Chloracne: a case series on cutaneous expression of CYP1A1 as diagnostic biomarker.

Chloracne, also known as metabolizing acquired dioxin-induced skin hamartomas (MADISH), is a rare disfiguring disease related to dioxin exposure. There is a paucity of literature on the clinical manifestations and pathogenesis of chloracne/MADISH. The aim of this study was to assess the clinical features of this very unusual acneiform eruption and to explore the pathogenesis of the disease. This was a retrospective, observational report study was conducted on five patients belonging to the same nuclear family (father, mother and three children) and a relative (father's brother) living in the same house. Histopathological, immunohistochemical, laboratory and toxicological analyses were performed for all patients. The results suggest that CYP1A1 in human skin is a diagnostic biomarker in chloracne, and was positive for all the patients in our sample. Tetrachlorodibenzo-p-dioxin is the most investigated dioxin responsible for chloracne; however, several other agonists, whether dioxin-like or not, can activate the aryl hydrocarbon receptor. To our knowledge, this Italian case series is the first study to suggest polychlorinated biphenyls as a possible cause of an overstimulation of aryl hydrocarbons causing the consequent acneiform eruption.

Microcomedones in non-lesional acne prone skin New orientations on comedogenesis and its prevention.

BACKGROUND: In non-lesional skin of acne patients, cyanoacrylate skin surface stripping can harvest a structure called microcomedone (MC) which is the earliest phase of comedogenesis; the root of any subsequent clinical lesion and a target for the prevention of acne relapses. More information is needed on the putative biochemical contributors (biomarkers) of comedogenesis expressed in MC. METHODS: Proteins expressed in MC were screened by proteomics, immunohistochemistry and Western blotting. The in vitro effects of a comedolytic Silybum marianum fruit extract (SMFE) were studied in sebocyte cultures by RNA-Seq and modulation of CYP1A1 by qPCR and enzymatic activity. MC severity was correlated to lesions counts and keratin expression during 48 weeks in 23 acne patients using a topical comedolytic formulation containing SMFE. RESULTS: Two infundibular keratins, K75 and K79, co-localized in MC with the sebocyte progenitor cell marker LRIG1 and were used as a biomarker of comedogenesis for the follow-up of patients. In cultured sebocytes exposed to SMFE (i) transcriptomic analysis showed an up-regulation by a factor of 15 of RNA coding for K75 and (ii) the gene expression and catalytic activity of CYP1A1 under exposure to dioxin was decreased. In the acne patients using SMFE, the MC index in non-lesional skin decreased over time and remained until the 48th week, significantly lower than that of the first week. There was a high correlation between the decrease of MC index and the decrease and stability of the clinical lesions counts over time. Importantly, a low MC index status was found to be associated with a significant higher K75 expression in microcomedones. DISCUSSION: These observations provide new orientations on the mechanism of comedogenesis and its prevention. Maintaining a low MC status in non-lesional skin is a sound target for the prevention of acne relapse and a good sentinel of acne remissions.

RASopathic comedone-like or cystic lesions induced by vemurafenib: a model of skin lesions similar but not identical to those induced by dioxins MADISH.

BACKGROUND: Patients treated with vemurafenib for metastatic melanoma often develop skin lesions similar to those observed after exposure to dioxin-like compounds. We previously called these lesions MADISH (metabolizing acquired dioxin-induced skin hamartoma) when analysing a case of acute dioxin poisoning. OBJECTIVE: We performed a clinical trial aimed at comparing the skin lesions observed under vemurafenib treatment with MADISH in order to bring to light a possible crosstalk between vemurafenib and dioxin pathways. METHODS: In this case series study, we explored the histological aspect of skin lesions in 10 cases treated with vemurafenib for malignant melanoma. We also analysed the ability of vemurafenib and tyrosine kinase inhibitors to induce dioxin-AhR pathway. RESULTS: All patients had skin lesions diagnosed as 'non-inflammatory acneiform eruption' by dermatologists. These were predominantly facial with notable retroauricular involvement and clinically compatible with chloracne/MADISH when assessed by dioxin expert. Histological analysis showed mostly comedone-like lesions and dermal cysts containing epithelial wall with basal or lateral epithelial projections and lamellar keratinization and alterations of remaining sebaceous glands. The expression of CYP1A1, a gene highly induced following dioxin exposure, was not observed in these lesions. Vemurafenib and the tyrosine kinase inhibitors erlotinib and gefitinib did not induce CYP1A1 activity. DISCUSSION: Although the skin lesions under vemurafenib treatment were morphologically similar to MADISH, the absence of CYP1A1 expression in dermal cysts of patients and the absence of CYP1A1 activation by vemurafenib led us consider that these skin lesions were different from true MADISH and not mediated by a crosstalk of AhR signalling, but rather to a hyperactivation of PI3K-Akt pathway as a consequence of vemurafenib treatment. A strong expression of CYP1A1 in the epithelial wall of dermal cysts must be required, parallel to the morphology of the lesions, to make the diagnosis of MADISH, the hallmark of an exposure to dioxin-like/chloracnegen compounds.

The potential depigmenting activity of retinaldehyde.

BACKGROUND: Retinoids have been reported to exert depigmenting activity. Unlike most depigmenting agents that target tyrosinase, they are not phenolic agents and may act via different mechanisms. OBJECTIVES: We analysed the properties of retinaldehyde (RAL), a precursor of retinoic acid (RA), as a skin-lightening agent in various models. METHODS: The viability and the depigmenting properties of RAL were assessed in murine melanocytes, in human reconstructed epidermis, and in mice and guinea pigs. The melanin content and cytotoxicity were assessed in melanocytes; in 3-dimensional models, the melanin concentration and the number of active melanocytes were determined. RESULTS: RAL was taken up by melanocytes and mostly metabolised to retinol and retinyl esters, and to a lesser extent to RA. RAL decreased the melanin concentration of guinea pig ears and mouse tails by 54 and 74%, respectively, and decreased the number of active melanocytes by 42 and 77%, respectively. In reconstructed epidermis the melanin concentration was increased by 52%, whereas the number of active melanocytes decreased by 44%. CONCLUSION: RAL exerts a significant depigmenting activity with a mode of action that looks different from that of RA. Our data suggest a skin-lightening effect related to a melanolytic action (i.e. a decrease in melanin concentration, whatever the mechanism) rather than to melanocytotoxicity, besides other still unknown actions of RAL on melanocytes.

The combination of a retinoid, a phenolic agent and an antioxidant improves tolerance while retaining an optimal depigmenting action in reconstructed epidermis.

BACKGROUND: Cutaneous pigmented lesions urge the need to find safe and effective treatments to lighten the skin. OBJECTIVE: The aim of this study was to combine a retinoid (retinaldehyde), a new phenolic agent (4-(1-phenylethyl)-resorcinol) and a proreducing agent (δ-tocopheryl-ß-D-glucopyranoside) to achieve synergistic actions for skin lightening. METHODS: The tolerance profile and the depigmenting properties of these agents were assessed in murine keratinocyte and melanocyte cell lines, as well as in a 3-dimensional model of reconstructed epidermis. RESULTS: Retinaldehyde and 4-(1-phenylethyl)-resorcinol induced a significant decrease of tissue viability in reconstructed epidermis, but this cytotoxicity was prevented by the addition of δ-tocopheryl-ß-D-glucopyranoside. The combination of the three agents was, however, efficient in decreasing the specific melanin content and the density of active melanocytes. CONCLUSION: A combination of various chemicals acting via different mechanisms allows a decrease in the toxicity of each compound alone while retaining optimal skin-lightening properties.

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) poisoning in Victor Yushchenko: identification and measurement of TCDD metabolites.

BACKGROUND: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a long half-life of 5-10 years in human beings as a result of its high lipophilicity, and little or no metabolism. We monitored TCDD, its form, distribution, and elimination in Victor Yushchenko after he presented with severe poisoning. METHODS: In late December, 2004, a patient presented with TCDD poisoning; the levels in his blood serum (108000 pg/g lipid weight) were more than 50 000-fold greater than those in the general population. We identified TCDD and its metabolites, and monitored their levels for 3 years using gas chromatography and high-resolution mass spectrometry in samples of blood serum, adipose tissue, faeces, skin, urine, and sweat, after they were extracted and cleaned with different organic solvents. FINDINGS: The amount of unmodified TCDD in the samples that were analysed accounted for about 60% of TCDD eliminated from the body during the same period. Two TCDD metabolites-2,3,7-trichloro-8-hydroxydibenzo-p-dioxin and 1,3,7,8-tetrachloro-2-hydroxydibenzo-p-dioxin-were identified in the faeces, blood serum, and urine. The faeces contained the highest concentration of TCDD metabolites, and were the main route of elimination. Altogether, the different routes of elimination of TCDD and its metabolites accounted for 98% of the loss of the toxin from the body. The half-life of TCDD in our patient was 15.4 months. INTERPRETATION: This case of poisoning with TCDD suggests that the design of methods for routine assessment of TCDD metabolites in human beings should be a main aim of TCDD research in the metabolomic era. FUNDING: University of Geneva Dermatology Fund, and Swiss Centre for Applied Human Toxicology.

Sirolimus-induced inflammatory papules with acquired reactive perforating collagenosis.

Sirolimus is an immunosuppressive macrolide with antineoplasic properties that is increasingly used in posttransplantation immunosuppression. The treatment is frequently associated with cutaneous side effects such as sirolimus-associated acneiform facial dermatitis, which has been observed in up to 50% of treated patients. We report a 51-year-old female with liver transplantation who developed inflammatory papules and nodules on the face and the upper chest 3 weeks after the initiation of sirolimus therapy. Sequential biopsies revealed lymphocytic infiltration of the dermis with a peculiar pattern of sebotropism, while older lesions showed acquired reactive perforating collagenosis. The lesions were responsive to hydroxychloroquine treatment despite continued sirolimus treatment.

Amicrobial pustulosis of the folds associated with auto-immune disorders. A case report with an analysis of cytokine expression profile in skin lesions of cutaneous neutrophilic lupus.

A new entity was described by Crickx et al. in 1991, associating amicrobial pustulosis of the folds with systemic lupus erythematosus in young females. It is proposed to regroup this entity under the name of 'neutrophilic cutaneous lupus'. We report a case of a 13-year-old girl with a pustular eruption of the cutaneous folds and scalp associated with undetermined connective tissue disease. We performed a screening for the expression of 174 cytokines in the pustules and compared it with other pustular diseases (acne flare, acute generalized exanthematous pustulosis, pustulosis of Sneddon and Wilkinson). Matrix metalloproteinase 9 and Siglec-5 (CD170) were highly expressed in all types of pustules and reflect high neutrophil density. Amicrobial pustulosis of the folds was characterized by a higher expression of interleukin (IL) 1alpha, IL-2 receptor alpha, macrophage colony-stimulating factor, insulin-like growth factor binding protein 1, brain-derived neurotrophic factor, tumour necrosis factor (TNF) alpha and a lower expression of CD14, IL-1beta, IL-12, soluble TNF receptors I and II, growth-regulated oncogene alpha, fibroblast growth factor 4 and vascular endothelial growth factor as compared to the controls.

Safety of topical methimazole for the treatment of melasma. Transdermal absorption, the effect on thyroid function and cutaneous adverse effects.

Methimazole is an oral antithyroid compound that exhibits a skin-depigmenting effect when used topically. However, the effect of topical methimazole on thyroid function has not been reported. This study was aimed at assessing the safety of topical methimazole used to treat pigmented lesions, without affecting thyroid hormones due to systemic delivery. The pharmacokinetics of methimazole, either applied in the form of a 5% topical formulation to facial skin or taken orally in the form of a 5-mg tablet by 6 volunteers, were determined. In addition, the effect of long-term topical applications of 5% methimazole on the function of the thyroid gland in 20 patients with epidermal melasma was determined following 6 weeks of once-daily application. Cutaneous adverse effects of topical methimazole were determined. From 15 min up to 24 h after application, methimazole was undetectable in the serum of the individuals receiving single topical methimazole dosing. Methimazole, however, was detected in serum after 15 min of oral administration and remained detectable in serum up to 24 h after administration. Long-term topical methimazole applications in melasma patients did not induce any significant changes in serum TSH, free thyroxine and free triiodothyronine levels. Topical methimazole was well tolerated by the patients and did not induce any significant cutaneous side effects. Present data together with the previously shown non-cytotoxic and non-mutagenic characteristics of methimazole indicate that this agent could be considered as a safe skin-depigmenting compound for topical treatment of skin hyperpigmentary disorders in humans.

Spectral properties of topical retinoids prevent DNA damage and apoptosis after acute UV-B exposure in hairless mice.

We showed in a recent study that topical retinyl palmitate prevented UV-B-induced DNA damage and erythema in humans. Given that retinyl palmitate is a precursor of retinoic acid, the biological form of vitamin A that acts through nuclear receptors, we wondered whether these protective effects toward UV-B exposure were either receptor dependent or linked to other properties of the retinoid molecule such as its spectral properties. We determined the epidermal retinoid profile induced by topical retinoic acid in hairless mice and analyzed its effect on markers of DNA photodamage (thymine dimers) and apoptosis following acute UV-B exposure; we compared these effects to those induced by other natural topical retinoids (retinaldehyde, retinol and retinyl palmitate) which do not directly activate the retinoid receptors. We then analyzed the direct action of these retinoids on UV-B-induced DNA damage and apoptosis in cultured A431 keratinocytes. Topical retinoic acid significantly decreased (approximately 50%) the number of apoptotic cells, as well as the formation of thymine dimers in the epidermis of mice exposed to acute UV-B. Interestingly, the other topical retinoids decreased apoptosis and DNA damage in a similar way. On the other hand, neither retinoic acid nor the other retinoids interfered with the apoptotic process in A431 keratinocytes exposed to UV-B, whereas DNA photodamage was slightly decreased. We conclude that the decrease of apoptotic cells in hairless mouse epidermis following topical retinoids and UV-B irradiation reflects a protection of the primary targets of UV-B (DNA) by a mechanism independent of the activation of retinoid nuclear receptors, rather than a direct inhibition of apoptosis.

Characterization of the glycogenolysis elicited by vasoactive intestinal peptide, noradrenaline and adenosine in primary cultures of mouse cerebral cortical astrocytes.

In recent years evidence has accumulated indicating the presence of functional receptors for most neurotransmitters on astrocytes. In particular, receptors coupled to adenylate cyclase have been demonstrated, in primary astrocyte cultures, for vasoactive intestinal peptide (VIP), noradrenaline (NA) and adenosine. Here we provide, in primary cultures of cerebral cortical astrocytes prepared from neonatal mice, a detailed characterization of a cAMP-dependent process elicited by VIP, NA and adenosine, i.e. the hydrolysis of glycogen. The EC50s for the glycogenolytic effect of VIP, NA and adenosine are 3, 20 and 800 nM, respectively. The initial rate of glycogen hydrolysis is, in nmol/mg prot/min, 9.1 for VIP and 7.5 for NA. The effect of NA is predominantly mediated by beta-adrenoceptors, although an alpha 1-adrenergic component, acting most likely through protein kinase C activation, is also present. The action of VIP is mimicked by peptides sharing sequence homologies such as PHI and secretin. Glutamate, GABA, carbachol and the peptides NPY and somatostatin do not influence glycogen levels. The glycogen content of the cultures can be markedly increased by anabolic factors present in fetal calf serum, by high (e.g. 25 mM) glucose in the medium and by 48-h pretreatment of the cultures with dibutyryl cAMP. These results indicate that the glycogen content of astrocytes is under the dynamic control of various factors, including certain neurotransmitters. They also further stress the notion of a functional interaction between neurons and glial cells aimed at maintaining local energy metabolism homeostasis.

VIP receptor subtypes in mouse cerebral cortex: evidence for a differential localization in astrocytes, microvessels and synaptosomal membranes.

The binding characteristics of a monoiodinated form of vasoactive intestinal peptide (M-[125I]VIP) to the membranes of astrocytes, intraparenchymal microvessels and synaptosomes were analyzed in mouse cerebral cortex. Binding to astrocytes, studied in primary cultures, indicates the presence of a single class of high affinity binding sites with a Kd of 3.3 nM and a Bmax of 565 fmol/mg protein. The structurally related peptide secretin does not compete for sites labeled by M-[125I]VIP. In cultured astrocytes, VIP has been previously shown to promote glycogenolysis. Secretin, despite its lack of interaction with sites labeled by M-[125I]VIP, stimulates glycogenolysis with an EC50 of 0.5 nM, thus demonstrating the presence in astrocytes of functional secretin receptors independent from those for VIP. Trypsinization of the primary astrocyte cultures followed by replating as secondary cultures, reveals a second class of low affinity binding sites, with a Kd of 41.3 nM and a Bmax of 881 fmol/mg protein. Secretin does not compete for this class of low affinity binding sites either. Binding of M-[125I]VIP to intraparenchymal microvessels reveals the presence of two classes of binding sites with Kd of 1.4 and 30.3 nM, and Bmax of 7.1 and 73.8 pmol/mg protein, respectively. Similar to what is observed in primary or secondary astrocyte cultures, secretin does not interact with these sites. In this cell type VIP stimulates cAMP formation with an EC50 of 18 nM, while secretin is ineffective. Finally, in agreement with previous reports in rat and guinea pig cerebral cortex, two classes of binding sites are observed in synaptosomal membranes: a high affinity class with a Kd of 4.9 nM and a Bmax of 316 fmol/mg protein, and a low affinity class with a Kd of 42.8 nM and a Bmax of 1578 fmol/mg protein. In contrast to what is observed in non-neuronal membranes, in synaptosomal membranes, secretin effectively competes for sites labeled by M-[125I]VIP with an EC50 of approximately 150 nM. These results indicate that secretin may represent a useful tool to discriminate between neuronal and non-neuronal VIP binding sites, since it competes with M-[125I]VIP exclusively for the neuronal class of binding sites.

Adenosine triphosphate and arachidonic acid stimulate glycogenolysis in primary cultures of mouse cerebral cortical astrocytes.

Adenosine triphosphate (ATP) promotes glycogenolysis in primary cultures of mouse cerebral cortical astrocytes with an EC50 of 1.5 microM. A pharmacological analysis indicates an involvement of purinergic P2Y receptors in this action of ATP. Application of either arachidonic acid (AA), or certain unsaturated fatty acids, also results in glycogen breakdown. The EC50 of AA is approximately 50 microM. Thus ATP and AA can be added to the list of neuroactive agents that control glycogen levels in astrocytes, which includes noradrenaline, vasoactive intestinal peptide (VIP), adenosine and histamine.

Topical delivery of retinoids counteracts the UVB-induced epidermal vitamin A depletion in hairless mouse.

UVB irradiation depletes all-trans-retinol (ROL) and all-trans-retinyl esters (RE) from the hairless mouse epidermis. Prevention of this may be of relevance in counter-acting the long-term side effects of UVB exposure. We studied the effects of a topical treatment with natural retinoids before and after UVB exposure on three parameters involved in vitamin A metabolism: the amount of epidermal ROL and RE, the level of functional cellular retinol-binding protein I (CRBP-I), which is likely to protect ROL from UVB, as well as the cytosolic and microsomal enzyme activities which generate ROL and RE, i.e. all-trans-retinaldehyde (RAL) reductase, acylCoA:retinol acyltransferase (ARAT) and retinyl-ester hydrolase (REH). Topical pretreatment with retinoids promoted a dramatic increase of epidermal ROL, RE and CRBP-I levels, a transient increase of RAL reductase and ARAT activities as well as a decreased activity of REH, indicating a direction of epidermal vitamin A metabolism toward storage. In untreated mice UVB irradiation induced a depletion of epidermal ROL and RE in 10 min and a 50% decrease of CRBP-I after 24 h. In mice treated with topical retinoids, and then exposed to UVB, epidermal RE levels were higher than in vehicle-treated, nonirradiated mice. In contrast, ROL was as much depleted after UVB in pretreated as in untreated animals in spite of an induction of CRBP-I, indicating that CRBP-I does not actually protect ROL from UVB-induced depletion in this model. However, the reconstitution of both epidermal ROL and RE, after their depletion induced by UVB, was accelerated by previous topical treatment with RAL. Our results indicate that topical delivery of retinoids partly counteracts UVB-induced vitamin A depletion and promotes recovery.

Effects of the naturally occurring food mycotoxin ochratoxin A on brain cells in culture.

The potential of ochratoxin A (OTA) to damage brain cells was studied by using a three-dimensional cell culture system as model for the developing brain. Aggregating cell cultures of foetal rat telencephalon were tested either during an early developmental period, or during a phase of advanced maturation, over a wide range of OTA concentrations (0.4 nM to 50 microM). By monitoring changes in activities of cell type-specific enzymes (ChAt and GAD, for cholinergic and GABAergic neurones, respectively, GS for astrocytes and CNP for oligodendrocytes), the concentration-dependent toxicity and neurodevelopmental effects of OTA were determined. OTA proved to be highly toxic, since a 10-day treatment at 50 nM caused a general cytotoxicity in both mature and immature cultures. At 10 nM of OTA, cell type-specific effects were observed: in immature cultures, a loss in neuronal and oligodendroglial enzyme activities, and an increase in the activity of the astroglial marker glutamine synthetase were found, Furthermore, at 2 and 10 nM of OTA, a clustering of microglial cells was observed. In mature cultures, OTA was somewhat less potent, but caused a similar pattern of toxic effects. A 24 h-treatment with OTA resulted in a concentration-dependent decrease in protein synthesis, with IC50 values of 25 nM and 33 nM for immature and mature cultures respectively. Acute (24 h) treatment at high OTA concentrations (10 to 50 microM) caused a significant increase in reactive oxygen species formation, as measured by the intracellular oxidation of 2',7'-dichlorofluorescin. These results suggest that OTA has the potential to be a potent toxicant to brain cells, and that its effects at nanomolar concentrations are primarily due to the inhibition of protein synthesis, whereas ROS seem not to be involved in the toxicity mediated by a chronic exposure to OTA at such low concentrations.

The naturally occurring food mycotoxin fumonisin B1 impairs myelin formation in aggregating brain cell culture.

The effects of subchronical applications of the mycotoxin Fumonisin B1 (FB1) were analyzed in vitro, using aggregating cell cultures of fetal rat telencephalon as a model. As cells in the aggregates developed from an immature state to a highly differentiated state, with synapse and compact myelin formation, it was possible to study the effects of FB1 at different developmental stages. The results showed that FB1 did not cause cell loss and it had no effects on neurons. However it decreased strongly the total content of myelin basic protein, the main constituent of the myelin sheath, during the myelination period (DIV 18-28). The loss of myelin was not accompanied by a loss of oligodendrocytes, the myelinating cells. However FB1 had effects on the maturation of oligodendrocytes, as revealed by a decrease in the expression of galactocerebroside, and on the compaction of myelin, as shown by a reduction of the expression of the mnyelin/oligodendrocyte glycoprotein MOG. The content of the cytoskeletal component glial fibrillary acidic protein (GFAP) was decreased in differentiated astrocytes, exclusively, while neurons were not affected by 40 microM of FB1 applied continuously for 10 days. In summary, FB1 selectively affected glial cells. In particular, FB1 delayed oligodendrocyte development and impaired myelin formation and deposition.