Teratogenicity of arotinoid ethyl ester (RO 13-6298) in mice

Arotinoid ethyl ester (RO 13-6298) is a new and very potent retinoid that exerts a profound influence on epithelial and mesenchymal differentiation in doses 500 times lower than those of compounds of the first and second retinoid generation. In the present study the teratogenicity of arotinoid ethyl ester was investigated in NMRI mice employing different treatment schedules. Recording of abnormalities was performed on day 18 (day 0 = day of conception) according to Wilson and with cleared skeletal preparations. Intraperitoneal application of the drug at a dosage of 10 micrograms/kg/day for three consecutive days (days 9-11 or 12-14) caused severe malformations, particularly in the skeletal system and the cavernous organs. Skeletal elements were reduced in number, shortened, or abnormally shaped. Ossification was diminished. Atresia of anus and urethra were frequent. Single application of 200 micrograms/kg between days 8 and 14 also caused multiple and severe malformations. However, no stage-specific pattern of abnormalities was detectable. Some skeletal malformations indicated more or less vulnerable stages that were in concordance with special developmental steps. Others, however, seemed to be equally susceptible over a longer period, eg, rays 1 and 5 of the hand or foot and the development of the mandibular joints. The pattern of abnormalities caused by these very low doses of RO 13-6298 is comparable to that obtained with other retinoids and is achieved within the same relative dose-response range. Preconceptional treatment of the animals did not induce any malformations.     

Suppression by cyclohexanetriones of retinoic acid-induced cartilage degradation in vitro and teratogenicity in vivo

In cultured fetal rat bones, cyclohexanetriones that stimulate prostaglandin synthesis inhibited retinoic acid-induced cartilage degradation in a dose-dependent manner. The inhibition by the cyclohexanetrione Ro 31-0521 was reversible, indicating that the effect was not due to cytotoxicity. Excess retinoic acid is teratogenic in rats and adversely affects the normal differentiation of various morphogenetic systems, depending on the time of administration. The following retinoic acid-induced malformations were suppressed by Ro 31-0521: malformations of long bones and of apical phalanges induced on days 13 and 15 of gestation, respectively; spina bifida and tail malformations induced on day 11 of gestation and cleft palate induced on day 15 of gestation. However, cleft palate and other head malformations including exencephaly induced by retinoic acid on day 11 of gestation were not suppressed but even increased by Ro 31-0521. At a high dose, Ro 31-0521 given alone on day 11 of gestation was embryolethal and teratogenic but was not on the tested other days, indicating that the cyclohexanetrione at specific stages and doses also interfered with normal morphogenesis like retinoic acid. Assuming that stimulation of prostaglandin synthesis is the main biological effect of the cyclohexanetriones, our findings suggest that prostaglandins may be involved in mediating retinoid action.     

Potentiation of IL-2-induced t-cell proliferation by retinoids.

We evaluated the capacity of retinoids to potentiate proliferative responses of murine T-cells to recombinant human interleukin 2 (rIL-2). Concanavalin A (Con A) prestimulated spleen cells responded in a dose-dependent manner to added rIL-2. All-trans-retinoic acid (RA) at 10(-8) M potentiated the proliferative response by fivefold at saturating levels of IL-2. In similar experiments, two closely related retinamides, all-trans-(phenyl)retinamide (PR) and N-(4-hydroxyphenyl)retinamide (4-HPR), also potentiated murine splenocyte rIL-2 responses. Potentiation of IL-2-induced proliferation was dose-responsive to the concentration of added retinoid with peak potentiation occurring at 10(-10) - 10(-8) M in the presence of 10 U/ml rIL-2. Significant potentiation was observed at retinoid concentrations as low as 10(-14) M. Fluorescence flow cytometry of the responding cells revealed that among L3T4+, Lyt-2+ or total T-cells, at 72 h following Con A stimulation, essentially all of the cells expressed IL-2 receptors (IL-2R). This apparently represents near maximum IL-2R expression and treatment of the cells with retinoids did not increase IL-2R expression at that time point. The potentiation of IL-2 responses by retinoids was also observed with IL-2-dependent HT-2 cells, 98% of which were IL-2R positive. HT-2 proliferative responses to rIL-2 were potentiated as much as fourfold by 10(-10) M RA. HT-2 proliferative responses to rIL-2 were potentiated by all three retinoids dose dependently. Significant potentiation was observed with as little as 10(-14) M retinoid. Retinoids in the absence of IL-2 induced no proliferative responses. These data suggest that retinoids can augment the capacity of IL-2 to induce T-cell proliferation using Con A-activated murine splenic T-cell blasts and a long-term-cultured T-cell line.     

Promotion of Wound Collagen Formation in Normal and Diabetic Mice by Quadrol

The rate of collagen deposition in implanted polytetrafluoroethylene (PTFE) tubing in non-diabetic and streptozotocin-induced (STZ) diabetic mice was measured during 14 days post-wounding. At the time of implantation, test groups received injections of either Quadrol [N,N,N',N'-tetrakis(2-hydroxypropyl)ethylenediamine], glucan, or buffer in an area adjacent to the wound site. The accumulation of collagen in the implants of Quadrol-treated non-diabetic animals was more than 200% above control on days 8 to 11 and was 50% above control on day 14. In Quadrol-treated STZ-diabetic mice, the collagen accumulation gradually increased from 50% above control on day 8 to 200% above control on day 14. Treatment with glucan increased the collagen accumulation in normal mice 200 to 300% above control from days 8 to 11 respectively and then 30% above control on day 14. Collagen accumulation in the implants of the glucan-treated STZ-diabetic mice was similar to the control group. These results indicate that Quadrol promotes in vivo collagen synthesis and that Quadrol may be effective as a stimulator of wound healing in diabetic and non-diabetic animals.     

Comparative teratogenic activities of two retinoids: effects on palate and limb development

Two closely related retinoids, all-trans and 13-cis retinoic acids, were assessed for their relative activities as teratogens in ICR mice by monitoring the frequency with which either isomer produced discrete dysmorphogenesis of the embryonic limb and the secondary palate. A single oral dose of all-trans retinoic acid at 100 mg/kg on either day 11.5 or 12.0 of gestation (plug day = day one) was maximally effective; more than 90% of the treated embryos developed reduction defects of the limb bones and an equally high percentage also had cleft palate. The limb development was most sensitive on day 11.5 of gestation while the peak susceptibility for palatal clefts began on day 12.0. Under identical experimental conditions, treatment with 100 mg/kg 13-cis retinoic acid produced no apparent teratogenic effects. By assessing the relative incidence of readily identifiable malformations of the limb and palate associated with various doses of the two isomers, we found that 13-cis retinoic acid was four to eight times less embryopathic than all-trans retinoic acid. Since the mechanism of teratogenic action of retinoids is still far from clear, it is suggested that further studies on causative factors will be greatly assisted by the use of these two closely related retinoids, which substantially differ from each other in their teratogenic potency.     

Differential in vitro effects of etretinate and retinoic acid on the PHA and con A induced lymphocyte transformation, suppressor cell induction and leukocyte migration inhibitory factor (LMIF) production

The in vitro influence of two retinoids, etretinate and retinoic acid, on the human lymphocyte transformation, on the induction of suppressor cells and on leukocyte migration inhibitory factor (LMIF) production were investigated. Nontoxic concentration of retinoic acid increased significantly the PHA response to suboptimal mitogen concentrations, but had no effect on the Con A response; it also abolished the PHA induced LMIF production. In corresponding assays etretinate was without effect. Etretinate augmented the PHA induced suppressor cell activity, while retinoic acid was ineffective. No effect was observed on Con A induced suppressor cells by either retinoid. The findings extend the information about the immunomodulatory effects of retinoids and demonstrate that retinoic acid and etretinate have different effects on PHA and Con A induced immune responses. The mode of action of retinoids is discussed.     

Effects of retinoids on macrophage function and IL-1 activity

The effects of three retinoids, all-trans-retinoic acid (RA), 13-cis-retinoic acid (cRA), and N-(4-hydroxyphenyl) retinamide (4-HPR), on macrophage function were evaluated. In vitro, RA, cRA, and 4-HPR caused a greater than twofold increase in phagocytosis of IgG-sensitized bovine erythrocytes (IgG-ORBC) by a mouse macrophage cell line (RAW). Significant increases in phagocytosis were produced by retinoid concentrations as low as 2 x 10(-10) M. RA also significantly increased phagocytosis of IgG-sensitized ORBC by BALB/c peritoneal macrophages in vitro. The ability of RAW macrophages to bind IgG-ORBC was significantly increased by 10(-6) to 10(-14) M RA. The potentiation of mitogenic responses of spleen cells to Con A and PWM by RA was relatively independent of macrophage function, i.e., splenocytes that were macrophage-depleted were responsive to the potentiating effects of RA. The effects of retinoids on T-cell-dependent B-cell mitogenesis induced by PWM appeared not to be dependent on their previously reported capacity to alter prostaglandin synthesis. Treatment of spleen cells with 10(-6) M indomethacin did not abolish the potentiating effects of RA. However, RA in a dose-dependent fashion increased IL-1 activity at the level of the target T-cell. The greatest potentiation of IL-1 activity was at 10(-8) M RA. These results show that retinoids can modulate macrophage function at two different levels: potentiation of phagocytosis and potentiation of IL-1 activity at the level of the T-cell.     

Retinoids in embryonal development

The key role of vitamin A in embryonal development is reviewed. Special emphasis is given to the physiological action of retinoids, as evident from the retinoid ligand knockout models. Retinoid metabolism in embryonic tissues and teratogenic consequences of retinoid administration at high doses are presented. Physiological and pharmacological actions of retinoids are outlined and explained on the basis of their interactions as ligands of the nuclear retinoid receptors. Immediate target genes and the retinoid response elements of their promoters are summarized. The fundamental role of homeobox genes in embryonal development and the actions of retinoids on their expression are discussed. The similarity of the effects of retinoid ligand knockouts to effects of compound retinoid receptor knockouts on embryogenesis is presented. Although much remains to be clarified, the emerging landscape offers exciting views for future research.     

The use of gemfibrozil in a patient with chronic myelogenous leukemia to successfully manage retinoid-induced hypertriglyceridemia

Summary Systemic retinoids are used in the management of chronic cutaneous conditions and lifethreatening dermatoses. Unfortunately, drug-induced hypertriglyceridemia may necessitate either dose reduction or discontinuation of therapy. The purpose of this article is to describe the successful management of isotretinoin-induced hypertriglyceridemia with gemfibrozil in a leukemia patient. Sequential serum chemistries were performed prior to, during, and following treatment with a systemic retinoid and a lipid-regulating agent. A prompt and sustained normalization of fasting triglycerides occurred following the initiation of gemfibrozil in a patient with isotretinoin-induced hypertriglyceridemia. When retinoids are being used in the management of serious conditions, the initiation of therapy with gemfibrozil to reduce the elevated triglycerides may be appropriate in those patients with retinoid-induced hypertriglyceridemia.     

Retinoid targets for the treatment of cancer

Retinoic acid (RA), the most potent natural retinoid, is essential for normal cell growth and differentiation. The RA signaling pathway is multistep, involving the precise regulation of retinoid levels and the control of RA-dependent gene expression in target cells. Within this complex scheme, there are many different aberrations in the RA signaling pathway of tumor cells that have been found to be associated with abnormal cell growth and tumorigenesis. This article reviews the normal pathways of RA signaling, followed by a discussion of the various sites that have been implicated in tumorigenesis and targeted for drug development. Currently, there are several retinoids and one rexinoid approved for the treatment of specific cancers. Future experimentation in drug discovery will continue to explore the efficacy of retinoids/rexinoids, either alone or in combination with other chemotherapeutic agents and/or chromatin remodeling agents, and the development of agents to modulate RA metabolism within cells. It is likely that different drug treatments will be developed that are specifically tailored to the unique point(s) in the RA signaling pathways that are aberrant in specific types of tumor cells.     

The effect of maternal antigenic stimulation upon the active immune responsiveness of their offspring.

Pregnant mice were stimulated by sheep erythrocytes (SRBC) and the active immune responses of their offspring were investigated. The offspring whose mothers were stimulated with SRBC did not develop either IgM or IgG plaque-forming cell (PFC) target cells. From the dose response of pregnant mice for inducing suppression, enough doses (10(8)-10(10) cells of SRBC) for inducing primary anti-SRBC PFC could establish suppression in the young. Both intravenous and intraperitoneal administration of SRBC induced almost complete suppression of the specific PFC response (98.6-95.2%), but only partial suppression (57.8%) was induced by subcutaneous injection. For suppression to take place, female mice had to be injected with SRBC from 2 days before fertilization to day 16 of gestation. Suppression of the PFC response was not obtained when SRBC were given 3 days before fertilization or just 24 hr before delivery. This suppressive effect on the PFC response persisted until the 15th week after birth. In these newborn mice, detectable amounts of specific anti-SRBC antibodies were found. After exchanging mothers and newborns, the stimulated newborns fostered by normal mothers were still unresponsive following antigenic stimulation, even though a specific antibody from the mother was not detected. However, normal newborns nursed by stimulated mothers could respond to SRBC injection, no matter how the specific antibodies were transferred. Possible mechanisms of immunosuppression of the PFC response by antibody transmitted by the mother to her offspring are discussed.     

Cellular retinol-binding protein-1 is expressed by distinct subsets of rat arterial smooth muscle cells in vitro and in vivo.

Previous work (M.-L. Bochaton-Piallat, P. Ropraz, F. Gabbiani, G. Gabbiani, Arterioscler Thromb Vasc Biol 1996, 16:815-820) has shown that a subset of smooth muscle cell (SMC) clones derived from the normal rat aortic media displays an epithelioid phenotype similar to that of the whole SMC population cultured from the intimal thickening 15 days after endothelial injury (IT-15). We show here that the whole IT-15 SMC population and the epithelioid clones, derived either from the normal media or from the IT-15, express cellular retinol-binding protein-1 (CRBP-1), a protein involved in retinoid metabolism. The expression of CRBP-1 is accompanied by the expression of cytokeratin 8. In both whole SMC population cultured from IT-15 and epithelioid clones, retinoic acid modulates the transition from the epithelioid phenotype to the spindle phenotype, typical of whole SMC populations cultured from the rat normal aortic media. Moreover, after endothelial injury in vivo, a CRBP-1 expressing SMC subset appears transiently in the IT and disappears, allegedly by apoptosis, when re-endothelialization takes place. Our results suggest that the expression of CRBP-1 is a marker of arterial SMC activation after endothelial injury in vivo and that CRBP-1 and probably retinoids participate in this process.     

Spinal nerve defects in mouse embryos prenatally exposed to valproic acid

To examine in detail spinal nerve defects induced by prenatal exposure to valproic acid in mice, pregnant ICR mice were subcutaneously injected with a single dose of 400 mg/kg valproic acid on gestational day 6, 7, 8, or 9, and their embryos were observed on gestational day 10. The whole-mount immunostaining using an anti-neurofilament antibody allowed us to identify spinal nerve defects, such as a loss of bundle, anastomosis among bundles arising from adjacent segment, and a disrupted segmental pattern of the dorsal root ganglia, in valproic acid-exposed embryos. The prevalence of spinal nerve defects was the highest in the embryos exposed to valproic acid on gestational day 8 among the experimental groups. Then, effects of the administration dose of valproic acid on the prevalence of spinal nerve defects were examined on gestational day 10 and found to be dose-dependently increased. It was noteworthy that all embryos exposed to 600 mg/kg of valproic acid on gestational day 8 suffered spinal nerve defects. Folic acid (3 mg/kg/day) supplementation during gestational day 6–10 suppressed the prevalence of valproic acid-induced neural tube defects, which are common malformations in offspring prenatally exposed to valproic acid, but not that of spinal nerve defects. Thus, the spinal nerve defects due to prenatal valproic acid exposure might be induced by mechanisms different from those of neural tube defects. Because spinal nerve defects were predicted to be caused by the disrupted segmental arrangement of the somites and/or that of neural crest cells, which was the origin of the dorsal root ganglia and/or abnormal polarity of the somite, this mouse model with spinal nerve defects at high incidence would be useful to examine the effects of valproic acid on the somitogenesis and morphogenesis of somite-associated structures.     

Effects of prenatal cocaine exposure and maternal separation on heart rate,orienting response habituation,and retention

Effects of the presence or absence of the dam during testing and the retention interval on pretone heart rate (HR) and habituation and retention of an HR orienting response to tone were examined in prenatally cocaine-exposed and nontreated Sprague Dawley rat pups in two experiments. On postnatal day 16, each pup received two test sessions, separated by a 4-hr retention interval during which pups were either isolated or placed with their dam and siblings. For testing, each pup was placed in the test apparatus in the presence or absence of an anesthetized dam where, after a 15-min adaptation period, 10 tone presentations were given, each separated by a 65-s intertrial interval, with HR measured during a 5-s pretone period and throughout the 10-s tone for each trial. Experiment 1 used offspring from the regular breeding colony and observed the typical HR lowering effect of maternal presence during testing, an effect that was surprisingly potentiated, however, following the retention interval in animals that were isolated during this interval. This apparent potentiation by prior isolation of the HR lowering effect of the dam was confirmed in Experiment 2 in nontreated offspring, but did not emerge convincingly in offspring of either dams subcutaneously injected with 40 mg/kg of cocaine HCl daily from gestational days 8 to 20 (C40) or dams injected with saline and pair-fed 4 days (PF4) to mimic the acute anorexic effects of cocaine administration. Consistent with prior work, C40 offspring also were found to exhibit better retention of the habituated orienting response than offspring of NT dams and to some extent PF4 dams as well, a retention effect that was not significantly influenced, however, by social context during the retention interval.     

Pathogenesis of craniofacial and body wall malformations induced by ochratoxin A in mice

Ochratoxin A (OA), a mycotoxin commonly found in soils and on moldy food such as cereal grains, is a potent teratogen. The present investigation was designed to examine the teratogenicity of OA administered acutely at early post-implantation stages in mice, with particular emphasis on the pathogenetic basis of induced malformations. Maternal OA administration on gestational day (GD) 7 or 8 resulted in excessive cell death in selected cell populations. After a single dose of 2–4 mg/kg, excessive amounts of cell death was notable within 6 hours, and persisted to 36 hours post-treatment. As observed in GD 14 or 18 fetuses, the spectrum of induced craniofacial malformations included exencephaly, midfacial clefting, cleft lip, as well as hypotelorism, and synophthalmia associated with holoprosencephaly. Body wall defects involved either the abdominal wall alone, or in combination with the thoracic wall, resulting in partial or complete exposure of the viscera. Potential mechanisms for OA-induced selective cell killing are discussed. © 1993 Wiley-Liss, Inc.     

Retinoid- and carotenoid-enriched diets influence the ontogenesis of the immune system in mice

Vitamin A (VA) has been identified as an important factor for the development of the immune system, especially during ontogenesis. It has been shown that antibody secretion and proliferation of lymphocyte populations depend on retinoids. In the present study we investigated the influence of a base VA diet and diets enriched with VA, beta-carotene and lycopene, on the ontogenesis of the immune system in mice. We examined the absolute and relative concentrations of splenic B lymphocytes (CD45R/B220), T lymphocytes (CD3+) and their subpopulations (CD4+ and CD8+), and measured serum immunoglobulin G (IgG) concentrations in the offspring of supplemented dams at different ages (1, 3, 5, 7, 14, 21 and 65 days). The experimental diets resulted in higher numbers of T and B lymphocytes after VA and carotenoid enrichment, when compared, at various time-points, with the base diet. Higher values of total serum IgG were found in the beta-carotene-enriched diet group on day 7. On days 7 and 14, the enriched diets induced significant alterations in the percentages and total numbers of splenic lymphocytes in comparison to the base diet. Our results confirm that supplementation with VA and carotenoids affect the immune-cell function during ontogenesis and suggest a possible role of these nutritional factors on the development of the immune system.     

Retinoic acid embryopathy

Retinoic acid, an analogue of vitamin A, is known to be teratogenic in laboratory animals and has recently been implicated in a few clinical case reports. To study the human teratogenicity of this agent, we investigated 154 human pregnancies with fetal exposure to isotretinoin, a retinoid prescribed for severe recalcitrant cystic acne. The outcomes were 95 elective abortions, 26 infants without major malformations, 12 spontaneous abortions, and 21 malformed infants. A subset of 36 of the 154 pregnancies was observed prospectively. The outcomes in this cohort were 8 spontaneous abortions, 23 normal infants, and 5 malformed infants. Exposure to isotretinoin was associated with an unusually high relative risk for a group of selected major malformations (relative risk = 25.6; 95 per cent confidence interval, 11.4 to 57.5). Among the 21 malformed infants we found a characteristic pattern of malformation involving craniofacial, cardiac, thymic, and central nervous system structures. The malformations included microtia/anotia (15 infants), micrognathia (6), cleft palate (3), conotruncal heart defects and aortic-arch abnormalities (8), thymic defects (7), retinal or optic-nerve abnormalities (4), and central nervous system malformations (18). The pattern of malformation closely resembled that produced in animal studies of retinoid teratogenesis. It is possible that a major mechanism of isotretinoin teratogenesis is a deleterious effect on cephalic neural-crest cell activity that results in the observed craniofacial, cardiac, and thymic malformations.     

孕期酒精接触对子鼠视皮质神经元凋亡的影响

目的 探讨孕期酒精接触对子鼠视皮质神经元凋亡的影响.方法 从妊娠母鼠第5d酒精灌胃直至小鼠出生;采用激活的半胱氨酸天冬氨酸蛋白3(Caspase-3)免疫组织化学和原位末端标记(TUNEL)法观察P0、P7和P14小鼠视皮质神经元的凋亡.结果 酒精实验组妊娠时间延长,出现死胎和畸形(小头畸形、无脑儿和脊柱脊髓裂等).酒精实验组Caspase-3阳性率和凋亡指数明显高于对照组(P＜0.001),高剂量酒精组明显高于低剂量组(P＜0.01).随着酒精剂量的增加,子鼠视皮质神经元的凋亡明显增加.结论 孕期酒精接触可导致子鼠视皮质神经元凋亡,且有剂量依赖性和长时程效应.