Search

Selegiline (SE) is a selective, irreversible monoamine oxidase-B inhibitor, used for reducing symptoms in early-stage Parkinson’s disease. The metabolites of SE include l-methamphetamine, l-amphetamine and desmethylselegiline (DSE). The stereoisomers of SE metabolites, d-methamphetamine and d-amphetamine are highly addictive psychostimulants and some of the most abused drugs in South Korea. In order to differentiate medical SE users form illicit methamphetamine abusers, it is important to distinguish between the l-isomers and d-isomers in urine samples. A 52-year-old male, seemingly under the influence of intoxication and demonstrating abnormal behavior, was reported to the police. The initial urine test using a methamphetamine detection kit demonstrated a positive result. Given the initial results, the police officer requested a further analysis of the urine sample. The urine sample was screened using headspace-solid phase microextraction-gas chromatography–mass spectrometry (HS-SPME-GC–MS). Both methamphetamine and amphetamine were detected, in addition to SE and DSE. To quantitate methamphetamine and amphetamine by HS-SPME-GC–MS, we performed a standard addition method due to the matrix effect of the case sample. Consistent with previous studies, our results indicated that the ratio of amphetamine to methamphetamine was 0.27, which was in the range of SE ingestion. Furthermore, we confirmed l-methamphetamine and l-amphetamine by chiral derivatization using (R)-(−)-α-methoxy-α-(trifluoromethyl) phenylacetyl chloride.

Veronica persica is a flowering plant belonging to the family Scrophulariaceae. Here, we aimed to evaluate the pharmacological activity of the ethanol extract of Veronica persica (EEVP) in an airway inflammation model. We examined airway responsiveness to aerosolized methacholine, serum immunoglobulin (Ig)E levels, and total cell numbers in the lung and bronchoalveolar lavage fluid (BALF). Histological analysis of the lung tissue was performed using hematoxylin-eosin, Masson trichrome, or periodic acid-Schiff staining. Fluorescence-activated cell sorting analysis in the lung and BALF was applied to clarify the changes in immune cell types. Enzyme-linked immunosorbent assay and quantitative real-time polymerase chain reaction were applied to investigate cytokine levels and gene expression related to airway inflammation. STAT-3/6 phosphorylation was examined in primary bronchial/tracheal epithelial cells using western blot analysis. EEVP significantly suppressed total IgE levels and methacholine-induced increase of Penh value in the HDM-challenged mouse model. EEVP also attenuated the severity of airway remodeling in lung tissues and decreased eosinophil and neutrophil infiltration in the lungs and BALF. EEVP significantly reduced the production of cytokines in BAL and splenocyte culture medium, and the expression of mRNAs related to airway inflammation in the lung tissue. EEVP suppressed IL-4/13-induced STAT-3/6 phosphorylation in the epithelial cells. We showed for the first time that EEVP effectively inhibits eosinophilic airway inflammation by suppressing the expression of inflammatory factors for T cell activation and polarization, and inhibits MCP-1 production of bronchial/tracheal epithelial cells by suppressing STAT-3/6 activation. EEVP may be a potential pharmacological agent to prevent inflammatory airway diseases.

Acute bronchitis and acute exacerbations of chronic bronchitis (AECB) have cough and sputum as the main symptoms with a high prevalence and substantial economic burden. Although the demand for bronchitis treatment increases due to causes, such as air pollution, the appropriateness of antibiotic prescriptions and the effects of current symptomatic treatments for bronchitis are unclear. GHX02, which is a combined formulation containing four herbs, and has been clinically used for bronchitis in South Korea. We conducted a phase II, randomized, double-blind, and placebo-controlled, multicenter trial to evaluate its efficacy and safety. Patients with acute bronchitis or AECB were recruited and randomized to receive high-dose GHX02 (1920 mg/day), standard-dose GHX02 (960 mg/day), or placebo for 7 days. The primary outcome measure was the change in Bronchitis Severity Score (BSS) from baseline to Day 7. The secondary outcomes were the frequency of coughing fits, Questionnaire of Clinical Symptoms of Cough and Sputum (QCSCS), Leicester Cough Questionnaire (LCQ), Integrative Medicine Outcome Scale (IMOS), and Integrative Medicine Patient Satisfaction Scale (IMPSS). A total of 117 patients were randomized to parallel groups (38 in the high-dose GHX02, 41 in the standard-dose GHX02 group, and 38 in the placebo group). The mean differences in BSS from baseline to Day 7 in the treatment groups (4.2 ± 2.0 and 4.5 ± 1.8 in the high-dose GHX02 and standard-dose GHX02 groups, respectively) were higher than the placebo group (3.8 ± 2.1), p = 0.028. The mean differences in the frequency of coughing fits from baseline to Day 7 and IMPSS were better in the GHX02 treatment group than in the placebo group (standard-dose GHX02 group vs placebo group, p = 0.036). The QCSCS, LCQ, IMOS, and GHX02 of the treatment groups also showed more improvement than the placebo group, but there were no statistically significant differences between the groups. There were no severe adverse effects during the trial. This study supports that GHX02 is effective and safe for patients with bronchitis and provides the basis for progression to a phase III study.Clinical Trial Registration: [https://cris.nih.go.kr] WHO International Clinical Trials Registry Platform, Clinical Research Information Service [KCT0003665].

Cicadae Periostracum (CP), derived from the slough of Cryptotympana pustulata, has been used as traditional medicine in Korea and China because of its diaphoretic, antipyretic, anti-inflammatory, antioxidant, and antianaphylactic activities. The major bioactive compounds include oleic acid (OA), palmitic acid, and linoleic acid. However, the precise therapeutic mechanisms underlying its action in asthma remain unclear. The objective of this study was to determine the antiasthmatic effects of CP in an ovalbumin (OVA)-induced asthmatic mouse model. CP and OA inhibited the inflammatory cell infiltration, airway hyperresponsiveness (AHR), and production of interleukin (IL)7 and Th2 cytokines (IL-5) in the bronchoalveolar lavage fluid and OVA-specific imunoglobin E (IgE) in the serum. The gene expression of IL-5, IL-13, CCR3, MUC5AC, and COX-2 was attenuated in lung tissues. CP and OA might inhibit the nuclear translocation of GATA-binding protein 3 (GATA-3) and retinoic acid receptor-related orphan receptor γt (RORγt) via the upregulation of forkhead box p3 (Foxp3), thereby preventing the activation of GATA-3 and RORγt. In the in vitro experiment, a similar result was observed for Th2 and GATA-3. These results suggest that CP has the potential for the treatment of asthma via the inhibition of the GATA-3/Th2 and IL-17/RORγt signaling pathways.

The aim of this study was to evaluate the efficacy and safety of CAEC (Canavalia gladiata arctium lappa extract complex) in adults with white blood cells between 3 and 8 × 103 cells/µL by assessing natural killer (NK) cell activity and immune-related biomarkers. Overall, 100 participants (CAEC group = 50, placebo group = 50) were randomised, and administered CAEC or placebo once daily for 8 weeks, with an evaluation visit performed every 4 weeks. NK cell activity, the primary outcome, significantly increased in CAEC when compared with the placebo at effector-to-target (E:T) ratios of 25:1 and 50:1 after 8 weeks. Compared with the placebo group, IL-10 demonstrated significant differences at week 8 in the CAEC group. Our findings demonstrate that CAEC is safe and enhances immune function by stimulating NK cell activity through IL-10 expression. Hence, we anticipate that CAEC can be developed as a functional food for stimulating immunity.

The consequences of increased industrialization increased the risk of asthma and breathing difficulties due to increased particulate matter in the air. We aim to investigate the therapeutic properties of Hypericum ascyron L. extract (HAE) in airway inflammation and unravel its mechanism of action. We conducted nitric oxide and cell viability assay, real-time PCR and western blot analyses along with in vitro studies. in vivo studies include a model of coal fly ash and diesel exhaust particle (CFD)-induced airway inflammation in mice. HAE reduced coal fly ash (CFA)-induced nitric oxide secretion without exhibiting cytotoxicity in MH-S cells. HAE also reduced the mRNA expression of pro-inflammatory cytokines and reduced the expression of proteins in the NFκB and MAPK pathways. In a mice model of CFD-induced airway inflammation, HAE effectively reduced neutrophil infiltration in bronchoalveolar lavage fluid (BALF) and increased the amount of T cells in the BALF, lungs, and blood while reducing all other immune cell subtypes to reduce airway inflammatory response. CXCL-1, IL-17, MIP-2, and TNF-α expression in the BALF were also reduced. HAE effectively reduced MIP-2 and TNF-α mRNA expression in the lung tissue of mice. In a nutshell, HAE is effective in preventing airway inflammation induced by CFA in MH-S cells, as well as inflammation induced by CFD in mice.

Background Atopic dermatitis is a chronic inflammatory skin disease in humans. Although Olea europaea leaf extract (OLE) and Spirodela polyrhiza extract (SPE) have been used to protect against skin damage, the effects of their combined administration on atopic dermatitis have yet to studied. Purpose In this study, we evaluated the potential therapeutic effects of an OLE and SPE combination on the progression of atopic dermatitis and the possible mechanisms underlying these effects in 1-chloro-2,4-dinitrobenzene (DNCB)-treated NC/Nga mice. Methods Atopic dermatitis was induced by topical application of 0.2% w/v DNCB prepared in an olive oil:acetone solution (1:3), and thereafter OLE, SPE and OLE + SPE were administered orally for 5 weeks. We determined atopic dermatitis symptoms, serum IgE levels, and levels of cytokine- and gene expression in the dorsal skin and splenocytes, and performed histological and immune cell subtype analyses. The expression of skin barrier-related proteins (filaggrin, sirtuin 1, and claudin 1) was also evaluated. Results The OLE + SPE combination significantly ameliorated atopic dermatitis symptoms, including dermatitis scores, and reduced epidermal thickness and infiltration of different inflammatory cells in mice with DNCB-induced atopic dermatitis. It also significantly reduced the number of CD4+, CD8+, and CD4+/CD69+ T cells; immunoglobulin E-producing B cells (CD23+/B220+) in the axillary lymph nodes; CD3+ T-cell eosinophils (chemokine–chemokine receptor 3+/CD11b+) in the skin; and CD3+ T cells, immunoglobulin E-producing B cells (CD23+/B220+), and eosinophils in peripheral blood mononuclear cells. Additionally, the experimental combination lowered levels of serum immunoglobulin E and histamine, as well as Th2-mediated cytokines, and interleukin-4, -5, and -13, whereas it increased the levels of Th1-mediated cytokine interferon-γ in splenocytes. Furthermore, the preparation significantly restored expression of the skin barrier-related proteins filaggrin, sirtuin 1, and claudin 1, and also reduced the expression of the inflammatory cytokine interleukin-6 and chemokine–chemokine receptor 3, as well as the pruritus-related cytokine interleukin-31 and interleukin-31 receptor, in atopic dermatitis skin lesions. Conclusion Taken together, our findings indicate that administration of a combination of OLE and SPE can alleviate atopic dermatitis symptoms by regulating immune balance and skin barrier function and may be an effective therapeutic option for the treatment of atopic dermatitis.

Industrial development, along with the rapid growth of the economy, has greatly improved the quality of life in humans. Moreover, advancements in medical technology have increased life expectancy. Small particles increase airway inflammation when they penetrate the alveoli. We observed that GHX02 decreased the frequency and delayed the onset time of citric acid-induced coughing in guinea pigs. A phenol red secretion assay indicated that the GHX02 extract exhibits potent expectorant activity. The GHX02 extract also greatly reduced leukocyte levels. Our results indicate that GHX02 inhibits airway inflammation, reduces sputum production, and relieves cough. The GHX02 extract suppressed histamine release from mast cells resulting from compound 48/80-induced degranulation. The extract exhibited antimicrobial activity against Streptococcus pneumoniae and significantly inhibited the formation of LTC4. At high concentrations, the GHX02 extract suppressed the formation of PGE2 (prostaglandin E2). Interleukin (IL)-4 and IL-13 levels decreased with an increasing dosage of GHX02. Oral administration of the GHX02 extract suppressed PM10D-induced inflammatory symptoms in the lung, including increased alveolar wall thickness, accumulation of collagen fibers, and cytokine release. Treatment with the GHX02 extract also resulted in lower levels of inflammatory cells, in bronchoalveolar lavage fluid and lung tissue. Our results indicate that GHX02 may be a useful therapeutic agent for treatment of respiratory diseases.

Chronic obstructive pulmonary disease (COPD) is caused by exposure to toxic particles, such as coal fly ash (CFA), diesel-exhaust particle (DEP), and cigarette smoke (CS), leading to chronic bronchitis, mucus production, and a subsequent lung dysfunction. This study, using a mouse model of COPD, aimed to evaluate the effect of herbal combinational medication of Glycyrrhiza glabra (GG), Agastache rugosa (AR) containing glycyrrhizic acid (GA), and tilianin (TN) as active ingredients. GA, a major active component of GG, possesses a range of pharmacological and biological activities including anti-inflammatory, anti-allergic, anti-oxidative. TN is a major flavonoid that is present in AR. It has been reported to have anti-inflammatory effects of potential utility as an anti-COPD agent. The COPD in the mice model was induced by a challenge with CFA and DEP. BALB/c mice received CFA and DEP alternately three times for 2 weeks to induce COPD. The herbal mixture of GG, AR, and TN significantly decreased the number of neutrophils in the lungs and bronchoalveolar lavage (BAL) fluid. It also significantly reduced the production of C-X-C motif chemokine ligand 2 (CXCL-2), IL-17A, CXCL-1, TNF-&alpha, symmetric dimethylarginine (SDMA) in BALF and CXCL-2, IL-17A, CXCL-1, MUC5AC, transient receptor potential vanilloid-1 (TRPV1), IL-6, COX-2, NOS-II, and TNF-&alpha, mRNA expression in the lung tissue. Notably, a combination of GG and AR was more effective at regulating such therapeutic targets than GG or AR alone. The histolopathological lung injury was alleviated by treatment with the herbal mixture and their active ingredients (especially TN). In this study, the herbal combinational mixture more effectively inhibited neutrophilic airway inflammation by regulating the expression of inflammatory cytokines and CXCL-2 by blocking the IL-17/STAT3 pathway. Therefore, a herbal mixture of GG and AR may be a potential therapeutic agent to treat COPD.

Canavalia gladiata (C. gladiata) and Arctium lappa (A. lappa), used in traditional medicine and food. However, their preventive effects in dextran sulphate sodium (DSS)-induced inflammatory bowel disease (IBD) remain unclear. We found that the C. gladiata and A. lappa mixture suppressed inflammation in LPS-induced RAW 264.7 cells. The mixture increased the population and activation of immune cells, up-regulation of cell cycle, and induction of IgA and IgG production in wild-type mice. Furthermore, the extract mixture prevented clinical IBD symptoms and restored IgA production in IBD mice. Similarly, a mixture of the marker compounds, chicoric acid and lupeol, ameliorated IBD-associated clinical signs, improved the population and activation of immune cells and functional defects in natural killer (NK) cells and reduced production of IgA in these mice. In conclusion, the C. gladiata and A. lappa mixture ameliorated progression of DSS-induced IBD by enhancing immune responses and recovering functional immune cell defects.

Ethnopharmacological relevance The modified gamgil-tang (GGX) is a mixture of four herbal medicine including Platycodi Radix, Glycyrrhizae Radix, Lonicerae Flos and Mori Radicis Cortex which has been traditionally used to treat lung and airway diseases to relieve symptoms like sore throat, cough, and sputum in Korea. Its major component chlorogenic acid had been reported to have antioxidant, antibacterial, hepatoprotective, cardioprotective, anti-inflammatory, antiviral, and anti-microbial activity. Aim of the study To identify the inhibitory effect of GGX in a particulate matter (PM) induced lung injury mouse model. Materials and methods We evaluated NO production, the release of TNF-α and IFN-γ in PM-induced MH-S cells, and the number of neutrophils, immune cell subtypes, and the secretion of TNF-α, IL-17, CXCL-1, MIP-2 in the PM-stimulated mouse model to assess the inhibitory effect of GGX against PM. In addition, as exposure to PM increases respiratory symptoms, typically cough and sputum, we attempted to evaluate the antitussive and expectorant activities of GGX. Results Our study provided evidence that GGX has inhibitory effects in PM-induced lung injury by inhibiting the increase in neutrophil and inflammatory mediators, deactivating T cells, and ameliorating lung tissue damage. Notably, GGX reduced PM-induced neutrophilic inflammation by attenuating the number of neutrophils and regulating the secretion of neutrophil-related cytokines and chemokines, such as TNF-α, IL-17, MIP2, and CXCL-1. In addition, GGX demonstrated an antitussive activity by significantly reducing citric acid-induced cough frequency and delaying the latent period and expectorant activities by the increased phenol red secretion compared to the control group. Conclusions GGX is expected to be an effective herbal remedy to prevent PM-induced respiratory disease.

Regulatory T cells play an important role in autoimmunity and have been shown to exert anti-inflammatory effects in allergic asthma. Mouse model of airway inflammation was used to examine the suppressive activity of luteolin-induced CD4+CD25+ regulatory T cells (Tregs) in vivo. In this study, BALB/c mice were sensitized with ovalbumin antigen (OVA) by aerosol challenge. Then, various biological processes were examined, including airway eosinophilia; mucus hypersecretion; elevation of OVA-specific IgE, expression of Th2 cytokines and chemokine levels; expression of eotaxin 2 and CCR3; and airway hyper responsiveness (AHR). Luteolin significantly inhibited OVA-induced increase in immune cell and eosinophil counts as well as IL-4, IL-5, IL-13, and eotaxin levels in bronchoalveolar lavage fluid (BAL Fluid). Luteolin and cyclosporine A (CsA) which was a positive control also substantially reduced OVA-specific IgE levels, eotaxin 2 levels, and CCR3 expression in BAL Fluid. In contrast, luteolin significantly increased IL-10 and IFN-γ protein levels, as well as IL-10 and TGF-β1 mRNA expression in the lung. In vitro studies showed that the number of luteolin-induced CD4+CD25+ Treg (iTreg) cells was higher, with elevated levels of TGF-β1 and foxp3 mRNA expression in lungs tissue. Transfer of iTreg cells into OVA-sensitized mice reduced AHR, eosinophil recruitment, eotaxin, IgE, and Th2 cytokine expressions, and increased IFN-γ production in BAL Fluid after allergen challenge. Furthermore, adoptive transfer of iTreg cells prevented disease in a CD25-depleted mouse asthma model. Luteolin via induction of foxp3 and CD4+CD25+ Treg cells may represent a new strategy in the development of therapies for managing asthma.

Background Bamboo (Phyllostachys pubescens) leaves and Japanese apricot (Mume fructus) fruit are traditionally recognized to be safe herbs broadly used for food and medicinal purposes in Southeast Asia. Our group previously explored their antiplatelet effects. This study was designed to confirm inhibition effects of PM21 (a 2:1 mixture of bamboo leaf extract and Japanese apricot fruit extract) on platelet aggregation and evaluate its potency to use as an herbal remedy to prevent and/or treat the diseases caused by platelet aggregation and thrombus formation. Methods Washed platelets were prepared and platelet aggregation was induced by adding 5 μg/mL collagen. Anti-platelet effects of PM21 (75 mg/kg, 150 mg/kg, and 300 mg/kg for ex vivo and in vivo assays, and 50, 100, 200 μg/mL for in vitro assays) were evaluated. In ex vivo assays, PM21 was orally administered to rats daily after overnight fasting for 3 days and blood was collected 1 h after the final treatment. In vivo antithrombotic effect of PM21 was observed from a carrageenan induced mouse tail thrombosis model. Results In ex vivo assay, PM21 inhibited platelet aggregation significantly. PM21 showed a strong antithrombotic effect by reducing significantly the length of mouse tail thrombus. PM21 increased intracellular cAMP level and reduced the release of ATP, TXA2, and serotonin. PM21 also reduced intracellular concentration of calcium ion, fibrinogen binding to integrin αIIbβ3, and phosphorylation of ERK2, p38, PLCγ2, and PI3 K. Conclusions PM21 showed remarkable inhibitory effects on platelet aggregation and thrombus formation. Its inhibitory function seems to influence on GPVI binding to its ligand and subsequent initiation of a signaling cascade that involves activation of effector proteins and secretion of effector molecules, such as ATP, TXA2, serotonin, and Ca2+. PM21 also appears to exert its anti-platelet effect by deactivation of ERKs activation pathway as well as inhibition of fibrinogen binding to integrin αIIbβ3.

Objectives: Allergic asthma generally presents with symptoms of wheezing, coughing, breathlessness, and airway inflammation. Seonpyejeongcheon-tang (SJT) consists of 12 herbs. It originated from Jeong-cheon-tang (JT), also known as Ding-chuan-tang, composed of 7 herbs, in She-sheng-zhong-miao-fang. This study aimed to evaluate the effects of local delivery of SJT via inhalable microparticles in an asthma mouse model. Methods: Microparticles containing SJT were produced by spray-drying with leucine as an excipient. SJT microparticles were evaluated with respect to their aerodynamic properties, in vitro cytotoxicity, in vivo toxicity, and therapeutic effects on ovalbumin (OVA)-induced asthma in comparison with orally-administered SJT. Results: SJT microparticles provided desirable aerodynamic properties (fine particle fraction of 48.9% +/- 6.4% and mass median aerodynamic diameter of 3.7 +/- 0.3 mu m). SJT microparticles did not show any cytotoxicity against RAW 264.7 macrophages at concentrations of 0.01 - 3 mg/mL. Inhaled SJT microparticles decreased the levels of IL-4, IL-5, IL-13, IL-17A, eotaxin and OVAIgE in bronchoalveolar lavage fluid (BALF) in mice with OVA-induced asthma. These effects were verified by histological evaluation of the levels of infiltration of inflammatory cells and collagen, destructions of alveoli and bronchioles, and hyperplasia of goblet cells in lung tissues. The effects of SJT microparticles in the asthma model were equivalent to those of orally-administered SJT extract. Conclusion: This study suggests that SJT is a promising agent for inhalation therapy for patients with asthma.

The effects of daucosterol have been identified in cancer therapy and neuronal diseases. However, the regulatory function of daucosterol in DSS-induced colitis has not yet been investigated. In this study, we evaluated the immunological and therapeutic effects of daucosterol in a mouse model of dextran sulfate sodium (DSS)-induced colitis. Unlike vehicle mice, mice pre- or post-treated with daucosterol showed inhibition of body weight loss and the decrease in the disease activity index (DAI). In addition, daucosterol treatment rescued the DSS-induced decrease in colon length and disruption of the epithelial lining. Furthermore, it reduced DSS-induced production of reactive oxygen species (ROS), infiltration of macrophages, and expression of pro-inflammatory cytokines such as TNF-α, IL-6, and IL-1β. Mice with colitis showed a decreased population of Foxp3+ cells, which was upregulated by daucosterol treatment. Furthermore, daucosterol increased natural killer (NK) cell activity and inhibited excessive IgA levels in mice with DSS-induced colitis. Collectively, our findings demonstrated that daucosterol significantly alleviated DSS-induced colitis, indicating the possibility of daucosterol as a therapeutic option for colitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Bleomycin (BLM) contributes to the induction of pulmonary inflammation and fibrosis in animals. Although skin and lung tissue inflammation is closely related in the pathogenesis of allergic diseases, a proper animal model for investigating the relationship between skin and lung inflammation is lacking. Therefore, we developed a mouse model of AD with relapsing dermatitis and pulmonary fibrosis caused by the administration of allergen and BLM. The present study determined whether lung injury caused by the bronchial application of BLM would exacerbate AD-like allergic inflammation induced by 2, 4-dinitrochlorobenzene (DNCB) in NC/Nga mice. NC/Nga mice treated with BLM and DNCB had increased severity of clinical symptoms and airway hyperresponsiveness as well as increased inflammatory cell infiltration and collagen deposition in the dorsal skin and lung. Compared to normal mice, interleukin (IL)-6 and tumor necrosis factor (TNF)-α production in bronchoalveolar lavage fluid were increased in NC/Nga mice treated with both DNCB and BLM and in animals treated with DNCB alone. Administration of BLM and DNCB increased the levels of IL-4 and IL-13 production in spleen cells and eotaxin-2 mRNA expression in dorsal skin, compared to NC/Nga mice treated with DNCB alone. The total cell numbers in axillary lymph node, bronchoalveolar lavage, and thymus were increased in DNCB-BLM mice compared to those in mice treated with DNCB alone. Administration of BLM and DNCB increased the numbers of cluster of differentiation 4 (CD4)+ T cells and CD11b+granulocyte-differentiation antigen-1 (Gr-1)+ cells among peripheral blood mononuclear cells, CD4+ cells in bronchoalveolar lavage, CD4+ and B220+CD23+ B cells in the axillary lymph node, and CD4+ cells in thymus, compared to DNCB-treated mice. The number of total, CD4+, and CD11b+Gr-1+ cells in the lung were increased in both DNCB and DNCB-BLM mice. These results demonstrate that BLM aggravates allergic skin inflammation and promotes airway hyperreactivity and lung inflammation when combined with DNCB in NC/Nga mice.

Hair is a highly relevant specimen that is used to verify drug exposure in victims of drug-facilitated crime (DFC) cases. In the present study, a new analytical method involving ultrahigh-performance liquid chromatography-tandem mass spectrometry was developed for determining the presence of model drugs, including zolazepam and tiletamine and their metabolites in hair specimens from DFCs. The incorporation of zolazepam and tiletamine into hair after a single exposure was investigated in Long-Evans rats with the ratio of the hair concentration to the area under the curve. For rapid and simple sample preparation, methanol extraction and protein precipitation were performed for hair and plasma, respectively. No interference was observed in drug-free hair or plasma, except for hair-derived diphenhydramine in blank hair. The coefficients of variance of the matrix effects were below 12 %, and the recoveries of the analytes exceeded 70 % in all of the matrices. The precision and accuracy results were satisfactory. The limits of quantification ranged from 20 to 50 pg in 10 mg of hair. The drug incorporation rates were 0.03 ± 0.01 % for zolazepam and 2.09 ± 0.51 % for tiletamine in pigmented hair. We applied the present method to real hair samples in order to determine the drug that was used in seven cases. These results suggest that this comprehensive and sensitive hair analysis method can successfully verify a drug after a single exposure in crimes and can be applied in forensic and clinical toxicology laboratories.

The continuing appearance of new synthetic cannabinoids has been a major issue in the field of forensic and clinical toxicology. In response to that, analytical methods for synthetic cannabinoids have been increasingly established in a variety of biological matrices. Since most of synthetic cannabinoids with structure similarity share some enzymatic metabolites, making the interpretation of analytical results and the discovery of the parent drug actually ingested very complicated, the investigation on metabolites of the first generation of synthetic cannabinoids with their relatively short side chains in chemical structure could be more important. Therefore, in the present study, we developed the analytical method for AM-2201, JWH-122 and MAM-2201 with JWH-018 as a precursor and their monohydroxylated metabolites in hair matrix. Also, using a rat model, AM-2201 and its monohydroxylated metabolites were identified and then the ratios of metabolite-to-parent drug were estimated to be used as criteria on external contamination. All analytes were extracted with methanol from washed and cut hair samples and the extracts were injected into LC-MS/MS with electrospray ion source in the positive ionization mode. Matrix effect and recovery were evaluated in hair matrices and no significant variations were observed. The validation results for precision and accuracy were satisfactory in both human and rat hair. The LOD and LOQ were 0.5 pg/10mg and 1.0 pg/10mg in human hair and 0.5 pg/20mg and 1.0 pg/20mg in pigmented and non-pigmented rat hair, respectively. Additionally, as a result of the animal study, there were not significant differences in the effect of pigmentation on the distribution of AM-2201 and its monohydroxylated metabolites in hair. Wide variations were observed for the concentrations of the naphthoylindole-based synthetic cannabinoids and metabolites in authentic hair samples from nine cases; those were 0.4-59.2 pg/mg for JWH-018, 0.1-0.8 pg/mg for JWH-073, 1.7-739.0 pg/mg for AM-2201, 0.1-402.0 pg/mg for JWH-122, 0.2-276.0 pg/mg for MAM-2201, 0.2-1.1 pg/mg for JWH-018 N-COOH, 0.3-37.2 pg/mg for JWH-018 N-5-OH, 0.3 pg/mg for JWH-073 N-COOH, 0.4 pg/mg for AM-2201 N-4-OH, 0.2-3.1 pg/mg for AM-2201 N-6-OHindole and 0.1-3.5 pg/mg for JWH-122 N-5-OH. This quantitative LC-MS/MS analytical method for five naphthoylindole-based synthetic cannabinoids and their metabolites was very useful to be applied to authentic hair samples, of which their analytical results suggested the incorporation of synthetic cannabinoids in the hair matrix and provided the information on ingested parent drugs.

Abuse of fluorinated synthetic cannabinoid analogs to avoid existing legal regulations has increased globally. The fluorinated JWH-122 analog, MAM-2201, was first reported in September 2012 as an ingredient in herbal mixtures in Korea. MAM-2201 is more potent than JWH-122 and a fatal intoxication case has been reported. In this study, we identified major MAM-2201 and JWH-122 metabolites from in vitro metabolism studies using human liver microsomes and compared the results with those of urine specimens from suspected MAM-2201 or JWH-122 users. MAM-2201 and JWH-122 produced common metabolites, N-5-hydroxylated, N-4-hydroxylated and carboxylated JWH-122 metabolites. Trace amounts of an N-4-hydroxylated MAM-2201 metabolite, a characteristic MAM-2201 metabolite, was detected in only a few urine specimens from MAM-2201 users. Both in vitro and in vivo studies demonstrated that N-5-hydroxylated JWH-122 metabolite was the primary metabolite of MAM-2201, whereas N-4-hydroxylated JWH-122 metabolite was predominant in JWH-122 metabolism. Based on these results, relative concentrations of N-5- and N-4-hydroxylated JWH-122 metabolites should be considered to verify MAM-2201 or JWH-122 users.

Background The roots of Korean red ginseng (Panax ginseng C.A.Mey.; KGC) have been used as an herbal supplement to enhance vital energy and immune capacity. Salvia plebeia R.Br. has been used to treat inflammatory diseases. Purpose The aim of this study was to examine the anti-asthmatic effects of a mixture of Korean red ginseng and Salvia plebeia R.Br. (KGC3P), its component nepetin, and their modes of action in alleviating ovalbumin (OVA)-induced asthma in mice. Method BALB/c mice were sensitized with OVA then subjected to intratracheal, intraperitoneal, and aerosol challenges. KGC3P and nepetin were administered orally for four weeks. Airway hyperresponsiveness (AHR), OVA-specific IgE levels, and Th2 cytokine- and gene expression levels in bronchoalveolar lavage fluid (BALF) and splenocytes were measured. Histological and immune cell subtype analyses were performed. PTEN and Akt phosphorylation levels were also evaluated. Results KGC3P reduced OVA-induced AHR, serum IgE levels, histological changes, and eosinophils infiltration but also the absolute number of immune cell subtypes including CD3+/CD4+, CD3+/CD8+, CD4+/CD69+, and Gr-1+/CD11b+ in the lungs, BALF, and mesenteric lymph nodes (MLN). KGC3P also lowered the Th2 cytokines IL-4, IL-5, and IL-13 in the BALF and splenocytes and downregulated the IL-4, IL-13, IL-17, TNF-α, and MUC5AC genes in the lung. KGC3P upregulated the peroxisome proliferator-activated receptor (PPAR)γ gene but downregulated the p-Akt and p-PTEN phosphorylation. Similar results were obtained with nepetin treatment. Conclusion KGC3P and nepetin are anti-asthmatic because they reduce various immune cells such as eosinophils and Th2 cell as well as Th2 cytokines. These mechanisms may be accompanied by the regulation of PPARγ expression and the PTEN pathway. Taken together, our results indicate that KGC3P and nepetin may potentially prevent and treat asthma.

Background Siraitia grosvenorii fruits are used in traditional medicine to treat cough, sore throat, bronchitis, and asthma. Purpose This study aimed to investigate the anti-inflammatory and anti-asthmatic effects of S. grosvenorii residual extract (SGRE) on ovalbumin (OVA)-induced asthma in mice. Methods Asthma was induced in BALB/c mice by systemic sensitization to OVA, followed by intratracheal, intraperitoneal, and aerosol allergen challenges. SGRE was orally administered for four weeks. We investigated the effects of SGRE on airway hyper-responsiveness, OVA-specific IgE production, histological analysis of lung and trachea, immune cell phenotyping, Th1/Th2 cytokine production in bronchoalveolar lavage fluid (BAL) fluid and splenocytes, and gene expression in the lung. Results SGRE ameliorated OVA-driven airway hyper-responsiveness, serum IgE production, and histopathological changes in the lung and trachea. SGRE reduced the total number of cells in the lung and BAL, the total number of lymphocytes, neutrophils, monocytes, and eosinophils in the lung and BAL, the absolute number of CD4+/CD69+ T cells in the lung, and the absolute number of CD4+/CD8+ T cells and CD11b+/Gr-1+ granulocytes in the lung and BAL. SGRE also reduced Th2 cytokines (IL-4, IL-5, and IL-13) and increased the Th1 cytokine IFN-γ in the BAL fluid and supernatant of splenocyte cultures. SGRE decreased the OVA-induced increase of IL-13, TARC, MUC5AC, TNF-α, and IL-17 expression in the lung. Conclusion SGRE exerts anti-asthmatic effects via the inhibition of Th2 and Th17 cytokines and the increase of Th1 cytokines, suggesting that SGRE may be a potential therapeutic agent for allergic lung inflammation, such as asthma.

Natural and synthetic opioids have efficient analgesic activity but can also be addictive. Thus, the determination of opioids and their metabolites in biological specimens is of interest in clinical and forensic toxicology laboratories. The analysis of drugs in hair provides valuable information on previous chronic drug use and has been successfully applied to the diagnosis of drug abuse, tolerance, compliance and gestational drug exposure. Despite the abuse of prescription opioids along with heroin and other illegal opiates, few studies have been conducted on the simultaneous determination of the broad range of opioids covering those drugs in hair. In the present study, an analytical method for the simultaneous detection in hair of 18 opioids and metabolites considered to have a high abuse risk based on the results of urine drug screening was established and validated using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the purpose of clinical and forensic applications. The drugs and metabolites were extracted from hair using methanol and analyzed using LC-MS/MS. The validation results proved that the method was selective, accurate and precise with acceptable linearity within calibration ranges. No significant variation was observed by different sources of matrices. The limits of detection and the limits of quantification ranged from 0.05 to 0.25ng/10mg hair and from 0.05 to 0.5ng/10mg hair, respectively. The developed method was successfully applied to 15 hair samples from opioids users. This method will be very useful for monitoring the inappropriate use of opioid drugs.

In recent years, the inappropriate use of antipsychotics by young Korean men has become a social problem. As military service exemptions are given for mental illness, some men pose as mental health patients to avoid military service. In order to verify the authenticity of mental illnesses, we developed simultaneous analytical methods for the detection of 15 antipsychotics and 2 of their metabolites in hair using liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. The target drugs were modafinil, atomoxetine, aripiprazole, benztropine, buspirone, duloxetine, gabapentin, oxcarbazepine, topiramate, escitalopram, paliperidone, ziprasidone, lamotrigine, clonazepam, levetiracetam, and metabolites of oxcarbazepine and clonazepam. To remove possible contaminants on the hair surface, hair samples were washed twice with methanol and distilled water, and then were extracted with methanol overnight at 38°C. Desipramine-d3 was used as an internal standard. LC-MS/MS analysis was performed on an Agilent 1290 Infinity UHPLC coupled to an AB Sciex Qtrap® 5500 MS/MS. The total chromatographic run time was 14min. The following validation parameters were evaluated: selectivity, linearity, limit of detection (LOD), limit of quantification (LOQ), precision, accuracy, matrix effect, and recovery. The LOD and LOQ values for all analytes, except modafinil, ranged from 0.2 to 10pg/mg hair and from 0.2 to 20pg/mg hair, respectively. Good linearity was achieved for most of the analytes in the range of 20-200pg/mg hair. The method showed acceptable precision and accuracy, which were less than 15%, as well as satisfactory matrix effects and recoveries. Furthermore, this method was also applied to the analysis of rat hair samples. The study in rats showed that the concentrations of atomoxetine and aripiprazole in pigmented hair were significantly higher than those in non-pigmented hair. However, no significant difference was observed in the concentration of topiramate between pigmented and non-pigmented hair. This method will be useful in monitoring the inappropriate use of antipsychotics in suspects posing as mental health patients. However, further research is necessary before applying this method to authentic hair samples from mental health patients.

Paramethoxyamphetamine (PMA) is a phenethylamine derivative that is structurally related to 3,4-methylenedioxymethamphetamine (MDMA), but has higher toxicity than MDMA. Here, we report a fatal intoxication case involving PMA. A 36-year-old man was found dead in a hotel room. Toxicological analysis revealed that PMA concentrations were 0.57 and 0.59mg/L in peripheral and heart blood, respectively. Ketamine and diazepam were also detected in his blood. Based on toxicological results and autopsy findings, the cause of death was determined to be acute fatal intoxication with PMA. Hair analysis using gas chromatography/mass spectrometry was performed and PMA was detected at a concentration of 20.1ng/mg after methanol extraction for 20h. This is the first report of the determination of PMA concentration in the hair from a drug abuser.

Ethnopharmacological relevance Illicium verum is a traditional herbal medicine with anti-inflammatory properties used in Asia. However, its usefulness in the treatment of allergic diseases remains unclear. This study evaluated the anti-inflammatory and antiallergic effects of I. verum extract (IVE) in a mouse model of atopic dermatitis. Materials and methods We investigated the effects of IVE on compound 48/80-induced histamine release, and phorbol 12-myristate13-acetate and calcium ionophore A23187-stimulated cytokines secretion in MC/9 mast cells. Atopic dermatitis was induced in NC/Nga mice by exposure to extract of house dust mite (Dermatophagoides farinae). After a topical application of IVE on ear and skin lesions, we evaluated the severity of skin symptoms, ear thickness, inflammatory cell infiltration, and serum levels of immunoglobulin E (IgE), histamine, interleukin (IL)-6, and intercellular adhesion molecule (ICAM)-1. In addition, we determined the expression of IL-4, IL-6, tumor necrosis factor (TNF)-α, interferon (IFN)-γ thymus- and activation-regulated chemokine (TARC), regulated on activation, normal T cell expressed and secreted (RANTES), ICAM-1, and vascular cell adhesion molecule (VCAM)-1 in ear tissues. Results IVE inhibited secretion of histamine, IL-4, IL-6, and TNF-α from mast cells in a dose-dependent manner. Topical application of IVE significantly reduced dermatitis scores, ear thickness, and serum levels of IgE, histamine, IL-6, and ICAM-1. Histopathological analysis demonstrated decreased epidermal thickening and dermal infiltration by inflammatory cells. In the ear lesions, IVE treatment reduced expression of IL-4, IL-6, TNF-α, TARC, RANTES, ICAM-1, and VCAM-1, but not IFN-γ. Conclusions These results indicate that IVE inhibits atopic dermatitis-like skin lesions by suppressing the expression of cytokines, chemokines, and adhesion molecules. These results suggest that IVE may be a potential therapeutic candidate for atopic dermatitis.

The metabolite-to-parent drug ratios were determined in the hair of 2444 methamphetamine (MA) abusers who had produced MA-positive hair results from 2001 to May 2005 and in the hair of 53 ecstasy abusers who had produced positive methylenedioxymethamphetamine (MDMA) hair results from 2002 to May 2005. For the hair analyses, hair strands were washed, cut into small pieces and extracted for 20 h in 1 mL methanol containing 1% HCl. Drugs in the extract were determined by gas chromatography-mass spectrometry (GC-MS) using selective ion monitoring after derivatization with trifluoroacetic anhydride. The six range groups were divided as follows on the basis of MA concentrations in hair (n = 2389): 0.5-5 ng/mg (n = 950), 5-10 ng/mg (n = 582), 10-20 ng/mg (n = 503), 20-30 ng/mg (n = 160), 30-40 ng/mg (n = 80), more than 40 ng/mg (n = 114) to assess the correlations between MA concentrations and metabolite-to-parent drug ratios. In groups of higher MA concentrations, lower ratios of AP/MA were found, and there was a statistically significant difference among six range groups. Comparisons of age groups (tens, twenties, thirties, forties, fifties, and sixties) and male and female subjects for the ratios of AP/MA showed a statistically significant difference. The detection of metabolites and the parent drug with reasonable ratios was found to be a useful indicator for distinguishing internal drug incorporation from external contamination. In our study, MA users can produce 0.4-116% (mean = 9%) of amphetamine (AP) concentrations in hair, and ecstasy users 1-110% (mean = 12%) of methylenedioxyamphetamine (MDA) in appropriately washed hair samples.

Ethnopharmacological Relevance.Morus albaL. leaves (MAE) have been used in fork medicine for the treatment of beriberi, edema, diabetes, hypertension, and atherosclerosis. However, underlying mechanism of MAE on cardiovascular protection remains to be elucidated. Therefore, we investigated whether MAE affect platelet aggregation and thrombosis.Materials and Methods. The anti-platelet activity of MAE was studied using rat platelets. The extent of anti-platelet activity of MAE was assayed in collagen-induced platelet aggregation. ATP and serotonin release was carried out. The activation of integrinαIIbβ3and phosphorylation of signaling molecules, including MAPK and Akt, were investigated with cytofluorometer and immunoblotting, respectively. The thrombus formationin vivowas also evaluated in arteriovenous shunt model of rats.Results. HPLC chromatographic analysis revealed that MAE contained rutin and isoquercetin. MAE dose-dependently inhibited collagen-induced platelet aggregation. MAE also attenuated serotonin secretion and thromboxane A2formation. In addition, the extractin vivoactivity showed that MAE at 100, 200, and 400 mg/kg significantly and dose-dependently attenuated thrombus formation in rat arterio-venous shunt model by 52.3% (P<0.001), 28.3% (P<0.01), and 19.1% (P<0.05), respectively.Conclusions. MAE inhibit platelet activation, TXB2 formation, serotonin secretion, aggregation, and thrombus formation. The plant extract could be considered as a candidate to anti-platelet and antithrombotic agent.

Ulmus macrocarpa Hance (Ulmaceae) has been used as a traditional oriental medicine for the treatment of edema, mastitis, gastric cancer and inflammation. The aim of this study was to investigate the effects of Ulmus macrocarpa extract (UME) on thrombus formation in vivo, platelet activation ex vivo and fibrinolytic activity in vitro. To identify the antithrombotic activity of UME in vivo, we used an arterial thrombosis model. UME delayed the occlusion time by 13.4 and 13.9 min at doses of 300 and 600 mg/kg, respectively. UME significantly inhibited ex vivo platelet aggregation induced by collagen and adenosine 5'-diphosphate (ADP), respectively, but did not affect the coagulation times following activated partial thromboplastin and prothrombin activation. Therefore, to investigate the antiplatelet effect of UME, the effect of UME on collagen and ADP-induced platelet aggregation in vitro was examined. UME exhibited antiplatelet aggregation activity, induced by ADP and collagen. Furthermore, the fibrinolytic activity of UME was investigated. The results showed that UME significantly increased fibrinolysis at 1,000 mg/ml. In conclusion, the results suggested that UME may significantly inhibit artery thrombus formation in vivo, potentially due to antiplatelet activity, and also exhibits potential as a clot‑dissolving agent for thrombolytic therapy.

Due to their cannabis-like effects, synthetic cannabinoids have attracted much public attention since 2008. Thus, elucidation of the metabolic pattern and the detection of the intake of these drugs have been of major concern. In order to suggest appropriate urinary biomarkers to prove JWH-018 or JWH-073 intake, we selected the major metabolites of JWH-018 and JWH-073, namely (ω)-, (ω-1)-hydroxy, carboxy and 6-hydroxyindole metabolites, and validated a method for the quantification of these metabolites using solid-phase extraction based on LC-MS/MS analysis. Authentic urine specimens obtained from drug offenders were screened via a synthetic cannabinoid ELISA kit and were analyzed by LC-MS/MS for confirmation. Twenty-one out of a total of 52 samples (40%) were found positive for at least one metabolite of JWH-018 or JWH-073. N-pentyl hydroxy metabolites of JWH-018 and carboxy metabolites of JWH-018 and JWH-073 were detected in all positive samples. However, the rest of the metabolites were either not detected or only a small amount of them were found. A considerable variation was observed in the concentration ratio of (ω) and (ω-1)-hydroxy metabolites of JWH-018. Based on the results, it may have some pitfalls to determine the ingestion of specific synthetic cannabinoids by detecting a few metabolites, considering the continuous emergence of structurally related synthetic cannabinoids. Thus, use of synthetic cannabinoids should be proven carefully through comprehensive investigation of analytical results of biological specimens.

Ethnopharmacological relevance Drynaria fortunei (Kunze) J. Sm has been widely used in traditional medicine for the treatment of inflammation, hyperlipidemia, arteriosclerosis, rheumatism, and bone healing. We investigated the anti-inflammatory effects of a 70% ethanol extract of Drynaria fortunei (DFE). Materials and methods We evaluated the anti-inflammatory effects of topically applied DFE on house dust mite Dermatophargoides farinae-induced atopic dermatitis-like skin lesions in NC/Nga mice. Results Treatment of NC/Nga mice with DFE reduced the dermatitis score, ear thickness, and serum levels of IgE, IgG1, and IL-6. Histopathological analyses of ear and skin lesions showed inhibition of the thickening of the epidermis and reduced epidermal/dermal infiltration of inflammatory cells. In ear lesions, mRNA expression levels of IL-4, IL-6, and tumor necrosis factor-α were reduced by DFE treatment. Conclusions DFE inhibited the development of dermatitis-like skin lesions in NC/Nga mice. These results suggest that DFE may be a therapeutic candidate for the treatment of AD.

Litchi chinensis Sonn. (lychee), which belongs to the family of Sapindaceae, is a subtropical evergreen tree that is cultivated throughout Southeast Asia, particularly in China. Litchi chinensis has been reported to have anti-inflammatory, antioxidant and antidiabetic activities. However, the antiplatelet and anticoagulant effects of Litchi chinensis have not been reported previously. In this study, we investigated the effects of a 70% ethanol extract from Litchi chinensis (LCE) on platelet aggregation, coagulation and fibrinolysis. LCE dose-dependently inhibited collagen- and ADP-induced platelet aggregation in rat platelet-rich plasma. LCE at 4 mg/ml had a maximal inhibitory effect on platelet aggregation. In particular, the LCE 4 mg/ml-treated group showed almost complete inhibition in the collagen-induced platelet aggregation assay. It also significantly prolonged coagulation times, such as the activated partial thromboplastin and prothrombin time, in rat platelet-poor plasma. We also investigated the fibrinolytic effects of LCE using the fibrin plate assay. LCE increased fibrinolytic activity in a dose-dependent manner. These results demonstrated the antithrombotic effects of LCE and suggest that Litchi chinensis may be a new natural source for the development of antiplatelet, anticoagulant and thrombolytic therapeutics for thrombotic and cardiovascular diseases.

The prevalence and age distribution of 3,4-methylenedioxymethamphetamine (MDMA) and 3,4-methylenedioxyamphetamine (MDA) in hair samples by gas chromatography/mass spectrometry (GC/MS) were studied. The recoveries obtained from hair were 97% and 99% for MDMA and MDA, respectively. The inter- and intra-assay precision and accuracy were determined. Out of 791 hair samples, 44 (5.6 %) contained MDMA and/or MDA. Out of these 44 subjects, urinalyses from 35 were negative for both MDMA and MDA, while only 9 were positive. We also evaluated concentrations of MDMA and MDA, and the metabolite-to-parent drug ratios. This study showed that the abuse of MDMA or MDA was found principally among young adults and male abusers. We found the epidemiology of ecstasy users in Korea between March 2002 and April 2003.

The effects of heterocycles including imidazole (IM), 1,2,4-triazole (TR) and thiazole (TH) on the expression of microsomal epoxide hydrolase (mEH) gene were examined in rats (200 mg/kg body weight/day, i.p.). Hepatic microsomes prepared from rats treated with IM for 3 days failed to exhibit an increase in mEH protein level whereas TR treatment resulted in an approximately 2- to 3-fold elevation in hepatic mEH levels relative to control, as assessed by both SDS-PAGE and immunoblot analyses. In contrast, thiazole-induced hepatic microsomes resulted in a substantial increase in mEH levels (i.e. approximately 5-fold). Slot and northern blot analyses, probed with an mEH cDNA, showed that the hepatic mEH mRNA levels in the animals treated with IM for 3 days were marginally increased by approximately 2-fold, as compared with untreated animals, whereas TR caused an approximately 8-fold increase in hepatic mEH mRNA levels after three consecutive daily treatments. TH treatment resulted in an approximately 22-fold increase in the mEH mRNA levels, demonstrating that TH is the most efficacious among these three azole heterocycles. Because TH was the most effective in increasing hepatic mEH protein and mRNA levels, the agent was chosen for further evaluation. Time course of mEH gene expression at early times after a single treatment with TH was determined and compared with that caused by pyrazine (PZ), a strong mEH inducer. Hepatic mEH mRNA levels were increased approximately 1-, 3-, 20- and 16-fold at 3, 6, 12 and 24 hr, respectively, following TH treatment, relative to control, whereas mEH mRNA levels were elevated approximately 1-, 1-, 22- and 18-fold, respectively, at the same time points after PZ treatment, as monitored by slot RNA hybridization analyses. Northern blot analyses using either total RNA or poly(A)+ RNA fractions exhibited comparable time courses in increasing mEH mRNA levels after TH or PZ treatment with maximal mRNA increases being noted at 12 hr post treatment. Although neither IM or TR failed to affect renal mEH gene expression to a notable extent, TH treatment caused 6- to 8-fold increases in kidney mEH mRNA levels, with a 2-fold increase in mEH protein detected. These results demonstrated that the azole heterocyclic compounds IM, TR and TH differentially induce mEH with TH as the most efficacious azole; and that the changes in mEH levels are primarily associated with increases in mRNA levels.