the mice in each group were given an intraperitoneal injection of 3H-leucine to label embryonic protein synthesis one hour after dosing with RA. These animals were sacrificed 1.5 hour later, and embryonic protein synthesis was determined by two-dimensional gel electrophoresis followed by autoradiography. Other animals in each group were sacrificed on day 17 of gestation, and fetuses were examined for the presence of malformations. Following treatment with RA on day 10 of gestation, malformations were observed in the forelimbs, the hindlimbs and the tail; heart defects were not observed. SPs of 20-25,000 and 90,000 relative molecular mass (Mr) were synthesized in the forelimb bud and tail; in addition, a second low molecular weight (20-25,000) and a 84,000 Mr SPs were synthesized in forelimb buds. No SPs were synthesized in the hindlimb bud or the heart. Following RA treatment on GD 13, cleft palate was observed in 58% of fetuses; no other malformations were found. Proteins of 34,000, 84,000 and 90,000 Mr were synthesized in craniofacial tissue; SPs were not observed in forelimb bud, hindlimb bud, heart or tail tissues at this time. Therefore, it appears that there may be a correlation between tissue-specific SP synthesis early in organogenesis and the presence of a malformation later in gestation.