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Biomedical subjects

R M Pratt

Publications and source records attributed to R M Pratt.

At least 37 records · Page 2Linked to original sources
PubMed1987

Human embryonic palatal epithelial differentiation is altered by retinoic acid and epidermal growth factor in organ culture.

Reports of adverse human pregnancy outcomes including cleft palate have increased as the clinical use of isotretinoin (13-cis-retinoic acid) and other retinoic acid (RA) derivatives have increased, but the mechanisms by which their effects are exerted are not understood. Research in craniofacial development is generally performed in rodents, and mouse palatal shelves exposed in organ cultures to retinoids and epidermal growth factor (EGF) display altered medial epithelial cell morphology blocking normal union of apposing shelves. In the present study, precontacting human palatal shelves were maintained in organ culture for 2, 3, or 6 days and exposed to labeled thymidine (3H-TdR) during the last 16 hr. Retinoids and EGF were included in the media so that each shelf was exposed to one of the following: control, EGF, trans-RA at 10(-5)M, cis-RA at 10(-7) or 10(-9) M, or RA + EGF. After exposure of cultured human embryonic palatal shelves to 13-cis-RA and trans-RA with or without EGF, medial epithelial cells do not degenerate, cell surface morphology shifts toward a nasal type, glycogen deposits decrease, smooth endoplasmic reticulum (SER) increases, and basal lamina appear altered. In shelves exposed to EGF and trans-RA early in their development, DNA synthesis appears to terminate prematurely as compared to shelves cultured in control media, and this effect is accompanied by excessive mesenchymal extracellular space expansion. Exposure of shelves to EGF alone is sufficient to block degeneration and induce hyperplasia of the medial epithelial cells but does not induce other ultrastructural changes seen with both EGF and RA. The observed alterations in medial cell morphology could interfere with adhesion of the palatal shelves and may play a role in retinoid-induced cleft palate in the human embryo.

Cell Differentiation↗
PubMed1986

Karyotype, growth, and cell cycle analysis of human embryonic palatal mesenchymal cells: relevance to the use of these cells in an in vitro teratogenicity screening assay.

Human embryonic palatal mesenchyme (HEPM) is an established cell line that is presently under investigation as an in vitro prescreening assay used to determine the teratogenic potential of chemicals. We describe here general growth characteristics, karyotype, and cell cycle analysis of these cells. HEPM cells had plating efficiencies of less than 95% and displayed notable contact growth inhibition following an exponential growth phase that lasted for approximately 6 days. These cells had the diploid karyotype of a female human embryo. The chromosomal complement showed no dramatic change between passage 5 and 14. Flow cytofluorometry analysis using bromodeoxyuridine (BrdU) pulse labeling and a direct immunofluorescence anti-BrdU FITC probe revealed that the total cell cycle transit time was approximately 22 hr: the duration of G1 was 12.2 hr, S was 6.1 hr, and G2-M lasted for 3.7 hr. The results indicate that HEPM cells met the criteria regarding karyotype stability that were assumed by the National Toxicology Program of the USA.

Bromodeoxyuridine↗
PubMed1986

Synergistic interaction of 2,3,7,8,-tetrachlorodibenzo-p-dioxin and hydrocortisone in the induction of cleft palate in mice.

Glucocorticoids cause cleft palate in sensitive mouse strains by interfering with the proliferation of mesenchymal cells in the palatal shelves; 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) also causes cleft palate, but its effects involve the epithelial cells. The purpose of this study was to examine the interaction of TCDD and the glucocorticoid hydrocortisone in the induction of malformations. Pregnant C57BL/6N mice were treated on gestation days 10-13 with TCDD (0 or 3 micrograms/kg, p.o.), hydrocortisone (0, 25, 50, or 100 mg/kg, s.c.) or a combination of TCDD and hydrocortisone. The dams were killed on gestation day 18 and the mice were analyzed for maternal and fetal toxicity and soft tissue malformations. TCDD alone had no effect on litter size, fetal weight or viability, or maternal weight gain. This dose of TCDD is essentially a threshold dose and it did not produce cleft palate in this study, but all the TCDD-treated fetuses had hydronephrosis, the most sensitive indicator of TCDD teratogenicity. Hydrocortisone alone caused dose-related decreases in fetal weight and maternal liver/body weight ratios, and dose-related increases in cleft palate (0, 5, 10, and 30%). No effects of hydrocortisone were detected on litter size or fetal viability, but maternal weights were affected. Combination of all doses of hydrocortisone with TCDD resulted in a 100% incidence of cleft palate, accompanied by a decrease in litter size and fetal weight and an increase in fetal mortality related to the dose of hydrocortisone. TCDD tended to reverse the decrease in liver/body weight ratio seen with hydrocortisone alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Abnormalities, Drug-Induced↗
PubMed1986

Isotretinoin teratogenicity in mouse whole embryo culture.

Recent clinical observations strongly suggest that isotretinoin [13-cis-retinoic acid (cis RA)] is a human teratogen causing primarily heart and craniofacial malformations including ear and palatal defects. The purpose of the present study was to determine if cis RA could induce similar craniofacial malformations in mouse embryo culture. Day 8 CD-1 mouse embryos were cultured for 48 hours in rat serum in the presence or absence of various concentrations of cis RA dissolved in DMSO. DMSO by itself had no effect on embryonic development; however, cis RA at 2 X 10(-5) M (6 micrograms/ml) was clearly toxic. At 2 X 10(-6) M cis RA, growth retardation was minimal, and approximately one-third of the embryos exhibited very specific defects including a dramatic reduction in the size of the first and second visceral arches, which eventually give rise to the maxilla, mandible, and ear. Similar observations were also made with 4-oxo-13-cis RA, which is a major metabolite of cis RA in the mouse and human. These malformations would be expected to result in defects similar to those observed in the human, and preliminary observations suggest these defects are due to cis RA-induced inhibition of cranial neural crest cell migration. Using day-10 mouse embryos cultured for 48 hours in Waymouth's medium containing 50% fetal calf serum, we observed that cis RA at 2 X 10(-5) M produced a high percentage of embryos with limb defects and median cleft lip. Our results demonstrate that labeled cis RA enters the tissues of the embryo both in vivo and in vitro. Cis RA inhibited proliferation of the frontonasal mesenchyme cells in primary culture with 31% inhibition occurring at 2 X 10(-5) M cis RA.

Abnormalities, Drug-Induced↗
PubMed1986

Studies on phosphatidylinositol metabolism and dexamethasone inhibition of proliferation of human palatal mesenchyme cells.

The relationship between dexamethasone (DEX)-induced phosphatidylinositol (PI) turnover and inhibition of cell proliferation was investigated in human embryonic palatal mesenchyme (HEPM) cells in culture. Evidence based on studies with the partial glucocorticoid agonist cortexolone suggests both the PI response and the inhibition of proliferation are mediated by the glucocorticoid receptor. The role of PI turnover in the mechanism of DEX-inhibited HEPM cell proliferation was investigated using two agents that stimulated PI turnover (serum and platelet-derived growth factor) and one that did not stimulate PI turnover (epidermal growth factor). DEX partially inhibited both serum-induced and platelet-derived growth factor-induced proliferation of HEPM but not epidermal growth factor-induced proliferation. These results suggest that DEX-induced alteration of PI metabolism may be involved in the mechanism by which DEX inhibits proliferation of cultured HEPM cells and results in cleft palate formation in rodents.

Cell Division↗
PubMed1985

Inhibition of arachidonic acid metabolism is not involved in dexamethasone-induced growth inhibition in embryonic palatal development.

Previous studies have shown that glucocorticoids induce cleft palate in susceptible strains of mice and inhibit proliferation of palatal mesenchyme cells in vivo and in culture. The present study shows that the synthetic glucocorticoid, dexamethasone (DEX), inhibits serum-stimulated arachidonic acid release in cultured mouse palatal mesenchyme cells. Arachidonic acid could neither prevent the DEX effect on cell proliferation when added in culture nor prevent glucocorticoid-induced cleft palate when administered in vivo. Furthermore, the time course for DEX-induced inhibition of arachidonic acid release (maximal by 5h) is markedly different from the time courses for both inhibition of cell proliferation in culture and cleft palate induction in vivo (3 to 4 days). These results suggest that both DEX-induced cleft palate formation and inhibition of palatal cell proliferation arise from some mechanism other than a DEX-induced inhibition of arachidonic acid metabolism.

Animals↗
PubMed1985

In vitro screening assay for teratogens using growth inhibition of human embryonic cells.

We have tested 35 teratogenic and 20 nonteratogenic chemicals or drugs in a short-term, in vitro assay that identifies teratogens by their ability to inhibit growth of an established line of human embryonic palatal mesenchymal cells. Only those chemicals that exhibited a dose-dependent inhibition of growth at concentrations less than 1 mM were classified as inhibitory. An Aroclor-induced rat liver S-9 system was effective in metabolizing cyclophosphamide to its teratogenic form in culture. We suggest that this assay, along with the complementary tumor cell-attachment assay of Braun et al. [Braun, A. G., Emerson, D. J. & Nichinson, B. B. (1979) Nature (London) 282, 507-509] may be useful as a short-term in vitro battery for assessment of the teratogenic potential in environmental agents and to prioritize those chemicals which merit further testing in vivo.

Biotransformation↗
PubMed1985

Effect of epidermal growth factor/urogastrone on glycosaminoglycan synthesis and accumulation in vitro in the developing mouse palate.

Epidermal growth factor/urogastrone (EGF-URO) has previously been implicated in murine secondary-palate formation. We report here that, in correlation with its effects on palate fusion, EGF-URO in physiological amounts (1.7 nmol/l) markedly affects glycosaminoglycan (GAG) production in organ cultures of mouse palate tissue; the effects of EGF-URO are dependent on the developmental stage of the palate. GAG production, particularly that of hyaluronic acid (HA), is stimulated two- to eight-fold by EGF-URO in cultures of palate tissue obtained between days 11-12 and 13-15 of development; by the time of birth, EGF-URO no longer stimulates GAG production in such cultures. EGF-URO increases the amount and alters the distribution of HA within the palate. The results suggest a role for EGF-URO and for HA in the process of normal palatal development.

Animals↗
PubMed1985

Receptor-dependent mechanisms of glucocorticoid and dioxin-induced cleft palate.

Glucocorticoids (triamcinolone) and dioxins (TCDD) are highly specific teratogens in the mouse, in that cleft palate is the major malformation observed. Glucocorticoids and TCDD both readily cross the yolk sac and placenta and appear in the developing secondary palate. Structure-activity relationships for glucocorticoid- and TCDD-induced cleft palate suggest a receptor involvement. Receptors for glucocorticoids and TCDD are present in the palate and their levels in various mouse strains are highly correlated with their sensitivity to cleft palate induction. Receptors for glucocorticoids appear to be more prevalent in the palatal mesenchymal cells whereas those for TCDD are probably located in the palatal epithelial cells. Glucocorticoids exert their teratogenic effect on the palate by inhibiting the growth of the palatal mesenchymal cells whereas TCDD alters the terminal cell differentiation of the medial palatal epithelial cells.

Animals↗
PubMed1984

2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced cleft palate in the mouse: evidence for alterations in palatal shelf fusion.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes a high percentage of cleft palate in fetuses when administered during organogenesis in certain strains of mice including the C57BL/6J, but not in certain other strains (AKR/J). The purpose of the present study was to examine various biochemical and morphological aspects of TCDD-induced changes in the developing palatal shelves. Our results indicate that when TCDD (100 micrograms/kg) was given on individual days between days 8 and 10 of gestation, a high percentage of cleft palate was observed. Receptors specific for TCDD were detected in the C57BL/6J but not AKR/J palatal shelves. The amount of TCDD receptors is highest in the palatal shelves on day 13 as compared to other embryonic tissues including the liver. Examination of cryostat sections taken from embryos during the time of palatal elevation and fusion demonstrated that TCDD does not interfere with growth, elevation, or initial contact of the palatal shelves, but does interfere with firm adhesion and/or degeneration of the medial epithelial cells. Our results suggest that TCDD exerts a direct effect on the embryonic palatal shelves which results in formation of cleft palate.

Animals↗
PubMed1984

Glucocorticoid teratogenesis in mouse whole embryo culture.

Glucocorticoids, such as triamcinolone acetonide (TAC-A) and triamcinolone hexacetonide (TAC-HA), are potent inducers of cleft palate in vivo in various mouse strains when administered on day 11 of gestation, whereas they are poor or ineffective inducers of cleft lip when given on day 7. The purpose of the present study was to determine whether glucocorticoids are capable of interfering with early embryonic development in culture. CD-1 mouse embryos were cultured for 48 hours starting either on day 8 (plug day 0) with the embryo inside the yolk sac, or on day 10 with the embryo exteriorized from its functional yolk sac. At the end of the culture period, embryos were examined grossly for malformations and biochemically for altered DNA and protein levels. With the day 8 cultures, TAC-A produced a dose-dependent inhibition of growth along with malformations consisting of cardiac irregularities, abnormal rotation, and irregular neural tube closure. With the day 10 cultures, these malformations were not observed, presumably due to the advanced stage of development when the embryos were exposed to TAC-A; however, TAC-A did produce growth inhibition along with cleft lip. When TAC-HA was administered in vivo to pregnant donor females on day 7, in combination with TAC-A added on day 10 to the culture medium, there was a dramatic increase in the frequency of cleft lip along with other alterations in craniofacial appearance. Our results demonstrate that glucocorticoids are capable of directly affecting embryonic growth and development during the early stages of organogenesis.

Abnormalities, Drug-Induced↗
PubMed1984

Influence of epidermal growth factor and cyclic AMP on growth and differentiation of palatal epithelial cells in culture.

A serum-free, hormonally defined medium was developed which supports growth and differentiation in primary culture of epithelial cells from prefusion embryonic mouse palatal shelves. Using this culture system, medial epithelial programmed cell death was investigated. In the absence of EGF, medial epithelial cells undergo cell death and detach from the substratum by 24 hr of culture. The addition of EGF alone or in combination with various agents which increase intracellular cyclic AMP levels prevented medial epithelial cell death in both cell and organ culture. EGF appeared to exert its most dramatic effect in cell culture on growth and differentiation of the squamous oral epithelial cells. In addition, EGF and agents such as 8-bromo-cyclic AMP, dibutyryl cyclic AMP, or cholera toxin synergistically stimulated the appearance of a long-lived, rapidly proliferating cell type by Day 4 of culture. Our results suggest that both EGF and cyclic AMP together may be important in regulating proliferation of embryonic palatal epithelial cells.

Animals↗
PubMed1984

Role of glucocorticoids and epidermal growth factor in normal and abnormal palatal development.

The purpose of this chapter has been to discuss glucocorticoid and EGF involvement in normal and abnormal palatal development. It is to be hoped that we have made clear the important point that these hormone/growth factors and their receptors are present during normal embryonic palatal development to provide for regulation of growth and cellular differentiation. When these hormone/growth factors are administered in pharmacological or large doses that result in teratogenesis, these potent chemicals and their receptors then become inducers of cleft palate. The primary reason for this is that the hormone/growth factor receptors have unique and special areas of localizations in target (embryonic and fetal) tissues, e.g., glucocorticoids in the palate. Therefore, large amounts of these chemicals are specifically bound to receptors in these target tissues and these high levels of hormone/growth factor-receptor complexes result in aberrant development, e.g., glucocorticoids cause inhibition of palatal mesenchymal cell growth. These effects are distinct from the interactions of physiological levels of these hormone/growth factors with their receptors in these target tissues during development, e.g., glucocorticoids cause induction of key enzymes and modulation of EGF receptor levels. The exact molecular mechanism(s) by which high levels of hormone/growth factors--receptor complexes exert harmful effects on embryos or fetuses is (are) unknown and remain(s) a challenge for the future. Interaction of hormone/growth factors and their receptors certainly cannot provide an explanation for the mechanism of all types of craniofacial teratogenesis, but this concept certainly appears capable of providing important information relating to the mechanisms of many animal and human teratogens. The fact that these chemicals and their receptors are involved in normal development makes them all the more important since subtle alterations in their levels or activities could result in teratogenesis without an exposure to pharmacological levels of these hormone/growth factors. It seems that progress in this area will develop quickly since the techniques of recombinant DNA research are available in conjunction with responsive in vitro cell systems such as the established line of human embryonic palatal mesenchymal cells. Clearly, the future looks very exciting for understanding the role that these hormone/growth factors and their receptors play in normal and abnormal palate development.

Animals↗
PubMed1984

Epidermal growth factor stimulates type-V collagen synthesis in cultured murine palatal shelves.

The effects of epidermal growth factor (EGF) on the synthesis of collagen and fibronectin in murine palatal shelves in serum-free organ culture have been examined. Palatal shelves grown in the presence of EGF were substantially larger and showed dramatic increases in immunofluorescent localization of fibronectin, possibly reflecting increased synthesis compared to controls cultured without EGF. EGF also prevented the normal dissolution of the medial palatal epithelium. Net protein synthesis in experimental palatal shelves increased by 18%, whereas net collagen synthesis was approximately 10% of protein synthesis in both conditions. There was, however, an apparent shift in collagen isotypes. Under the influence of EGF, there was a significant increase in synthesis of the alpha 1 (V) and alpha 2 (V) chains of type-V collagen.

Animals↗
PubMed1984

Immunocytochemical localization of glucocorticoid receptors in midgestation murine embryos and human embryonic cultured cells.

Glucocorticoid receptors have been localized immunocytochemically in the developing mouse secondary palatal shelves and in cultured human embryonic palatal mesenchyme cells. In the midgestation embryo, receptors are found in the highest concentration in the palatal mesenchymal cells, suggesting that they play a major role in normal development as well as in glucocorticoid-induced cleft palate. The presence of these receptors in cultured human embryonic palatal cells also suggests that development of the human secondary palate may be dependent on glucocorticoids.

Animals↗
PubMed1983

Dexamethasone affects phosphatidylinositol synthesis and degradation in cultured human embryonic cells.

Dexamethasone (DEX), a glucocorticoid which induces cleft palate, causes marked alterations in the synthesis and degradation of phosphatidylinositol (PI) but not phosphatidylcholine in an established fibroblastic cell line derived from a human embryonic palate. Incorporation of radiolabeled inositol into phosphatidylinositol as well as degradation of prelabeled phosphatidylinositol is stimulated by DEX. The dose-response curves for the DEX-induced effect on PI synthesis and DEX-induced inhibition of cell proliferation are nearly identical, with the maximal responses occurring at 10(-8)M DEX. Our results suggest that DEX-induced inhibition of human embryonic palatal mesenchyme cell proliferation and alterations in synthesis and degradation of phosphatidylinositol are related.

Cell Division↗
PubMed1983

Specific craniofacial defects induced by jervine in the cultured rat embryo.

The Veratrum-derived steroidal alkaloid, jervine, induces cyclopia and limb malformations in sheep, and various other craniofacial malformations in several other mammalian and avian species. In the present study, the question whether jervine acts directly or indirectly on mammalian embryos to produce malformations and the nature of the target tissue or cells were examined using whole-embryo cultures of the CD rat. Embryos were explanted into culture at the presomite, early neurula stage and cultured in the presence or absence of jervine for 48 hours. Jervine (at 1-5 micrograms/ml) induced an oblong-head appearance and ventrally displaced optic vesicles, with little or no other effects observed on overall growth and development. The specific target tissue in the embryo was found to be the cranial neuroepithelium. This specificity of action is quite unusual since most teratogens examined in whole-embryo culture to date have various nonspecific effects on embryonic growth and differentiation.

Animals↗