Comparison of dental students' performances and perceptions in preclinical and clinical pharmacology in an Irish Dental School.

INTRODUCTION: Knowledge of pharmacology is essential for dental students and for its safe application in the clinical environment. AIMS: The goals of our study were to assess dental students' performances in pharmacology with and without shared instruction, to investigate correlations between students' final grades in pre-clinical and clinical pharmacology, to determine if gender affects test performance and to explore characteristics of effective dental student learning. METHODS: A 9 year mixed method study was undertaken comprising (i) quantitative analysis of undergraduate performances in basic and clinical pharmacology (n = 320) and (ii) quantitative and qualitative exploration of student perceptions on teaching and learning in pharmacology. RESULTS: Mean basic pharmacology scores were not significantly different when dental students were co-taught with medical and pharmacy students. Regression analysis showed a statistically significant correlation (r = 0.582, P < 0.01) between basic and clinical pharmacology dental student scores. Interestingly, correlation was independent of gender with female student scores demonstrating a correlation of r = 0.480, P < 0.01 and males a correlation of 0.684, P < 0.01. Quantitative and qualitative feedback highlighted four thematic areas of effective dental student learning namely: (i) quality of instructors, (ii) lecture content, (iii) assessment type and (iv) learning environment. CONCLUSIONS: Teaching basic pharmacology in a multidisciplinary environment did not adversely affect dental students' examination performances. Dental students who perform well in basic pharmacology perform similarly well in clinical pharmacology. However, whether students' understanding and the application of pharmacology affects the quality of patient care in the clinical environment awaits further investigation.

Protein kinase C isozymes, novel phorbol ester receptors and cancer chemotherapy.

Recent years have seen extensive growth in the understanding of the role(s) of the various PKC isozymes and novel receptors for the phorbol ester tumor promoters. The PKC family of serine-threonine kinases is an important regulator of signaling cascades that control cell proliferation and death, and therefore represent targets for cancer therapy. While past interests have focused on PKC-selective inhibitors, more recently, intensive research has been underway for selective activators and inhibitors for each individual PKC isozyme. In the past few years a large number of PKC activators and inhibitors with potential as anticancer agents have been developed. A number of these compounds are already in Phase II clinical testing. As a new generation of cancer chemotherapeutic agents are designed, developed and put through a series of rigorous clinical trials, we can anticipate achieving exquisite control over PKC-mediated regulatory pathways, leading ultimately to a greater understanding of different cancers.

Constitutive ERK1/2 activation in esophagogastric rib bone marrow micrometastatic cells is MEK-independent.

In this study, we examined the mitogen-activated protein kinase (MAPK) cascade in micrometastatic cell lines generated from rib bone marrow (RBM) of patients undergoing resection of esophagogastric malignancies. The molecular mechanism(s) involved in esophagogastric MAPK activation have not previously been investigated. Constitutive activation of both ERK1 and -2 isoforms was evident in each of the five RBM cell lines. Elk-1, a transcription factor activated by the ERK1/2 pathway was also found to be constitutively activated. Cell lines generated from metastases of involved lymph nodes (OC2) and ascites (OC1) of patients with esophageal cancer do not display, however, hyperphosphorylation of ERK1/2. Constitutive RBM ERK1/2 activation is protein kinase C and phosphatidylinositol 3-kinase dependent. Surprisingly, constitutive ERK1/2 activation is MEK-independent. Pharmacological inhibition of MEK with two specific inhibitors, PD 98059 and U0126, were both ineffective in blocking ERK activation. Similarly, the use of a dominant negative MEK mutant was without effect. Interestingly, experiments overexpressing two different dominant negative Pak1 mutants significantly reduced RBM ERK1/2 activation, albeit not to the same extent for all cell lines. We also examined the role of three different phosphatases, PAC1, MKP-1, and -2. While RBM ERK1/2 activation was found to be PAC1- and MKP-2-independent, surprisingly, MKP-1 was down-regulated in all five RBM cell lines. In conclusion, we provide evidence for the first time for a MEK-independent constitutive ERK1/2 activation pathway in esophagogastric RBM cell lines. These findings have important implications for drug treatment strategies which currently target MEK in other forms of cancer.

Effects of vitamin E on lipid peroxidation in healthy persons.

CONTEXT: Oxidative stress may play a role in the development or exacerbation of many common diseases. However, results of prospective controlled trials of the effects of antioxidants such as vitamin E are contradictory. OBJECTIVE: To assess the effects of supplemental vitamin E on lipid peroxidation in vivo in healthy adults. DESIGN: Randomized, double-blind, placebo-controlled trial conducted March 1999 to June 2000. SETTING: A general clinical research center in a tertiary referral academic medical center. PARTICIPANTS: Thirty healthy men and women aged 18 to 60 years. INTERVENTIONS: Participants were randomly assigned to receive placebo or alpha-tocopherol dosages of 200, 400, 800, 1200, or 2000 IU/d for 8 weeks (n = 5 in each group), followed by an 8-week washout period. MAIN OUTCOME MEASURES: Three indices of lipid peroxidation, urinary 4-hydroxynonenal (4-HNE) and 2 isoprostanes, iPF(2alpha)-III and iPF(2alpha)-VI, measured by gas chromatography/mass spectrometry and compared among the 6 groups at baseline, 2, 4, 6, and 8 weeks, and 1, 3, and 8 weeks after discontinuation. RESULTS: Circulating vitamin E levels increased in a dose-dependent manner during the study. No significant effect of vitamin E on levels of urinary 4-HNE or either isoprostane was observed. Mean (SEM) baseline vs week 8 levels of iPF(2alpha)-III were 154 (20.1) vs 168 (22.3) pg/mg of creatinine for subjects taking placebo; 165 (19.6) vs 234 (30.1) pg/mg for those taking 200 IU/d of vitamin E; and 195 (26.7) vs 213 (40.6) pg/mg for subjects taking 2000 IU/d. Corresponding iPF(2alpha)-VI levels were 1.43 (0.6) vs 1.62 (0.4) ng/mg of creatinine for subjects taking placebo; 1.64 (0.3) vs 1.24 (0.8) ng/mg for those taking 200 IU/d of vitamin E; and 1.83 (0.3) vs 1.94 (0.9) ng/mg for those taking 2000 IU/d. Baseline vs week 8 levels of 4-HNE were 0.5 (0.04) vs 0.4 (0.05) ng/mg of creatinine for subjects taking placebo; 0.4 (0.06) vs 0.5 (0.02) ng/mg with 200 IU/d of vitamin E; and 0.2 (0.02) vs 0.2 (0.1) ng/mg with 2000 IU/d. CONCLUSIONS: Our results question the rationale for vitamin E supplementation in healthy individuals. Specific quantitative indices of oxidative stress in vivo should be considered as entry criteria and for dose selection in clinical trials of antioxidant drugs and vitamins in human disease.

Alcohol-induced generation of lipid peroxidation products in humans.

To address the hypothesis that elevated blood alcohol increases systemic oxidant stress, we measured urinary excretion of isoprostanes (iPs), free radical-catalyzed products of arachidonic acid. Ten healthy volunteers received acute doses of alcohol (Everclear-R) or placebo under randomized, controlled, double-blind conditions. Urinary iPF2a-III increased in a time- and dosage-dependent manner after dosing with alcohol, with the peak urinary iPF2a-III excretion correlating with the rise in blood alcohol. To determine whether oxidant stress was associated with alcohol-induced liver disease (ALD), we then studied the excretion of iP in individuals with a documented history of alcohol-induced hepatitis or alcohol-induced chronic liver disease (AC). Both urinary iPF2a-III and urinary iPF2a-VI were markedly increased in patients with acute alcoholic hepatitis. In general, urinary iPF2a-III was significantly elevated in cirrhotic patients, relative to controls, but excretion was more pronounced when cirrhosis was induced by alcohol than by hepatitis C. Excretion of iPF2a-VI, as well as 4-hydroxynonenal and the iPF2a-III metabolite, 2,3-dinor-5, 6-dihydro-iPF2a-III, was also increased in AC. Vitamin C, but not aspirin, reduced urinary iPs in AC. Thus, vasoactive iPs, which serve as indices of oxidant stress, are elevated in the urine in both acute and chronic ALD. Increased generation of iPs by alcohol in healthy volunteers is consistent with the hypothesis that oxidant stress precedes and contributes to the evolution of ALD.

Iron-dependent human platelet activation and hydroxyl radical formation: involvement of protein kinase C.

BACKGROUND: Iron is an important modulator of lipid peroxidation, and its levels have been associated with the progression of atherosclerosis. Little is known about the possibility that this metal, when released from tissue stores, may modulate the reactivity of blood cell components, in particular platelets. Therefore, we investigated a possible link between iron, oxygen free radical formation, and platelet function. METHODS AND RESULTS: Human whole blood was stimulated with collagen 2 micrograms/mL, and an irreversible aggregation with thromboxane (Tx)B2 formation was observed (15+/-4 versus 130+/-10 ng/mL). Deferoxamine (DSF), a specific iron chelator, and catalase, an H2O2 scavenger, inhibited collagen-induced whole-blood aggregation. The aggregation was accompanied by an increase in hydroxyl radical (OH.) levels (30+/-8 versus 205+/-20 nmol/L dihydroxybenzoates), which were reduced by DSF and by 2 specific OH. scavengers, mannitol and deoxyribose. Iron (Fe2+) dose-dependently induced platelet aggregation, TxB2 formation (6+/-2 versus 135+/-8 ng/mL), and protein kinase C (PKC) translocation from the cytosol to the cell membrane when added to platelets that have been primed with a low concentration of collagen (0.2 micrograms/mL). In the same system, an increase in OH. levels was observed (37+/-12 versus 230+/-20 nmol/L dihydroxybenzoates). Mannitol and deoxyribose, but not urea, were able to reduce OH. formation, PKC activation, and platelet aggregation. Selective inhibition of PKC activity by GF 109203X prevented iron-dependent platelet aggregation without influencing OH. production. CONCLUSIONS: The present study shows that iron can directly interact with human platelets, resulting in their activation. Its action is mediated by OH. formation and involves PKC activity. Our findings provide an additional contribution to the understanding of the mechanism(s) by which iron overload might promote atherosclerosis and coronary artery disease.

Arachidonic acid in platelet microparticles up-regulates cyclooxygenase-2-dependent prostaglandin formation via a protein kinase C/mitogen-activated protein kinase-dependent pathway.

Activation of platelets results in shedding of membrane microparticles (MP) with potentially bioactive properties. Platelet MP modulate platelet, monocyte, and vascular endothelial cell function, both by direct effects of MP arachidonic acid (AA) and by its metabolism to bioactive prostanoids. We have previously reported that platelet MP induce expression of cyclooxygenase (COX)-2 and prostacyclin production in monocytes and endothelial cells. To elucidate further the molecular mechanisms that underlie MP-induced up-regulation of COX-2 expression, we investigated the response of a human monocytoid (U-937) cell line to platelet MP stimulation. In U-937 cells, MP-induced COX-2 expression and eicosanoid formation is prevented by pharmacological inhibitors of protein kinase C (PKC), PI 3-kinase, mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, and p38 kinase. Treatment with the PI 3-kinase inhibitors wortmannin and LY294002 also blocked MP-induced p42/p44 MAPK, p38, and JNK1 phosphorylation. Conversely, platelet MP stimulation of U-937 cells results in direct activation of PKC, p42/p44 MAPK, p38 kinase, and c-Jun N-terminal kinase (JNK) as well as activation of the transcription factors c-Jun and Elk-1. However, MP failed to activate the cAMP response element. Activation of U-937 cells by MP induces translocation of classical (PKCbeta), novel (PKCdelta) and atypical (PKCzeta and PKClambda) isozymes of PKC from the cytosol to the membrane, with concomitant activation of downstream MAPK. While MP-induced activation of p42/p44 MAPK and p38 kinase is transient, a sustained activation of JNK1 was observed. Although PKC activation is required for MP-induced p42/p44 MAPK, activation of the stress kinases p38 and JNK1 was PKC-independent. The fatty acid fraction of the MP accounted for these effects, which were mimicked by MP AA. Rather than acting directly via nuclear receptors, MP AA activates COX-2-dependent prostaglandin production by a PKC/p42/p44 MAPK/p38 kinase-sensitive pathway in which PI 3-kinase plays a significant role. MP AA also stimulates transcriptional activation of COX-2 as well as c-Jun and Elk-1.

Modulation of monocyte-endothelial cell interactions by platelet microparticles.

Platelets, activated by various agonists, produce microparticles (MP) from the plasma membrane, which are released into the extracellular space. Although the mechanism of MP formation has been clarified, their biological importance remains ill defined. We have recently shown that platelet-derived MP influence platelet and endothelial cell function. In this study, we have further examined the mechanism of cellular activation by platelet MP. To address the possibility that they may influence monocyte-endothelial interactions, we used an in vitro assay to examine their effects on the adhesion of monocytes to human umbilical vein endothelial cells (HUVEC). Platelet MP increased the adhesion of monocytes to HUVEC in a time- and dose-dependent manner. Maximal adhesion of monocytes to resting HUVEC was observed after 24 h of stimulation with MP. Similar kinetics were observed with U-937 (human promonocytic leukemia) cells, used as a model for the blood-borne monocyte. Maximal adhesion of resting monocytes to MP-stimulated HUVEC was observed after 5 h of stimulation with MP. The EC50s for MP-induced increases in HUVEC, monocyte, and U-937 cell adhesion is 8.74, 43.41, and 10.83 microg/ml of MP protein, respectively. The induction of monocyte-endothelial adhesion was mimicked by arachidonic acid isolated from MP. The observed increased cellular adhesiveness correlated with MP-induced upregulation of cell adhesion molecules. MP-stimulated HUVEC increased intracellular cell adhesion molecule-1 (ICAM-1) but not vascular cell adhesion molecule-1 (VCAM-1), P-, or E-selectin expression. Monocyte and U-937 lymphocyte function-associated antigen-1 (CD11a/CD18) and macrophage antigen-1 (CD11b/ CD18, alpham/beta2) were both upregulated upon MP stimulation, but an increase in p150,95 (CD11c/CD18), very late antigen-1, or ICAM-1 expression was not observed. The functional importance of these changes was demonstrated with blocking antibodies. MP also induced the chemotaxis of U-937 cells in a dose-dependent manner with an EC50 of 4.40 microg/ml of MP protein. Similarly, arachidonic acid isolated from MP mimicked the chemotactic response. A role for PKC was implicated in both adhesion and chemotaxis. GF 109203X, a specific inhibitor of PKC, significantly reduced monocyte-endothelial adhesion, as well as U-937 chemotaxis. The demonstration that platelet MP may modulate important aspects of endothelial and monocyte function provides a novel mechanism by which platelets may interact with such cells in human atherosclerosis and inflammation.

IPF2alpha-I: an index of lipid peroxidation in humans.

Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2alpha, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product, prostaglandin F2alpha (PGF2alpha), has exhibited promise as an index of oxidant stress in vivo. We have developed a quantitative method to measure isoprostane F2alpha-I, (IPF2alpha-I) a class I isomer (8-iso-PGF2alpha is class IV), using gas chromatography/mass spectrometry. IPF2alpha-I is severalfold as abundant in human urine as 8-iso-PGF2alpha, with mean values of 737 +/- 20.6 pg/mg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper in vitro. However, IPF2alpha-I, unlike 8-iso-PGF2alpha, is not formed in a COX-dependent manner by platelets activated by thrombin or collagen in vitro. Similarly, COX inhibition in vivo has no effect on IPF2alpha-I. Neither serum IPF2alpha-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF2alpha-I, an index of actual generation in vivo, is depressed by aspirin or indomethacin. In contrast, both serum thromboxane B2 and urinary excretion of its 11-dehydro metabolite are depressed by the COX inhibitors. Although serum 8-iso-PGF2alpha formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF2alpha-I is elevated in cigarette smokers compared with controls (1525 +/- 180 versus 740 +/- 40 pg/mg creatinine; P < 0.01) and is highly correlated with urinary 8-iso-PGF2alpha (r = 0.9; P < 0.001). Urinary IPF2alpha-I is a novel index of lipid peroxidation in vivo, which can be measured with precision and sensitivity. It is an abundant F2-isoprostane formed in a free radical- but not COX-dependent manner. Although 8-iso-PGF2alpha may be formed as a minor product of COX, this pathway contributes trivially, if at all, to levels in urine. Urinary excretion of both isoprostanes is elevated in cigarette smokers.

Transcellular activation of platelets and endothelial cells by bioactive lipids in platelet microparticles.

Microparticles are released during platelet activation in vitro and have been detected in vivo in syndromes of platelet activation. They have been reported to express both pro- and anticoagulant activities. Nevertheless, their functional significance has remained unresolved. To address the mechanism(s) of cellular activation by platelet microparticles, we examined their effects on platelets and endothelial cells. Activation of human platelets by diverse stimuli (thrombin, 0.1 U/ml; collagen, 4 microg/ml; and the calcium ionophore A23187, 1 microM) results in shedding of microparticles. Pretreatment of these particles, but not membrane fractions from resting platelets, with (s)PLA2 evokes a dose-dependent increase in platelet aggregation, intracellular [Ca2+] movement, and inositol phosphate formation. These effects localize to the arachidonic acid fraction of the microparticles and are mimicked by arachidonic acid isolated from them. However, platelet activation requires prior metabolism of microparticle arachidonic acid to thromboxane A2. Thus, pretreatment of platelets with the cyclooxygenase (COX) inhibitor, indomethacin (20 microM), the thromboxane antagonist SQ29,548 (1 microM), or the protein kinase C inhibitor GF109203X (5 microM) prevents platelet activation by microparticles. However, platelet microparticles fail to evoke an inositol phosphate response directly, via either of the cloned thromboxane receptor isoforms stably expressed in human embryonic kidney (HEK) 293 cells. Prelabeling platelets with [2H(8)] arachidonate was used to demonstrate platelet metabolism of the microparticle-derived substrate to thromboxane. Platelet microparticles can also induce expression of COX-2 and prostacyclin (PGI2) production, but not expression of COX-1, in human endothelial cells. These effects are prevented by pretreatment with actinomycin D (12 microM) or cycloheximide (5 microg/ml). Expression of COX-2 is again induced by the microparticle arachidonate fraction, which it may then use to synthesize PGI2. Both PGE2 and iloprost, a stable PGI2 analog, evoke human umbilical vein endothelial cell COX-2 expression, albeit with kinetics that differ from the response to platelet microparticles. These studies indicate a novel mechanism of transcellular lipid metabolism whereby platelet activation may be amplified or modulated by concentrated delivery of arachidonic acid to adjacent platelets and endothelial cells.