Oleic acid exhibits an expressive anti-inflammatory effect in croton oil-induced irritant contact dermatitis without the occurrence of toxicological effects in mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Cutaneous inflammatory diseases, such as irritant contact dermatitis, are usually treated with topical corticosteroids, which cause systemic and local adverse effects limiting their use. Thus, the discovery of new therapeutic alternatives able to effectively treat skin inflammatory disorders, without causing adverse effects, is urgently needed. AIM OF THE STUDY: To investigate the topical anti-inflammatory effect of oleic acid (OA), a monounsaturated fatty acid, into Pemulen® TR2-based semisolid dosage forms, employing a croton oil-induced irritant contact dermatitis model in mice. MATERIALS AND METHODS: Male Swiss mice were submitted to skin inflammation protocols by acute and repeated applications of croton oil. The anti-inflammatory activity of Pemulen® TR2 hydrogels containing OA was evaluated by assessing oedema, inflammatory cell infiltration, and pro-inflammatory cytokine IL-1ß levels. The mechanisms of action of OA were evaluated using cytokine IL-1ß application or pretreatment with the glucocorticoid antagonist mifepristone. Possible toxic effects of OA were also assessed. RESULTS: Pemulen® TR2 3% OA inhibited the acute ear oedema [maximal inhibition (Imax) = 76.41 ± 5.69%], similarly to dexamethasone (Imax = 84.94 ± 2.16%), and also inhibited ear oedema after repeated croton oil application with Imax = 85.75 ± 3.08%, similar to dexamethasone (Imax = 81.03 ± 4.66%) on the day 7 of the experiment. Croton oil increased myeloperoxidase activity, which was inhibited by Pemulen® TR2 3% OA (Imax = 71.37 ± 10.97%) and by 0.5% dexamethasone (Imax = 96.31 ± 3.73%). Pemulen® TR2 3% OA also prevented the increase in pro-inflammatory cytokine IL-1ß levels induced by croton oil (Imax = 94.18 ± 12.03%), similar to 0.5% dexamethasone (Imax = 87.21 ± 10.58%). Besides, both Pemulen® TR2 3% OA and 0.5% dexamethasone inhibited IL-1ß-induced ear oedema with an Imax of 80.58 ± 2.45% and 77.46 ± 1.92%, respectively. OA and dexamethasone anti-inflammatory effects were prevented by 100% and 91.43 ± 5.43%, respectively, after pretreatment with mifepristone. No adverse effects were related to Pemulen® TR2 3% OA administration. CONCLUSIONS: OA demonstrated anti-inflammatory efficacy similar to dexamethasone, clinically used to treat skin inflammatory conditions, without presenting adverse effects.

Cariniana domestica fruit peels present topical anti-inflammatory efficacy in a mouse model of skin inflammation.

To investigate the topical anti-inflammatory activity of the crude extract of Cariniana domestica fruit peels (CdE), its dichloromethane, n-butanol, and ethyl acetate (EtAc) fractions, and steroids (ß-sitosterol, lupeol, and stigmasterol) isolated from the EtAc fraction in models of irritant contact dermatitis (ICD) croton oil-induced in mice. We induced skin inflammation by single (acute; 1 mg/ear) and multiple (chronic; 0.4 mg/ear) croton oil application. We topically applied C. domestica (CdE, fractions, and gel formulations) and ß-sitosterol, lupeol, and stigmasterol immediately after applying croton oil. HPLC-DAD chromatography of the EtAc fraction and stability of the gel formulations were verified. HPLC-DAD of the EtAc fraction revealed the stigmasterol, lupeol, and ß-sitosterol presence. CdE and EtAc fraction gels showed no organoleptic or pH changes at room temperatures. CdE and dichloromethane, n-butanol, and EtAc (1 mg/ear) fractions decreased the acute ear edema with maximum inhibition (Imax) of 97 ± 2, 86 ± 1, 81 ± 4, and 95 ± 2%, respectively. CdE and EtAc fraction gel presented similar effects, with respective Imax of 85 ± 6% (3%;15 mg/ear) and 82 ± 2% (1%;15 mg/ear). ß-sitosterol (7.5 µg/ear), lupeol (10 µg/ear), and stigmasterol (5.7 µg/ear) also reduced this parameter by 46 ± 8, 51 ± 7, and 62 ± 7%, respectively. All topical treatments reduced the inflammatory cells' infiltration in the acute ICD model. CdE reduced the ear edema by 77 ± 4% (1 mg/ear) and the inflammatory cell infiltration in the chronic ICD model. CdE's anti-inflammatory effect was accompanied by a minimum development of adverse effects. C. domestica demonstrates a promising potential for the development of a topical anti-inflammatory agent. Graphical abstract Cariniana domestica, popularly known as jequitibá-roxo, presented topical anti-inflammatory activity in an acute and chronic irritant contact dermatitis croton oil-induced in mice. The crude extract (solutions and gel formulations) and different fractions obtained from fruit peels of C. domestica showed topical antiinflammatory activity on skin inflammation models with minimum adverse effects in preliminary toxicological studies (behavior and biochemical parameters). Moreover, the HPLC analysis revealed the presence of ß-sitosterol, stigmasterol and lupeol, which also presented topical anti-inflammatory effect in the acute irritant contact dermatitis croton oil-induced. Our findings support the use of this species as a promising topical antiinflammatory agent.

Differential effects of functionally different histamine H4 receptor ligands on acute irritant dermatitis in mice.

The anti-inflammatory effects of histamine H4 receptor (H4R) antagonists opened new therapeutic options for the treatment of inflammatory/allergic diseases, but the role of H4R in inflammation is far from being solved. Aim of the present study was to investigate the role of structurally related H4R ligands of the aminopyrimidine class with different efficacies and functionalities (neutral antagonist ST-994, partial agonist ST-1006, inverse agonist ST-1012, and partial inverse agonist ST-1124) on croton oil-induced ear edema and pruritus in mice. The H4R ligands were administered subcutaneously before topical application of croton oil. While ST-1006 and ST-1124 were ineffective at any dose tested (10-100 mg/kg), both ST-994 and ST-1012 (30 and 100 mg/kg) significantly reduced croton oil-induced ear edema. Moreover, ST-994, ST-1006, and ST-1124, but not ST-1012, significantly inhibited croton oil-induced ear pruritus at 30 mg/kg. In accordance with results obtained with the reference H4R antagonist JNJ7777120 (100 mg/kg), histological examination of inflamed ear tissue indicated that treatment with ST-994 (30 mg/kg) led to a significant reduction in the inflammatory severity score and in the number of eosinophils infiltrating the tissue, while the number of degranulated mast cells in inflamed tissues was increased in comparison with the number of intact mast cells. These data indicate that croton oil-induced ear inflammation and pruritus seem to be clearly, but variably, affected by the H4R ligands tested. The potential advantage of dual effect of the H4R neutral antagonist ST-994 has to be carefully considered as a new therapeutic approach to the treatment of inflammatory diseases.

Anti-irritant and anti-inflammatory effects of glycerol and xylitol in sodium lauryl sulphate-induced acute irritation.

BACKGROUND: Glycerol is known to possess anti-irritant and hydrating properties and previous studies suggested that xylitol may also have similar effects. OBJECTIVE: Our aim was to study whether different concentrations of these polyols restore skin barrier function and soothe inflammation in sodium lauryl sulphate (SLS)-induced acute irritation. METHODS: The experiments were performed on male SKH-1 hairless mice. The skin of the dorsal region was exposed to SLS (5%) for 3 h alone or together with 5% or 10% of glycerol respectively. Further two groups received xylitol solutions (8.26% and 16.52% respectively) using the same osmolarities, which were equivalent to those of the glycerol treatments. The control group was treated with purified water. Transepidermal water loss (TEWL) and skin hydration were determined. Microcirculatory parameters of inflammation were observed by means of intravital videomicroscopy (IVM). Furthermore, accumulation of neutrophil granulocytes and lymphocytes, the expression of inflammatory cytokines and SLS penetration were assessed, as well. RESULTS: Treatment with the 10% of glycerol and both concentrations of xylitol inhibited the SLS-induced elevation of TEWL and moderated the irritant-induced increase in dermal blood flow and in the number of leucocyte-endothelial interactions. All concentrations of the applied polyols improved hydration and prevented the accumulation of lymphocytes near the treatment site. At the mRNA level, neither glycerol nor xylitol influenced the expression of interleukin-1 alpha. However, expression of interleukin-1 beta was significantly decreased by the 10% glycerol treatment, while expression of tumour necrosis factor-alpha decreased upon the same treatment, as well as in response to xylitol. Higher polyol treatments decreased the SLS penetration to the deeper layers of the stratum corneum. CONCLUSION: Both of the analysed polyols exert considerable anti-irritant and anti-inflammatory properties, but the effective concentration of xylitol is lower than that of glycerol.

Decrease of serum adenine nucleotide hydrolysis in an irritant contact dermatitis mice model: potential P2X7R involvement.

Extracellular adenosine 5'-triphosphate (ATP) has significant effects on a variety of pathological conditions and it is the main physiological agonist of P2X7 purinergic receptor (P2X7R). It is known that ATP acting via purinergic receptors plays a relevant role on skin inflammation, and P2X7R is required to neutrophil recruitment in a mice model of irritant contact dermatitis (ICD).The present study investigated the effects of chemical irritant croton oil (CrO) upon ATP, ADP, and AMP hydrolysis in mice blood serum, and the potential involvement of P2X7R. The topical application CrO induced a decrease on soluble ATP/ADPase activities (~50 %), and the treatment with the selective P2X7R antagonist, A438079, reversed these effects to control level. Furthermore, we showed that CrO decreased cellular viability (52.6 % ± 3.9) in relation to the control and caused necrosis in keratinocytes (PI positive cells). The necrosis induced by CrO was prevented by the pre-treatment with the selective P2X7R antagonist A438079. The results presented herein suggest that CrO exerts an inhibitory effect on the activity of ATPDase in mouse serum, reinforcing the idea that ICD has a pathogenic mechanism dependent of CD39. Furthermore, it is tempting to suggest that P2X7R may act as a controller of the extracellular levels of ATP.

Topical herbal extract (Huangdang mixture) exhibits both preventive and therapeutic effects in murine acute irritant contact dermatitis.

BACKGROUND: Contact dermatitises, including allergic contact dermatitis and irritant contact dermatitis, are among the most common skin disorders in humans. Chinese herbal medicines (CHM) have been used in treating contact dermatitises for centuries. Systemic administration of CHM, including ingredients in huangdang mixture containing Chinese angelica, radix Paeonlae rubra, cat nut, and phelloden dron, rhizoma alismatis, rhizoma smilacis glabrae, and rhizome of swordlike, improves allergic contact dermatitis induced by l-fluoro-2,4-dinitrobenzene. Whether topical applications of these herbal extracts display preventive and/or therapeutic effects on contact dermatitis, thereby avoiding the potential side effects of systemic CHM, remains largely unknown. AIMS: To determine whether this topical CHM extract exerts preventive and/or therapeutic effects, we assessed its efficacy in both allergic contact dermatitis and irritant contact dermatitis murine models. MATERIALS AND METHODS: Allergic contact dermatitis and irritant contact dermatitis murine models were established by topical oxazolone and a phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA), respectively. Ear thickness was assessed in both dermatitis models. RESULTS: Our results demonstrate that this topical CHM extract exhibits both therapeutic and preventive effects in acute irritant contact dermatitis but no demonstrable efficacy in murine allergic contact dermatitis. CONCLUSION: These results suggest that this topical CHM extract could provide an alternative regimen for the prevention and treatment of irritant contact dermatitis.

Insights into the mechanism of Piper betle leaf-induced contact leukomelanosis using C57BL/6 mice as the animal model and tyrosinase assays.

BACKGROUND/OBJECTIVES: Steamed piper betle leaves (PBL) were once used by many Taiwanese women to treat pigment disorders on the face. Most women claimed a quick, favourable response at first, only to be overcome with facial leukomelanosis later. METHODS: C57BL/6 mice were randomly assigned to different groups to study if PBL could cause the following effects: contact dermatitis, leukomelanosis, or hair bleaching. Intracellular melanin content was measured by tyrosinase assays. RESULTS: Most steamed PBL-treated mice developed contact dermatitis and postinflammatory hyperpigmentation (PIH) on their shaved backs. About half developed bleached hair to varying extents. The steamed PBL did not only bleach the hairs, but also, unexpectedly, stimulated melanocyte replication, indicated by the fact that the number of functional melanocytes in the tail epidermis increased significantly after treatment (P = 0.007). Using tyrosinase assays PBL extract at the undiluted concentration showed limited inhibition of melanogenesis, probably via melanocytotoxicity. CONCLUSIONS: The leukomelanosis observed in patients might be the consequence of PIH combined with a mixed reaction (hyper- and hypopigmentation), probably due to the different volatile chemicals that surface after steaming the PBL. This conflicting mixed reaction suggests that counteractive ingredients might exist in PBL. PBL, if purified, might be a promising source of a novel bleaching agent.

Self-treatment with bleach by a Latino farmworker.

Skin disease is common among migrant Latino farmworkers. These workers rarely use formal health care services but commonly engage in self-treatment of their skin disease. We present a patient with dermatitis who self-treated with bleach. This patient illustrates a common practice that exacerbates skin disease and sheds light on social and cultural factors of which health care providers serving this community should be aware.

Anti-irritants II: Efficacy against cumulative irritation.

So-called anti-irritants (AI) are widely used in cosmetic formulations, with the aim of reducing irritation from substances in the formulation. It may also be claimed that they are 'soothing' and 'healing' ingredients. However, the proof for these claims is circumstantial. The dose-response effect of 4 alleged AI (nifedipine, (-)-alpha-bisabolol, canola oil and glycerol) was studied on experimentally induced acute irritation in healthy volunteers, and only glycerol showed dose-related response and effects potentially better than no treatment. The acute irritation model only allowed a small window of opportunity in which to demonstrate efficacy. Therefore, the effect of AI was studied in a cumulative irritation model by inducing irritant dermatitis with 10 min daily exposures for 5+4 days (no irritation on weekend) to 1% sodium lauryl sulfate on the right and 20% nonanoic acid on the left volar forearm. AI ointments were applied twice daily. Clinical scoring was performed daily, evaporimetry (Trans Epidermal Water Loss), hydration and colourimetry were measured at baseline (D0), in the middle and at the end of treatment. The glycerol ointment was the only treatment statistically better than both 'no treatment' and vehicle.

Anti-irritants I: Dose-response in acute irritation.

The term 'anti-irritant' (AI) was coined in 1965 by Goldemberg to describe a diverse group of topical product ingredients, which were able to reduce the irritation potential of other more irritating ingredients in the same product. 'AIs' are being added to cosmetic formulations in order, allegedly, to benefit tolerability of the products and allow claims such as 'soothing' and 'healing' ingredients. Limited documentation in favour of the efficacy of AIs is published. We studied the dose-related effect of 4 alleged AIs (nifedipine, (-)-alpha-bisabolol, canola oil and glycerol) on experimentally induced acute irritation in healthy volunteers. Each AI was used in 3 concentrations. Acute irritation was induced by occlusive tests with 1% sodium lauryl sulfate and 20% nonanoic acid in N-propanol. The irritant reactions were treated twice daily with AI-containing formulations from the time of removal of the patches. Evaluation of skin irritation and efficacy of treatments were performed daily for 4 days using clinical scoring, evaporimetry (transepidermal water loss), hydration measurement and colourimetry. Only glycerol showed dose-response and effects potentially better than no treatment. There was no significant effect and no difference between the three other AIs.

Neutral endopeptidase terminates substance P-induced inflammation in allergic contact dermatitis.

Sensory nerve-derived neuropeptides such as substance P demonstrate a number of proinflammatory bioactivities, but less is known about their role in inflammatory skin disease. The cell surface metalloprotease neutral endopeptidase (NEP) is the principal proteolytic substance P-degrading enzyme. This study tests the hypothesis that the absence of NEP results in dysregulated inflammatory skin responses. The effector phase of allergic contact dermatitis (ACD) responses was examined in NEP(-/-) knockout and NEP(+/+) wild-type mice and compared with the irritant contact dermatitis response in these animals. NEP was found to be normally immunolocalized in epidermal keratinocytes and dermal blood vessels. The ACD ear swelling response was 2.5-fold higher in animals lacking NEP and was accompanied by a significant increase in plasma extravasation and infiltration of inflammatory leukocytes. The augmented ACD response in NEP(-/-) animals was abrogated by either administration of a neurokinin receptor 1 antagonist or by repeated pretreatment with topical capsaicin. Similar to NEP(-/-) mice, the acute inhibition of NEP in NEP(+/+) animals resulted in an augmented ACD response. In contrast to the ACD responses, little differences were observed in the irritant contact dermatitis response of NEP(-/-) compared with NEP(+/+) animals after epicutaneous application of the skin irritants croton oil or SDS. Thus, these results indicate that NEP and cutaneous neuropeptides have a significant role in the pathogenesis of ACD.

Contact leukomelanosis induced by the leaves of Piper betle L. (Piperaceae): a clinical and histopathologic survey.

BACKGROUND: In April 1997, an unusual pigmentary disorder was noticed by dermatologists in Taiwan. All patients had a history of using facial dressings with steamed leaves of Piper betle L. (Piperaceae). OBJECTIVE: Our purpose was to clarify the evolution and the origin of this unique leukomelanosis. METHODS: Fifteen patients with an unusual pigmentary disorder, who visited our clinic in September and October 1997, were asked to complete a questionnaire designed to elicit the history related to the disorder. Eight of these 15 patients underwent skin biopsies: 6 on the mottled hyperpigmented area (group A) and 2 on the hypopigmented area (group B). All 8 specimens were prepared with hematoxylin-eosin, Masson-Fontana, and S-100 stains. RESULTS: The results of the questionnaire revealed that these patients had all experienced a temporary erythematous reaction in the first few days of the use of the facial dressing, and 9 of them also complained of an accompanying stinging sensation. A bleaching effect became noticeable approximately 1 week to 1 month later. Eight patients reported that the hyperpigmentation and confetti-like hypopigmentation occurred after overexposure to the sun. In both groups, histopathologic examination revealed some melanophages in the dermis. Masson-Fontana staining of specimens from group A showed local interspersed depigmentation and hyperpigmentation in the basal epidermis and pigmentary incontinence in the dermis. This picture was different from the homogeneous depigmentation within basal epidermis in specimens from group B. In both groups, S-100 staining was negative for melanocytes in the depigmented area. CONCLUSION: The clinical course and histopathologic findings suggest that the evolution of this pigmentary disorder can be divided into 3 stages. The first stage is the immediate bleaching stage, when an irritant reaction is usually conspicuous. The second stage consists of prominent hyperpigmentation visible both grossly and microscopically. The final stage is characterized by confetti-like depigmentation. It may be induced by chemicals in the betel leaves such as phenol, catechol, and benzene derivatives, perhaps through inhibition of melanin synthesis or melanocytotoxicity.

Short-term biomarkers of tumor promotion in mouse skin treated with petroleum middle distillates.

Topical application of certain petroleum middle distillates (PMD) to mice produces skin tumors after long latency, and initiation/promotion protocols indicate that this effect is associated with their tumor promoting activity. Since induction of sustained, potentiated epidermal hyperplasia is predictive of promoting activity, five compositionally distinct PMD [hydrodesulfurized kerosene (API 81-07); hydrodesulfurized PMD (API 81-10); odorless light petroleum hydrocarbons; severely hydrotreated light vacuum distillate (LVD); and lightly refined paraffinic oil (LRPO)] were assessed for their effects on epidermal hyperplasia. PMD were administered (2 x/week for 2 weeks) to skin of CD-1 mice. Four quantitative biomarkers of epidermal hyperplasia were evaluated: epidermal thickness, number of nucleated epidermal cells per unit length of basement membrane, labeling (BrdUrd) index of epidermal cells, and induction of epidermal ornithine decarboxylase (ODC) activity. As positive controls, 12-O-tetradecanoylphorbol-13-acetate (TPA) and n-dodecane were utilized. PMD-induced skin irritation was evaluated visually and/or histopathologically. All five PMD produced dose-dependent, skin irritation and epidermal hyperplasia. On a weight basis the magnitude of the maximal PMD-induced effects was similar to that produced by n-dodecane, but > 1000-fold less than that produced by TPA. Epidermal hyperplasia and subacute skin irritancy produced by the five PMD were similar. Of the four short-term markers of tumor promotion assessed, labeling index and epidermal ODC activity were predictive of the relative promoting activities of those PMD for which tumorigenicity bioassay data are available, i.e., API 81-07 > API 81-10 > LRPO. An apparent discrepancy to the predictability of epidermal ODC activity occurred with LRPO:toluene [1:1 (v/v)]. This mixture is nontumorigenic, yet significantly induced epidermal ODC activity. This mixture, however, produced severe epidermal toxicity that precluded any meaningful analysis of short-term biomarkers in relationship to biological activity.

An integrated model for the differentiation of chemical-induced allergic and irritant skin reactions.

Contact and photocontact allergic as well as irritant and photoirritant skin reactions represent a major problem in clinical dermatology and during the development of new pharmaceuticals. Furthermore, there is a lack of in vitro and in vivo assays that provide a clear differentiation between allergic and irritant skin reactions. Here, we describe an integrated model to differentiate between chemical-induced allergic and irritant skin reactions by measuring objective and easy-to-determine parameters within both skin and skin-draining lymph nodes. Dose-response studies with standard contact and photocontact allergens as well as irritants and photoirritants revealed that irritants predominantly induced skin inflammation, which in turn stimulated draining lymph node cell proliferation. In contrast, the induction phase of contact or photocontact allergy was characterized by marginal skin inflammation, but a marked activation and proliferation of skin-draining lymph node cells. Therefore, a differentiation index (DI) was defined describing the relation between skin-draining lymph node cell activation (lymph node cell count index) and skin inflammation (ear swelling). A DI > 1 indicates an allergic reaction pattern whereas DI < 1 demonstrates an irritant potential of a chemical. Experiments with the contact allergen oxazolone, the photocontact allergen TCSA + UVA, the irritant croton oil, and the photoirritant 8-methoxypsoralen + UVA confirmed the predictive value of DI. Furthermore, flow cytometric analysis of lymph node-derived T- and B-cell subpopulations revealed that contact sensitizer, but not irritant, induced the expression of CD69 on the surface of I-A+ cells. In conclusion, further studies with a broad range of irritants and allergens will be required to confirm general applicability.

Developmental toxicity of dermally applied crude oils in rats.

Two crude oils, differing in viscosity (V) and nitrogen (N) and sulfur (S) content, were evaluated for pre- and postnatal developmental toxicity. In Crude I (low V, low N, low S) studies, the material was applied neat to the clipped backs of pregnant rats at dose levels of 0, 125, 500, 1000 (postnatal only), and 2000 (prenatal only) mg/kg. In Crude II (high V, high N, moderate S) studies, the oil was applied similarly but at dose levels of 0, 30, 125, and 500 mg/kg. Rats were exposed to the crude oils on gestation days (GD) 0-19; application sites were not covered. "Prenatal" rats were killed on GD 20. "Postnatal" rats were allowed to deliver naturally; surviving dams and litters were killed 3-4 wk postpartum. Both crude oils produced maternal and developmental toxicity. Adverse fetal effects included increased in utero death, decreased body weight, and reduced ossification of skeletal elements. Parturition was delayed in Crude II dams at 500 mg/kg. The 4-d viability index was decreased in all Crude II-exposed groups. Pup body weights were decreased by each oil, but at the high dose only. Prenatal effects are probably related to polynuclear aromatic compounds (PAC) found in petroleum. The cause(s) of delayed parturition and postnatal toxicity have not been determined.

Coin-rubbing injuries.

Two cases of injuries caused by "coin rubbing" (Kuasha) are presented. In one case these injuries were confined to the neck, raising the possibility of strangulation, and in the other to the trunk and limbs, suggesting torture. Coin rubbing is practiced by most South-East Asian cultures, which believe that it relieves the symptoms of headache, fever, and flu. The causation and characteristics of these injuries and their medicolegal importance are discussed.