Identification of contact allergens in C.I. Solvent Red 23 (commercial Sudan III) by chemical analysis and animal testing

C.I. Solvent Red 23, commercial Sudan III, is widely used in cosmetic products. Chemical analyses and guinea pig sensitization tests were carried out to identify its contact allergens. In the Magnusson & Kligman guinea pig maximization test, C.I. Solvent Red 23 showed 20% positive reactions. By conducting chemical analyses with HPLC and GLC, 2-naphthol (82 ppm), azobenzene (48 ppm), Sudan I (570 ppm) and many unknown impurities, as well as the main constituent pigment Sudan III (87%), were found. The chemical structure of one unknown impurity was identified as an isomer of Sudan III. We found that purified Sudan III showed no positive reaction, while the isomer elicited 30% positive reactions, in the same guinea pig test. Furthermore, cross-sensitization with p-phenylenediamine was investigated using the guinea pig test. Animals sensitized with p-phenylenediamine also showed positive elicitation reactions with purified Sudan III. From these results, the contact allergenicity of C.I. Solvent Red 23 is considered to be due to impurities, including the isomer of Sudan III, 1-(o-phenylazophenylazo)-2-naphthol. Positive reactions to Sudan III previously demonstrated in hairdressers are due to cross-sensitivity with p-phenylenediamine.     

A modified technique of guinea pig testing to identify delayed hypersensitivity allergens

A modified guinea pig testing technique was developed For the detection of weak allergens and allergenicity of materials unsuitable for testing by intradermal injection. This test involved the use of Freund's complete adjuvant to stimulate the immune system of the animal, and external application instead of intradermal injection of the test compound in the induction stage. The allergenicity of Sudan III, Brilliant Lake Red R and Sudan I was tested by this procedure.
In the dose-effect study of Sudan I, the dose dependency of a positive reaction of the induction and challenge concentrations was recognised.
The test was compared with three other guinea pig sensitization tests. The results obtained with this test correlated well with those obtained with the guinea pig maximization test.     

Modified short-term guinea pig sensitization tests for detecting contact allergens as an alternative to the conventional test

The conventional adjuvant and patch test (APT) method of guinea pig sensitization testing was modified in 2 ways, s-APT and s-APT(2), in order to shorten the test period. These short-term test methods consist of 72-h closed application of test material with intradermal injection of emulsified Freund's complete adjuvant (E-FCA) for 1st induction, 48-h closed application of test material with (s-APT) or without (s-APT(2)) intradermal injection of E-FCA on the 7th day for 2nd induction, and open application on the 14th day for challenge. They were compared with conventional APT by using 8 allergenic chemicals (formaldehyde, nickel sulfate, cobalt sulfate, ethyl-p-aminobenzoate (benzocaine), isoeugenol, 2-mercaptobenzothiazole, 2,4-dinitrochlorobenzene (DNCB) and 1-phenylazo-2-naphthol (Sudan I)). The short-term methods gave similar results to those of conventional APT in terms of mean response, sensitization rate and sensitization potency (challenge concentration that induces a mean response equal to 1.0). Thus, our short-term methods, which are capable of evaluating skin sensitization within 17 days, are sufficiently sensitive to detect potentially hazardous contact allergens.     

Experimental study on skin sensitization potencies and cross-reactivities of hair-dye-related chemicals in guinea pigs

In screening patch testing of hairdressers with occupational contact dermatitis, multiple positive reactions to hair dye-related chemicals, such as p-phenylenediamine (PPD), p-toluenediamine x 2HCl (PTD) and p-aminophenol (PAP), a fabric dye p-aminoazobenzene (PAB), and a tar dye Sudan III, were frequently encountered. To investigate individual skin sensitization potency and the cross-reactivities among above chemicals, a guinea pig maximization test with the above 5 chemicals was performed. In each group, 6 animals were induced with one of the chemicals at 0.1% concentration by intradermal injection and at 1.0% by topical application. The animals were challenged with all 5 chemicals in concentrations of dilution by 10 from 0.1% to 0.001%. Under the conditions of 0.1% challenges, similar sensitization potencies were observed in PPD (6/6), PTD (6/6), PAP (5/6) and PAB (6/6) groups, but no positive reactions were elicited in the Sudan III group. The cross-reactivities to PPD were confirmed in the animals challenged with PTD (6/6), PAP (6/6), PAB (6/6) and Sudan III (3/6). In the PTD-induced group, positive responses to cross-challenges were elicited by PPD (5/6), PAP (3/6), PAB (5/6) and Sudan III (1/6). The cross-reactivities to PAP were observed only with PPD (2/5) and PAB (5/5). PAB-induced animals responded only to PPD (1/6). The results indicate that all these chemicals except Sudan III are strong sensitizers. Their cross-reactivities are different in sensitized conditions, respectively. The cross-reactivities to PPD were higher than those to PTD, PAP and PAB.     

Pigmented contact dermatitis from azo dyes

Eight patients suffering from pigmented contact dermatitis caused by the commercial Brilliant Lake Red R were patch tested with purified Sudan I and its several chemical analogues. Positive reactions were observed to Sudan I, Orange SS, Brilliant Lake Red R, Vacanceine Red, Yellow OB and Sudan II. Negative patch tests were obtained with Toluidine Red, Permanent Orange, Lithol Red and Sudan III.
We found that the commercial Brilliant Lake Red R which had been said to be the most important causative agent of pigmented contact dermatitis, contained Sudan I as a major impurity. The patients reacting to Brilliant Lake Red R always gave a positive reaction to Sudan I, hut these reacting to Sudan I did not always give a positive reaction to Brilliant Lake Red R.
This suggests that Sudan I is a potent sensitizer and may induce contact sensitivity and that Brilliant Luke Red R itself is a weak sensitizer or a cross-reacting substance.     

Antiflammin 1 peptide delivered non-invasively by iontophoresis reduces irritantinduced inflammation in vivo

Abstract The potential of an anti-inflammatory peptide (antiflammin 1) to reduce irritation when delivered transdermally by iontophoresis was examined. A model drug irritant, chlorpromazine, was co-delivered with and without antifiammin 1 by iontophoresis to hairless guinea pigs transdermally. Quantitative skin irritation measurements were obtained by monitoring erythema by skin color reflectance with the Minolta Chromameter. Antifiammin 1 delivered by iontophoresis significantly decreased, but did not eliminate, the erythema associated with co-delivery of an irritating drug compound. Lesion formation was also reduced in the presence of antiflammin 1. In vitro flux across hairless guinea pig skin demonstrated no significant differences in flux of the irritant compound in the presence or absence of antiflammin 1. In vivo generation and efflux of the inflammation mediator Prostaglandin E₂ increased during 24-h application of irritant and was unchanged in the presence of antiflammin 1. This result is discussed with respect to recent evidence that antiflammins may act on the lipo-oxygenase pathway. In summary, antiflammin 1, an antiflammatory peptide, can be delivered transdermally by iontophoresis with retention of its biological activity in vivo.     

Mating type gene for isolates of Trichophyton mentagrophytes from guinea pigs

Trichophyton mentagrophytes were isolated from 19 of 20 guinea pigs in a children's corner of a zoo. The nucleotide sequence identity of the internal transcribed spacer region among 19 guinea pig isolates was 99%, including the reference strain of animal type 3 of T. mentagrophytes. The genomic DNA of all isolates were investigated for the mating (MAT) gene by specific polymerase chain reaction. The alpha‐box gene was detected in all 19 isolates, while the high‐mobility‐group (HMG) gene was detected in only one of 19 isolates. Therefore, the guinea pig population harbored at least 2 MAT types of Arthroderma vanbreuseghemii. The T. mentagrophytes that was prevalent in this population may constitute a constant source of infection for persons coming into contact with guinea pigs.     

Detection of circulating antibodies to purified keratinolytic proteinase in sera from guinea pigs infected with Microsporum canis by enzyme-linked immunosorbent assay

Summary A keratinolytic proteinase (KPase) which is regarded as an important factor in the pathogenesis of dermatophytosis was isolated and purified from Microsporum (M.) canis culture filtrates. Enzyme-linked immunosorbent assay (ELISA) was used to determine the occurrence of circulating antibodies to this enzyme in sera samples from guinea pigs with superficial fungal infections caused by M. canis. Of sera samples from guinea pigs infected with M. canis, 75% were reactive within 10 weeks, however, those ELISA values were relatively low compared with those from guinea pigs immunized with KPase. The presence of circulating antibodies was first detected 2 weeks post inoculation with M. canis, corresponding to the period when the lesions were most severe. The titers of the ELISA antibodies reached a peak at 4 weeks; at that time the lesions had disappeared completely.     

Reverse iontophoresis: monitoring prostaglandin E2 associated with cutaneous inflammation in vivo

Abstract In response to topical application of irritants, increased concentrations of prostaglandin E₂ (PGE₂) are found in human skin exudate and in cultured dermal fibroblasts. In this study, PGE₂ generated in response to transdermal delivery of irritant drug compounds was monitored in hairless guinea pig (HOP) by a non-invasive method, reverse iontophoresis. Reverse iontophoresis is the movement of molecules from the skin under the influence of an applied electric field. Irritant drug compounds were applied with iontophoresis (electrotransport), and reverse iontophoresis of PGE₂ from skin was monitored by radioimmunoassay (RIA) after extraction from the delivery system. Chlorpromazine was used as a model drug irritant. When chlorpromazine and saline were applied over a range of current densities from 0 to 200 μA/cm², visual scores of erythema and edema yielded a correlation with measured skin efflux of PGE₂ (r=0.86). Delivery of chlorpromazine resulted in greater efflux of PGE₂ than delivery of non-irritant saline controls under the same delivery conditions. Five drug compounds, chloroquine, promazine, chlorpromazine, tetracaine, metoclopramide, and saline were applied to hairless guinea pig skin. The 6 agents were similarly rank ordered by visual erythema/edema scores and by PGE₂ efflux, indicating that the quantity of PGE₂ effluxed reflects the intensity of skin irritation. In contrast, vasoconstriction or vasodilation produced by the local delivery of vasoactive agents did not correlate with PGE₂ skin efflux, indicating that this measurement is specific for an inflammatory response. In summary, PGE₂ generated in response to transdermally applied drug irritants can be monitored non-invasively in vivo by reverse iontophoresis.     

The impact of diabetes and metabolic syndromes to the effectiveness of cyclosporine a pharmacotherapy in psoriatic patients

Metabolic diseases concurrent with psoriasis may considerably affect the intensity of its symptoms and therapy efficacy. Cyclosporine A (CsA) is one of the medicines used in conventional therapy of psoriasis. The aim of the study was to determine whether Diabetes 2 and metabolic syndromes influence the efficacy of the CsA therapy in psoriatic patients. The sample group was composed of 32 patients with diagnosed moderate to severe forms of psoriasis vulgaris. The group was divided into subgroups, with regard to concurrently occurring Diabetes 2 and metabolic syndromes. The subgroups were composed of as follows: with diabetes—7 patients, without diabetes—25, with metabolic syndrome—15, without concurrent metabolic syndrome—17, with a metabolic syndrome without diabetes—8 and with both a metabolic syndrome and diabetes—7 patients. The efficacy of therapy was evaluated in each subgroup on the basis of the following scales: PASI, BSA, DLQI on the day of therapy commencing, after 42 and 84 days of the CsA therapy. The statistical analysis was performed with the use of STATISICA 12 (Cracow, Poland; p < .05). The following tests were used: The ANOVA Friedeman test, the posthoc test for ANOVA Friedeman test, the Mann–Whitney U test. We observed clinical improvement measured with PASI BSA scales in each subgroup. The patients themselves also reported improved comfort in their lives, which is confirmed by the lower score in the DLQI scale after 42 and 84 days of the pharmacotherapy. Differences in the values of each scale in a given subgroup turned to be statistically significant. The biggest differences occur after the first 42 days of therapy and they last in the later period of observations. We did not determine any statistically significant differences as a response to treatment in the subgroups subject to comparison. Diabetes and a metabolic syndrome concurrent with psoriasis vulgaris do not affect the efficacy of CsA therapy, which indicates no necessity to modify the standard dosage of the medicine and therapy regime.     

Comparative studies of the sensitization potential of morpholine, 2-mercaptobenzothiazole and 2 of their derivatives in guinea pigs

The aim of this study was to determine the sensitization potential of morpholine (M), 4,4'-dithiodimorpholine (DTDM), morpholinyl-mercaptobenzothiazole (MMBT) and 2-mercaptobenzothiazole (MBT) in guinea pigs. 5% and 10% DTDM, MMBT and MBT produced irritation reactions. M up to 10% failed to irritate. Sensitization tests showed that all the guinea pigs treated with DTDM and MMBT were sensitized. Cross-sensitization tests showed that 60% of the DTDM-sensitized animals reacted to challenge with MMBT; 30% of the animals sensitized to DTDM reacted to challenge with M; 80% of the MMBT-sensitized animals reacted to challenge with MBT and 10% to challenge with M; 42% of the MBT-sensitized animals reacted to MMBT. The rank order of sensitization potential in guinea pigs observed from this study is DTDM, MMBT and then MBT. It appears that the disulfide bond, sulfur linkage and the sulfhydryl group may each play a role in the sensitizing capacity of these compounds.     

HYDROALCOHOLIC HUMAN PLACENTAL EXTRACT: SKIN PIGMENTING ACTIVITY AND GROSS CHEMICAL COMPOSITION

Background. Vitiligo is a pigmentary disorder of the skin of unknown etiology. It is thought to be of autoimmune origin after demonstration of antibody-mediated destruction of melanocytes. Photochemotherapeutic PUVA therapy is widely used in vitiligo with about 33% success. Aqueous or hydroalcoholic extracts of human placenta of ill-defined composition have also been used therapeutically for vitiligo. A hydroalcoholic human placental extract has been developed by us with pigmenting activity based on experimental therapies. Its chemical analysis was the primary objective of this study. Methods. For the guinea pig experiment, 20 drops of the extract or vehicle (60% alcohol) as control was topically applied around the nipples covering the areola zones of male immature white guinea pigs (wt. 175–250 g) daily for 60 days with 15 minutes infrared (IR) exposure used for vascular dilatation and enhancement of the absorption of the extract. Standard methods have been followed for all chemical analyses. Results. The guinea pig experiment showed clear pigmentation and hypertrophy of the experimental nipples to varying degrees. Chemical analysis of the extract revealed the presence of small-molecular-weight proteins/peptides, lipids (including glycosphingolipids), carbohydrates, sialic acids, cholesterol, triglycerides, high density lipoproteins (hdl ), and others, including amino acids, nucleotides, carotenes, vitamins, etc. Conclusion. Glycosphingolipids, known modulators of B and T cells, were reported capable of inducing adhesion, spreading, and motility of melanoma. It is present in the extract and, therefore, may lead to skin pigmentation through induction of melanocytes. Endothelin, a 21-amino acid pep-tide, detected in human placenta and possibly extractable by our process, has been reported to be indispensable for melanocyte growth.     

Efficacy of NVC‐422 in the treatment of dermatophytosis caused by Trichophyton mentagrophytes using a guinea pig model

Objectives Dermatophytes, belonging to genera including Trichophyton, Epidermophyton, and Microsporum, are the causative agents of superficial fungal infections, prevalences of which are estimated to be as high as 25% in the worldwide population. This study evaluated the activity of topical formulations of NVC‐422 (sodium 2‐[dichloroamino]‐2‐methylpropane‐1‐sulfonate), the lead compound in a new class of antimicrobials that consist of broad‐spectrum, fast‐acting, nonantibiotic antimicrobial molecules based on the endogenously produced N‐chlorotaurines. Methods The antifungal efficacy of NVC‐422 was investigated using a guinea pig model of infection with Trichophyton mentagrophytes. Infected guinea pigs were randomly assigned to four treatment and two control groups. The efficacy of the treatments was assessed clinically and mycologically at 72 hours after the final topical dose. Results The test compound 2% NVC‐422 in 1% Noveon Gel demonstrated the highest level of clinical efficacy. Outcomes of treatment with all other test compounds differed significantly from outcomes in the untreated control group (P = 0.003, P = 0.029, P = 0.012, and P < 0.0001, respectively). Fungal elements were detectable in skin sections from untreated guinea pigs but not in skin sections obtained from any of the treatment groups. Conclusions Evaluation of the efficacy of NVC‐422 in the treatment of dermatophytosis using an experimental guinea pig model showed that this compound possesses potent antifungal efficacy as measured by mycological and clinical endpoints. The highest degree of clinical and mycological efficacy was demonstrated by 2% NVC‐422 in 1% Noveon Gel. These data show that NVC‐422 has potent antifungal activity in vivo. Clinical evaluation of NVC‐422 in the treatment of superficial infections caused by dermatophytes, including onychomycosis, is warranted.     

Epidermal hyperplasia induced in guinea pig flank skin by intradermal injection of Sudan red.

Studies of dermal-epidermal interactions were conducted with guinea pig flank skin and intradermal injections of the irritant, Sudan IV dye in olive oil. These injections led to epidermal hyperplasia in areas overlying the irritant and the effect was most significant when the irritant was placed in the upper dermis. Basal cell mitotic activity and thymidine uptake reached a peak by 24 h and thereafter dropped rapidly. Maximal epidermal thickness (4.3 times the control) resulting from an increase in cell number occurred within 2-4 days. Despite the very short period of increased cell growth, epidermal thickness returned to control values only after a 24-day period. A similar growth response could not be induced by saline injections. A single topical application of the irritant showed a qualitatively and quantitatively different epidermal response. These experiments indicate that an intradermal irritant can lead to epidermal hyperplasia and a long-lasting epidermal thickening.     

Increased permeability of psoriatic skin to the protein,plasminogen activator inhibitor 2

The penetration and permeation of the recombinant protein plasminogen activator inhibitor type 2 (PAI-2) in two formulations, one containing a penetration enhancer, into the psoriatic and uninvolved skin of eight patients with plaque-type psoriasis were investigated. Penetration and permeation of PAI-2 were measured by gamma counting and imaging following radiolabelling of a fraction of the applied PAI-2 with ¹²³I. The feasibility of topical delivery of drug to psoriatic plaques was confirmed by the finding that the permeability of psoriatic plaques to radiolabelled PAI-2 (P=0.007) and free ¹²³I (P=0.001) was approximately tenfold higher than the permeability of uninvolved skin. The addition of a penetration enhancer improved the permeation of PAI-2 into psoriatic plaques from an average of 35% to 46% (P=0.005). Occlusion decreased the permeation amount of PAI-2 from 46% to 15% due to losses on the occlusive dressing (P=0.001).Abbreviations PA Plasminogen activator - PAI-2 Plasminogen activator inhibitor, type 2 - tPA Tissue plasminogen activator - uPA Urinary plasminogen activator     

The effect of UV radiation and sun blockers on free radical defence in human and guinea pig epidermis

Summary Defence against oxidative damage by UV-generated free radicals in both guinea pig and human skin has been found to be mediated by the ubiquitous thioprotein, thioredoxin reductase. Human keratinocytes contain approximately 5% thioredoxin reductase in their total acidic protein fraction and also express membrane-associated enzyme activity in cells cultured in synthetic medium. The thioredoxin reductase/thioredoxin system has been shown to reduce superoxide anion radicals through hydrogen peroxide to water. However, both UVA and UVB radiation, below the minimal erythemal dose, generate a sufficiently high concentration of oxygen radicals to deactivate thioredoxin reductase considerably. In albino guinea pigs, enzyme deactivation was up to 70% for UVA and 66% for UVB (n=10 animals/protocol). The application of sun blockers SPF₄, SPF₈ and SPF₁₅ to albino guinea pig skin offered no significant protection for the deactivation of thioredoxin reductase by either UVA or UVB radiation. In the human population (n=15), thioredoxin reductase was deactivated by 54% with UVA and 36% with UVB radiation, although the degree of enzyme inhibition depended on skin phototype (I–VI, Fitzpatrick Classification). SPF₂₄ offered considerable protection for thioredoxin reductase against both UVA and UVB for skin types I and II. However, SPF₂₄ yielded no significant protection with UVA for skin types III–VI, and enhanced the enzyme inhibition with UVB additively. These results indicate that UVB photo-oxidation of oxybenzone (the UVA filter in SPF₂₄) may deactivate thioredoxin reductase in more pigmented members of the population by Michael addition of oxybenzone semiquinone to the thiolate active site of this enzyme.     

Retroperitoneal undifferentiated pleomorphic sarcoma having microsatellite instability associated with Muir‐Torre syndrome: case report and review of literature

Muir‐Torre syndrome represents a rare autosomal dominant familial cancer predisposition disorder defined by the occurrence of cutaneous sebaceous tumors and an internal malignancy, most commonly gastrointestinal carcinoma. Most examples of hereditary non‐polyposis cancer syndrome (Lynch syndrome), including the Muir‐Torre syndrome, are associated with microsatellite instability (MSI) and germline mutations in mismatch repair genes—most commonly MLH1 or MSH2. We present a 58‐year‐old man with Muir‐Torre syndrome and a large retroperitoneal mass (14.3 cm in greatest dimension) encompassing the left adrenal gland. Sections showed a cellular malignant tumor composed of spindle cells with a high mitotic index and lacking morphologic evidence of adipocytic differentiation. It was weakly reactive for smooth muscle actin (SMA) and negative for desmin, CD117, CD31, CD34, S100 protein and pan‐cytokeratin. Further immunohistochemical analysis revealed intact expression of MLH1 but loss of MSH2 in tumor nuclei. Compared to non‐neoplastic tissue, the tumor showed MSI in five of seven dinucleotide markers. Fluorescence in situ hybridization (FISH) failed to reveal 12q15 amplification, effectively excluding dedifferentiated liposarcoma as a diagnostic consideration. This is a rare case of a patient with Muir‐Torre syndrome who developed a related high‐grade undifferentiated pleomorphic sarcoma as the associated internal malignancy.     

INDUCTION OF PSORIASIFORM CHANGES IN GUINEA PIG SKIN BY PROPRANOLOL

Background. The ability of β-adrenergic blocking agents to induce psoriasis as an adverse effect prompted us to use such an agent to induce psoriasis in guinea pigs. Methods. Thirty female albino guinea pigs were divided into four groups. Group 1 received propranolol, 0.1 mg/day, dissolved in 2 mL of normal saline, orally by gavage for 30 days. Group 2 was given the same treatment, but in addition intradermal injections of propranolol with Freund's complete adjuvant, injected at weekly intervals. Group 3 (five animals) received 2 mL saline, and group 4 additional injections of adjuvant without propranolol. Groups 3 and 4 served as normal controls. Results. All animals of group 2 (which received propranolol orally and in addition intradermal injections of adjuvant) developed psoriasiform epidermal hyperplasia with acanthosis. Parakeratosis, papillomatosis, and formation of microabscesses, all characteristic signs of psoriasis, have not been seen in any of the skin samples of this group. Skin samples from group 1 animals receiving propranolol orally showed normal epidermis and dermis. They showed exactly the same histologic picture as the control groups 3 and 4. Conclusions. Beta-blockers given orally for 30 days do not cause any significant skin changes in guinea pigs. When given with a weekly intradermal injection of Freund's complete adjuvant, they cause psoriasiform epidermal hyperplasia. Although the overall histologic appearance of the skin of group 2 resembled psoriasis, it lacked important histologic features characteristic of this disease. It seems, therefore, that the model, per se, does not fulfill the initial expectations as an experimental model for psoriasis; however, this model has potential in the study of adverse drug reactions. Perhaps by introducing modifications to the experimental protocol, we may succeed also in developing a better model for experimental psoriasis.