Xenobiotics in vitro: the influence of L-cystine, pantothenat, and miliacin on metabolic and proliferative capacity of keratinocytes.

To investigate the effect of cell growth-stimulating agents on human epidermal keratinocytes, we exposed monolayers of normal human keratinocytes derived from foreskin to different concentrations of the amino acid L-cystine, the member of the vitamin B family D-pantothenat, the phytosterol miliacin, and a combination thereof in keratinocyte growth medium. As a test system for the metabolic capacity, we used the activity of mitochondrial deyhdrogenases as measured by XTT, and for the cell proliferation, we determined the BrdU-uptake. The additives, active ingredients of the hair growth drug PRIORIN, were added in the presence of fully supplemented keratinocyte growth medium or a deficient medium without L-cystine, L-methionine, L-histidin, D-pantothenat, epidermal growth factor, and bovine pituary gland extract. Deficient medium itself reduced the metabolic capacity of keratinocytes to 35% compared with keratinocytes in fully supplemented growth medium. In deficient medium cell, proliferation was not measurable. Increasing doses of L-cystine restored the reduced metabolic capacity from 46% (0.009 mg/L) to 54% (0.09 mg/L) and 92% (0.45 mg/L) in deficient medium. Addition of D-pantothenat (0.43 mg/L) enhanced the metabolic capacity to 150% only in fully supplemented growth medium, compared with untreated controls with growth medium. Miliacin (6 mg/mL) increased not only the metabolic capacity (162%) but also stimulated cell proliferation (215%) as measured by BrdU-uptake in growth medium. The combination of all three additives increased the metabolic capacity (245%) synergistically in growth medium. We were able to show effects of D-panthenol, L-lysine, and miliacin on proliferation and metabolic capacity of keratinocyte monocell culture, which was further increased by combination of the three substances. These basic results suggest a beneficial effect on keratinocyte growth and stimulation by products combining these substances (e.g., Priorin). Furthermore, this work emphasizes the suitability of keratinocyte monolayers for pharmacological testings.

An improved and rapid method to construct skin equivalents from human hair follicles and fibroblasts.

To produce sufficient amounts of high quality skin equivalents (SE), either allogenic for dermatopharmacological and dermatotoxicological studies or autologous for transplantation purposes, we established a rapid, easy and cost effective three-dimensional SE model on the basis of human dermal fibroblasts, collagen and freshly plucked hair follicles. Acidic liquid collagen was polymerized with sodium hydroxide in the presence of fibroblasts to form a dermal equivalent (DE) resembling normal human dermis. At 24 h later, freshly plucked hair follicles were implanted into the surface of these DEs after cutting their bulbs off. Another 48 h later, the surface of the SEs was lifted to the air-liquid interface. Fourteen days after implantation, outgrowing keratinocytes from the outer root sheath of the hair follicles completely covered the surface of the SE and built a fully developed, multi-layered and cornified epidermis. Histology and immunofluorescence studies with specific antibodies directed against components of keratinocytes, fibroblasts, cell-adhesion molecules, different extracellular matrix and basement membrane proteins revealed the similarity of our three-dimensional SEs to the in vivo situation in normal human skin. Using autologous cell sources and cell culture media enriched with serum from the respective cell donor, it will be possible to use these SEs for autologous transplantation, thereby reducing the risk of transplant rejection.

Expression of multiple cytochrome p450 enzymes and multidrug resistance-associated transport proteins in human skin keratinocytes.

Cytochrome P450 enzymes metabolize various endogenous and exogenous small molecular weight compounds. Transport-associated proteins, such as P-glycoprotein, multidrug resistance-associated protein and lung resistance protein are overexpressed in drug-resistant cell lines, as well as in human tumors from various histologic origins, including malignant melanoma. Little is known about the expression and function of cytochrome enzymes and multidrug resistance-associated transport proteins in human skin; therefore, the aim of this study was to analyze the expression pattern of cytochrome enzymes and multidrug resistance-associated transport proteins in proliferating human epidermal keratinocytes under constitutive conditions and after induction with various inducers. Reverse transcription-polymerase chain reaction revealed constitutive expression of cytochromes 1A1, 1B1, 2B6, 2E1, and 3A5 in keratinocytes and showed expression of cytochrome 3A4 after incubation with dexamethasone. The expression of cytochrome 1A1 was enhanced on the mRNA level after induction with benzanthracene. Reverse transcription-polymerase chain reaction analysis of the multidrug resistance-associated transport proteins revealed constitutive expression of multidrug resistance-associated proteins 1 and 3-6, and lung resistance protein in human epithelial keratinocytes and was negative for multidrug resistance 1 and 2. Expression of 1 was seen after induction with dexamethasone. Reverse transcription-polymerase chain reaction results were confirmed by immunoblots which showed expression of cytochromes 1A1, 2B6, 2E1, and 3A, multidrug resistance-associated proteins 1, 3, and 5 as well as multidrug resistance 1 after induction with dexamethasone. Immunohistology showed positive immunofluorescence in skin specimens for cytochromes 1A1, 2B6, 2E1, and 3A and multidrug resistance-associated protein 1 and multidrug resistance 1. Constitutive activity of cytochrome 1A1, 2B, 2E1, and 3A enzymes was measured by catalytic assays. These results show that keratinocytes of the human skin express various transport-associated enzymes and detoxifying metabolic enzymes. Previous studies have revealed that cytochrome enzymes and transport-associated proteins play complementary parts in drug disposition by biotransformation (phase I) and anti-transport (phase III) and act synergistically as a drug bioavailability barrier.

Erythropoietic protoporphyria: identification of novel mutations in the ferrochelatase gene and comparison of biochemical markers versus molecular analysis as diagnostic strategies.

BACKGROUND: Erythropoietic protoporphyria (EPP) results from an inherited deficiency of the last enzyme of the heme biosynthetic pathway, ferrochelatase (FC). EPP is usually inherited in an autosomal dominant fashion, and the mutations in the FC gene on chromosome 18q21.3 detected in EPP patients are heterogeneous. METHODS: In this study, we screened the FC gene for mutations in 12 patients from 10 unrelated families with EPP and their family members using heteroduplex analysis, automated sequencing, and restriction enzyme digestion. RESULTS: We detected 8 different mutations in these patients, including 1 missense mutation, 5 frameshift mutations, and 2 splice site mutations, 6 of which are previously undescribed. CONCLUSIONS: We have established the molecular basis of EPP in 10 unrelated families, thereby providing further evidence for the heterogeneity in this disorder. Importantly, molecular diagnosis allowed revisions in the status of several clinically unaffected silent mutation carriers within the families. We compare the value of genetic research strategies with the combination of biochemical data and clinical phenotype as diagnostic tools to confirm a putative diagnosis in EPP.

Inhibition of allergic contact dermatitis to DNCB but not to oxazolone in interleukin-4-deficient mice.

The role of interleukin-4 as a regulator of immune responses in the skin is investigated with regard to the outcome of contact hypersensitivity reaction in interleukin-4-deficient BALB/C mice. In previous studies conflicting results were obtained concerning the role of interleukin-4 in contact hypersensitivity reactions supporting either a proinflammatory or rather an inhibitory function of this cytokine. Interleukin-4 deficient BALB/C mice sensitized to 2,4-dinitrochlorobenzene showed after challenge a significant reduction in magnitude and duration of the contact hypersensitivity response in comparison with wild-type mice. This attenuation was accompanied by a significant reduction of edema and cellular infiltrates in the dermis and a lacking induction of IL-10 mRNA expression in skin. Also, adoptive transfer experiments revealed that BALB/C mice failed to exhibit contact hypersensitivity after injection of lymph node cells obtained from sensitized interleukin-4 deficient mice. To examine further the role of the contact allergen used to induce the contact hypersensitivity response, mice were also sensitized and challenged with Oxazolone. Here a similar magnitude and duration of contact hypersensitivity in both the interleukin-4 deficient mice and BALB/C control mice was observed. This indicates that the contact hypersensitivity response to 2,4-dinitrochlorobenzene and Oxazolone may partly evolve on different pathways being dependent and independent of interleukin-4. Our results clearly show that the complete loss of endogenous interleukin-4 expression in BALB/C mice is associated with an impaired manifestation of contact hypersensitivity response to 2,4-dinitrochlorobenzene, implying an important proinflammatory function of this cytokine.

C73R is a hotspot mutation in the uroporphyrinogen III synthase gene in congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) results from profoundly deficient activity of the fourth enzyme of the haeme biosynthetic pathway, uroporphyrinogen III synthase (UROIIIS). CEP is a rare, recessively inherited disorder, and mutations in the UROIIIS gene detected in CEP patients are heterogeneous. The notable exception to this rule is a single missense mutation, designated C73R, which represents over 40% of all mutant UROIIIS alleles. In this study, we investigated three separate families with CEP from different ethnic backgrounds. We performed haplotype analysis using two microsatellite markers that closely flank the UROIIIS gene on chromosome 10q24, spanning a region of 4 cM on the GB4 linkage panel. Haplotype analysis revealed the occurrence of C73R on different haplotypes in four out of four disease chromosomes studied. The results are consistent with the hypothesis that C73R is a hotspot mutation for CEP, and does not represent wide dispersion of a single ancestral mutant C73R allele.

Cytochrome P450 1B1: a major P450 isoenzyme in human blood monocytes and macrophage subsets.

In this study, cytochrome P450 (CYP; EC 1.14.14.1)-dependent activities and P450 isoenzyme patterns were determined in human monocytes and macrophages, which play a major role in antigen processing including small molecular weight compounds which cause contact dermatitis or drug-allergic reactions. Using reverse transcriptase-polymerase chain reaction (RT-PCR) we determined the mRNA expression of eight CYPs (1A1, 1A2, 1B1, 2B6/7, 2E1, 3A3/4, 3A7 and 4B1) in human blood monocytes and macrophage subsets 27E10 and RM3/1. To study the influence of known P450 inducers, monocytes were incubated in vitro with ethanol, dexamethasone, cyclosporin A (CSA), benzanthracene (BA), phenobarbital (PB), lipopolysaccharide (LPS) and 12-O-tetradecanoyl-phorbol-13-acetat (TPA) for 24 hr. Percoll density gradient isolated monocytes as well as the pro-inflammatory macrophage subtype 27E10 expressed 1B1, 2E1 and 2B6/7. On the other hand, in the anti-inflammatory macrophage subtype RM3/1, predominantly 1B1 and to some extent 2B6/7 were found. Treatment with cyclosporin A, phenobarbital, benzanthracene or ethanol resulted in induction of the expression of 3A3/4. CYP1B1 was the predominant isoenzyme in all monocytes and macrophages. In monocytes purified by adherence or induced by benzanthracene, lipopolysaccharide or 12-O-tetradecanoyl-phorbol-13-acetat, 1A1 was also expressed. Northern blot analysis confirmed the presence of CYP1B1 in monocytes and macrophages, a presence which was also demonstrated on the protein level by immunoblot and by immunohistochemical staining of the cells. The expression of several CYPs in monocytes/macrophages suggests that these cells may be important in the metabolism of small molecular weight compounds, which play a role in allergic contact dermatitis and drug reactions. Of particular interest is the remarkably strong expression of the recently identified dioxin inducible CYP1B1, known to be present in a wide range of malignant tumors.

Vitamin D3 and its synthetic analogue secocholestra-trien-1,2, 24-triol influence the metabolism and the isomerization of retinoic acid in human keratinocytes.

Vitamin D and vitamin A acid share metabolic pathways thereby influencing their benefit as a given drug. Little is known concerning their metabolic interactions in epidermal cells. We compared the influence of 1,25-dihydroxycholecalciferol (vitamin D3 - VD3) and its synthetic analogue secocholestra-trien-1,3,24-triol (tacalcitol - TAC) in combination with different calcium concentrations (Ca) on the metabolism and the isomerization of retinoic acid (RA) in cultured primary human keratinocytes. After preincubation with 0.09, 0.6 and 1.2 mM Ca for 24 h, followed by the addition of 10(-6), 10(-8) or 10(-10) M VD3 or TAC, we added 10(-5) M 13-cis-RA (isotretinoin). 24 h later, concentrations of RA isomers and oxidated RA metabolites were measured by RP-HPLC. VD3 (10(-6) M) inhibited the isomerization of 13-cis-RA to all-trans-RA (tretinoin) and 9-cis-RA independently from the Ca concentration in the culture medium. 10(-6)-10(-10) M TAC equally inhibit the 4-hydroxylation of all-trans-RA significantly (12.8 vs. 6.7% of total RA), thereby reducing the amount of irreversible inactivated 4-oxo-all-trans-RA, leading to a higher persistence of all-trans-RA, the active hormone. Both VD3 and its analogue TAC influence the metabolism of RA, a well-known regulator of epithelial cell proliferation and differentiation processes, in two distinct ways. Further studies are necessary to test the hypothesis that the hormone activity of RA can be increased by concomitant treatment with VD3 which prolongs the persistence of 13-cis-RA, and TAC elevating the intracellular concentration of all-trans-RA.

Recurrent missense mutation in the protoporphyrinogen oxidase gene underlies variegate porphyria.

The porphyrias represent a heterogeneous group of disorders of porphyrin or porphyrin-precursor metabolism, resulting from the inherited or acquired dysregulation of one of the eight enzymes in the porphyrin-heme biosynthetic pathway. Variegate porphyria, one of the acute hepatic porphyrias, is characterized by a partial reduction in the activity of the penultimate enzyme in the heme biosynthetic pathway, protoporphyrinogen oxidase (PPO). Recently, VP has been linked to the PPO gene on chromosome 1q22-23, and several disease-causing mutations have been described. In this study, we identified the underlying genetic lesion in two unrelated patients with VP and investigated all available family members by polymerase chain reaction, heteroduplex analysis, automated sequencing, and restriction enzyme digestion. Mutation analyses in both families revealed a G-to-A transition in exon 6 of the PPO gene resulting in the substitution of arginine by histidine at position 168 of the protein (R168H). This arginine residue is evolutionarily conserved in human, mouse, and Bacillus subtilis, indicating the importance of this residue in PPO function. Our study establishes a recurrent missense mutation as the underlying genetic defect in two unrelated patients with VP and explains the occurrence of the phenotype in their families.

Variegate porphyria: identification of a nonsense mutation in the protoporphyrinogen oxidase gene.

The porphyrias are disorders of porphyrin metabolism that result from inherited or acquired aberrations in the control of the heme biosynthetic pathway. Variegate porphyria is characterized by a partial reduction in the activity of protoporphyrinogen oxidase. In this study, we identified the first nonsense mutation in a family with variegate porphyria. The mutation consisted of a previously unreported G-to-T transversion in exon 5 of the protoporphyrinogen oxidase gene, resulting in the substitution of glutamic acid by a nonsense codon, designated E133X. Our investigation establishes that a nonsense mutation in the protoporphyrinogen oxidase gene is the underlying mutation in this family with variegate porphyria.

CD8+ dermal T cells from a sulphamethoxazole-induced bullous exanthem proliferate in response to drug-modified liver microsomes.

There is evidence that T lymphocytes play a critical role in the pathogenesis of drug-induced bullous exanthems. Sulphonamides are known to be among the most frequent aetiological agents in these severe drug-induced cutaneous hypersensitivity reactions. Several studies indicate that cytochrome P450-dependent metabolites of sulphonamides act as the nominal allergens. A 70-year-old woman with a severe blistering exanthem caused by cotrimoxazole (sulphamethoxazole and trimethoprim) was studied. We employed an in vitro approach to determine whether cytochrome P450-dependent enzymes activated drug-specific T lymphocytes from this patient. Immunohistochemical analysis of involved skin revealed a majority of epidermal CD8+ T lymphocytes, whereas the dermal infiltrate was composed of both CD4+ and CD8+ T cells. Dermal T lymphocytes isolated from lesional skin proliferated in response to sulphamethoxazole, but not to trimethoprim, in the presence of autologous mononuclear cells used as antigen-presenting cells. The antigen-specific response of sulphamethoxazole-specific T cells was significantly augmented in the presence of murine liver microsomes with P450-dependent catalytic activities. Our observations suggest that some cutaneous hypersensitivity reactions to sulphamethoxazole are due to drug-specific T lymphocytes. Cytochrome P450-dependent enzymes may play a critical role in the formation of the nominal antigen, which is recognized by antigen-specific T cells.

Routine intraoperative application of high-dose aprotinin in open heart surgery in adults: antibody formation after first exposure.

High-dose aprotinin is now routinely used in cardiac surgery to reduce postoperative blood loss and transfusion requirements, although several cases of anaphylactic reactions to the proteinase inhibitor have been reported. As part of a multi-centre study to evaluate the immunological response to aprotinin after first exposure 61 cardiac surgical patients were treated with the Hammersmith regimen. Patients with previous aprotinin exposure were excluded from the study. To determine specific IgG and IgE antibodies blood samples were taken pre-operatively, within 3 to 4 weeks and 6 to 7 months after operation. Determinations were made by using Western Blot and ELISA methods. Fifty-six patients were followed up for a 6-month period, 26 (46.4%) of them developed IgG antibodies to aprotinin determined by Western Blot, whereas only 14 (26.8%) patients with IgG antibodies were found by the ELISA. IgE antibodies were not found in any of the patients. On hospital admission and 6 months post-operatively additional intradermal prick tests were performed. No clear-cut positive reaction to the skin test was found in any patient.

Multiple cytochrome P450 isozymes in murine skin: induction of P450 1A, 2B, 2E, and 3A by dexamethasone.

Cytochrome P450s (P450s) are a supergene family of enzymes responsible for the metabolism of a wide range of endogenous and foreign compounds. P450 isozymes possess overlapping substrate specificity. Systemic administration of dexamethasone, a widely used topical agent in dermatologic practice, to animals is known to result in the induction of multiple P450 isozymes in liver. In this study the effect of topical application of dexamethasone to mice on P450-dependent monooxygenase activities, expression of P450 isozymes, and P450 mRNA levels in skin was assessed. The treatment of mice with dexamethasone resulted in significant induction of 7-ethoxyresorufin O-deethylase (2.3 times), 7-pentoxyresorufin O-depentylase (19.2 times), para-nitrophenol hydroxylase (7.5 times), and erythromycin N-demethylase (2.2 times) activities; the monooxygenases catalyzed preferentially by P450 isozymes 1A1, 2B1, 2E1, and 3A, respectively. Immunoblot analysis of cutaneous microsomes, employing antibodies directed against purified P450s 1A1/2, 2B1/2, 2E1, and 3A, showed that dexamethasone treatment results in an increased immunoreactivity (1.8-13.9 times). In immunohistochemical staining of skin with antibody against P4502B1/2, topical application of dexamethasone resulted in an increased reactivity towards microsomal protein in the suprabasal layer of the epidermis and with the cells of the hair follicles. Whereas constitutive expression of mRNAs for CYP1A1 and CYP2E1 was evident in murine skin, any change in the levels of these mRNAs following treatment with dexamethasone was not apparent. The results of our study indicate that the application of dexamethasone to murine skin results in the induction of several families of P450 isozymes, suggesting that murine skin contains multiple inducible P450 isozymes capable of participating in the metabolism of a wide range of xenobiotics and endogenous compounds.

Evidence for multiple inducible cytochrome P450 isozymes in SENCAR mouse skin by pyridine.

Pyridine, an amphipathic solvent, is widely used in industry and is also a constituent of tobacco and its smoke. Since, in addition to inhalation and ingestion, pyridine is also readily absorbed through skin, we assessed the effect of skin application of pyridine on monooxygenase activities and cytochrome P450 (CYP) isozymes and CYP mRNA levels in the skin of SENCAR mice. Compared to controls, a single topical application of pyridine (30 or 50 mg/100 g body weight) resulted in induction of cutaneous 7-ethoxyresorufin O-deethylase, 7-pentoxyresorufin O-depentylase, and erythromycin N-demethylase activities. Pyridine treatment also resulted in an increase in reactivity with monoclonal antibodies directed against CYP 1A1, 2B1 and 3A. In Northern blot analysis, treatment of pyridine also showed a significant increase in mRNA for Cyp1a-1 in the skin. These data indicate that murine skin contains multiple inducible CYP isozymes, and that pyridine results in the induction of at least three families of CYP in murine skin.