Andreas Kistler

Fingertip temperature as an indicator for sympathetic responses

Changes of acral skin blood flow are a commonly used indicator for sympathetic reflex responses to various stimuli. The goal of the present study was to determine whether decreases in fingertip temperature are indicative for sympathetic induced changes in microcirculation. Infrared thermography demonstrated that various stimuli triggering the sympathetic nervous system induced decreases in cutaneous microcirculation, most prominently in fingertip skin. Various such stimuli induced almost immediate temporary vasoconstriction, measured by laser Doppler flux and photoplethysmography. With a lag phase of approximately 15 s, reduced microcirculation was also reflected by a transient decrease in fingertip temperature. Vasoconstrictions were easily demonstrable by fingertip temperature when the starting fingertip temperature was above 32 degrees C and vasoconstriction lasted at least 5 s. Temperature measurement offers the advantages of ease and simplicity of performance and analysis, compared with the more complex analyses of flux and pulse volume.

Limb bud cell cultures for estimating the teratogenic potential of compounds

Mesenchyme cells, derived from embryonic limb buds, cultured at high cell density, multiply and differentiate into chondrocytes. Using alcian blue, a stain specific for cartilage proteoglycans, the degree of chondrogenesis can be visualized in the micromass cultures as well as quantified by extraction of the stain and spectrophotometric determination of its absorbance. In the presence of active retinoids chondrogenesis is concentration-dependently inhibited. For comparison of the activity of the various retinoids the concentration needed to reduce alcian blue staining by 50% was estimated. In order to validate whether the activity in limb bud cells can predict the teratogenic potential in vivo, the in vitro activity of 25 retinoids was compared with their in vivo teratogenicity observed mainly in rodents. For retinoids which were already in the biologically active form like those with a free carboxylic acid endgroup, there was a good quantitative correlation between the in vitro and in vivo activity. In contrast, the ethylester analog etretinate was slightly active and the ethylamine analog motretinide inactive in vitro but both were teratogenic in vivo. This finding may indicate that these retionoids were not metabolized to the active form in vitro. In conclusion, these results suggest that the limb bud cell culture system may be useful for a preliminary testing to select non-teratogenic retinoids. For the risk assessment in humans, however, the in vitro result should be verified in animal studies.

Teratogenesis and reproductive safety evaluation of the retinoid etretin (Ro 10-1670).

Reproduction and teratology studies were performed with etretin, the free acid analog of the retinoid etretinate. The lowest teratogenic dose of etretin in mice, rats and rabbits was 3, 15 and 0.6 mg/kg, respectively. In all three species, the lowest dose which was embryolethal, fetolethal or reduced the survivability of the pups during lactation was 2–3 times higher than the above doses. In rats, the lowest effective dose of etretin was 3 mg/kg in both the study for fertility and general reproductive performance and the peri- and postnatal study. The main adverse effect in these two experiments was a reduced survival of the F1-generation.

Inhibition and reversion of chondrogenesis by retinoic acid in rat limb bud cell cultures

SummaryMesenchyme cells derived from embryonic rat limb buds cultured at high density differentiated into chondrocytes. The degree of chondrogenesis was assessed by alcian blue staining, a stain specific for cartilage matrix. The addition of retinoic acid on day 1 of culture inhibited chondrogenesis in a dose-dependent fashion. When retinoic acid was added to the cultures on day 5, the cartilage nodules, consisting of newly differentiated cartilage cells, disappeared during the following 6 days. Coinciding with this process the histochemically demonstrable alkaline phosphatase activity, localized in the internodular areas, also disappeared. This indicated that retinoic acid not only inhibited chondrogenesis but also induced resorption of cartilage cells and that at least two cell types were affected, the cartilage cells and the cells bearing alkaline phosphatase.Actinomycin D and cycloheximide, inhibitors of RNA and protein synthesis, suppressed the retinoic acid effect in day 5 limb bud cell cultures. This result indicated that the effect of retinoic acid required RNA and protein synthesis and is compatible with the view that vitamin A may act in a hormone-like way.

Structure-activity relationship of retinoids in fetal rat bone cultures

SummaryThe structure-activity relationship of 29 retinoids was investigated in fetal rat bone organ cultures. Retinoids induced the release of proteoglycan followed by cartilage tissue breakdown. In this study the loss of RNA was used as a parameter for cartilage resorption. During 6 days of incubation RNA decreased up to 80% in presence of active retinoids. Thus the ED40 was determined from dose-response curves of the various retinoids. The new compounds, called arotinoids, which contained the retinoic acid carbon skeleton in a fixed cisoid geometric conformation, were up to 200 times more active than all-trans-β-retinoic acid. The most active compound contained a tetramethylated tetralin ring and a second aromatic ring in the side chain. Several lines of evidence indicated that the carboxylic acid end group was essential for the activity of retinoids in fetal bone cultures. The new, highly active retinoids described here might be an excellent tool to investigate whether the retinoid action is mediated by specific cellular retinoid binding proteins.

Embryolethality of new herbicides is not detected by the micromass teratogen tests

New herbicidal compounds (11 pyrimidine-diones, 3 benzoates and 1 sulfonamide) were found to be embryolethal but not teratogenic in rats. The range of the embryolethal dose varied from 0.2 to >200 mg/kg. This broad range enabled us to validate whether proposed in vitro teratogen tests can detect the embryolethality of these herbicides. The IC50 values (inhibition concentration 50%) for both differentiation and proliferation of midbrain and limb bud cells of rat embryos were determined and found to be above 50 μg/ml in all cases, confirming that the herbicides were not teratogenic. No correlation, however, was observed between the embryolethality in vivo and the activities in these cells. In order to test whether the potential to cause embryolethality could be predicted and detected as a general cytotoxic effect, the inhibition of colony forming ability in V79 cells was determined. The results indicated that cytotoxicity in V79 cells may be useful for preliminary testing of the embryolethal effect of herbicides.

Teratogenicity of arotinoids (retinoids) in vivo and in vitro

The effect of structural modifications on the arotinoid molecule, a new class of retinoids, on their teratogenicity in mice was studied. Animals were treated on days 8 and 9 of gestation, the most susceptible stages to retinoid-induced malformations in rodents. The teratogenic potency of the 13 arotinoids tested varied over a dose range of more than five orders of magnitude. Next, we tested whether the quantitative differences in the teratogenicity of these arotinoids correlates with their activity in high density (micromass) cultures of rat embryonic limb bud and midbrain cells. There was a good quantitative correlation between the in vivo teratogenicity and the in vitro activity in limb bud cells but no correlation was found in midbrain cells. Thus, the limb bud cell culture system may be useful for a preliminary testing to select non-teratogenic retinoids. For the risk assessment in humans, however, the in vitro results should be verified in animals studies.

Retinoic acid induced proteoglycan release and cartilage resorption in rat bone cultures are age dependent and inhibited by EDTA

SummaryIn explanted humeri of late fetal rats, retinoic acid was found to induce the release of proteoglycan followed by cartilage resorption. Tissue breakdown, which was demonstrated by losses of DNA, RNA, and protein, coincided with the appearance of necrotic cells. In control humeri chondrocytes were the main cell type, but in humeri treated for 4 days with retinoic acid the surviving cells were chondroblastlike. Sensitivity of proteoglycan release and tissue breakdown to retinoic acid decreased with age.The proteinase inhibitors cysteine, Trasylol, and soya and lima bean trypsin inhibitors did not antagonize the effects of retinoic acid. Phenylmethanesulfonyl fluoride suppressed cartilage resorption more effectively than proteoglycan release, while pepstatin merely suppressed cartilage resorption. The inhibition by EDTA of both the release of proteoglycan and cartilage resorption induced by retinoic acid was dose dependent. Zn2+ abolished these effects, whereas Mn2+ only relieved the release of proteoglycan induced by retinoic acid; this indicates that these two effects of retinoic acid are not necessarily linked.In view of our recent demonstration that the release of proteoglycan induced by retinoic acid requires RNA and protein synthesis, we suggest that the degradation of proteoglycans in response to retinoic acid is dependent upon continued synthesis of metalloproteinases.

Structure-Activity Relationship of Retinoids on the Differentiation of Cultured Chick Foot Skin

SummaryThe influence of retinoids (vitamin A and its analogues) on epithelial differentiation was examined in explants of foot skin from chick embryos. In the presence of retinoic acid (10 μM) keratinization and differentiation of scale-like structures, which occurred in tarsometatarsal skin explants, was inhibited and a mucous metaplasia developed. Retinoic acid caused club-shaped feathers in skin explants taken from the anterior surface of the tibia — skin which was determined to differentiate into feathers. In skin explants taken from a breed with feathered feet, the differentiation of tarsometatarsal skin was completely blocked; in tibial skin, club-shaped feathers resulted in response to retinoic acid. These findings indicated that skin of the two origins reacted differently to the retinoid, as was noted in the breed with scaly feet. The structure-activity relationship of 22 retinoids with marked differences in their biological activity was investigated in tarsometatarsal skin explants. Comparing the concentration of various retinoids needed to completely inhibit the differentiation of scale-like structures, retinoids containing tetramethylated indane or tetraline were 100 and 1,000 times more active than retinoic acid. Retinoids with a sulphur-containing end group were also active but less so than the corresponding compound with a carboxylic acid end group. The inactive ethyl, ester analogue, etretinate, was activated in the presence of esterase, indicating that the free carboxylic acid group was important for the activity of retinoids. The retinoid-induced inhibition of keratinization followed by mucous metaplasia in cultured chick embryo skin is a simple and useful model system to test new retinoids which may be helpful in the treatment of dermatological and oncological diseases.

Comparative teratogenicity of three retinoids: The arotinoids Ro 13-7410, Ro 13-6298 and Ro 15-1570

Three retinoids of the arotinoid series, namely the free carboxylic acid Ro 13-7410, its ethyl ester Ro 13-6298, and the new arotinoid ethyl sulfone Ro 15-1570, were tested for their embryotoxic and teratogenic activity in rats. The retinoids were administered orally on either day 9 or 13 of gestation. Treatment on day 9 of gestation resulted mainly in malformations of the head and the trunk; whereas, on day 13 limb malformations were prominent. Ro 13-7410 and Ro 13-6298 were about 1000 times more embryotoxic and teratogenic than retinoic acid but induced a similar malformation pattern to retinoic acid. In contrast, the sulfur-containing arotinoid Ro 15-1570 was active at similar dose levels to retinoic acid but caused a peculiar malformation pattern on day 13 of gestation. This finding supports the hypothesis that the arotinoid ethyl sulfone Ro 15-1570 has uniqe biological properties, inducing no bone toxicity in adult rats and distinctly affecting limb development.