Chada S. Reddy

Developmental effects of fumonisin B1 in mice

Developmental and toxic effects of aqueous extracts of F. moniliforme culture material containing known levels of fumonisin B1 were recently reported in mice and included maternal hepatotoxicity and lethality, maternal body weight gain reduction, increased embryonic resorptions, reduced offspring body weights, and fetal malformations including cleft palate, hydrocephalus, malformed ribs and incomplete digital and sternal ossification. These studies also suggested that the effects of the fungal extract on the mouse offspring may be mediated via maternal effects. The contribution of fumonisin B1 (FB1), a major toxic metabolite of F moniliforme, in the induction of these effects was evaluated in this study by administering 0 to 100 mg pure FB1/kg of body weight on gestational days (GD) 7 through 15 to pregnant Charles River CD1 mice and assessing maternal health and fetal development till the end of gestation. Doses of 25 mg/kg or higher of pure FB1 induced maternal liver lesions (mostly necrotic changes), associated with ascites and increased hepatocytic nuclear diameter. Fumonisin doses of 50 mg/kg or higher also resulted in significantly increased maternal ALT on GD12, and reduced offspring bodyweights on GD 18. Increased resorptions and decreased numbers of live offspring were only evident at 100 mg FB1/kg body weight. Offspring exhibited dose-dependent increase in the incidence and severity of hydrocephalus of both the lateral and third ventricles at doses of 25 mg/kg or higher. Doses of 25 mg/kg or higher also increased the sphinganine/sphingosine (Sa/So) ratios in maternal but not fetal livers. These results suggest that FB1 may be a developmental toxicant accounting for most but not all earlier reported effects of F. moniliforme culture extract. Association of FB1 effects on the offspring with maternal hepatoxicity and with alteration of Sa/So ratio in maternal but not fetal liver supported the earlier claim that FB1 effects on the mouse offspring are mediated by maternal hepatotoxicity.

Developmental effects of fumonisin B1-containingFusarium moniliforme culture extract in CD1 mice

Pregnant Charles River CD1 mice were treated with a semipurified extract ofFusarium moniliforme culture containing 0, 12.5, 25, 50 or 100 mg FB1/kg each day orally (diluted in distilled water) between gestational days (GD) 7 and 15 to evaluate the developmental toxicity of FB1. Following sacrifice of dams on GD 18, litters were examined for gross abnormalities and divided equally for skeletal or visceral examination by routine techniques. Significant maternal mortality was observed at doses of 50 and 100 mg FB1/kg. Dose-dependant decreases in maternal body weight gains, number of live offsprings per litter, and mean body weight of the offspring were produced at FB1 doses of 25 mg/kg or higher. The percentage of implants resorbed increased at all doses in a dose-dependant manner. A dose-dependant increase, except at the lowest dose tested, in the incidence of ossification deficits involving digits and sternum, short and wavy ribs, and hydrocephalus of lateral and third ventricles was also evident. Cleft palate was seen only at the highest FB1 dose. Maternal intoxication manifested as a dose-dependant increase in the severity of ascites associated mainly with increased histopathologic scores reflecting hepatocellular damage at day 18. Concommittant increases in serum alanine amino transferase (ALT) on GD 12, reflecting parenchymal liver cell damage, was also observed at all doses above 12.5 mg of FB1/kg. These results suggest that FB1-containingF. moniliforme culture extract is developmentally toxic in mice, and that this toxicity may be mediated by maternal hepatotoxicity.

Cell cycle proteins in normal and chemically induced abnormal secondary palate development: a review

Cell cycle progression and thus proper cell number is essential for normal development of organs and organisms. Craniofacial tissues including the secondary palate are vulnerable to disruption of cell cycle progression and proliferation by many chemicals including mycotoxin, secalonic acid D (SAD), glucocorticoids, retinoic acid and 2,3,7,8-tetrachlorodibenzodioxin. Induction of cleft palate (CP) by SAD in mice occurs from a reduction in the size of developing palatal shelves. This is associated with an inhibition of proliferation of murine and human embryonic palatal mesenchymal (MEPM and HEPM) cells as well as a G1/S block of cell cycle. In murine embryonic palates and HEPM cells, SAD inhibited G1/S-phase-specific cyclin-dependent kinase (CDK)2 activity, reduced the level of cyclin E and increased the level of the CDK2 inhibitor, p21. These results, together with those from other laboratories, suggest that common cell cycle protein targets (biomarkers), relevant to the pathogenesis of CP by multiple chemical exposures, that can form the basis for the diagnosis and the development of preventive strategies, are likely to exist.

Developmental stage specificity and dose response of secalonic acid D-induced cleft palate and the absence of cytotoxicity in developing mouse palate

Incidence of cleft palate (CP) in full-term mouse fetuses was evaluated following administration of 25 mg/kg of the mycotoxin, secalonic acid D (SAD), to groups of female mice on each of Days 10, 11, 12, 13, 14, or 15 of pregnancy. Although the highest numerical incidence (45.3%) of cleft palate resulted following SAD exposure on Day 12 of pregnancy, and the response tapered off to 16.9% on Day 10 and 0% on Day 15 of pregnancy, similar responses were produced also following exposures on Days 11 (38.4%) and 13 (39.9%) of pregnancy. Maternal exposure to doses of 0, 15, 20, 25, or 30 mg/kg of SAD, given on Day 12 of pregnancy indicated that although fetuses in the 30-mg/kg group had the highest incidence (51.9%) of CP, the effect was associated with increased resorptions and decreased fetal weights. The 25-mg/kg dose was optimally teratogenic (45.3% cleft palate) and maximally tolerable with neither an increase in resorptions nor a decrease in fetal body weights. Cytotoxicity of the optimally teratogenic dose of SAD (25 mg/kg given ip) on Day 12 of pregnancy was evaluated as a possible mechanism of SAD teratogenicity using indices such as mesenchymal cell density, mitotic index, and the uptake of [3H]thymidine in the developing palatal shelves. No evidence of SAD cytotoxicity was obtained in palatal shelves indicating a possible role for nonlethal cellular effects of SAD in the pathogenesis of CP. These studies also suggest the suitability of the maternal 25-mg/kg dose of SAD to study cellular biochemical effects in the developing embryo without the complicating influence of cytotoxic effects.

Alterations in protein kinase A signalling and cleft palate: a review

Cyclic AMP is an important second messenger mediating the actions of many hormones and other ligands in a variety of cells. Cells of the developing organism are no exception. Once generated, it releases the catalytic subunit of protein kinase A (PKA) from the inhibitory influence of its regulatory subunit, which then migrates into the nucleus to phosphorylate and enhance the binding of relevant transcription factors to the promoter element CRE of genes involved in above cellular responses. This review summarizes the available data on the essential role of this pathway in embryonic development as well as the functionality, ontogeny and consequences of genetic and chemical disruption of this pathway in the developing orofacial structures, especially the secondary palate as influenced by the mycotoxin, secalonic acid D.

Role of p21 and cyclin E in normal and secalonic acid d-inhibited proliferation of human embryonic palatal mesenchymal cells

Secalonic acid D (SAD), a cleft palate-inducing teratogen, has been shown to inhibit proliferation/cell cycle progression in association with alteration in the levels of cell cycle regulators, p21 and cyclin E. These studies were conducted to test the hypotheses that p21 and cyclin E play an important functional role in normal human embryonic palatal mesenchymal (HEPM) cell cycle and that their up- and down-regulation, respectively, by SAD is functionally significant to its cell cycle block. Using small interfering RNA (siRNA) to silence p21gene and transient transfection to overexpress cyclin E in control & SAD-treated HEPM cells, cell proliferation was assessed using a combination of cell numbers, thymidine uptake, CDK2 activity and Ki-67 expression. The results showed that silencing of p21 gene, although increased cell proliferation/numbers and CDK2 activity in normal HEPM cells, failed to counteract SAD-induced anti-proliferative effect despite inducing partial recovery of CDK2 activity. Similar effects were apparent with cyclin E overexpression. It is concluded that p21 and cyclin E are important for normal HEPM cell proliferation. However, SAD-induced deregulation of either protein, singly, may not be sufficient to induce anti-proliferative effect. Involvement of other cell cycle proteins such as cyclin D1 or of multiple proteins in SAD-induced cell cycle block needs to be examined.

Neurotoxic effects of secalonic acid D in mice during subchronic postnatal exposure

To establish a dose-response of neurotoxic effects to daily oral doses of the mycotoxin secalonic acid D (SAD), as well as to correlate the neonatal behavioral responses to smaller doses of SAD with the attendant neurochemical effects in mice, 5 neonates of each sex were placed with each mother and 4 litters were treated orally with 0 to 5 mg/kg of SAD daily from postnatal day (PND) 3 through 35. Body weights, toxic signs, and mortality were used to arrive at no observable effect level (NOEL). Performance in several behavioral tests and changes in regional norepinephrine and dopamine levels in the brains of neonates treated with SAD at NOEL (1.25 mg/kg/day) or below were evaluated at selected times during SAD exposure. Doses as low as 1.25 mg/kg/day reversibly reduced body weights in both sexes on PND 12 and 13 compared to controls, whereas doses of 2.5 mg/kg/day or greater were lethal (LD50 of 2.5 mg/kg/day). Toxic signs observable in neonates receiving 2.5 mg/kg/day or more of SAD included fine body tremors, uncoordinated movements, hindlimb weakness, circling, loss of righting reflex, paddling, and terminal coma. Ontogeny of cliff avoidance (PND 5, 7, and 9), hindlimb grip response (PND 14, 17, and 20), olfactory discrimination (PND 8 through 11) and swimming (PND 13 through 21) were significantly delayed by SAD exposure: some even at 0.625 mg/kg/day. Dopamine levels significantly increased on PND 13 and decreased on PND 20 only in the olfactory lobe of SAD-exposed neonates. Norepinephrine levels were unchanged in all the brain regions examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C and chemical-induced abnormal palate development

The protein kinase C (PKC) family of proteins mediates the action of growth factors and other ligands by activating a network of transcription factors that bind to TRE sequences in the promoters of many genes that regulate cell proliferation, differentiation, extracellular matrix synthesis, apoptosis and others in a cell type-, isozymeand context-specific manner. The critical role of PKC in embryonic development is indicated by early death of embryos in which one or more of these isozymes are inactivated. Our studies together with others show that palatal PKC signalling is functional and may be essential for normal palate development. Although single gene knockouts have failed to exhibit the cleft palate (CP) phenotype, owing to compensation by other kinases, many chemicals including the mycotoxin, secalonic acid D, disrupt palatal PKC signalling leading to altered palatal mesenchymal gene expression. The potential relevance of such effects to chemical-induced CP is discussed.

Evaluation of Sinclair Miniature Swine as an Osteopenia Model

Postmenopausal osteoporosis is a chronic, disabling disease. The high prevalence of osteoporosis in elderly people, particularly women, is becoming increasingly important as baby boomers are reaching retirement age and life expectancy in industrialized countries is increasing.1,2 In addition to the reduced quality of life suffered by affected people, osteoporosis is a substantial contributor to the burden of health care costs frequently due to prolonged treatment and hospitalization from osteoporotic fractures.1,2 Although risk factors contributing to osteoporotic fractures, such as gonadal failure following menopause, lower peak bone mass, lack of physical activity, genetic and nutrition, are well identified, the deficit in understanding of osteoporosis pathogenesis makes the search for therapeutic agents difficult.3,4

Vaccination Study with the Sinclair Miniature Swine - Effect of Vaccine Dose and Litter

Prophylactic protection of miniature swine, critical to the outcome of research using these animals, received little attention in the past. Lack of approved commercially available vaccines contributed to our lack of knowledge of the efficacy of vaccination and the observed anaphylactoid reactions following the use of conventional swine bacterin. To evaluate the serologic response and clinical effect of various doses of a commercial vaccine (Actinobacillus pleuropneumoniae-Erysipelothrix rhusiopathiae), 20 titer negative male Sinclair miniature swine (between 41 and 43 da old) from 5 litters were divided randomly in 4 groups based on their litter of origin and were administered 2 IM doses of the vaccine (21 da apart) at 2 and 2, 1 and 2, 1 and 1, and 0.5 or 1 ml. Behavior changes and rectal temperatures were monitored following vaccination. Two different muscle vaccination sites were used to detect any immediate or delayed local reactions. Body weights and blood samples were collected at 0, 21 and 42 da. Serology titers were determined using an ELISA assay specific to Actinobacillus pleuropneumoniae serotypes 1, 5 and 7, which are present in the commercial vaccine. The highest dose regimen yielded a higher serologic titer than the other regimens only 21 da after the second vaccination. The litter of origin had a strong effect on post vaccination serologic titers with some litters having very low titers mainly after the first vaccination. No clinical effects were noted following vaccination, but nodules were palpated at the vaccination site which gradually regressed within 2 mo. Body weights and rectal temperature elevation did not affect the titers. The litter of origin heavily influences the outcome of the vaccination and the highest dose regimen is necessary to provide the best protection.