Ebselen oxide attenuates mechlorethamine dermatotoxicity in the mouse ear vesicant model.

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard mimetic. Topical exposure to HN2 is associated with tissue blistering. Previous work in our laboratory has shown that ebselen (EB-1) possesses anti-vesicant, anti-inflammatory, anti-bacterial, anti-fungal, and cytoprotective properties, both in vivo and in vitro. We recently reported that ebselen oxide (EB-2), an analog of EB-1 with a tetravalent selenium atom, also possesses anti-bacterial and anti-fungal activity and confers cytoprotection against HN2 in vitro. The purpose of the present study was to determine the vesicant countermeasure potential of EB-2 using the mouse ear vesicant model (MEVM). Compared to control ears, mouse ears exposed to a single dose of HN2 (0.500 µmol/ear) showed an increase in wet weights, ear thickness, hyperplasia, vesication, and inflammatory cell infiltration after 24 h. Fluorescence microscopy of terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL)-stained sections showed that the occurrence of apoptosis extended from the epidermis of the HN2-treated side, all the way to the contralateral epidermis. In contrast, HN2-exposed ears treated topically with EB-2 at a test dose of 0.250 mg/ear showed a significant decrease in wet weight (12% less vs. HN2 alone), morphometric thickness (13% less vs. HN2 alone), and vesication. In addition, TUNEL staining revealed that HN2 ears treated with EB-2 (0.250 mg/ear) showed a decrease in apoptosis as compared to the HN2 group. EB-2 also reduced the abundance of matrix metalloproteinase-9 (MMP-9) in ear tissues exposed to HN2. Taken together, our study demonstrates that EB-2 is an efficacious countermeasure to HN2.

Ebselen reduces the toxicity of mechlorethamine in A-431 cells via inhibition of apoptosis.

A series of test compounds were evaluated for an ability to reduce the toxicity of the nitrogen mustard mechlorethamine (HN2) in vitro. The test compounds included resveratrol, pterostilbene, vitamin C, ebselen, ebselen diselenide, and ebselen-sulfur. Among them, ebselen demonstrated the highest degree of protection against HN2 toxicity. To this end, pretreatment of the cells with ebselen offered protection against the toxicant whereas no protection was observed when cells were first incubated with HN2 and then treated with ebselen. Significant increases in caspase 3 and caspase 9 activities were observed in response to HN2, and ebselen was found to reduce these effects. Taken together, the data presented here indicate that ebselen is an effective countermeasure to nitrogen mustard in vitro, which is worthy of future investigation in vivo.

Counter irritant activity of Carthamus oxycantha.

Many locally occurring species of Asteraceae are used as medicinal plants by various tribal and ethnic communities in Pakistan. Carthamus oxycantha is often occurs as weed in cultivated fields. Folk medicines indicated its use as an anti inflammatory and wound healing plant. It is used for wound healing by the local population in the form of powder paste. No scientific Report, about the behavior of this plant has so far been published. The counter irritant studies of locally occurring Carthamus oxycantha was carried out. The main objectives of the project were to evaluate its wound healing effects on animal skin and the identity and characterization of chromatographically isolated fractions. For this purpose, different solvents with a broad range of polarity were successively used to extract non-polar compounds (petroleum ether extract), constituents intermediate polarities (chloroform extract) and polar constituents (methanol extract) from the whole herb of Carthamus oxycantha. The counter irritant activity of the crude extracts and isolated fractions was evaluated on rabbit's skin. Five fractions Co-1 to Co-5 were isolated from the active chloroform extract by column and thin layer chromatography. Co-1, Co-3 and Co-5 appeared to be the most potent counter irritant than others. A possible structure-activity relationship of these active compounds was investigated by using spectroscopy (UV and FTIR analysis).

Menthol attenuates respiratory irritation responses to multiple cigarette smoke irritants.

Menthol, the cooling agent in peppermint, is added to almost all commercially available cigarettes. Menthol stimulates olfactory sensations, and interacts with transient receptor potential melastatin 8 (TRPM8) ion channels in cold-sensitive sensory neurons, and transient receptor potential ankyrin 1 (TRPA1), an irritant-sensing channel. It is highly controversial whether menthol in cigarette smoke exerts pharmacological actions affecting smoking behavior. Using plethysmography, we investigated the effects of menthol on the respiratory sensory irritation response in mice elicited by smoke irritants (acrolein, acetic acid, and cyclohexanone). Menthol, at a concentration (16 ppm) lower than in smoke of mentholated cigarettes, immediately abolished the irritation response to acrolein, an agonist of TRPA1, as did eucalyptol (460 ppm), another TRPM8 agonist. Menthol's effects were reversed by a TRPM8 antagonist, AMTB. Menthol's effects were not specific to acrolein, as menthol also attenuated irritation responses to acetic acid, and cyclohexanone, an agonist of the capsaicin receptor, TRPV1. Menthol was efficiently absorbed in the respiratory tract, reaching local concentrations sufficient for activation of sensory TRP channels. These experiments demonstrate that menthol and eucalyptol, through activation of TRPM8, act as potent counterirritants against a broad spectrum of smoke constituents. Through suppression of respiratory irritation, menthol may facilitate smoke inhalation and promote nicotine addiction and smoking-related morbidities.

Identification and evaluation of counter-irritant potential of crude extract of Malva parviflora L. by WHO recommended methods.

Plenty of medicinal plants are available in Pakistan and are in human use as herbal medicines from ancient time. Present work is based on the evaluation of the use of Malva parviflora in skin irritation problems. For this purpose, powdered plant material (The aerial part and roots separately) was extracted by using successive solvent extraction method using petroleum ether, chloroform and methanol. Resulting three crude fractions were subjected to counter-irritant investigations on rabbit's ear. Petroleum ether fraction exhibited prominent counter-irritant potential. Five compounds named, as MP-1, MP-2, MP-3, MP-4 and MP-5 were isolated from petroleum ether extract by column and thin layer chromatography. These compounds were subjected to UV spectrophotometer for detection of absorption of light, then FTIR for specific functional group identification and counter-irritant potentials was evaluated on rabbit's ear skin. The MP-1 and MP-2 exhibited excellent counter-irritant activity in different dilutions than others. However, dilution 100 µg/ml showed maximum activity.

Protective effect of ß-(1,3 → 1,6)-D-glucan against irritant-induced gastric lesions.

ß-(1,3)-D-Glucan with ß-(1,6) branches has been reported to have various pharmacological activities, such as anti-tumour and anti-infection activities, which result from its immunomodulating effects. Gastric lesions result from an imbalance between aggressive and defensive factors. In the present study, we examined the effect of ß-(1,3)-D-glucan with ß-(1,6) branches isolated from Aureobasidium pullulans on the gastric ulcerogenic response in mice. Oral administration of ß-glucan ameliorated gastric lesions induced by ethanol (EtOH) or HCl. This administration of ß-glucan also suppressed EtOH-induced inflammatory responses, such as infiltration of neutrophils and expression of pro-inflammatory cytokines, chemokines and cell adhesion molecules (CAM) at the gastric mucosa. Of the various defensive factors, the levels of heat shock protein (HSP) 70 and mucin but not PGE(2) were increased by the administration of ß-glucan. ß-Glucan-dependent induction of the expression of HSP70 and mucin proteins and suppression of the expression of pro-inflammatory cytokines, chemokines and CAM were also observed in cultured cells in vitro. The results of the present study suggest that ß-glucan protects the gastric mucosa from the formation of irritant-induced lesions by increasing the levels of defensive factors, such as HSP70 and mucin.

Dimethylthiourea protects against chlorine induced changes in airway function in a murine model of irritant induced asthma.

BACKGROUND: Exposure to chlorine (Cl2) causes airway injury, characterized by oxidative damage, an influx of inflammatory cells and airway hyperresponsiveness. We hypothesized that Cl2-induced airway injury may be attenuated by antioxidant treatment, even after the initial injury. METHODS: Balb/C mice were exposed to Cl2 gas (100 ppm) for 5 mins, an exposure that was established to alter airway function with minimal histological disruption of the epithelium. Twenty-four hours after exposure to Cl2, airway responsiveness to aerosolized methacholine (MCh) was measured. Bronchoalveolar lavage (BAL) was performed to determine inflammatory cell profiles, total protein, and glutathione levels. Dimethylthiourea (DMTU;100 mg/kg) was administered one hour before or one hour following Cl2 exposure. RESULTS: Mice exposed to Cl2 had airway hyperresponsiveness to MCh compared to control animals pre-treated and post-treated with DMTU. Total cell counts in BAL fluid were elevated by Cl2 exposure and were not affected by DMTU treatment. However, DMTU-treated mice had lower protein levels in the BAL than the Cl2-only treated animals. 4-Hydroxynonenal analysis showed that DMTU given pre- or post-Cl2 prevented lipid peroxidation in the lung. Following Cl2 exposure glutathione (GSH) was elevated immediately following exposure both in BAL cells and in fluid and this change was prevented by DMTU. GSSG was depleted in Cl2 exposed mice at later time points. However, the GSH/GSSG ratio remained high in chlorine exposed mice, an effect attenuated by DMTU. CONCLUSION: Our data show that the anti-oxidant DMTU is effective in attenuating Cl2 induced increase in airway responsiveness, inflammation and biomarkers of oxidative stress.

Gastroprotective actions of bombesin, L-DOPA, and mild irritants: roles of prostaglandins and sensory neurons.

BACKGROUND: Bombesin and dopamine prevent gastric injury by an unknown mechanism. Sensory neurons and endogenous prostaglandins play an important role in gastric mucosal defense. This study was designed to assess the role of these two local defense mechanisms in bombesin and dopamine-induced gastroprotection, as well as mild irritant-induced adaptive cytoprotection. METHODS: Conscious, fasted rats were given either capsaicin (125 mg/kg subcutaneously) to ablate sensory neurons or indomethacin (5 mg/kg intraperitoneally) to inhibit prostaglandin synthesis, 2 weeks and 30 minutes, respectively, before administration of bombesin (100 micrograms/kg subcutaneously), the dopamine precursor L-DOPA (25 mg/kg intraperitoneally), or the mild irritant 25% ethanol (1 mL orogastric). A 1-mL orogastric bolus of acidified ethanol (150 mmol/L HCl/50% ethanol) was given 30 minutes after pretreatment with these peptides and 15 minutes after administration of the mild irritant. Rats were killed 5 minutes later and the total area of macroscopic gastric injury quantified. RESULTS: Ablation of sensory neurons with capsaicin negated the protective actions of bombesin but failed to reverse gastroprotection by L-DOPA or 25% ethanol. Cyclooxygenase inhibition with indomethacin partially reversed bombesin and mild irritant-induced gastroprotection but did not diminish the protective actions of L-DOPA. CONCLUSIONS: Bombesin requires intact sensory neurons to exert its protective actions through a mechanism mediated, at least in part, by endogenous prostaglandins. Adaptive cytoprotection by the mild irritant 25% ethanol requires the presence of endogenous prostaglandins but not sensory neurons. L-DOPA-induced gastroprotection is independent of both local defense mechanisms.

Mecamylamine inhibits nicotine but not capsaicin irritation on the tongue: psychophysical evidence that nicotine and capsaicin activate separate molecular receptors.

Using a two-alternative forced-choice (2-AFC) discrimination test coupled with category intensity ratings, we investigated the effect of mecamylamine, an antagonist of neuronal nicotinic acetylcholine receptors (nAchRs), on oral irritation elicited by nicotine or capsaicin. Mecamylamine (0.075%) was first delivered to one side of the tongue with distilled H2O delivered to the other side. After 10 min either capsaicin (1 ppm) or nicotine (0.12%) was applied bilaterally to the tongue, and subjects were asked to choose which side yielded a stronger sensation (2-AFC) as well as to provide a rating of the irritation intensity difference between the two sides of the tongue. When nicotine was given after mecamylamine, a significant proportion of subjects chose the mecamylamine-untreated side as yielding stronger irritation. When capsaicin was given after mecamylamine, both sides of the tongue were chosen in equal numbers. These data indicate that mecamylamine reduced irritation elicited by nicotine but not capsaicin, and provide further evidence that nicotine oral irritation is mediated via a neuronal nAchR while capsaicin activates trigeminal fibers via a separate molecular receptor.

A modified HET-CAM assay approach to the assessment of anti-irritant properties of plant extracts.

Hen's egg--chorioallantoic membranes were used to screen for and assess anti-irritant properties among aqueous extracts of plants (HET-CAM tests), in connection with searches for plant-derived substances with topical anti-irritant action. The main question to be answered was whether CAM-assay screening of plant extracts could provide a useful route to identifying promising anti-irritant extracts for follow-up clinical testing. To be useful, the method would have to flag materials with strong anti-irritant properties, and would have to avoid registering false negatives. The tests conducted provided positive indications. We measured the delays in onset of three manifestations of membrane irritation-vascular hemorrhaging, membrane lysis and membrane coagulation-observed with test substances relative to positive controls. Aqueous 15% lactic acid, a commonly used irritant in direct tests on human skin, was employed as the test irritant in this study. The ratio [irritation onset times after test substance pre-treatment]:[onset times without test substance pretreatment] was used to measure the anti-irritant power of test substances. A scoring notation was devised for this which treats the delay parameters as independent effects. Most tested plant extracts showed no significant irritant or anti-irritant effects. Among the apparently anti-irritant plant extracts (approx. 10% of all those tested), most showed their greatest effect against hemorrhaging. Lesser but still readily measurable effects against membrane lysis and coagulation were also observed in nearly all the apparently anti-irritant extracts. Two of the tested extracts proved to be membrane irritants. Some key CAM assay results were compared with results obtained in direct tests on human skin using the same test irritant (15% lactic acid). In these comparative tests on skin, an essentially similar pattern of efficacy was obtained, with the plant extract deemed best in the CAM screenings, outperforming the benchmark anti-irritant hydrocortisone. From these initial results it appears that physiological CAM assays may prove useful in screening natural materials for anti-irritant properties, as alternatives to mechanism-dependent biochemical assays, or expensive direct screening tests on human subjects. Further work remains to extend the CAM screening approach to irritants other than lactic acid, and to assess its quantitative powers of prediction of topical anti-irritancy.

3-Methoxy-5,7,3',4'-tetrahydroxyflavan (ME): a new compound to prevent gastric irritation. A gastric potential difference analysis.

The protective effect of 3-Methoxy-5,7,3',4'-tetrahydroxyflavan (ME) on the gastric irritation induced by aspirin has been established using the non-invasive method of determination of the gastric potential difference (GPD) in male volunteers. 500 mg and 1000 mg ME (approx. 6.0-16.7 mg/kg b.w.) showed protective activity against induced gastric irritation in this model. The possible mechanism of action of this agent is discussed.

Coprecipitation of nonoxynol-9 with polyvinylpyrrolidone to decrease vaginal irritation potential while maintaining spermicidal potency.

The aim of this study was to test the hypothesis that polyvinylpyrrolidone (PVP) would increase the critical micelle concentration (CMC) of nonoxynol-9 (N-9), providing a reduction in its irritation potential, while maintaining essential spermicidal activity. Solid coprecipitates of N-9 with PVP were manufactured with the use of a modified lyophilization process. The irritation potential of N-9 was estimated by an in vitro assay, monitoring the extent of hemolysis of red blood cells. CMCs of N-9 were measured in the presence of various concentrations of PVP. A modified Sander-Cramer assay was implemented to measure the spermicidal activity of N-9 and the N-9/PVP coprecipitates. With the use of the lyophilization process and more suitable solvents, solid coprecipitates of N-9/PVP were manufactured with no residual organic solvents. The irritation potential of N-9 was reduced when in the presence of PVP-50% hemolysis values increased from 0.054 mM to more than 0.2mM. N-9 CMC values increased in the presence of PVP from 0.085 mM (0% PVP) to 0.110 mM (3.5% PVP) and 0.16 6mM (10% PVP). However, spermicidal activities ranged from 0.213 mM to 0.238 mM, N-9 remaining steady regardless of the amount of PVP. By use of N-9/PVP coprecipitates, the self-association properties and irritation potentials of N-9 were altered. This result suggests a process to produce a spermicidal product that reduces the detrimental implications to the vaginal epithelium while maintaining the essential spermicidal activity.

Capsaicin-induced joint inflammation is not blocked by local anesthesia.

The purpose of this study was to evaluate the effect of local anesthetic blockade of afferent innervation on the development of capsaicin-induced edema in the rat temporomandibular joint (TMJ) region and on reflex jaw muscle activity. Under halothane anesthesia, 64 male Sprague-Dawley rats were prepared for monitoring of edema development by lateral movement of a needle overlying the left TMJ region and for acute recording of electromyographic activity in ipsilateral digastric and masseter muscles. A double-barrel catheter was inserted into the TMJ region for delivery of saline or 0.5% bupivacaine from 1 needle, followed with the injection of 1% capsaicin, 0.1% capsaicin, or vehicle control from the other needle 5 minutes later. Application of capsaicin into the saline pretreated TMJ region led to dose-dependent edema development and reflex jaw muscle activity; however, only 1% capsaicin solution resulted in significant tissue expansion and muscle activity when compared with the vehicle control. Pretreatment of the rat TMJ region with bupivacaine failed to inhibit capsaicin-induced edema development, although successful blockade of nerve conduction was confirmed with the absence of reflex jaw muscle activity. Capsaicin-induced edema of the rat TMJ region developed independent of axonal conduction, suggesting neurogenic inflammation may arise regardless of functional nerve conduction.

Cytokine regulation by MAPK activated kinase 2 in keratinocytes exposed to sulfur mustard.

Uncontrolled inflammation contributes to cutaneous damage following exposure to the warfare agent bis(2-chloroethyl) sulfide (sulfur mustard, SM). Activation of the p38 mitogen activated protein kinase (MAPK) precedes SM-induced cytokine secretion in normal human epidermal keratinocytes (NHEKs). This study examined the role of p38-regulated MAPK activated kinase 2 (MK2) during this process. Time course analysis studies using NHEK cells exposed to 200µM SM demonstrated rapid MK2 activation via phosphorylation that occurred within 15 min. p38 activation was necessary for MK2 phosphorylation as determined by studies using the p38 inhibitor SB203580. To compare the role of p38 and MK2 during SM-induced cytokine secretion, small interfering RNA (siRNA) targeting these proteins was utilized. TNF-α, IL-1ß, IL-6 and IL-8 secretion was evaluated 24h postexposure, while mRNA changes were quantified after 8h. TNF-α, IL-6 and IL-8 up regulation at the protein and mRNA level was observed following SM exposure. IL-1ß secretion was also elevated despite unchanged mRNA levels. p38 knockdown reduced SM-induced secretion of all the cytokines examined, whereas significant reduction in SM-induced cytokine secretion was only observed with TNF-α and IL-6 following MK2 knockdown. Our observations demonstrate potential activation of other p38 targets in addition to MK2 during SM-induced cytokine secretion.

Inhibition of erythema induced by pro-inflammatory esters of 12-deoxyphorbol.

The pro-inflammatory tigliane esters 12-deoxyphorbolphenylacetate (12-DOPPA) and 12-deoxyphorbolphenylacetate-20-acetate (12-DOPPAA) at a dose of 0.1 microgram induced erythema in the mouse ear. Observations of ear redness were made both two and four hours after application. Indomethacin was only partly successful as an antagonist since 10% inhibition of 12-DOPPA and no inhibition of 12-DOPPAA induced erythema was produced four hours after application. The free radical scavengers, phenol, thioanisole and sodium benzoate all produced less than 30% inhibition of 12-DOPPA induced erythema and less than 15% inhibition of 12-DOPPAA, whereas aminopyrine produced 70% and 25% inhibition of 12-DOPPA and 12-DOPPAA respectively. The fact that free radical scavengers (with the exception of aminopyrine) and indomethacin, failed to markedly change the mouse ear reaction to 12-deoxyphorbol esters, indicated that this erythema is not entirely mediated via cyclooxygenase products. Mepyramine and cyproheptadine also failed to inhibit the erythema, whereas hydrocortisone produced a 55% inhibition of the 12-DOPPA and a 20% inhibition of the 12-DOPPAA reaction. The membrane stabilising agents trifluoperazine, promethazine, imipramine and desmethylimipramine were the most successful compounds used in inhibiting both 12-DOPPA and 12-DOPPAA induced erythema. In addition propranolol, which inhibits stimulus activation of phospholipase A2, produced 70% and 55% inhibition of the reaction of mice ears to 12-DOPPA and 12-DOPPAA.

Acetylsalicylic acid inhibits non-immunologic contact urticaria.

To investigate the mechanisms of non-immunologic contact urticaria (NICU), the effects of 1g + 1g of acetylsalicylic acid (ASA) on contact reactions to methyl nicotinate, diethyl fumarate, benzoic acid, cinnamic acid, cinnamic aldehyde and dimethyl sulfoxide were studied in 21 test subjects. Erythema and edema reactions were observed visually, and the changes in the skin blood flow were monitored using laser-Doppler flowmetry. ASA had a significant inhibitory effect on erythema from all 6 agents and also on edema from all substances except dimethyl sulfoxide. The mechanism of the effect may be a result of the inhibitory influence of ASA on prostaglandin bioformation. Thus, to avoid false negative test results, non-steroidal anti-inflammatory drugs should not be used during NICU tests.

[Effect of various types of analgesic drugs on an experimental model of chronic inflammatory pain in mice].

We attempted to develop an experimental model of chronic inflammatory pain in mice. The mice were injected intradermally at the base of the tail with various kinds of irritants (yeast, carrageenin, mustard and adjuvant). The pain threshold was measured by the pressure method every 60 min for 5 hr and once a day at the same time throughout the experimental period. The group of 10% yeast-injected mice exhibited the most intensive hyperalgesia. The analgesic effect of various types of analgesic drugs were studied, comparing the effects in normal mice and various kinds of irritants-induced hyperalgesia mice. It was demonstrated by observing ED50 values that nonsteroidal antiinflammatory drugs (NSAIDs), narcotic analgesic drugs and agonist/antagonist type of analgesic drugs were effective, but CNS-acting drugs were ineffective in yeast hyperalgesia mice. In comparison with yeast hyperalgesia mice, larger doses of analgesic drugs were required in normal mice and other irritants-treated mice. Especially, acidic NSAIDs were more effective in yeast hyperalgesia mice than normal mice. It was suggested that acidic NSAIDs specifically inhibit inflammatory pain. Moreover, yeast hyperalgesia mice are useful for the quantitative measurement of analgesic drugs.

Comparison of the concentration-effect relationship of a local antiinflammatory agent and oral acetylsalicylic acid: the value of local application.

Using a pharmacological model, the comparison between acetylsalicylic acid (ASA), administered orally, and a solution combining two salicylate derivatives (ethyl 5-methoxy-salicylate and 3-phenyl-propyl-salicylate), applied locally, demonstrated the value of the local application. Indeed, the pharmacological activity was highly significant and directly related to the tissue concentration of salicyl ions, which was higher after local application of the solution than after oral administration of ASA. The local solution also resulted in a lower plasma concentration of salicylate ions, allowing high plasma salicylate concentrations to be avoided.

Synthesis and structure--activity relationships of aroylpyrrole alkylamide bradykinin (B2) antagonists.

The synthesis and structure-activity relationships of a novel series of aroylpyrrole alkylamides as potent selective bradykinin B(2) receptor antagonists are described. Several members of this series display nanomolar affinity at the B(2) receptor and show activity in an animal model of antinociception.