TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin.     

Systemic allergic contact dermatitis to fiberglass in a factory worker of wind turbine blades

Fiberglass is extensively used due to its properties of thermal, acoustic and electrical insulation, and also to reinforce other materials such as plastics. Irritant contact dermatitis to fiberglass is a well established occupational dermatose and is due to penetration of small fragments in the cornified layer of the skin. On the other hand, allergic contact dermatitis (ACD) is rare and is more often triggered by sensitivity to the additives and resins used in the manufacture of fiberglass products.

We report a case of ACD to fiberglass in a factory worker of fiberglass reinforced products.     

Systemic allergic contact dermatitis to fiberglass in a factory worker of wind turbine blades

Fiberglass is extensively used due to its properties of thermal, acoustic and electrical insulation, and also to reinforce other materials such as plastics. Irritant contact dermatitis to fiberglass is a well established occupational dermatose and is due to penetration of small fragments in the cornified layer of the skin. On the other hand, allergic contact dermatitis (ACD) is rare and is more often triggered by sensitivity to the additives and resins used in the manufacture of fiberglass products. We report a case of ACD to fiberglass in a factory worker of fiberglass reinforced products.     

Cornified envelope formation by anthralin, simple analogues, and related anthracenones

The ability of the antipsoriatic anthralin to induce HaCaT keratinocyte differentiation was investigated and correlated with its potency to inhibit proliferation of keratinocytes. To determine the structural requirements for this effect, anthralin and seventeen simple analogues or related anthracenones were examined for their ability to induce the formation of cornified envelope as a marker of terminal differentiation. Covalently cross-linked protein was measured as a key feature of this process. Induction of keratinocyte differentiation was significant at a concentration of 0.5 microM anthralin after 48 h exposure. The presence of the 1,8-dihydroxy groups is a critical determinant of cross-linking activity, since removing or exchanging these groups prevented the induction of keratinocyte differentiation. Furthermore, at least one hydrogen atom at the 10-position of anthralin is required. Moreover, anthralin, anthralin dimer, and anthralin triacetate exhibited antiproliferative and antirespiratory activity at concentrations required to induce keratinocyte differentiation, suggesting a causality between these effects. In addition, cornified envelope formation was observed for a number of related anthracenones at concentrations as low as 1-5 microM. In general, compounds containing benzoyl substituents, independent of the position in the anthralin nucleus, were more potent than those having benzyl substituents. Only marginal differences in cross-linking potency were observed within a number of phenylpropionyl substituted analogues, suggesting that the ability to induce keratinocyte differentiation is independent of the nature of substituents at the side chain.     

Structural identification of skin ceramides containing ω-hydroxy acyl chains using mass spectrometry

The stratum corneum (SC) acts as a barrier that protects organisms against the environment and from transepidermal water loss. It consists of corneocytes embedded in a matrix of lipid metabolites (ceramides, cholesterol, and free fatty acids). Of these lipids, ceramides are sphingolipids consisting of sphingoid bases, linked to fatty acyl chains. Typical fatty acid acyl chains are composed of α-hydroxy fatty acids (A), esterified ω-hydroxy fatty acids (EO), non-hydroxy fatty acids (N), and ω-hydroxy fatty acids (O). Of these, O-type ceramides are ester-linked via their ω-hydroxyl group to proteins in the cornified envelope and can be released and extracted following mild alkaline hydrolysis. Tandem mass spectrometry (MS/MS) analysis of O-type ceramides using chip-based direct infusion nanoelectrospray-ion trap mass spectrometry generated the characteristic fragmentation pattern of both acyl and sphingoid units, suggesting that this method could be applied to the structural identification of O-type ceramides. Based on the MS/MS fragmentation patterns of O-type ceramides, comprehensive fragmentation schemes are proposed. In addition, we have also developed a method for identifying and profiling O-type ceramides in the mouse and guinea pig SC. This information may be used to identify O-type ceramides in the SC of animal skin.     

Comparison of in vitro and in vivo human skin responses to consumer products and ingredients with a range of irritancy potential.

Human skin equivalent cultures were investigated as possible pre-clinical skin irritation screens to aid safety assessments for chemicals and product formulations, and to facilitate design of safe and efficient human studies. In vitro responses in human skin equivalent cultures were compared directly to in vivo human skin responses from historic or concurrent skin tests for representative chemicals and products, including surfactants, cosmetics, antiperspirants, and deodorants. The in vivo data consisted of visual scores (i.e., erythema and edema) from skin-patch tests and diary accounts of skin irritation from product-use studies. In the in vitro studies, cornified, air-interfaced human skin cultures (EpiDerm) were evaluated using methods designed to parallel human clinical protocols with topical dosing of neat or diluted test substances to the stratum corneum surface of the skin cultures. The in vitro endpoints have previously been shown to be relevant to human skin irritation in vivo, including the MTT metabolism assay of cell viability, enzyme release (lactate dehydrogenase and aspartate aminotransferase), and inflammatory cytokine expression (Interleukin-1alpha). For surfactants, dose-response curves of MTT cell-viability data clearly distinguished strongly-irritating from milder surfactants and rank-ordered irritancy potential in a manner similar to repeat-application (3x), patch-test results. For the antiperspirant and deodorant products, all the in vitro endpoints correlated well with consumer-reported irritation (r, 0.75-0.94), with Interleukin-1alpha (IL-1alpha) release, showing the greatest capacity to distinguish irritancy over a broad range. IL-1alpha release also showed the best prediction of human skin scores from 14-day cumulative irritancy tests of cosmetic products. These results confirm the potential value of cornified human skin cultures as in vitro pre-clinical screens for prediction of human skin irritation responses. A preliminary report of these results has been published.     

Transdermal delivery of drugs and enhancement of percutaneous absorption

This paper describes 1) the drug delivery through the skin to produce systemic effects, 2) the enhancement of percutaneous absorption by absorption enhancers, heating and complex formation, 3) the mechanism for the enhancement effect by enhancers, 4) the percutaneous absorption of peptides, and 5) the pharmacokinetic analysis for percutaneous absorption. 1,3-Dinitroglycerin, indomethacin (IND) and many drugs were efficiently absorbed via rat and rabbit skins in the presence of some enhancers, and using a microporous membrane therapeutic plasma concentrations were maintained for a long time. Enhancement of percutaneous absorption by the complex formation with fatty acid was observed for propranolol (PL) in vitro and in vivo. Heating at 42-45 degrees C also enhanced the percutaneous absorption dramatically, with decreased activation energies. The following mechanisms for the enhancement effect by enhancers were found: a) an increase in the fluidity of the stratum corneum lipids and reduction in the diffusional resistance to permeants, b) the removal of intercellular lipids and dilation between adherent cornified cells, c) an increase in the thermodynamic activity of drugs in vehicles, d) the exfoliation of stratum corneum cell membranes, the dissociation of adherent cornified cells and elimination of the barrier function. Peptides such as enkephalin, elcatonin and insulin were effectively absorbed through the skin in the presence of some enhancers and specific inhibitors, with no proteolytic degradation. The pharmacokinetic model with two parallel absorption processes, lipidic and aqueous pore transport pathways, in skin could adequately describe the percutaneous absorption of IND, PL and valproic acid. With peptides, a kinetic model including zero-order input rate, first-order permeation rate and first-order degradation rate was able to describe well the steady-state flux of peptides.     

New in-situ gelling biopolymer-based matrix for bioavailability enhancement of glimepiride; in-vitro/in-vivo x-ray imaging and pharmacodynamic evaluations

Glimepiride (Gmp) a third generation of sulphonylurea is a weakly acidic hypoglycemic drug that belongs to Biopharmaceutical Classification System (BCS) class II. It suffers from poor solubility as well as erratic and variable therapeutic effect. The authors investigated the feasibility of utilizing two nontoxic and biodegradable biopolymers (casein (CA) and chitosan (CT)) as a new in-situ gelling tablet matrix to circumvent this limitation. Both polymers in different ratios were combined with constant dose of the drug and compressed by direct compression to produce constant weights of different tablet matrices. Basic tromethamine (Tris) was also included in each matrix as a pH modifier. Swelling indices, rheological properties of the swollen matrices, and their in-vitro drug release in simulating gastric fluid were assessed. The higher the ratio of casein in the tablet matrix, the lower its swelling index and the higher its viscosity indicate a shear thickening property. Intuitively, zero order drug diffusion in 0.1?N HCl prevailed for more than 8?hours from this gelled matrix. Both reduction of blood glucose level up till 11?hours and x-ray imaging of the selected tablets in the GIT of rabbits correlated well with the shear thickening properties. These findings propose a new stable, simple and affordable price matrix with large versatility.     

Tributyltin impairs the reproductive cycle in female rats

Triorganotins are environmental contaminants, commonly used in antifouling agents for boats, that bioaccumulate and thus are found in mammals and humans due to ingestion of contaminated seafood diets. The importance of triorganotins as environmental endocrine disruptors and consequent reproductive toxicity in different animal models is well known; however, the adverse effects on reproductive cycle are less well understood. The potential reproductive toxicity of tributyltin (TBT) on regular reproductive cycling of female rats was examined. Wistar female rats (12 wk old, weighing approximately 230 g) were divided into two groups: control (vehicle, ethanol 0.4%) and tributyltin (100 ng/kg/d, 7 d/wk, for 16 d by gavage). Tributyltin significantly decreased the cycle regularity (%), duration of the reproductive cycle, the proestrus and diestrus phases, and number of epithelial cell in proestrus phase. TBT also increased the duration of metestrus and the number of cornified cells in this phase. Ovary weight and serum 17β-estradiol levels decreased markedly, accompanied by a significant increase in progesterone levels. Histological analysis showed apoptotic cells in corpus luteum and granulosa cells layer, with cystic follicles after TBT exposure. Tributyltin also elevated number of atretic follicles and corpoa lutea. The micronucleus (MN) test, using Chinese hamster ovary cells, demonstrated a concentration-dependent mutagenic effect of TBT, and at 2.0?×?10(-2)ng/ml most of the cells were nonviable. The toxic potential of TBT over the reproductive cycle may be attributed to changes found in the ovarian weight, unbalanced levels of sexual female hormones, and number of ovarian follicles and corpora lutea.     

The use of agar as a novel filler for monolithic matrices produced using hot melt extrusion.

The use of filler materials in an extended release monolithic polymer matrix can lead to a vastly altered release profile for the active pharmaceutical ingredient. A range of excipients for use in monolithic matrices have been discussed in the literature. The body of work described in this research paper outlines the use of agar as a novel filler material in a hot melt extruded polymer matrix. Several batches of matrix material were prepared with Diclofenac sodium used as the active pharmaceutical ingredient (API). Agar and microcrystalline cellulose were used as the filler materials in varying ratios, to examine the effect of % filler content as well as filler type on the properties of the hot melt extruded matrix. The resultant extrudates were characterised using steady state parallel plate rheometry, differential scanning calorimetry (DSC) and dissolution testing. The rheometry analysis concluded that the fillers used resulted in an increase in the matrix viscosity. The DSC scans obtained showed negligible effects on the melting behavior of the matrix as a result of the filler inclusion. Dissolution analysis showed that the presence of the fillers resulted in a slower release rate of API than for the matrix alone. The results detailed within this paper indicate that agar is a viable filler for extended release hot melt produced dosage forms.     

Influence of Different Shellfish Matrices on the Separation of PSP Toxins Using a Postcolumn Oxidation Liquid Chromatography Method

The separation of PSP toxins using liquid chromatography with a post-column oxidation fluorescence detection method was performed with different matrices. The separation of PSP toxins depends on several factors, and it is crucial to take into account the presence of interfering matrix peaks to produce a good separation. The matrix peaks are not always the same, which is a significant issue when it comes to producing good, reliable results regarding resolution and toxicity information. Different real shellfish matrices (mussel, scallop, clam and oyster) were studied, and it was seen that the interference is not the same for each individual matrix. It also depends on the species, sampling location and the date of collection. It was proposed that separation should be accomplished taking into account the type of matrix, as well as the concentration of heptane sulfonate in both solvents, since the mobile phase varies regarding the matrix. Scallop and oyster matrices needed a decrease in the concentration of heptane sulfonate to separate GTX4 from matrix peaks, as well as dcGTX3 for oysters, with a concentration of 6.5 mM for solvent A and 6.25 mM for solvent B. For mussel and clam matrices, interfering peaks are not as large as they are in the other group, and the heptane sulfonate concentration was 8.25 mM for both solvents. Also, for scallops and oysters, matrix interferences depend not only on the sampling site but also on the date of collection as well as the species; for mussels and clams, differences are noted only when the sampling site varies.     

Quantitative structure-activity relationships. V. A simple simple algorithm for Fujita-Ban and Free-Wilson analyses.

For quantitative structure-activity analyses a simple algorithm for the calculation of de novo group contributions by Fujita-Ban analysis is given. This algorithm corresponds in all details to a computer program for linear multiple regression analysis. However, the transformation of the original matrix to the normal equations matrix is easily achieved without a computer. The normal equations matrix can be solved with a modern desk calculator being equipped with a matrix ROM. Two examples are given to explain the algorithm; the calculation of all important statistical parameters is illustrated. As for quantitative structure-activity analyses this algorithm can be applied as well for the calculation of other additive parameters, such as pi from log P-values or omega from equilibrium constants.     

Calcium induced differentiation of SV40 immortalized human epidermal keratinocytes cultured in a defined medium.

Human epidermal keratinocytes, immortalized by the introduction of SV40-adenovirus recombinants (9), were maintained in a low calcium (0.15 mM) defined medium. When differentiation was induced by a higher extracellular calcium concentration (1.5 mM), these cells altered in shape and developed stratification with formation of desmosomes, while they demonstrated limited terminal keratinization. The expression of involucrin, one of the precursor proteins of the cornified envelope, became elevated as the cell density increased, but was not affected by calcium. These results indicate that the SV40 immortalized human keratinocytes, maintained in a low calcium defined medium, partially respond to changes in extracellular calcium concentration.     

Sigmoidal release of indomethacin from pectin matrix tablets: effect of in situ crosslinking by calcium cations

Sigmoidal release pattern is therapeutically beneficial for timed release and colonic drug delivery, and is always observed in coated systems. In this study, sigmoidal release from pectin matrix tablets with indomethacin as a model drug was investigated. The underlying mechanisms are calcium cation-induced in situ crosslinking that retard the initial drug release to a limited percentage. Power law equation n values were estimated for sigmoidal release profiles. Results indicated that calcium chloride incorporated in pectin matrix functioned as retarding mechanisms on drug release. Larger amount of calcium chloride led to slower drug release and matrix erosion. Even at extremely high levels, retarding on drug release and matrix erosion rate was obvious, which highlighted the effect of calcium-induced in situ crosslinking as calcium chloride was a freely water-soluble salt. The sigmoidal release profiles were characterized by power law equation with high correlation coefficients of about 0.99 or over. Power law n values increased up to as high as 1.20 when calcium chloride content kept increasing. Erosion correlated well with release in almost all pectin matrix tablets indicating erosion-controlled mechanisms. It is concluded that large amount of calcium induces in situ crosslinking of pectin matrix and leads to sigmoidal release of indomethacin, and power law n values, sometimes larger than 1.0, are suitable to be used to describe sigmoidal release profiles.     

Passive transdermal drug delivery systems

The skin has evolved as a formidable barrier against invasion by external microorganisms and against the prevention of water loss. Notwithstanding this, transdermal drug delivery systems have been designed with the aim of providing continuous controlled delivery of drugs via this barrier to the systemic circulation. There are numerous systems now available that effectively deliver drugs across the skin. These include reservoir devices, matrix diffusion-controlled devices, multiple polymer devices, and multilayer matrix systems. This review article focuses on the design characteristics and composition of the main categories of passive transdermal delivery device available. Mechanisms controlling release of the active drug from these systems as well as patch size and irritation problems will be considered. Recent developments in the field are highlighted including advances in patch design as well as the increasing number of drug molecules now amenable to delivery via this route. From the early complex patch designs, devices have now evolved towards simpler, matrix formulations. One of the newer technologies to emerge is the delivery-optimized thermodynamic (DOT) patch system, which allows greater drug loading to be achieved in a much smaller patch size. With the DOT technology, drug is loaded in an acrylic-based adhesive. The drug/acrylic blend is dispersed through silicone adhesive, creating a semi-solid suspension. This overcomes the problem with conventional drug-in-adhesive matrix patches, in which a large drug load in the adhesive reservoir can compromise the adhesive properties or necessitate a large patch size. Transdermal drug delivery remains an attractive and evolving field offering many benefits over alternative routes of drug delivery. Future developments in the field should address problems relating to irritancy and sensitization, which currently exclude a number of therapeutic entities from delivery via this route. It is likely that further innovations in matrix composition and formulation will further expand the number of candidate drugs available for transdermal delivery.