The use of solvent relaxation technique to investigate headgroup hydration and protein binding of simple and mixed phosphatidylcholine/surfactant bilayer membranes.

The subject of this report was to investigate headgroup hydration and mobility of two types of mixed lipid vesicles, containing nonionic surfactants; straight chain Brij 98, and polysorbat Tween 80, with the same number of oxyethylene units as Brij, but attached via a sorbitan ring to oleic acid. We used the fluorescence solvent relaxation (SR) approach for the purpose and revealed differences between the two systems. Fluorescent solvent relaxation probes (Prodan, Laurdan, Patman) were found to be localized in mixed lipid vesicles similarly as in pure phospholipid bilayers. The SR parameters (i.e. dynamic Stokes shift, Deltanu, and the time course of the correlation function, C(t)) of such labels are in the same range in both kinds of systems. Each type of the tested surfactants has its own impact on water organization in the bilayer headgroup region probed by Patman. Brij 98 does not modify the solvation characteristics of the dye. In contrast, Tween 80 apparently dehydrates the headgroup and decreases its mobility. The SR data measured in lipid bilayers in presence of Interferon alfa-2b reveal that this protein, a candidate for non-invasive delivery, affects the bilayer in a different way than the peptide melittin. Interferon alfa-2b binds to mixed lipid bilayers peripherally, whereas melittin is deeply inserted into lipid membranes and affects their headgroup hydration and mobility measurably.

A new colorimetric assay for studying and rapid screening of membrane penetration enhancers.

PURPOSE: This work aims to demonstrate a novel chemical assay for rapid screening and analysis of the mode of action of membrane interaction by penetration enhancers. METHODS: The new bio-mimetic membrane assembly, consisting of supramolecular aggregates of lipids and conjugated polydiacetylene, undergoes visible and quantifiable blue-red color transitions upon interaction with penetration enhancers. RESULTS: The new colorimetric model has been employed to examine various classes of penetration enhancers, including 1-dodecylhexahydro-2H-azepin-2-one (Azone), oleic acid, propylene-glycol, menthol, ethoxyglycol-diethyleneglycol-monoethyl-ether (Transcutol), polysorbate-polyethylenesorbitan-monolaurate (Tween-20), and the drug 7-chloro-1-methyl-5-phenyl-3H-1,4-benzodiazepin-2-one (Diazepam). The assay enables to evaluate the validity of various observations and hypotheses proposed in previous studies regarding permeation enhancement activities. Our results suggest, for example. that propylene glycol (PG) by itself does not interfere with membranes, but rather exhibits synergistic effect in combination with other penetration enhancers. Similarly, our data demonstrate that Transcutol does not independently interact with membranes. The colorimetric system also indicates that interaction of penetration enhancers with membranes depend upon the lipid phase, as well as the self-assembly properties of the enhancer molecules. CONCLUSIONS: The new biomimetic model membrane system can be applied for rapid screening of the activities of penetration enhancers, and provides insight into the mechanisms of permeability of membrane-active compounds.

Hyperthermia in the chick embryo: HSP and possible mechanisms of developmental defects.

Although hyperthermia is an established teratogen in all species studied and the cellular heat shock response is well known, the mechanisms of developmental deviation remain obscure. We have used a chick model system in which fertilized eggs containing embryos at presomite and/or early somite stages (HH 4-10) were exposed to 45 degrees C for 180 min. Six hours following treatment we did not observe any overt morphological disturbance, but at twelve hours following exposure (when controls reached HH 11-13) embryos exposed at late streak stages (HH 4-6) exhibited severe malformation of the head. Embryos exposed later (HH 6-9) manifested spina bifida at the thoracic and lumbosacral levels. Mirror image heart looping was also observed in 20% of these embryos. Paraxial mesoderm was apparently unaffected. Changes in cell proliferation and induced cell death preceded morphological changes. We used acridine orange and confocal laser microscopy to demonstrate that hyperthermia induced cell death in neural folds starting 6 h following treatment. To assess cell proliferation, we used BrdU incorporation for 4 h. Immunodetection on paraffin sections demonstrated that proliferation was inhibited 6 h after treatment. Heat-exposed embryos exhibited the heat shock response, with protein expression reaching a maximum 4-6 h following heat treatment. Malformed embryos showed an intense heat shock response for a further 6 h. The levels of induced heat shock proteins were similar in the affected neural tube and in the heart, where neither induced cell death nor malformations were observed.

Teratogenic effects of bilirubin--a study using chick embryotoxicity screening test (CHEST).

Using the Chick Embrotoxicity Screening Test (CHEST), two samples of bilirubin of different commercial origin were tested on 2, 3 and 4- day old chick embryos. Water soluble Bilirubin Lachema (containing 20 mg albumin per 1 ml) had no teratogenic effect. On the opposite, Bilirubin Merck (containing 8 mg albumin per 1 ml) manifested an apparent teratogenic potential when single doses 0.2 and 0.6 micrograms were administered intraamniotically on day 4. Dose-dependent malformations of brain and eyes, cleft beak and reduction deformities of limbs were observed. No such effects could be produced by administration of Bilirubin Merck on either day 2 and 3. A tentative explanation of the difference between teratogenic properties of Merck and Lachema bilirubin preparations may be sougth in the different proportion of the free and albumin bound fractions.

Experimental models for drug teratogenicity.

Teratogenicity a specific manifestation of embryotoxicity, is explored in the course of the preclinical phase of drug testing. The official routine rat-rabbit procedures employ an integral experimental model--the whole animal with its species-specific metabolism. Although it has been widely accepted that pharmaco-kinetics of drugs represents the most important source of interspecies differences in teratology, the official procedure is still considered satisfactory. The reason why a new thalidomide affair has not occurred can be explained by the extremely low expression of teratogenic potential at the human population level--a condition inherent in the principles of teratogenesis. On the other hand, many partial experimental models have been developed that use mainly suborganismic objects (i.e. isolated morphogenetic systems, explanted tissues and cell populations). These objects possess many limitations disgracing their use as candidates for replacing the whole-animal experiments. Nevertheless, some of the partial models can be used and must be used in deeper analysis of the teratogenic potential of drugs with the aim of improving the predictive power of embryotoxicity risk assessment. (Fig. 1, Ref. 7.)

Use of chick embryo in screening for embryotoxicity.

In vitro systems afford a unique opportunity for investigating the direct interaction of substances with developing morphogenetic systems (MGSs), since maternal influences are excluded. As a carrier of a complete set of MGSs, the chick embryo in ovo manifests an advantage over those in vitro systems that employ isolated embryos or embryonic tissues that have only limited survival. Under controlled experimental conditions including standardization of subjects, administration technique and mode of evaluation according to the basic principles of teratology, the chick embryo test was demonstrated to be reliable and to afford quantifiable endpoints for evaluation (e.g., specific embryotoxicity effect level, positive dose response, and a stage response and effect). Individual compounds, mixtures of compounds, and even poorly identified extracts can easily be tested. The chick embryo possesses its own basic enzyme-catalyzed drug-transformation capacity and, moreover, it can be used for screening specific human metabolites. Comparative studies have indicated a high predictive value of the chick embryo system relative to other in vitro systems as well as to whole-animal testing.

Embryotoxicity of 9-(S)-(2,3-dihydroxypropyl)adenine.

Embryotoxic properties of a novel nucleoside analog, 9-(S)-(2,3-dihydroxypropyl)adenine (DHPA), with powerful antiviral activity were estimated using the Chick Embryotoxicity Screening Test (CHEST). Exerting no substantial influence upon function of the caudal morphogenetic system on Day 1.5 (stages HH 10-11), the substance manifested strong embryotoxic action when administered in 30- and 100 microgram doses on Days 2 or 3. Deviant morphogenesis involved brain vesicles eyes, the face, body wall, extremities, the heart, and great vessels. The malformations often combined into the characteristic triad comprising microphthalmia, cleft beak, and reduction deformities of the limbs. Embryotoxic properties of an enantiomer (R)-DHPA were less frequently expressed, when compared with those of (S)-DHPA, parallelling their known antiviral activity. The effects of DHPA can be substantially reduced by simultaneous administration of adenine nucleosides. The distribution analysis of (S)-DHPA after application on Day 3 revealed most of the drug unchanged in the yolk and the embryo proper indicating that for expression of the embryotoxic properties no metabolic activation of DHPA was needed. As the embryotoxicity of DHPA was confirmed, even in mice, the biological effect of the drug seems to be of a general character.

Morphogenetic systems and screening for embryotoxicity.

While mutagenesis appears to be a single-cell phenomenon, the target for teratogenesis are the groups of cell populations constituting the morphogenetic system (MGS). The sensitivity of MGSs is of two kinds. First, it is the sensitivity to general cytotoxic agents. It is non-specific and reaches its maximum during early embryogenesis. The second type of sensitivity manifests itself at the more advanced stages of development, and it is intimately connected with the specific cell differentiation. The basic parameters of embryotoxicity can be estimated from the direct exposure of the selected MGSs both to the test substance and to its metabolites occurring in man. Based upon these principles a rapid and inexpensive screening test (CHEST) has been introduced using the embryonic chick. The test is capable of demonstrating the embryotoxicity effect level, the gross dose-effect relationships as well as estimating the general type of embryotoxic action. The predictive value of this procedure is comparable to that of the current routine techniques. A multilevel combined test for embryotoxicity is proposed in which CHEST is engaged as a priority selection system.

An evaluation of the embryotoxic effects of blighted potatoes on chicken embryos.

The embryotoxic properties of an ethanol extract of boiled potatoes infected with Phytophthora infestans were investigated in White Leghorn chicken embryos, using controlled subgerminal injection on the second day of incubation and intraamniotic injection on the third and fourth days. A dose of 0.3 mg of sublimation-dried extract interfered at the somite stages with the function of the caudal morphogenetic system and induced various degrees of the caudal regression syndrome. Administration on the third and fourth days led to the development of a malformation syndrome comprising cranioschisis, celosoma, and cardiac septal defects. An equivalent amount of extract of healthy potatoes of the same variety and injection of pure solanine had the same effect. The results warrant the claim that the main factor responsible for the direct embryotoxicity of potatoes attacked by P. infestans is solanine, which evokes tonic contraction of the smooth muscle elements of the amnion.