An Academic Service Learning (AS-L) Activity within an Undergraduate Course in Pharmacology

Academic service learning (AS-L) is a type of active learning in which a student demonstrates knowledge and understanding through service to the community and reflection. The present report describes an activity in which AS-L was implemented as part of an undergraduate pharmacology course. The course is common to the curricula of the Doctor of Pharmacy, Physician Assistant and Toxicology programs at St. John’s University. In the AS-L project, students were charged to develop a presentation which they would then give to members of the community who were unfamiliar with the presentation topics. Students worked in teams and formed their presentations around discussion topics such as drugs versus natural substances, the medical benefits of drugs, the possible toxicities of drugs both legal and illegal, or the mechanisms by which drugs enter or leave our bodies. The student teams then traveled to various service sites throughout the greater university community with the goal of community outreach through education. In the present report, strengths and limitations of the AS-L project have been noted. The major strength of the project, as indicated from student reflection papers, was that each student in the team became an active learner and the otherwise “passive learning” environment of the classroom became an active one at the service site. All students in the team presented and answered questions. A major limitation of the activity was finding a suitable instrument for the assessment of student learning. Future AS-L courses of this type are anticipated to include pre and post surveys

Evaluacija citotoksičnosti resveratrola i piceatanola u makrofazima, T-stanicama i stanicama kože

The cytotoxicity of resveratrol and of piceatannol, a structural analog of resveratrol, was examined in cultured cells. Using a MTT-based assay, which measures the conversion of 3-[4,5-dimethylthiazol-2- yl]-2,5-diphenyl tetrazolium bromide (MTT) to a colored formazan product in living cells, resveratrol was found to inhibit the viability of transformed mouse macrophages, tumor-derived human T cells and human epidermoid carcinoma cells in a concentration-dependent manner, with the effect decreasing in the order: T cells (LC50 ~27 µmol L-1, 24 h; ~9 µmol L-1; 48h) > macrophages (LC50 ~29 µmol L-1, 24 h; 39 µmol L-1, 48 h) > skin cells (LC50 ~91 µmol L-1, 24 h ; ~66 µmol L-1, 48 h). Paradoxically, a high concentration of resveratrol (50 µmol L-1) inhibited the proliferation of all three cell types, and a low concentration (5 µmol L-1) stimulated the proliferation of macrophages. The viability of macrophages was also decreased by piceatannol in a concentration-dependent manner. The stimulation of macrophages with zymosan lowered the cytotoxicity of both resveratrol and piceatannol. Scanning electron microscopy of cells treated with resveratrol revealed changes in cellular morphology that were consistent with toxicity. In macrophages and skin cells, resveratrol (50 µmol L-1) induced a time-dependent increase in reduced glutathione levels but did not alter the background levels of thiobarbituric acid-reactive substances. Taken together, the present data indicate that resveratrol is toxic to cultured macrophages, T cells and skin cells at concentrations ≥25 µmol L-1, and that the cytotoxicity occurs via a mechanism that does not involve oxidative stress. Furthermore, the degree of toxicity of both resveratrol and piceatannol towards macrophages depends on the activation status of these cells, with zymosan-activated cells appearing more resistant than nonstimulated cells.Citotoksičnost resveratrola i piceatanola, strukturnog analoga resveratrola, ispitivana je u uzgojenim stanicama. Primjenom MTT-testa koji mjeri pretvorbu 3-[4,5-dimetiltiazol-2-il]2,5-difenil-tetrazolijeva bromida (MTT) u obojeni formazan produkt u živim stanicama, na|eno je da resveratrol inhibira preživljavanje transformiranih makrofaga miša, ljudskih tumorskih T-stanica i humanih stanica epidermoidnog karcinoma u ovisnosti o koncentraciji, a učinak opada redom: T-stanice (LC50 ~27 µmol L-1, 24 h; ~9 µmol L-1; 48 h) > makrofazi (LC50 ~29 µmol L-1, 24 h; 39 µmol L-1, 48 h) >stanice kože (LC50 ~91 µmol L-1, 24 h; ~66 µmol L-1, 48 h). Paradoksalno, pri visokoj koncentraciji resveratrola (50 µmol L-1) inhibirana je proliferacija svih triju tipova stanica, a pri niskim koncentracijma (5 µmol L-1) stimulirana je proliferacija makrofaga. Preživljavanje makrofaga bilo je tako|er smanjeno primjenom piceatanola ovisno o koncentraciji. Stimulacija makrofaga zimosanom smanjila je citotoksičnost i resveratrola i piceatanola. Skenirajuća elektronska mikroskopija stanica tretiranih resveratrolom pokazala je promjene u morfologiji stanica, što je bilo u skladu s toksičnosti. U makrofazima i stanicama kože resveratrol (50 µmol L-1) inducirao je porast smanjenja razina glutationa ovisan o vremenu, ali nije mijenjao osnovne razine reaktivnih spojeva tiobarbiturne kiseline. Gledajući skupno, prikazani rezultati indiciraju da je resveratrol toksičan za uzgojene makrofage, T-stanice i stanice kože pri koncentracijama ≥25 µmol L-1 i da se citotoksičnost zbiva mehanizmom koji ne uključuje oksidativni stres. Nadalje, stupanj toksičnosti resveratrola i piceatanola prema makrofagima ovisi o aktivacijskom statusu tih stanica, pri čemu su stanice aktivirane zimosanom rezistentnije od nestimuliranih stanica

Analozi epselena smanjuju toksičnost 2-kloroetil etilnog sulfida u A-431-stanicama

Vesicants are potent blistering agents. The prototype vesicant is sulphur mustard gas, first used in World War I, which still has no effective antidote. We used a mustard gas surrogate 2-chloroethyl ethyl sulphide (CEES) to study the ability of resveratrol (RES) and pterostilbene (PTS), two well-established stilbene antioxidants, ebselen (EB-1), an organoselenium compound, and three EB-1 analogues (EB-2, EB-3, and EB-4) to reduce CEES toxicity in human epidermoid carcinoma cells (A-431). Following a 24-hour incubation of a toxic concentration of CEES (1000 μmol L-1), we used the MTT [3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide] test to analyse cell viability. Different concentrations of test antioxidants alone (15 μmol L-1, 30 μmol L-1 or 60 μmol L-1) did not decrease cell viability. Treatment with CEES and test antioxidants for 24 h showed that only EB-1 and its analogues EB-2, EB-3, and EB-4 but not the stilbene compounds could rescue the cells from death. EB-1 and EB-4 were the most effective at reducing CEES cytotoxicity and did so in a concentration-dependent manner, while EB-2 and EB-3 demonstrated the least protective effect. In summary, the data described herein indicate that organoselenium antioxidants, especially EB-4, may prove useful as countermeasures to blistering agents.Plikavci izazivaju izražene mjehuriće na koži. Najpoznatiji je svakako sumporni iperit, koji se prvi put uporabio u 1. svjetskom ratu i do današnjega dana nema djelotvornog protuotrova. U ispitivanju smo rabili zamjenu za iperit, 2-kloroetil etilni sulfi d (CEES) da bismo testirali sposobnost resveratrola (RES) i pterostilbena (PTS), dvaju poznatih stilbenskih antioksidansa, organoselenijeva spoja epselena (EB-1) te njegovih triju analoga (EB-2, EB-3 i EB-4) da smanji toksičnost CEES-a u humanih stanica epidermoidnog karcinoma (A-431). Vijabilnost stanica testirali smo s pomoću 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazol bromida (MTT) nakon 24-satne inkubacije s toksičnom koncentracijom CEES-a (1000 μmol L-1). Antioksidansi davani sami u različitim koncentracijama (15 μmol L-1, 30 μmol L-1, odnosno 60 μmol L-1) nisu smanjili vijabilnost stanica. Dvadesetčetverosatna primjena CEES-a i testiranih antioksidansa pokazala je da samo EB-1 i njegovi analozi EB-2, EB-3 i EB-4 mogu spriječiti smrt stanica, ali ne i stilbenski spojevi. EB-1 i EB-4 pokazali su se najdjelotvornijima u ublažavanju toksičnosti CEES-a u skladu s koncentracijom, dok su se EB-2 i EB-3 pokazali najmanje djelotvornima. Ovdje prikazani podaci upućuju na to da organoselenijevi antioksidansi, a napose EB-4, mogu biti korisni protuotrovi plikavcima

Assessment of the time-dependent dermatotoxicity of mechlorethamine using the mouse ear vesicant model

Mechlorethamine (HN2) is an alkylating agent and sulfur mustard gas mimetic which is also used in anticancer therapy. HN2 is associated with skin inflammation and blistering which can lead to secondary infections. The purpose of the present study was to investigate the time-dependent dermatotoxicity of HN2 using the mouse ear vesicant model (MEVM). To this end, our operational definition of dermatotoxicity included tissue responses to HN2 consistent with an increase in the wet weights of mouse ear punch biopsies, an increase in the morphometric thickness of H&E stained ear sections and histopathologic observations including tissue edema, inflammatory cell infiltration and vesication. The ears of male Swiss Webster mice were topically exposed to a single dose of HN2 (0.5 µmol/ear) or DMSO vehicle (5 µl/ear) or left untreated (naive). Mice were then euthanized at 15 min, 1, 2, 4, 8 or 24 hr following HN2 exposure. Compared to control ears, mouse ears exposed to HN2 at all time points showed an increase in wet weights, morphometric thickness, edema, inflammatory cell infiltration and signs of vesication. The incidence in tissue vesication sharply increased between 4 and 8 hr after exposure, revealing that tissue vesication is well established by 8 hr and remains elevated at 24 hr after exposure. It is noteworthy that, compared to control ears, mouse ears treated with DMSO vehicle alone also exhibited an increase in wet weights and morphometric thickness at 15 min, 1, 2 and 4 hr following treatment; however, these vehicle effects begin to subside after 4 hr. The results obtained here using the MEVM provide a more holistic understanding of the kinetics of vesication, and indicate that time points earlier than 24 hr may prove useful not only for investigating the complex mechanisms involved in vesication but also for assessing the effects of vesicant countermeasures

Minimal amidine structure for inhibition of nitric oxide biosynthesis

Pharmacological modulation of nitric oxide synthase activity has been achieved using structural analogs of arginine. In the present studies, we demonstrated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cultures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a concentration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expressing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidine had no effect on gamma-interferon-induced expression of nitric oxide synthase protein, its enzymatic activity was blocked. Kinetic analysis revealed that formamidine acts as a simple competitive inhibitor with respect to arginine (K(i) formamidine approximately 800 microM). Using a polarographic microsensor to measure real-time flux of nitric oxide release from RAW 264.7 macrophages, formamidine was found to require 30-90 min to inhibit enzyme activity, suggesting that cellular uptake of the drug may limit its biological activity. Our data indicate that formamidine is an effective inhibitor of nitric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased production of nitric oxid