Teratogenicity screening in standardized chick embryo culture: Effects of dexamethasone and diphenylhydantoin

Summary Teratogenic and toxic effects of DXM and DPH were tested using a standardized chick embryo culture. Survival, growth and malformations were scored with respect to the drug concentrations used. DXM (>10^–8 mol/l) inhibited the differentiation of the extraembryonic blood circulation and induced craniofacial anomalies. DPH (>1.5 10^–5 mol/l) induced cardiomegaly, craniofacial and somitic anomalies. Both drugs were lethal at 10^–3 mol/l. Comparison of results obtained with 8 drugs shows that the method has a good discriminative power and specificity and that it can be used as a simple, reliable and economical primary screening test, making it possible to reduce the use of animals in toxicological studies.Supported by the grant 4.790.0.84.17 from the Swiss National Science Foundation.     

Dexamethasone-induced neutrophilia. Negative correlation with increased plasma adrenaline concentrations

Maximal increments in adrenaline and dexamethasone (DXM) plasma concentrations were observed c15 (T50 40 min) and 30 (T50 210-240 min) minutes after an i.v. DXM dose (6 mg/m2 BSA) in man. There appears, however, to be no direct interaction between these agents in the development of induced neutrophilia, which occurs c240 min postinjection.     

Action of diphenylhydantoin on chromosomes

The effect of diphenyl hydantoin, (DPH) a nonbarbituate anticonvulsant drug, on chromosomes and fertility was tested in cultured human lymphocytes, mouse fertility, and rat maternal marrow chromosomes and fetal development. Whole human blood from 5 male and 5 female subjects was cultured for 68 hours with phytohemagglutinin, then incubated for 2 hours in isotonic salts with .05-.3 mg per ml DPH, .02 mcg per ml colchicine, or .4 mg per ml sodium diethylbarbiturate. The mean number of metaphases per 1000 stimulated cells was 10.0 in controls, 40.3 with colchicine, 27.9 with diethylbarbiturate, and 30.5 with .25 mg DPH per ml. Both diphenylbarbiturate and DPH produced linear dose effect curves. These results were demonstrated not to be due to urea, since there were no differences in urea content, with a 2 hour urease micromethod. Mouse fertility was totally inhibited in 6 virgin mice given .1mg DPH daily for 10 days compared to 41 pups both of 6 control mice. In 6 pregnant rats given 25 mg DPH per 100 gm/orally 4 times daily for 2 days on gestation Days 7 and 8, there were 5 rats with all fetuses resorbed and 1 rat with 3 living and several resorptions. 6 controls had 6-14 normal fetuses each. 50 metaphase plates from each rat's femoral marrow and each fetus were examined 2 hous after injecting .3 mg colchicine per 100 gm. 30% of the metaphase cells from treated females and fetuses showed strongly contracted chromosomes and reduced number os "pulverized" chromosomes. These phenomena may have been due to inhibition by DPH of folic acid metabolism which is involved in purine synthesis.     

In vitro modulation of rat adipocyte ghost membrane fluidity by cholesterol oxysterols

The effects of cholesterol and cholesterol-derived oxysterols (cholestanone, cholestenone, coprostanone and epicoprostanol) on adipocyte ghost membrane fluidity were studied using a fluorescence depolarization method. The fluorescence anisotropy of the treated membranes was determined using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH). Cholestanone and cholesterol decreased membranes fluidity at both the concentrations tested (10 & 50 M) while the rest of the sterols did not exert any significant effect on membrane fluidity. In the presence of epinephrine, cholestanone partitioned more towards the lipid core but cholesterol partitioning was not affected. The fusion activation energies (E) obtained for membranes preincubated with cholestanone (8.6 kcal/mol) and cholesterol (8.2 kcal/mol) were not significantly different from that of untreated membranes (8.3 kcal/mol). Membranes preincubated with cholestanone and cholesterol did not exhibit any change in lipid phase throughout the temperature range (10–45°C) tested. The sterols were found to inhibit fisetin-induced phospholipid methylation in isolated rat adipocytes in the rank order of cholesterol > epicoprostanol > cholestanone=cholestenone=coprostanone, while basal methylations was unaffected. When adipocytes were preincubated with the sterols before the addition of fisetin, cholestanone and cholestenone showed 74% and 66% inhibition of maximal methylation respectively. These results indicated that cholesterol oxysterols interact differently with rat adipocyte membranes, with cholestanone interacting more with phospholipids located at the inner lipid bilayer (e.g. phosphatidylethanolamine) while cholesterol interacts more with phosphatidylcholine located at the outer lipid bilayer. This differential interaction may cause selective changes in membrane fluidity at different depths of the bilayer and thus may modulate the activities of membrane-bound proteins such as enzymes and receptors.     

Enhanced heparan sulfate proteoglycan-mediated uptake of cell-penetrating peptide-modified liposomes

Protein transduction domains (PTDs) are used to enhance cellular uptake of drugs, proteins, polynucleotides or liposomes. In this study, functionalized Antennapedia (Antp, aa 43–-58) and HIV Tat (aa 47–57) peptides were coupled to small unilamellar liposomes via thiol-maleimide linkage. Modified liposomes showed higher uptake into a panel of cell lines including tumor and dendritic cells than unmodified control liposomes. Liposome uptake was time and concentration dependent as analyzed by flow cytometry and live-cell microscopy. At least 100 PTD molecules per small unilamellar liposome (100 ± 30 nm) were necessary for efficient translocation into cells. Cellular uptake of PTD-modified liposomes was 15- to 25-fold increased compared to unmodified liposomes and was inhibited by preincubation of liposomes with heparin. Glycosaminoglycan-deficient CHO cells showed dramatically reduced cell association of PTD-modified liposomes, confirming the important role of heparan sulfate proteoglycans in PTD-mediated uptake. Antp-liposomes used as carriers of the cytotoxic drug N⁴-octadecyl-1--D-arabinofuranosylcytosine-(5- 5)-3-C-ethinylcytidine showed a reduction of the IC₅₀ by 70% on B16F1 melanoma cells compared with unmodified liposomes. PTD-functionalized liposomes, particularly Antp-liposomes, represent an interesting novel carrier system for enhanced cell-specific delivery of a large variety of liposome-entrapped molecules.Received 16 April 2004; received after revision 13 May 2004; accepted 25 May 2004     

Dexamethasone-induced neutrophilia. Negative correlation with increased plasma adrenaline concentrations

Summary Maximal increments in adrenaline and dexamethasone (DXM) plasma concentrations were observed c15 (T₅₀ 40 min) and 30 (T₅₀ 210–240 min) minutes after an i.v. DXM dose (6 mg/m² BSA) in man. There appears, however, to be no direct interaction between these agents in the development of induced neutrophilia, which occurs c240 min postinjection.Acknowledgments. The authors wish to thank Mrs C. Ditzler, Merck Sharp & Dohme Research Laboratories, USA, for determining the plasma dexamethasone concentrations J. M. M. is currently on sabbatical leave from McGaw Laboratories, USA. C. R. B. is a Rhodes scholar.     

The influence of prostacyclin (PGI2) on contractile properties of isolated right ventricle of rat heart

The mechanism of the positive inotropic effect of prostacyclin (PGI₂) (2.6×10^?6 mol/l) on the isolated right ventricle of rat heart was studied. Our results show that the positive inotropic effect of prostacyclin is produced indirectly through beta adrenoceptors and slow Ca²⁺ channels, because blockade of slow Ca²⁺ channels with verapamil (10^?6 mol/l) and beta adrenoceptors with propranolol (10^?6 mol/l) abolishes this effect. Alpha adrenoceptors do not mediate the action of PGI₂.     

Dexamethasone suppression of ovulation in PMS-treated immature rats

Summary A single injection of 2.0 mg/kg dexamethasone (DXM) administered at 51 h after pregnant mare serum gonadatropin (PMS) treatment inhibited both ovulation and luteinization. S.c. injection of human chorionic gonadotropin (HGG) caused ovulation ond luteinization in DXM-PMS-treated rats, whereas treatment with ACTH failed to overcome the DXM inhibitory effect. These findings are interpreted to indicate that DXM inhibits ovulation through a mechanism which might involve the central nervous system.     

Dexamethasone suppression of ovulation in PMS-treated immature rats.

A single injection of 2.0 mg/kg dexamethasone (DXM) administered at 51 h after pregnant mare serum gonadatropin (PMS) treatment inhibited both ovulation and luteinization. S.c. injection of human chorionic gonadotropin (HGG) caused ovulation and luteinization in DXM-PMS-treated rats, whereas treatment with ACTH failed to overcome the DXM inhibitory effect. These findings are interpreted to indicate that DXM inhibits ovulation through a mechanism which might involve the central nervous system.     

Polarographic activity of the antitumor drug cis-dichlorodiammineplatinum (II). The effect of hydrolysis and trans-isomerization of the drug

Electrochemical activities of cis-dichlorodiammineplatinum (II) (cis-DDP) and its trans isomer were studied by classical and differential pulse polarography (d.p.p.). It was shown that both isomers yielded a polarographic step or peak at about -1.6 V (vs. Ag/AgCl), which corresponded to electroreduction of the complex and to catalytic hydrogen evolution. This signal was easily measurable with the aid of d.p.p. and was suitable for investigation of the extent of hydrolysis and trans-isomerization of cis-DDP leading to the formation of toxic products. The detection limit for determination of cis-DDP and its trans isomer by d.p.p. was 1 X 10(-6) mol/l.     

Denaturation map of the ribosomal DNA ofLytechinus variegatus sperm

Summary Electron microscopy of the partially heat denatured ribosomal DNA (rDNA) from sea urchin (Lytechinus variegatus) sperm has demonstrated that it consists of repeating units of 3.6±0.2 m, corresponding to a mol.wt of 7.2±0.4×10⁶. Based on differential denaturability, each repeat unit is divided into 2 regions. The larger region of 2.47±0.11 m (mol.wt 4.9±0.22×10⁶) corresponds in length to the ribosomal precursor RNA of sea urchins and the smaller, GC-rich, subunit of 1.16±0.09 m (mol.wt 2.3±0.18×10⁶) is presumed to contain non-transcribed spacer sequences.Acknowledgments. This paper is dedicated to the memory of Dr D. P. Costello whose insprration made this work possible. I gratefully acknowledge the valuable advice of Drs D.W. Stafford and M.A. Bleyman.     

Postimplantation embryo culture for the assessment of the teratogenic potential and potency of compounds

Whole rat embryos cultured during the early stages of organogenesis were subjected to a panel of selected chemicals. Of seventeen known in vivo teratogens, seventeen also induced specific malformations in embryos grown in culture. Of ten chemicals which were reported to be negative in in vivo rat teratogenicity studies, eight also did not provoke dysmorphogenic effects in vitro. Of five additionally tested retinoids, all induced multiple malformations. However, concentrations used to induce these effects varied considerably, isotretinoin inducing malformations at 10(-5) M and arotinoid at 10(-11) M. The results indicate qualitatively as well as quantitatively a high predictability of this in vitro system and suggest that the postimplantation embryo culture system may also be useful in the prospective testing of new drugs and environmental chemicals.     

Dexamethasone protection against the acute lethality of ethanol in mice

Summary The administration of dexamethasone (DXM, 2.00 mg/kg) 1 h prior to the injection of lethal doses of ethanol was found to offer complete protection against ethanol toxicity at doses up to 5.25 g/kg and partial protection using higher doses. It is suggested that DXM central action might be involved in the protection against ethanol toxicity.Supported by a grant from U.S. National Aeronautics and Space Administration.     

Role of protein kinase C and Ca2+ in glucose-induced sensitization/desensitization of insulin secretion

The role of protein kinase C and Ca²⁺ in glucose-induced sensitization/desensitization of insulin secretion was studied. A 22–24h exposure of mouse pancreatic islets to glucose (16.7 mmol/l) in TCM 199 culture medium, with 0.26 mmol/l or 1.26 mmol/l Ca²⁺, reduced total islet protein kinase C activity to approx. 85% and 60% of control values, respectively. At 0.26 mmol/l Ca²⁺ in TCM 199 medium, exposure to glucose (16.7 mmol/l) led to a potentiation of both phase 1 and phase 2 of glucose-induced insulin secretion, and caused a shift in the dose-response curve with 10 mmol/l and 16.7 mmol/l glucose exhibiting equipotent effects in stimulation of insulin secretion. In glucose-sensitized islets, the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (0.16 μmol/l) did not further potentiate induction of secretion by 10 mmol/l or 16.7 mmol/l glucose. At 3.3 mmol/l glucose, however, phorbol ester-induced secretion was augmented, and was characterized by a faster onset of secretion in glucose-sensitized islets relative to control islets. In contrast, a partial reduction in arachidonic acid (100 μmol/l)-induced insulin release was observed in glucose-sensitized islets in the absence of extracellular Ca²⁺. Increasing the Ca²⁺ concentration to 1.26 mmol/l in TCM 199 during the 22–24h exposure to glucose (16.8 mmol/l) led to inhibition of phase 1 and abolition of phase 2 of glucose (10 mmol/l, 16.7 mmol/l)-induced insulin secretion. In addition, this treatment abolished phorbol ester-induced and arachidonic acid-induced insulin secretion at 3.3 mmol/l glucose. Altogether, these data suggest that sensitization of insulin secretion is caused by a preferential down-regulation of the inhibitory effects of protein kinase C, leading to an increased first phase, and an increased coupling of glucose to the stimulatory effects of protein kinase C during the second phase of glucose-induced insulin secretion. Desensitization of insulin secretion appears to be a consequence of sustained Ca²⁺ influx, inducing extensive down-regulation of protein kinase C and also causing deleterious effects on islet cell function in protein kinase C-deprived islets.     

Denaturation map of the ribosomal DNA of Lytechinus variegatus sperm.

Electron microscopy of the partially heat denatured ribosomal DNA (rDNA) from sea urchin (Lytechinus variegatus) sperm has demonstrated that it consists of repeating units of 3.6 +/- 0.2 micron, corresponding to a mol.wt of 7.2 +/- 0.4 x 10(6). Based on differential denaturability, each repeat unit is divided into 2 regions. The larger region of 2.47 +/- 0.11 micron (mol.wt 4.9 +/- 0.22 x 10(6)) corresponds in length to the ribosomal precursor RNA of sea urchins and the smaller, GC-rich, subunit of 1.16 +/- 0.09 micrometer (mol.wt 2.3 +/- 0.18 x 10(6)) is presumed to contain non-transcribed spacer sequences.     

Retention of topical liposomal formulations on the cornea

Summary The ability of liposomes composed of different kinds of phospholipid materials to adhere to the surface of the cornea was studied in the rabbit. The liposomes were labelled with tracer amounts of an I¹²⁵-labelled phosphatidylethanolamine derivative and were instilled in 10 l drops onto the cornea. The retention of radioactivity was monitored. The results show that liposomes containing positively charged phospholipids are better retained than an albumin control. Thus, it may be possible to develop a drug delivery, liposome system which would permit long-term sustained release of ophthalmic drugs onto the cornea.     

L-arginine regulates asymmetric dimethylarginine metabolism by inhibiting dimethylarginine dimethylaminohydrolase activity in hepatic (HepG2) cells

An increase in circulating asymmetric dimethylarginine (ADMA) and a decreased L-arginine/ADMA ratio are associated with reduced endothelial nitric oxide (NO) production and increased risk of vascular disease. We explored relations between ADMA, L-arginine and dimethylarginine dimethylaminohydrolase (DDAH) in liver (HepG2) cells. DDAH is the principle enzyme for the metabolism of ADMA. HepG2 cells metabolised 44.8 nmol/h of ADMA per 3.6 × 10⁶ cells in the absence of L-arginine. The metabolism of ADMA at physiological (1μ mol/l, p < 0.01) and at pathological (100μmol/l, p < 0.01) levels was inhibited dose-dependently by L-arginine (0–400μmol/l) in cultured HepG2 cells and increased intracellular ADMA (p = 0.039). L-arginine competitively inhibited DDAH enzyme activity to 5.6 ± 2.0% of the untreated level (p < 0.01). We conclude that L-arginine regulates ADMA metabolism dose-dependently by competing for DDAH thus maintaining the metabolic balance of L-arginine and ADMA, and endothelial NO homeostasis. Received 9 June 2006; received after revision 16 July 2006; accepted 19 September 2006     

Biological activity of enantiomerically pure forms of insect juvenile hormone I and III inBombyx mori

Summary Biological activity of enantiomerically pure juvenile hormones was assayed by topical application on allatectomizedBombyx fourth instar larvae. JHs tested were (10R)-JH I [methyl (2E,6E,10R,11S)-10,11-epoxy-3,11-dimethyl-7-ethyl-2,6-tridecadienoate], (10S)-JH I [methyl (2E, 6E, 10S, 11R)-10,11-epoxy-3,11-dimethyl-7-ethyl-2,6-tridecadienoate], (10R)-JH III [methyl (2E,6E,10R)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate] and (10S)-JH III [methyl (2E,6E,10S)-10,11-epoxy-3,7,11-trimethyl-2,6-dodecadienoate]. Among these compounds, natural (10R)-JH I was most active and the dose needed to induce 50% larval molting was 0.04 g/larva; it was approximately 12,000 times more active than unnatural (10S)-JH I. Though natural (10R)-JH III showed slight biological activity, it was only one three-thousandth of that of (10R)-JH I. Unnatural (10S)-JH III exhibited no biological activity at the levels assayed.     

Effects of retinoic acid on embryonic development of mice in culture

The effects of all-trans-retinoic acid (RA) (tretinoin) on the craniofacial development of mouse embryos were examined using whole embryo culture. In day 8 embryos cultured for 48 h, embryonic growth was inhibited concentration-dependently by all-trans-RA treatment. Most of the treated embryos exhibited hypoplasia of the primary palatal processes and a reduction in the development of the first visceral arches. In day 10 embryos cultured for 48 h, although embryonic growth was not inhibited at any concentrations of all-trans-RA, median cleft lip (93%), hypoplasia of the primary palatal processes (37%) and limb reduction deformities (48%) occurred commonly. Furthermore, RA treatment greatly reduced the size of the secondary palatal processes. The incorporation of 3H-thymidine in the treated maxillary processes was decreased to 65% of the control value at 1.0 x 10(-7) M all-trans-RA. These findings indicate that all-trans-RA is teratogenic in mouse whole embryo culture.     

Toxicity of statins on rat skeletal muscle mitochondria

We investigated mitochondrial toxicity of four lipophilic stains (cerivastatin, fluvastatin, atorvastatin, simvastatin) and one hydrophilic statin (pravastatin). In L6 cells (rat skeletal muscle cell line), the four lipophilic statins (100 micromol/l) induced death in 27-49% of the cells. Pravastatin was not toxic up to 1 mmol/l. Cerivastatin, fluvastatin and atorvastatin (100 micromol/l) decreased the mitochondrial membrane potential by 49-65%, whereas simvastatin and pravastatin were less toxic. In isolated rat skeletal muscle mitochondria, all statins, except pravastatin, decreased glutamate-driven state 3 respiration and respiratory control ratio. Beta-oxidation was decreased by 88-96% in the presence of 100 micromol/l of the lipophilic statins, but only at higher concentrations by pravastatin. Mitochondrial swelling, cytochrome c release and DNA fragmentation was induced in L6 cells by the four lipophilic statins, but not by pravastatin. Lipophilic statins impair the function of skeletal muscle mitochondria, whereas the hydrophilic pravastatin is significantly less toxic.