6'-substituted naphthalene-2-carboxylic acid analogs, a new class of retinoic acid receptor subtype-specific ligands.

The biological response to retinoic acid (RA) and synthetic derivatives (retinoids) is mediated by three nuclear retinoic acid receptors, RAR alpha, beta and gamma. To explore the potential of retinoids as receptor subtype selective activators, we employed a transcriptional activation assay. Hybrid receptors that recognize an estrogen response element were used to avoid measuring activities of endogenous retinoic acid receptors. In response to retinoic acid, the three hybrid receptors ER-RAR alpha, ER-RAR beta and ER-RAR gamma exhibited the same induction profile as the corresponding wild type receptors RAR alpha, RAR beta, and RAR gamma. Three different retinoids, analogs of 6'-substituted naphthalene-2-carboxylic acid, elicited strong transcriptional activation of gamma receptor while no activation of alpha receptor was observed. Conversely, two retinobenzoic acid analogs showed a limited alpha selectivity. We conclude that retinoids with unique profiles of retinoic acid receptor subtype selectivity can be defined and tested for their impact on cellular differentiation and for therapeutical applications.

[General process of aging]. / Processus généraux du vieillissement.

Genetic instability is considered to be a fundamental factor in the ageing process. This instability results from the accumulation of damage caused to DNA, and this is demonstrated by the relationship between longevity and DNA repair. However, the accumulation of simple somatic mutations does not explain all the experimental findings. We are forced to take into account, not only the instability of the genes, but also the instability of the expression of the genes. Knowing that the expression of genes depends on interactions between DNA and proteins, we can see that genetic instability is based, at least in part, on protein synthesis. Attention has recently been drawn to the mechanism of transcription and notably of messenger DNA division. At the level of translation, the rate at which messenger DNA is degraded and various elongation factors necessary for ribosome activity have also been implicated. Post-translational changes of proteins by glycosylation or oxidation may be involved in the progressive accumulation of errors, which finally leads to cell death.

Unscheduled DNA synthesis: a quantitative indicator of residual immunodetectable pyrimidine dimers in human fibroblasts after ultraviolet-B irradiation.

We have addressed the question whether the level of UV-B induced DNA damage can be accurately assessed by the measurement of the rate of unscheduled DNA synthesis (UDS). Cultured human fibroblasts were irradiated with UV radiation at 290, 313 or 365 nm. The LD50 was 85 J/m2 at 290 nm, 4500 J/m2 at 313 nm, and 70 kJ/m2 at 365 nm. The analysis of UDS measurements indicate complete arrest of repair processes within 24 h after irradiation, irrespective of the dose (in the range 10-60 J/m2 at 290 nm, and 250-1000 J/m2 at 313 nm). Irradiation at 365 nm failed to yield detectable evidence of UDS. Incubation of irradiated cells with an antiserum directed against both 6-4 type and cyclobutane-type pyrimidine dimers shows a clear parallelism between the disappearance of the antibody-binding determinants and the variation of the rate of UDS vs time after the end of the irradiation. Thus it is concluded that in UV-B irradiated normal cultured human fibroblasts, the lack of UDS reflects the absence of immunodetectable pyrimidine dimers.

Changes in glutathione content in human hair follicle keratinocytes as a function of age of donor: relation with glutathione dependent enzymes.

Synopsis Glutathione (GSH) plays an important role in cellular protection during ageing. The hair plucking technique is a non-invasive method for the direct biochemical study of keratinocytes. Hair was taken from the suboccipital area of 63 volunteers (men and women whose ages ranged from 13 to 103 years). The results show a diminution in the glutathione content as a function of age. We compare two groups of population: group A (less than 80) and group B (more than 80). A remarkable fact is observed: group B displays a weak dispersion of the values as compared to A. The glutathione content (nmol 10(-3) mg DNA) is 5.42 +/- 0.60 for A and 1.86 +/- 0.35 for B. A reduction of 88% was observed in glutathione reductase activity and of 78% in the activity of glutathione-S-transferase from group A to group B. The glutathione peroxidase activity remains relatively constant. The decrease in the GSH concentration and the constancy of the glutathione peroxidase suggest that the capacity of the cell to protect itself from peroxides remains unchanged but that the GSH concentration may become the limiting factor.

Evidence for an age-correlated change in glutathione metabolism enzyme activities in human hair follicle.

In this investigation, glutathione peroxidase (GSH-PX), glutathione reductase (GSSG-RD), glutathione-S-transferase (GSH-S-T), gamma-glutamyl transpeptidase (gamma-GT) and glucose-6-phosphate dehydrogenase (G6PDH) were measured in human hair follicle obtained by plucking as source of keratinized cells. This non-invasive method was used on 27 men and women volunteers ranging from 19 to 102 years. Our results show that GSSG-RD, GSH-S-T, gamma-GT and G6PDH activities decrease as a function of age, whereas GSH-PX activity does not vary. We discriminated 2 groups: a first one from 19 to 60 years with a large dispersion of the enzymatic activities and a second one corresponding to elderly people (up to 70) with a smaller dispersion of the values. This study suggests the keratinocytes possess an age-correlated enzymatic detoxification response potential.

Assessment of surfactant cytotoxicity: comparison with the Draize eye test.

Synopsis We have compared the in vitro cellular toxicity and the in vivo ocular irritation potency of 16 surfactants (7 non ionics, 3 anionics, 2 amphoterics, 4 cationics) ranking from very weakly irritant to strongly irritant. In vitro, the cellular toxicity was estimated on Chinese hamster lung fibroblasts (V79) using a cell mortality test and a cell growth inhibition test. For each surfactant, a lethal concentration 50% without foetal calf serum (LC.50-0) and with 10% foetal calf serum (LC50-I0), as well as the concentration required to reduce 50% of the growth (CI.50) were determined. In vivo, each surfactant was applied directly to the cornea of six albino rabbits. The maximal ocular irritation score (I0 max) and the ocular irritation score obtained seven days later (I0 J7) were collected. Comparison of in vitro results with those obtained in vivo showed good correlations, particularly when I0 max and the difference (LC.50-10 - LC50-0) were considered (r= 0.845, P<0.001). These results suggest that the use of cell culture tests as pre-screening systems to appreciate eye irritation potency of surfactants could be a reliable alternative method in order to reduce the use of the Draize rabbit eye test. They can provide a better knowledge of the irritative process induced by surfactants (cellular toxicity and protein interaction potency).

An immunohistological study of the revascularization process in human skin transplanted onto the nude mouse.

The revascularization of human skin transplanted onto the nude mouse was studied by performing double-labeling immunofluorescence microscopy on human skin before transplantation and at different stages, ranging from one week to six months after grafting. With a crossreacting anti-factor-VIII antigen antibody used to identify the endothelial cells, and with human-specific monoclonal antibodies directed against vimentin or HLA-DR antigen, it appeared that the original human endothelial cells of transplanted skin progressively disappear, while murine endothelial cells invade the graft. Moreover, in double-labeling experiments with a crossreacting anti-factor-VIII antibody and a human-specific anti-type-IV-collagen antibody, anastomosis between host and graft vessels and a constant codistribution of graft endothelial cells with human type IV collagen were observed. Finally, double staining with species-specific antibodies directed against murine or human type IV collagen showed that mouse type IV collagen appeared progressively in the graft and was constantly colocalized with human type IV collagen. From these observations, it was concluded that revascularization of human skin transplanted onto the nude mouse proceeds as follows: inoculation; disappearance of human endothelial cells and migration of mouse endothelial cells into the graft over the basement membrane of preexisting human vessels; and production of a new vascular basement membrane by mouse endothelial cells on the original basement membrane of human graft vessels.

Wound healing of human skin transplanted onto the nude mouse. II. An immunohistological and ultrastructural study of the epidermal basement membrane zone reconstruction and connective tissue reorganization.

The reconstruction of human epidermis during healing of human skin wounded after grafting onto the nude mouse was described in a previous paper (M. Démarchez, P. Sengel, and M. Pruniéras, 1986, Dev. Biol. 113, 90-96). The regeneration of the epidermal basement membrane zone (BMZ) and the reorganization of the connective tissue are the subjects of the present study. They were investigated by two complementary methods: electron microscopy to analyze the BMZ reorganization, and indirect immunofluorescence with species-specific and cross-reacting antibodies directed against laminin, bullous pemphigoid antigen, mouse or human collagens of types I or IV, human elastic fibers, fibronectin, fibrin, actin, and human vimentin, to examine the species origin and distribution of BMZ and connective tissue components during the regeneration process. It is reported that grafted human skin preserves its own immunological markers not only in the epidermis but also in the BMZ and dermis as well, and that, after injury, its regeneration proceeds according to the following sequence of overlapping events: production of a mouse granulation tissue; reepidermization by human cells; reconstruction of a BMZ with human characteristics; formation of a human neodermis. It is concluded that human skin grafted onto the nude mouse is able to regenerate its three structural compartments, namely, the epidermis, BMZ, and dermis. Interestingly, it appeared, also, that the connective tissue regeneration would be a two-step mechanism including the sequential formation of two tissues of distinct sources, namely, a granulation tissue and a neodermis.

Onset of epidermal differentiation in rapidly proliferating basal keratinocytes.

Stratified epithelia such as epidermis are classically considered to comprise 2 cell compartments, one consisting of undifferentiated proliferative cells occupying the basal layer, and the other consisting of differentiated postmitotic cells occupying the suprabasal layers. It is also generally assumed that the 58K basic-50K acidic couple of keratins is expressed in basal cells, while the 67K basic-56K acidic couple appears in suprabasal cells. In the present work we demonstrate that the population of basal keratinocytes is heterogeneous, since 8% of them are found to express the 67-56K "suprabasal" set of keratins. The morphology of these transitional cells suggests that they are in the process of detaching from the basement membrane to move upward to the epidermis. Cytoflow-fluorometric studies showed that the fraction of cells in S plus G2/M phases is 4 times higher in transitional keratinocytes than in basal or suprabasal keratinocytes. Altogether, these results suggest that the onset of terminal differentiation occurs in human epidermis in a subpopulation of keratinocytes which are still located in the basal layer, and that a transient increase in proliferation occurs when the cells engage in terminal differentiation and are ready to move toward the suprabasal layers.

Eye-derived growth factor isolated from bovine retina and used for epidermal wound healing in vivo.

Eye-derived growth factor (EDGF) has been found in several ocular tissues and shown to be able to stimulate the in vitro proliferation of cells from various tissues and organisms. It had already been shown that EDGF differs biochemically and biologically from other growth factors such as epidermal growth factor (EGF) and fibroblast growth factor in that it is the only one that can stimulate the in vitro growth of human adult keratinocytes. Moreover EDGF stimulates reepithelialization and neovascularization. In this paper we report data concerning the effect on the rate of epidermal wound healing in guinea pigs of different extracts obtained from adult bovine retina. Our results show that EDGF can significantly increase the rate of reepithelialization when epidermis is detached from dermis and removed after induction of a blister. The doses used were comparable to the ones used to obtain maximal increase of cell proliferation in vitro. However no attempt was made to further investigate the mechanism accounting for the observed wound healing. At 24 h, control wounds maintained under occlusive dressing had only about 50% of their surface covered with cells as opposed to EDGF-treated wounds which were covered up to about 80% (p = 0.05). On the other hand, EGF does not increase the rate of wound healing in this model even at 1000-fold higher doses than those used in in vitro bioassays. Although EDGF is still not purified to homogeneity and another 10- to 100-fold purification might be necessary to achieve homogeneity, our results suggest that EDGF may find therapeutic applications as a potent in vivo epidermal wound healing agent.

Expression of high molecular weight (67K) keratin in human keratinocytes cultured on dead de-epidermized dermis.

The influence of living dermal tissue upon epidermal differentiation during embryonic development as well as in vitro culture has been documented. Living dermal tissue contains both cellular and matricial elements. In the present study, third-passage subcultured adult human keratinocytes were either seeded on plastic dishes or recombined with dead de-epidermized dermis and further cultured for 3 weeks. After this time, keratins were extracted and analysed by one- and two-dimensional gel electrophoresis. The 67K keratin subunit, which is thought to be involved in the process of in vivo type skin differentiation, was absent in ordinary cultures; however, it was expressed in air-exposed cultures on dead de-epidermized dermis. Quantitatively, however, it did not reach the in vivo level. This suggests that in principle, the induction of the expression of this protein does not require the presence of living dermal cells.

Adult human keratinocytes migrating over nonviable dermal collagen produce collagenolytic enzymes that degrade type I and type IV collagen.

Human adult keratinocytes migrating on a nonviable dermal substrate in cultures without fibroblasts induce thinning and degradation of the collagen substrate beneath the migrating epithelium. Further, unconcentrated conditioned medium from the cultures exhibit collagenolytic activity against both type I and type IV collagen which is inhibited by EDTA but not by phenylmethylsulfonyl fluoride or N-ethylmaleimide. Since the migrating epithelium and dermal substrate do not contain fibroblasts, this study shows that migratory keratinocytes in contact with interstitial collagen are capable of producing collagenases against type I and type IV collagen. Moreover, migratory keratinocytes appear to be similar to highly metastatic cells in their ability to degrade basement membrane collagen.

Human epidermis reconstructed by culture: is it "normal"?

Human keratinocytes were grown on a dermal equivalent (or lattice) at the liquid-air interface in an attempt to reconstitute a functional epidermis in vitro. Although the multilayered epithelium thus obtained is well differentiated, as shown by the presence of keratohyaline granules and horny layer, several differences from its in vivo counterpart were also observed: In the reconstructed epidermis, basal keratinocytes do not have the cuboidal shape found in vivo; they synthesize bullous pemphigoid antigen and laminin, but the distribution of these antigens is not linear as in vivo; they contain the plasma-membrane antigens restricted to the basal layer in vivo (VM1, BC1), but these antigens are not polarized; lack of polarization is also evidenced by the distribution of actin. Differentiation markers appear but with a topography slightly different from that of epidermis in vivo; the 67-kD keratin does not appear in the first suprabasal layer as in vivo but above; involucrin, which appears in the granular layers in vivo appears as soon as the cells leave the basal layer. psi 3 antigen and fibronectin found in vivo only in hyperproliferative epidermis (wound healing, psoriasis) are detected. Hyperproliferation would also explain the unexpected straining of basal cells by KL1 monoclonal antibody. Because of the potential clinical or pharmacologic use of artificial epidermis, the question of whether the epidermis obtained in vitro can be considered as "normal" is discussed.

Wound healing of human skin transplanted onto the nude mouse. I. An immunohistological study of the reepithelialization process.

Two months after transplantation of human skin onto the nude mouse, excisional wounds were made through the entire thickness of the skin, at the center of the graft, using a 2-mm punch. At various time intervals thereafter, ranging from 2 days to 9 weeks, the graft sites were harvested and processed for an immunohistological study. With a monoclonal antibody directed against HLA-ABC antigens, it was demonstrated that the healing epidermis is of human origin. Moreover, with three different monoclonal antibodies directed against human keratins, named respectively AE1, AE3, and KL1 and with an anti-involucrin antiserum, it is reported that the keratinization and involucrin distribution patterns observed in normal human epidermis are reconstituted, 2 months after transplantation, in the major part of the grafted epidermis, undergo changes during the reepithelialization process, and are restored in the healed epidermis 9 weeks after injury. This study indicates that the nude mouse/human skin model could be a valuable tool to study a major aspect of regeneration such as the reepidermization of human skin without recourse to human volunteers.

Alternative method for checking toxicity of hair dyes.

Synopsis We have compared the in vitro and in vivo toxicities of 19 hair dyes. In vitro, the toxic effect was estimated in Chinese hamster lung fibroblasts (V79), using a cell growth inhibition test. For each compound, the concentration required to reduce 50% of the growth (CI(50)) was determined. In vivo, compounds were administered by i.p. route to groups of 10 Swiss mice per dose. The LD(50) values obtained were used to estimate the acute toxicity. The comparison of the results in vivo and in vitro showed that there is a good correlation between both ranking orders (correlation coefficient r= 0.90 with a first kind error P= 10(-4)). These results suggest that the use of cell culture tests is valuable as pre-screening systems to appreciate the toxicity level of hair dyes, in order to reduce the use of animals in safety evaluation.