Erigeron annuus Extract Improves DNCB-Induced Atopic Dermatitis in a Mouse Model via the Nrf2/HO-1 Pathway.

Atopic dermatitis (AD) is a persistent inflammatory skin condition resulting from an intricate interplay among genetic, immunological, and environmental factors. Erigeron annuus (EA), an annual winter plant belonging to the family Asteraceae, possesses anti-inflammatory, cytoprotective, and antioxidant activities. In this study, we hypothesized that Erigeron annuus extract (EAE) could be an effective agent for ameliorating AD-like symptoms. To confirm this hypothesis in vitro, we used H2O2-stimulated human keratinocytes (HaCaT cells) to demonstrate that pre-treatment with EAE protected against oxidative stress. HaCaT cells pretreated with EAE and stimulated with H2O2 showed decreased intracellular malondialdehyde content, increased superoxide dismutase activity, and reduced intracellular reactive oxygen species accumulation. To verify the in vivo hypothesis based on the intracellular results, an AD disease mouse model was induced with 1-chloro-2,4-dinitrobenzene (DNCB), and EAE was orally administered at a non-toxic concentration according to the toxicity evaluation results. The results showed that AD disease models in BALB/c mice exhibited reduced ear epidermal thickness, scratching behavior, and mast cell infiltration. In conclusion, our results indicate that EAE has the potential to improve AD by upregulating the nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway.

Exosomes derived from human dermal fibroblasts (HDFn-Ex) alleviate DNCB-induced atopic dermatitis (AD) via PPARα.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Skin barrier dysfunction is the initial step in the development of AD. Recently, exosomes have been considered as potential cell-free medicine for skin defects such as aging, psoriasis and wounds. The aim of this study was to investigate the effects of human dermal fibroblast-neonatal-derived exosome (HDFn-Ex) on AD. HDFn-Ex increased the expression of peroxisome proliferator activated receptor α (PPARα) and alleviated the 1-chloro-2,4-dinitrobenzene (DNCB)-mediated downregulation of filaggrin, involucrin, loricrin, hyaluronic acid synthase 1 (HAS1) and HAS2 in human keratinocyte HaCaT cells. However, these effects were inhibited by the PPARα antagonist GW6471. In the artificial skin model, HDFn-Ex significantly inhibited DNCB-induced epidermal hyperplasia and the decrease in filaggrin and HAS1 levels via a PPARα. In the DNCB-induced AD-like mouse model, HDFn-Ex administration reduced epidermis thickening and mast cell infiltration into the dermis compared to DNCB treatment. Moreover, the decreases in PPARα, filaggrin and HAS1 expression, as well as the increases in IgE and IL4 levels induced by DNCB treatment were reversed by HDFn-Ex. These effects were blocked by pre-treatment with GW6471. Furthermore, HDFn-Ex exhibited an anti-inflammatory effect by inhibiting the DNCB-induced increases in IκBα phosphorylation and TNF-α expression. Collectively, HDFn-Ex exhibited a protective effect on AD. Notably, these effects were regulated by PPARα. Based on our results, we suggest that HDFn-Ex is a potential candidate for treating AD by recovering skin barrier dysfunction and exhibiting anti-inflammatory activity.

Altered endocannabinoidome bioactive lipid levels accompany reduced DNBS-induced colonic inflammation in germ-free mice.

BACKGROUND: Gut microbiota are involved in the onset and development of chronic intestinal inflammation. The recently described endocannabinoidome (eCBome), a diverse and complex system of bioactive lipid mediators, has been reported to play a role in various physio-pathological processes such as inflammation, immune responses and energy metabolism. The eCBome and the gut microbiome (miBIome) are closely linked and form the eCBome - miBIome axis, which may be of special relevance to colitis. METHODS: Colitis was induced in conventionally raised (CR), antibiotic-treated (ABX) and germ-free (GF) mice with dinitrobenzene sulfonic acid (DNBS). Inflammation was assessed by Disease Activity Index (DAI) score, body weight change, colon weight-length ratio, myeloperoxidase (MPO) activity and cytokine gene expression. Colonic eCBome lipid mediator concentrations were measured by HPLC-MS /MS. RESULTS: GF mice showed increased levels of anti-inflammatory eCBome lipids (LEA, OEA, DHEA and 13- HODE-EA) in the healthy state and higher MPO activity. DNBS elicited reduced inflammation in GF mice, having lower colon weight/length ratios and lower expression levels of Il1b, Il6, Tnfa and neutrophil markers compared to one or both of the other DNBS-treated groups. Il10 expression was also lower and the levels of several N-acyl ethanolamines and 13-HODE-EA levels were higher in DNBS-treated GF mice than in CR and ABX mice. The levels of these eCBome lipids negatively correlated with measures of colitis and inflammation. CONCLUSIONS: These results suggest that the depletion of the gut microbiota and subsequent differential development of the gut immune system in GF mice is followed by a compensatory effect on eCBome lipid mediators, which may explain, in part, the observed lower susceptibility of GF mice to develop DNBS-induced colitis.

Use of a Silkworm (Bombyx mori) Larvae By-Product for the Treatment of Atopic Dermatitis: Inhibition of NF-κB Nuclear Translocation and MAPK Signaling.

Atopic dermatitis (AD) is a long-lasting inflammatory skin disease that contributes to the global health burden and impacts 10-20% of the world's population. In this study, we determined the anti-AD effect of a by-product of silkworm (Bombyx mori) larval powder, strain Yeonnokjam (SLPY), as a sustainable, natural source for the development of therapeutic agents for AD. HaCaT cells were used to assess the in vitro anti-inflammatory activity of SLPY, and a 1-chloro-2,4-dinitrobenzene (DNCB)-induced mouse model was used to study the in vivo anti-AD effects. SLPY treatment downregulated the expression of the inflammatory cytokines TNF-α, IL1ß, IL-8, and Cox-2 in stimulated HaCaT cells. Similarly, the topical application of SLPY in DNCB-treated mice downregulated the expression of inflammatory cytokines and proteins while ameliorating the clinical features of AD. Further, SLPY treatment inhibited the nuclear translocation of NF-κb p65, thereby supporting the efficacy of SLPY in the treatment of AD.

Integrated transcriptomic and metabolomic analyses of DNCB-induced atopic dermatitis in mice.

AIMS: Atopic dermatitis (AD) is a common chronic inflammatory skin disorder that affects up to 20 % of children and 10 % of adults worldwide; however, the exact molecular mechanisms remain largely unknown. MATERIALS AND METHODS: In this study, we used integrated transcriptomic and metabolomic analyses to study the potential mechanisms of 1-chloro-2,4-dinitrobenzene (DNCB)-induced AD-like skin lesions. KEY FINDINGS: We found that DNCB induced AD-like skin lesions, including phenotypical and histomorphological alterations and transcriptional and metabolic alterations in mice. A total of 3413 differentially expressed metabolites were detected between DNCB-induced AD-like mice and healthy controls, which includes metabolites in taurine and hypotaurine metabolism, phenylalanine metabolism, biosynthesis of unsaturated fatty acids, tryptophan metabolism, arachidonic acid metabolism, pantothenate and CoA biosynthesis, pyrimidine metabolism, and glycerophospholipid metabolism pathways. Furthermore, the differentially expressed genes associated (DEGs) with these metabolic pathways were analyzed using RNA sequencing (RNA-seq), and we found that the expression of pyrimidine metabolism-associated genes was significantly increased. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the glycolysis/gluconeogenesis, glucagon signaling pathway and pentose phosphate pathway-associated metabolic genes were dramatically altered. SIGNIFICANCE: Our results explain the possible mechanism of AD at the gene and metabolite levels and provide potential targets for the development of clinical drugs for AD.

Sargassum horneri extract containing polyphenol alleviates DNCB-induced atopic dermatitis in NC/Nga mice through restoring skin barrier function.

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by skin barrier dysfunction. Sargassum horneri (S. horneri) is a brown alga that has been widely used in traditional medicine of eastern Asian countries. Recent studies proved that a brown alga S. horneri has anti-inflammatory activity. In this study, we investigated the effect of S. horneri ethanol extract (SHE) against AD in 2,4-dinitrobenzene (DNCB) induced AD in NC/Nga mice. We observed that SHE treatment decreased the epidermal thickness and epidermal hyperplasia that had been worsened through DNCB application. Moreover, SHE significantly inhibited the proliferation of mast cells and decreased the expression of IL-13 on CD4⁺ cells prompted by elevated thymic stromal lymphopoietin (TSLP) expression in DNCB-induced AD in mice. We also demonstrated that SHE directly inhibited the expression of keratinocyte-produced TSLP known to exacerbate skin barrier impairment. Especially, the decrease of filaggrin, an integral component of proper skin barrier function through a function in aggregating keratin filaments, observed in DNCB-induced AD mice was significantly improved when treated with SHE. More importantly, we proved that SHE was able to decrease the serum levels of IgG1 and IgG2ₐ, two crucial factors of AD, indicating the protective effect of SHE. Taken together, our findings suggest that SHE may protect NC/Nga mice against DNCB-induced AD via promoting skin barrier function.

Tenebrio molitor Larva Trypsin Hydrolysate Ameliorates Atopic Dermatitis in C57BL/6 Mice by Targeting the TLR-Mediated MyD88-Dependent MAPK Signaling Pathway.

Atopic dermatitis (AD) is a widely researched chronic inflammatory skin disease with a complex etiology. The increased prevalence of AD necessitates exploration of natural sources as potential therapeutic agents with limited side effects. In the current study, a 1-chloro-2,4-dinitrobenzene (DNCB)-induced AD mouse model was used to examine the anti-AD effects of Tenebrio molitor trypsin hydrolysate (TMTH) and its underlying molecular mechanism. DNCB-treated mice were treated with TMTH (1 and 10 mg/kg), and prednisolone (3 mg/kg) was used as the positive control. Serum and skin tissue samples were collected for subsequent analyses. The expression levels of proteins linked to the myeloid differentiation primary response 88 (MyD88)-dependent mitogen-activated protein kinase (MAPK) signaling pathway and serum IgE levels were estimated via Western blotting technique and ELISA (enzyme-linked immunosorbent assay), respectively. Inflammatory cell infiltration and thickening of the dorsal skin were measured using toluidine blue and hematoxylin and eosin staining, respectively. Oral administration of TMTH significantly reduced mast cell infiltration and dermal and epidermal thickness. Moreover, TMTH treatment reduced serum IgE levels. Western blotting confirmed that TMTH treatment suppressed the MyD88-dependent MAPK signaling pathway. Therefore, TMTH substantially inhibited AD-like skin lesion formation via immunomodulation, showing considerable potential for AD treatment.

Cancer incidence among workers occupationally exposed to dinitrotoluene in the copper mining industry.

PURPOSE: Epidemiological and toxicological studies point to a potential carcinogenic effect of dinitrotoluene (DNT), particularly with respect to renal and urothelial cancer. METHODS: The cohort comprised all men born between 1920 and 1974 (n = 16,441) who were gainfully employed between 1953 and 1990 in one of two underground copper mines in Mansfeld, Saxony-Anhalt, former German Democratic Republic, and who were followed up for cancer incidence, 1961-2005. Incident cancer cases were identified by record linkage with the Common Cancer Registry of the New Laender. Standardized incidence ratios (SIR) were calculated with the general population of Saxony-Anhalt as the reference. RESULTS: Standardized incidence ratios for all cancers were not significantly elevated in the cohort (SIR = 1.04; 95 % confidence intervals (CI) 0.96-1.14). We found an increase in lung cancer (SIR = 1.29; 1.13-1.46), but not in kidney cancer (SIR = 1.01; 95 % CI 0.79-1.27) or bladder cancer (SIR = 1.04; 95 % CI 0.82-1.30). Standardized incidence ratios stratified by duration of employment with DNT exposure indicated moderately increased risks for kidney and bladder cancer in cohort members with longer exposure. CONCLUSIONS: The SIR analysis of workers in the copper mining industry in comparison with the general population of Saxony-Anhalt overall did not indicate increased risks for renal or bladder cancer. However, results by years of exposure to DNT suggested weakly increased risks for outcomes of a priori interest, bladder and kidney cancer. A subsequent case-cohort analysis including expert assessment of DNT exposure and identification of additional cancer cases from a network of pathology institutes will provide further insight into a potential etiologic role of DNT in renal and urothelial cancer.

Photopatch testing in Asians: a 5-year experience in Singapore.

BACKGROUND: Photopatch testing is important for diagnosing photoallergic contact dermatitis. We aimed to evaluate the use of photopatch test at the National Skin Centre, Singapore. METHODS: All patients who had photopatch tests done between 2007 and 2011 at the National Skin Centre were included. RESULTS: Twenty-two patients were included. The mean age was 40.2. Female : male ratio was 3.4. The ethnic groups were Chinese (68%), Malay (4%), Indian (14%) and others (14%). Ten out of 22 patients (45.5%) had a positive photopatch test. There were 20 positive photopatch test reactions found in these 10 patients, and all 20 positive reactions were of current relevance. The frequencies of the positive photopatch test reactions were 2-hydroxy-4-methoxybenzophenone (oxybenzone) (n = 6), 2-hydroxymethoxymethylbenzophenone (mexenone) (n = 3), 2-ethylhexyl-4-dimethylaminobenzoate (n = 1), ketoprofen gel (n = 1) and the patient's own product (n = 9). CONCLUSIONS: Our study suggests that sunscreen is the most common photoallergen to date as opposed to musk ambrette, which was the most common photoallergen in our earlier study in 1991-1993. This finding is similar to the recent European Multicentre Photopatch Test Study.

Involvement of microtubules in rhizoid differentiation of Spirogyra species.

Some species of Spirogyra form rosette-shaped or rod-shaped rhizoids in the terminal cell of the filaments. In the present study, we analyzed an involvement of microtubules (MTs) in rhizoid differentiation. Before rhizoid differentiation, cortical MTs were arranged transversely to the long axis of cylindrical cells, reflecting the diffuse growth. At the beginning of rhizoid differentiation, MTs were absent from the extreme tip of the terminal cell. In the other area of the cell, however, MTs were arranged transversely to the long axis of the cell. In the fully differentiated rosette-shaped rhizoid, MTs were randomly organized. However, at a younger stage of rosette-shaped rhizoids, MTs were sometimes arranged almost transversely in the lobes of the rosette. In the rod-shaped rhizoid, MTs were arranged almost transversely. MT-destabilizing drugs (oryzalin and propyzamide) induced swelling of rhizoids, and neither rosette-shaped nor rod-shaped rhizoids were formed. The role of MTs in rhizoid differentiation was discussed.

Exposure to nitroaromatic explosives and health effects during disposal of military waste.

AIMS: To investigate the exposure to dinitrotoluene (DNT) and trinitrotoluene (TNT) and the resulting effects in workers which occur during the disposal of military waste. METHODS: Eighty two employees from a mechanical plant in Germany were studied, of whom 51 were regularly exposed to ammunition containing TNT and DNT, 19 occasionally, and 12 not at all. RESULTS: Air analyses yielded maximum concentrations of 20 micro g/m(3) for 2,4-DNT and 3250 micro g/m(3) for 2,4,6-TNT, respectively. The maximum concentrations in the urine of workers regularly exposed amounted to 5.0 micro g/l of 2,4,6-TNT, 1464.0 micro g/l of 2-amino-4,6-dinitrotoluene, 6693.0 of micro g/l 4-amino-2,6-dinitrotoluene, 2.1 micro g/l of 2,4-DNT, 95.0 micro g/l of 2,4-dinitrobenzoic acid, and 3.6 micro g/l of 2,6-DNT. There was a highly significant linear correlation between the urinary concentrations of the two main metabolites of TNT, 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene. In 63 persons TNT or DNT or metabolite concentrations above the analytical detection limit were found in urine. These persons reported more frequently symptoms like bitter taste, burning eyes, and discoloration of the skin and hair than persons (n = 19) without detectable TNT and/or DNT exposure. CONCLUSION: During the disposal of military waste containing relevant TNT and DNT, exposure can occur of occupational-medical relevance. Biological monitoring is suitable for the early detection of possible adverse effects at workplaces exposed to TNT. Protective measures should be improved, together with adequate occupational-medical surveillance of persons exposed to nitroaromatic explosives. Further studies are necessary to exclude possible long term effects.

Nephrotoxicity and nephrocarcinogenicity of dinitrotoluene: new aspects to be considered.

Technical dinitrotoluene (DNT) is a mixture of 2,4- and 2,6-DNT. In humans, industrial or environmental exposure can occur orally, by inhalation, or by skin contact. The classification of DNT as an 'animal carcinogen' is based on the formation of malignant tumors in kidneys, liver, and mammary glands of rats and mice. Clear signs of toxic nephropathy were found in rats dosed with DNT, and the concept was derived of an interrelation between renal toxicity and carcinogenicity. Recent data point to the carcinogenicity of DNT on the urinary tract of exposed humans. Between 1984 and 1997, 6 cases of urothelial cancer and 14 cases of renal cell cancer were diagnosed in a group of 500 underground mining workers in the copper mining industry of the former GDR and having high exposures to explosives containing technical DNT. The incidences of both urothelial and renal cell tumors in this group were 4.5 and 14.3 times higher, respectively, than anticipated on the basis of the cancer registers of the GDR. The genotyping of all identified tumor patients for the polymorphic enzymes NAT2, GSTM1, and GSTT1 identified the urothelial tumor cases as exclusively 'slow acetylates'. A group of 161 miners highly exposed to DNT was investigated for signs of subclinical renal damage. The exposures were categorized semi-quantitatively into 'low', 'medium', 'high', and 'very high'. A straight dose-dependence of the excretion of urinary biomarker proteins with the ranking of exposure was seen. Biomarker excretion (alpha1-microglobulin, glutathione S-transferases alpha and pi) indicated that DNT-induced damage was directed toward the tubular system. New data on DNT-exposed humans appear consistent with the concept of cancer initiation by DNT isomers and the subsequent promotion of renal carcinogenesis by selective damage to the proximal tubule. The differential pathways of metabolic activation of DNT appear to apply to the proximal tubule of the kidney and to the urothelium of the renal pelvis and lower urinary tract as target tissues of carcinogenicity.

Pathological excretion patterns of urinary proteins in miners highly exposed to dinitrotoluene.

A cohort of 161 underground miners who had been highly exposed to dinitrotoluene (DNT) in the copper-mining industry of the former German Democratic Republic was reinvestigated for signs of subclinical renal damage. The study included a screening of urinary proteins excreted by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and quantitations of the specific urinary proteins alpha 1-microglobulin and glutathione-S-transferase alpha (GST alpha) as biomarkers for damage of the proximal tubule and glutathione-S-transferase pi (GST pi) for damage of the distal tubule. The exposures were categorized semiquantitatively (low, medium, high, and very high), according to the type and duration of professional contact with DNT. A straight dose-dependence of pathological protein excretion patterns with the semiquantitative ranking of DNT exposure was seen. Most of the previously reported cancer cases of the urinary tract, especially those in the higher exposed groups, were confined to pathological urinary protein excretion patterns. The damage from DNT was directed toward the tubular system. In many cases, the appearance of Tamm-Horsfall protein, a 105-kD protein marker, was noted. Data on the biomarkers alpha 1-microglobulin, GST alpha, and GST pi consistently demonstrated a dose-dependent increase in tubular damage, which confirmed the results of screening by SDS-PAGE and clearly indicated a nephrotoxic effect of DNT under the given conditions of exposure. Within the cluster of cancer patients observed among the DNT-exposed workers, only in exceptional cases were normal biomarker excretions found.

Transcriptional activation of stress genes and cytotoxicity in human liver carcinoma cells (HepG2) exposed to 2,4,6-trinitrotoluene, 2,4-dinitrotoluene, and 2,6-dinitrotoluene.

The CAT-Tox (L) assay has recently been developed and validated for detecting and quantifying the specific molecular mechanisms that underlie toxicity of various xenobotic chemicals. We performed this assay to measure the transcriptional responses associated with 2,4,6-trinitrotoluene (TNT) and 2 of its byproducts [2,4 and 2,6-dinitotoluenes (DNTs)] to 13 different recombinant cell lines generated from human liver carcinoma cells (HepG2) by creating stable transfectants of mammalian promoter chloramphenicol acetyltransferase (CAT) gene fusions. Cytoxicity test with the parental HepG2 cells, using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide]-based assay for cell viability, yielded LC50 values of 105 +/- 6 mg/mL for TNT in 1% dimethyl sulfoxide (DMSO), and > 300 mg/mL for DNTs, upon 48 h of exposure. TNT appeared to be more toxic than 2,4-DNT, which also showed a higher toxicity compared to 2,6-DNT. Of the 13 recombinant constructs evaluated, 8 (CYP 1A1, GST Ya, XRE, HMTIIA, c-fos, HSP70, GADD153, and GADD45), 5 (c-fos, HSP70, GADD153, GADD45, and GRP78), and none showed inductions to significant levels (p < 0.05), for TNT, 2,4-DNT, and 2,6-DNT, respectively. For most constructs, the induction of stress genes was concentration-dependent. These results show the potential for TNT and 2,4-DNT to cause protein damage and/or perturbations of protein biosynthesis (HSP70 and GRP78), alterations in DNA sequence or its helical structure (c-fos, GADD153, GADD45), and the potential involvement of TNT in the biotransformation process (CYP 1A1, GST Ya, XRE), and in the toxicokinetics of metal ions (HMTIIA). Within the range of concentrations tested (0-300 mg TNT or DNT/mL in 1% DMSO), no significant inductions (p > 0.05) of NFKBRE, p53RE, CRE, and RARE were found.

Occurrence of urinary tract tumors in miners highly exposed to dinitrotoluene.

Between 1984 and 1997, six cases of urothelial cancer and 14 cases of renal cell cancer occurred in a group of 500 underground mining workers in the copper-mining industry of the former German Democratic Republic, with high exposures to explosives containing technical dinitrotoluene. Exposure durations ranged from 7 to 37 years, and latency periods ranged from 21 to 46 years. The incidences of both urothelial and renal cell tumors in this group were much higher than anticipated on the basis of the cancer registers of the German Democratic Republic by factors of 4.5 and 14.3, respectively. The cancer cases and a representative group of 183 formerly dinitrotoluene-exposed miners of this local industry were interviewed for their working history and grouped into four exposure categories. This categorization of the 14 renal cell tumor cases revealed no dose-dependency concerning explosives in any of the four exposure categories and was similar to that of the representative group of employees, whereas the urothelial tumor cases were predominantly confined to the high-exposure categories. Furthermore, all identified tumor patients were genotyped by polymerase chain reaction, using lymphocyte DNA, regarding their genetic status of the polymorphic xenobiotic metabolizing enzymes, including the N-acetyltransferase 2 and the glutathione-S-transferases M1 and T1. This genotyping revealed remarkable distributions only for the urothelial tumor cases, who were exclusively identified as "slow acetylators." This points to the possibility of human carcinogenicity of dinitrotoluene, with regard to the urothelium as the target tissue.

Persistent light reaction with erythroderma caused by musk ambrette: a case report.

Persistent light reaction is an uncommon type of photodermatitis caused mainly by musk ambrette, a synthetic fragrance material commonly used in foods and cosmetics. Erythrodermic persistent light reaction is rare. We report a case of erythroderma with underlying persistent light reaction due to musk ambrette. A 71-year-old man showed a photodermatitis that waxed and waned for five years before it became more persistent and finally evolved into erythroderma. Positive results of a photopatch test to musk ambrette and a low minimal erythema dose to ultraviolet B were noted. A biopsy specimen of the erythrodermic lesion revealed spongiotic dermatitis. The erythroderma and photodermatitis responded to systemic steroids and psoralen/ultraviolet A therapy (total dose: 90 J/cm2). We suggest that persistent light reaction be included in the differential diagnosis of erythroderma.