Computer-automated structure evaluation (CASE) of retinoids in teratogenesis bioassays

alpha-acetoxynitrosamines have been used as model compounds to study biological activity of N-nitrosamines. After hydrolytic cleavage they yield an "active intermediate" (the hydroxynitrosamine) which presumably also arises as metabolite of N-nitrosamines. We tested eight N-nitrosamines mono-substituted at the alpha-carbon with an acetoxy group for their teratogenic potential in a mouse limb bud culture system. The following results were obtained: 1. The methyl compound (MOAc-MNA) - a derivative of dimethynitrosamine - proved to be the strongest teratogen in this group of chemicals. 2. The tertiary-butyl derivative - releasing a carbonium ion of low chemical reactivity - possessed the lowest activity in our test system. 3. The primary alpha-acetates with unbranched side chains (POAc-MNA or BOAc-MNA) were more active than the corresponding derivatives with branched side chains. 4. The secondary alpha-acetate (EOAc-ENA) was clearly less active than the primary alpha-acetate (EOAc-MNA). 5. A teratogenic potential could also be demonstrated in the organ culture system with the cyclic derivative: N-nitroso-alpha-acetoxy-pyrrolidine (NAPYR). 6. With the use of limb buds from 12-day-old mouse embryos the explants showed the highest susceptibility to the teratogens on the first day of culture. No effect could be produced if the substances were added to the culture medium at the third day of culture or later. 7. When initiating the culture with limb buds from 11-day-old mouse embryos the concentrations needed to induce typical effects were lower than those tested with 12-day-old explants. 8. Typical and pronounced impairment with morphogenetic differentiation could be induced by MOAc-MNA if the substance was present in the medium for less than 60 min - the shortest period tested was 15 min. 9. A different abnormality pattern could be induced with the various substances tested. This may partly be explained by a quite different stability of the compounds in the test system. 10. N-Nitrosamines must be expected to be highly teratogenic if they can be "activated" in embryonic tissues. Such an activation into potent electrophilic agents does not occur in rodents under normal conditions. It will be interesting to study if such activity can be "induced" in embryonic tissues of rodents or if it is already present in embryonic tissues of primates.

Valproic acid (VPA) is a branched-chain carboxylic acid with broad anticonvulsant activity. Its spectrum of activity is thought to be mediated by combined molecular effects on intercellular Na+ currents, neuron K+ channels, and inhibition of gamma-aminobutyric acid (GABA) transaminase (1,2). VPA is metabolized in the liver by cytochrome P-450 oxidase. Hepatotoxicity related to valproate therapy has been reported in certain individuals and appears to be caused by the accumulation of toxic metabolites, which may include 4-en-valproate (3). The hepatotoxicity has been ascribed to an inherited or acquired deficiency in the cytochrome P-450 enzyme-dependent beta-oxidation pathway, and is inducible by other drugs such as phenobarbital or phenytoin (4). Fatal hepatic failure can result, with a disproportionately large number of such cases observed in patients less than two years old with neurologic abnormalities, severe seizures, and multiple anti-epileptic drug therapy (3,5,6,7). However, neonatal hepatic toxicity in humans coincident with intrauterine VPA exposure has rarely been reported (8,9,10); these cases were never reversible, andconfounding etiologic variables could not be excluded. In pregnancy, VPA readily crosses the placenta and accumulates with fetal blood concentrations greater than those in the mother (11). It is a known human teratogen primarily associated with neural tube developmental defects such as spina bifida. A specific "fetal valproate syndrome," marked by fetal growth deficiency, developmental delay, and an increased incidence of craniofacial, cardiovascular, and digital abnormalities, has also been reported (1,3,12,13,14). Furthermore, a dose-dependent relationship in the occurrence of major malformations and minor anomalies has been suggested (15). Major malformations reported include persistent patent ductus arteriosus, trigonocephaly, aplasia of the first ribs, dysplasia of the sternum, meningomyelocele, aplasia of the radius, and congenital hip dislocation. Minor malformations included brachycephaly, dysmorphic facial features, long thin fingers, and inverted or accessory nipples. The teratogenic potential is thought to be mediated by a secondary zinc deficiency induced by complexing with VPA (16), as well as by inhibition of microsomal epoxide hydrolase (17), which may enhance the teratogenic potential of concomitant anticonvulsant therapy. Numerous animal models have been developed for the study of VPA. VPA has been shown to have equal teratogenic potential as trimethadione in mice (18). Skeletal abnormalities, delayed parturition, and postnatal growth (5) have been reported in both mice and rats receiving VPA. In addition, Paulson and Paulson (19) noted defects of the palate, eyes, heart, and limb buds in animal models. Animal models for a VPA-associated cholestasis, however, are lacking.

Caspases are key mediators in the regulation and execution of apoptosis, a crucial part of the morphogenetic process during limb development. Caspase-8 and -9 are upstream caspases. Caspase-8 mediates the extrinsic pathway of apoptosis triggered by signaling through TNF-R1 family receptors. Caspase-9 is activated during the intrinsic pathway downstream of mitochondria. Caspase-3 is an effector caspase that initiates degradation of the cell in the final stages of apoptosis. Vitamin A is a potent teratogen that causes limb reduction defects in embryos exposed during organogenesis. Previous in vitro studies have shown that exposure of the organogenesis-stage murine limb to vitamin A results in excessive levels of apoptosis. The goal of this work was to characterize the involvement of caspase-3, -8, and -9, as well as cytochrome-c release from the mitochondria, in the apoptotic cascade induced by vitamin A. Limb buds from gestational day 12 CD-1 mice were cultured in a chemically defined medium in the absence or presence of vitamin A. Cultures were terminated after 6 days to examine the effect of the drug on gross morphology. Apoptosis was detected by TUNEL staining after culture for 24 hr. Caspase activation was determined by Western blotting and localized by immunohistochemistry of control and treated limbs. The release of cytochrome-c into the cytoplasm was assessed by Western blotting after cell-fractionation. Limbs cultured in the presence of vitamin A showed a dose-dependent growth reduction and dysmorphogenesis of the cartilaginous anlagen. Apoptosis was increased in the interdigital, anterior, and posterior marginal zones and in the apical ectodermal ridge. Western-blotting confirmed the presence of activated caspase-3 that increased with time in culture and vitamin A concentration. Cleaved caspase-3 immunoreactivity colocalized with TUNEL stained limb regions and increased dramatically with increasing drug concentrations. In contrast, procaspase-8 and -9 were not activated. Exposure to high concentrations of vitamin A did, however, increase cytoplasmic cytochrome-c, suggesting mitochondrial involvement. Caspase-3 is a key effector caspase in the apoptotic pathway induced by Vitamin A. While caspases-8 and -9 are not responsible for the activation of caspase-3 in response to the drug, cytochrome-c release from mitochondria may play an upstream role.

1) A method was developed to measure the activity of DNA dependent RNA polymerase reactions I (alpha-amanitin-insensitive) and II (alpha-amanitin-sensitive) in homogenates of very small amounts of tissue. This technique was used to study the activity of these polymerase reactions in limb buds from mouse embryos developing in vivo or in an organ culture system. This culture system allows the morphogenetic differentiation of limb buds in early stages of embryonic development (day 10-11 of gestation greater than or equal to 37 - 45 pairs of somites) from a blastema stage to well recognizable cartilaginous bone analgen. 2) An increase in the activities of both types of RNA polymerases was found to occur in vivo in limb buds on day 12 of gestation when compared with the activity measured on day 11 of gestation. A similar increase with a maximum about 24-48h after initiation of the cultures could be observed in the explants differentiating in organ culture. To our knowledge this is the first time that induction processes proceeding in mammalian embryonic tissues could be correlated with an increased activity of RNA polymerases. 3)A rough estimate shows that the rate of transcription due to RNA polymerase I measured in homogenates or in isolated nuclei (measured with substrate concentrations giving about 80% of the maximal rate) would account for the assumed rate of rRNA synthesis in vivo. The measurement of the RNA polymerase activity in isolated nuclei, thus, may give some information about the transcriptional processes occurring under "physiological" conditions. 4) The mammalian organ culture system presented provides a suitable model for studying embryonic differentiation processes.

Purpose: To investigate the effects of an electromagnetic field (EMF) on limb bud development in vitro, an organ culture system was applied.Materials and methods: Three test groups of amputated mouse limb buds included the experimental (E) group which received EMF (50 Hz/13.1 mT, for 2 h), a sham (Sh) group exposed to no EMF treatment and the control (C) group. The limb buds of E and Sh groups (n = 20 per group) were amputated from mouse embryos on day 11.5 of development and cultured in minimum essential medium Eagle (MEM Eagle), supplemented with 15% human embryo cord serum, for 2 days, while those of group C (n = 20) were removed on day 13.5 of development. All samples were fixed in Bouin's fluid, embedded in paraffin, serially sectioned (5 μm thick) and stained with Hematoxylin and Eosin. Limb bud measurements were performed using a scaled graticule.Results: Morphological and histological examinations showed significant changes in the experimental limb bud group as compared with the sham and control groups. The growth rate in both fore and hindlimb buds in proximal-distal (P-D) and anterior-posterior (A-P) axes were significantly increased. Chondrocyte counts and mitotic figures of mesenchymal and red blood cells were significantly increased as compared with those of sham and control groups. There was also a significant reduction of mesenchymal cell counts, while no significant difference was observed in the degenerated cell counts among the three groups.Conclusions: These findings suggest that EMF, under the conditions applied, has progressive effects on the limb bud development and that both proliferation and differentiation can be stimulated in vitro.

EE422 Healthcare Costs Associated with Severe VS Moderate and Mild COVID-19 - a US Payer Perspective

PR_044: Psychologic Adjustment Has a Greater Effect on Health-Related Quality of Life Than on Severity of Disease in Parkinson’s Disease

Using a whole-embryo culture system valproic acid (VPA) and some of its metabolites (2-en-VPA, 4-en-VPA, 4,4'-dien-VPA) and analogues (ethyl-propyl-acetic acid, propyl-butyl-acetic acid, di-butyl-acetic acid, 2-methyl-2-ethyl-hexanoic acid, 1-methyl-1-cyclohexanoic acid) were tested for their potential to induce abnormal development. With regard to embryonic growth, development and abnormality rate, the tested compounds showed a wide range of "teratogenic potency" in vitro. In order to verify some of the in vitro results, in vivo experiments were performed. Pregnant rats were treated subcutaneously on day 10 of gestation with 2 x 330 mg VPA/kg, or 2 x 400 mg 2-en-VPA/kg, respectively. Evaluation of the embryos was performed on day 11.5 of gestation, corresponding to the in vitro experiments. VPA showed a high potential to induce abnormal development in vivo as well as in vitro, whereas 2-en-VPA was inactive under our experimental conditions. Problems connected with the evaluation of the predictive value of an in vitro test system for the detection of embryotoxic effects, such as "validation" and significance of pharmacokinetic data, are discussed.

There is widespread interest today in the use of in vitro methods to study normal and abnormal development. The limb is attractive in this context since much is known about pattern formation during limb development. The murine limb bud culture technique described in this chapter was developed and refined in the 1970s. In this culture system, limb development mimics the in vivo process, although at a slower rate, where growth and cartilage differentiation lead to the formation of proximal and distal structures with an "in vivo-like" 3D shape. Uniform developmental stages are selected for assessment, exposures are controlled precisely, and the confounding influences of maternal metabolism and transport are avoided. The existence of transgenic mice with fluorescent markers for the different stages of endochondral ossification adds a further dimension to the technique by allowing striking time course observations of the developing limb. Today, limb bud cultures are used to study the roles of genes during embryogenesis and the mechanisms by which chemicals interfere with critical signalling pathways.

A zebrafish (Danio rerio) teratogenicity assay has been developed and evaluated for its ability to predict the teratogenic potential of chemicals. Zebrafish embryos were dechorionated and then exposed to a test solution from 4-6 hours post-fertilization, and embryos or larvae were assessed up to 5 days post-fertilization (dpf) for viability and morphology. In preliminary experiments, the potential time points for assessment of compound-induced dysmorphology and general toxicity parameters were evaluated, and 5 dpf was found to be the optimum developmental stage for evaluation. Additionally, a morphological scoring system was devised to identify the developmental no-observed-adverse-effect level (NOAEL). For assay evaluation, 34 compounds with adequate in vivo developmental toxicity data were chosen. The compound set represented diversity in teratogenic potencies, structural classes, and pharmacologic targets. For 31 test compounds, each was evaluated over a concentration range, while 3 others were insufficiently aqueous-soluble to be fully tested. For each of the 31 tested compounds, the 5 dpf NOAEL was determined, and the concentration resulting in 25% lethality (LC25) was calculated by curve-fitting. Teratogenic potential of each compound was predicted based on the ratio of the LC25 to the NOAEL. LC25/NOAEL ratios of 10 or greater were considered predictive of teratogenicity. The model successfully categorized 87% of the compounds as teratogens or non-teratogens, with only 2 false-positives (dimethyl phthalate and a Bristol-Myers Squibb (BMS) investigative compound) and 2 false-negatives (valproic acid and a BMS compound). The results indicate that this assay is promising for screening compounds for teratogenic potential. Birth Defects Res (Part B) 89:66-77, 2010. (c) 2010 Wiley-Liss, Inc.

Comparative Study of the Effect of Different Weed Management Strategies on Disease Severity and Marketable Yield of Paprika (Capsicum AnnuumL.) in the Smallholder Farming Sector of ZimbabweOn-farm trials were conducted in the Chinyika Resettlement Area of Zimbabwe under dryland conditions to investigate the effects of different weed management methods on disease incidence, severity and paprika (Capsicum annuum) pod yield. The weed control treatments included hand weeding at 2 and 6 weeks after transplanting (WAT); ridge re-moulding at 3,6 and 9 WAT; application 4l/ha Lasso (alachlor) immediately after transplanting, and Ronstar (oxidiazinon) at 2l/ha tank mixed with Lasso at 2l/ha one day before transplanting. The herbicide-water solution was applied at the rate of 200l/ha using a knapsack sprayer. Major diseases identified were bacterial leaf spot (Xanthomonas campestrispv.vesicatoria), cercospora leaf spot (Cercospora unamunoi), grey leaf spot (Stemphylium solani) and powdery mildew (Leveillula taurica) in both seasons. For the 2000/2001 season hand weeding at 2 and 6 WAT and ridge re-moulding at 3, 6 and 9 WAT had the greatest reduction effect on the area under disease progress curve (AUDPC) and the highest marketable fruit yield. In the 2001/2002 season, both herbicide treatments had the same effect as hand weeding and ridge re-moulding on AUDPC and marketable fruit yield. The least weed density was obtained by ridge re-moulding at 3, 6, and 9 WAT in the 2000/2001 season. Weed density was statistically the same across all treatments except the check treatment in 2001/2002 season. Hand weeding operations were significantly (p < 0.05) effective and consequently gave the highest added profits mainly because of their effect on major weeds such asDatura stramonium.

The interaction of pregnancy and the rheumatic diseases varies, ranging from life-threatening conditions such as thromboembolic events and progressive renal disease in some autoimmune disorders, to minor flares of peripheral arthritis in inflammatory rheumatic disease. As a consequence, treatment strategy will vary according to the maternal or fetal compromise expected. All nonsteroidal anti-inflammatory drugs (NSAIDs), including high dose aspirin (acetylsalicylic acid), can cause adverse effects during pregnancy related to the inhibition of prostaglandin synthesis. Prolongation of gestation and labour, constriction of the ductus arteriosus, persistent fetal circulation, impairment of renal function and bleeding are risks of third trimester exposure of pregnant women to all inhibitors of cyclo-oxygenase. Most of these adverse effects can be prevented by discontinuing NSAIDs 8 weeks prior to delivery. Low dose aspirin has not been associated with fetal or neonatal toxicity. Some corticosteroids such as prednisone and prednisolone do not readily cross the placenta and can be safely used during pregnancy as immunosuppressive drugs. Maternal complications related to corticosteroids may occur and close monitoring is therefore mandatory. There is limited information on the safety of disease-modifying antirheumatic drugs including gold, antimalarials, penicillamine (D-penicillamine), sulfasalazine and cyclosporin. Of these agents, sulfasalazine has the best record for tolerability and can be used by pregnant patients. Gold compounds and penicillamine should be discontinued when pregnancy is recognised. Hydroxychloroquine has not been associated with congenital malformations and seems preferable to chloroquine in patients requiring treatment with antimalarials. Use of cyclosporin may be an alternative to other therapy in pregnant patients with severe rheumatic disease. Indications for treatment with colchicine during pregnancy are few, except for familial Mediterranean fever. Azathioprine can be used when the maternal condition requires a cytotoxic drug during the first trimester. Cyclophosphamide, chlorambucil and methotrexate are contraindicated during pregnancy because of their teratogenic potential. Their use may be considered in late pregnancy if the mother has a life-threatening condition.

Several in vitro cellular systems designed to screen agents for teratogenic potential are described in this report. These assays were selected from a review of literature published through the spring of 1986 that generated over 100 references on teratological research using cell-based systems. Some of the assays have a broader application than others, but most require confirmation by one or more additional complementary tests because of the specificity of the teratogenic mechanism the assays are investigating. Included are systems that use analysis of tumor cell attachment; intercellular communication; growth of human embryonic palatal mesenchyme cells; progesterone production in porcine granulosa cells; differentiation of embryonic neural crest, limb bud, midbrain, and Drosophila cells; and differentiation of tumor cells. Because of the dynamic nature of cell culture work, the group of assays listed here should not be viewed as encompassing all cell systems of value with regard to teratogenicity testing; instead, the list represents several of the more prominent systems now being evaluated by the scientific community.

Background: Psoriasis is common, chronic disease of the skin. The disease has psychosocial impact and associated with impairments in Quality of Life (QOL). Aim of the study: It was to define the psychosocial impact of psoriasis and to determine QOL of the psoriasis patients. Patients and methods: This study was conducted on 100 adult patients with psoriasis, attending Dermatology Clinic, Al-Hussein University Hospital and an equal number of normal adults as controls. An analytic, case-control, clinic based study design was selected to conduct this research. An interviewing form and Middle Sex Hospital Questionnaire were used to survey psychosocial impact of the disease and psychiatric symptoms. Dermatology life quality index was used to assess disease impact on the patients’ QOL. Results: The most common statistically significant impact was the interference with daily activities (59.0%). This was higher in patients with severe disease (96.2%) and with disease affected visible areas of the body (70.6%). The depression was the commonest psychiatric symptom (27.0%); 46.2% and 35.3% in patients with severe disease and with disease affected visible areas of the body, respectively. QOL was poor in 73.0% of the patients; the most common affected QOL mean domains scores were symptoms and feelings (15.8 ± 5.4), personal relationships (14.6 ± 5.2), and daily activities (14.4 ± 4.9). All QOL mean domain scores were statistically significantly higher in patients with disease affected visible areas of the body and with severe disease. Conclusions and recommendations: Psoriasis has a great effect on the patients’ QOL. It disrupting psychosocial aspects and interfering with symptoms and feelings, daily activities, etc. The highest mean domain was symptoms and feelings (15.8) and the lowest mean was treatment (13.6). It could be recommended that more researches should be carried out on big number of patients in Egypt and on different clinical types to understand the impact of this disease

Metabolic consequences of obesity as an "outside in" mechanism of disease severity in asthma.