Free radicals and related reactive species as mediators of tissue injury and disease: implications for Health.

A radical is any molecule that contains one or more unpaired electrons. Radicals are normal products of many metabolic pathways. Some exist in a controlled (caged) form as they perform essential functions. Others exist in a free form and interact with various tissue components. Such interactions can cause both acute and chronic dysfunction, but can also provide essential control of redox regulated signaling pathways. The potential roles of endogenous or xenobiotic-derived free radicals in several human pathologies have stimulated extensive research linking the toxicity of numerous xenobiotics and disease processes to a free radical mechanism. In recent years, improvements in analytical methodologies, as well as the realization that subtle effects induced by free radicals and oxidants are important in modulating cellular signaling, have greatly improved our understanding of the roles of these reactive species in toxic mechanisms and disease processes. However, because free radical-mediated changes are pervasive, and a consequence as well as a cause of injury, whether such species are a major cause of tissue injury and human disease remains unclear. This concern is supported by the fact that the bulk of antioxidant defenses are enzymatic and the findings of numerous studies showing that exogenously administered small molecule antioxidants are unable to affect the course of most toxicities and diseases purported to have a free radical mechanism. This review discusses cellular sources of various radical species and their reactions with vital cellular constituents, and provides examples of selected disease processes that may have a free radical component.

Lipocalin-2: pro- or anti-apoptotic?

Survival and apoptosis signaling pathways are altered concomitantly in response to numerous endogenous and exogenous stressors. The lipocalin family of small soluble proteins has been implicated in modulating apoptosis. However, the overall effect of these proteins has been variable, showing both pro- and anti-apoptotic activities. The goal of this minireview is to summarize the studies on lipocalins and apoptosis and consider what roles lipocalin-2 may play in cell death and survival.

Cytotoxicity of a non-cyclooxygenase-2 inhibitory derivative of celecoxib in non-small-cell lung cancer A549 cells.

Lung cancer is one of the most common causes of cancer death worldwide. Although recent advances in chemotherapy and radiation therapy have yielded modest improvements in patient outcomes, overall survival remains poor. Therefore, new therapeutic targets are needed. Phosphoinositide-dependent kinase-1 (PDK1) is one potential target. The aim of the present studies was to investigate the potential of a celecoxib-derived PDK1 inhibitor (OSU03013), that does not inhibit cyclooxygenase-2, to kill lung cancer cells in vitro. Using human non-small-cell lung cancer A549 cells, OSU03013 dose-dependently induced apoptosis. After 6 h of treatment with 7.5 microM OSU03013, 26% of the cells were apoptotic, compared to 4% of the control cells as determined by measuring the sub-G1 peak of propidium iodide stained cells with flow cytometry. A similar increase in apoptosis was evident using the Cell Death ELISA assay. OSU03013-induced apoptosis was accompanied by a reduction in the mitochondrial membrane potential, the release of cytochrome c and the cleavage of caspase-3. Surprisingly, the phosphorylation of Akt at serine 473 was increased in A549 cells treated with 7.5 microM OSU03013. However, the toxicity of OSU03013 was reduced in A549 cells expressing a constitutively active form of Akt. These data demonstrate that OSU03013 induces apoptosis in A549 cells via the mitochondrial pathway. Inhibition of the Akt pathway appears uninvolved in this toxicity, although Akt can provide protection. These results also suggest the potential of celecoxib-derived agents to treat some forms of lung cancer.

Neutrophil gelatinase-associated lipocalin as a survival factor.

NGAL (human neutrophil gelatinase-associated lipocalin) and its mouse analogue 24p3 are members of the lipocalin family of small secreted proteins. These proteins are up-regulated in a number of pathological conditions, including cancers, and may function as transporters of essential factors. Although previous publications have suggested that 24p3 has pro-apoptotic functions, other data are more suggestive of a survival function. The current study was designed to determine whether NGAL is pro- or anti-apoptotic. Apoptosis induced in human adenocarcinoma A549 cells by the 5-lipoxygenase-activating-protein inhibitor MK886, or several celecoxib-derived PDK1 (phosphoinositide-dependent kinase 1) inhibitors that are devoid of cyclo-oxygenase-2 inhibitory activity, was accompanied by a dose- and time-dependent increase of NGAL mRNA levels, as was reported previously with 24p3. A similar induction of NGAL mRNA was observed in human breast cancer MCF7 cells treated with MK886, indicating this was not a cell-specific effect. Treatment of A549 cells with up to 150 mug/10(6) cells of purified recombinant NGAL protein had no effect on viability, whereas antisera against the full-length NGAL protein induced apoptosis in these cells. The stable overexpression of NGAL in A549 cells had no effect on proliferation or viability. However, the cell death induced by a PDK1 inhibitor was reduced by 50% in NGAL-overexpressing cells. Decreasing NGAL mRNA and protein expression with siRNA (small interfering RNA) in A549 cells increased the toxicity of a PDK1 inhibitor by approx. 45%. These data indicate that, although the induction of NGAL correlates with apoptosis, this induction represents a survival response. Because NGAL is a secreted protein, it may play an extracellular role in cell defence against toxicants and/or facilitate the survival of the remaining cells.

The Role of Pharmacists and Pharmacy Education in Point-of-Care Testing.

Point-of-care testing (POCT) is defined as laboratory testing conducted close to the site of patient care. Although performed originally primarily by clinical staff for acute conditions, recent advances in technology have made such testing possible for disease screening and prevention across a wide range of conditions in virtually any setting, and often by individuals with little or no training. With the ongoing evolution in POCT, numerous concerns have arisen about the quality and accuracy of the tests, comparability between multiple tests for the same endpoint, interpretation of test results, and whether and how results should be used for therapeutic decisions and included in a patient's medical record. The pharmacist is well-positioned to manage and interpret POCT performed outside of the usual clinical settings. However, educational and regulatory changes are needed to enable pharmacists to take on this emerging activity effectively.

Free radicals and apoptosis: relationships with glutathione, thioredoxin, and the BCL family of proteins.

Cellular fate is controlled by a number of factors within the cell, including an abundance of, and defenses against, free radicals generated both endogenously and exogenously. Free radical species are involved in regulating various growth, differentiation and death processes including apoptosis. Apoptosis is a preferred form of cell death because it is highly ordered resulting in the death of a cell with minimal effects on surrounding cells or tissues. Radicals generated during apoptosis directly modulate signaling cascades by activating or inhibiting survival transcription factors (i.e. NF-kappa B and AP-1), or more indirectly affecting such signaling by changing the cellular redox status [i.e. glutathione (GSH) and thioredoxin (Trx)]. At high levels, free radicals, including reactive oxygen species and various unwanted and harmful byproducts of reactions with tissue macromolecules, particularly lipids, can cause acute injury if not hindered by cellular antioxidants. These antioxidant protective systems are not only involved in preventing stress, but also maintaining the normal functioning of specific transcription factors and the bcl proteins. This review will discuss the association of reactive oxygen species with GSH, Trx and bcl proteins in apoptosis.

Dissociation of oxidant production by peroxisome proliferator-activated receptor ligands from cell death in human cell lines.

Ligands of peroxisome proliferator-activated receptors (PPARs) come from a diverse group of chemicals that include pharmaceutical drugs, phthalate plasticizers, steroids, and pesticides. PPAR ligands exhibit a number of effects, including an ability to induce apoptosis in some systems. The mechanism(s) underlying the induction of apoptosis is not known. The current study examined the ability of Wy14643, a fibrate and PPARalpha agonist, and ciglitazone, a thiazolidinedione and PPARgamma agonist, to induce apoptosis as well as the production of oxidants in human Jurkat T cells that express all PPAR isoforms. Treatment with increasing doses of Wy14643 caused a substantial time-dependent increase in the overall oxidant status (as reflected by increased dichlorofluorescein fluorescence) of Jurkat cells without any change in viability except at the highest dose and longest time. Ciglitazone also caused a dose- and time-dependent increase in oxidant production. However, although the extent of this production was less than that seen with Wy14643, ciglitazone caused a dose- and time-dependent increase in apoptosis that could not be inhibited by antioxidants. Confocal micrographs of Jurkat cells loaded with dichlorofluorescein diacetate or dihydrorhodamine 123 and treated with Wy14643 or ciglitazone revealed a punctate pattern of fluorescence at early time points suggestive of a mitochondrial origin for these oxidants. Rotenone and antimycin A prevented Wy14643- but not ciglitazone-induced oxidant production. Other relatively specific PPARgamma agonists (15delta-PGJ2, and troglitazone), but not nonspecific agonists (bezafibrate and conjugated linoleic acid), were also able to induce oxidant production in Jurkat cells. These data, as well as the findings that oxidant production could be induced by Wy14643 in A549 cells that lack PPARalpha, and could not be blocked in Jurkat cells by the PPARalpha inhibitor MK886, indicate oxidant formation is unrelated to PPARalpha. These data also suggest that oxidant production induced by PPARalpha ligands originates in the mitochondria.

Thioredoxin and its role in toxicology.

Thioredoxins (Trx) are members of an evolutionarily conserved family of redox-active proteins containing a conserved active site dithiol motif. Trx supports diverse reduction reactions, including several of direct toxicologic interest, but relatively little information is available concerning the roles of Trx under specific toxicologic conditions. Accumulating evidence suggests that Trx serves a partially overlapping and highly complementary role to the glutathione (GSH) system in protecting against toxicity. GSH and Trx both function in the reduction of peroxides through the action of multiple GSH peroxidases and Trx peroxidases (peroxiredoxins), respectively. However, GSH is a small molecule that is present at millimolar concentrations, thereby providing a potential mechanism for elimination of alkylating electrophiles. In contrast, even though Trx is only present at micromolar or submicromolar concentrations, its dithiol motif makes it suited to reverse oxidative changes to proteins, including reduction of protein disulfides, methioninyl sulfoxides, and cysteinyl sulfenic acids. Moreover, Trx functions in redox-sensitive signal transduction, transcriptional activation of stress response genes, ribonucleotide reduction in synthesis of deoxyribonucleotides for DNA repair, and post-injury cell proliferation. Molecular studies show that the predominant cytoplasmic/nuclear form, Trx-1, and the mitochondrial form, Trx-2, both protect against oxidative stress, that both are essential for embryonic development, and that Trx-1 is inducible in response to oxidative stress. Because of the differences between GSH and Trx in distribution, catalytic activities and reactivities with electrophiles, particularly with the important role to be played by glutathione S-transferases, considerable research is needed to clarify their respective roles in protection against specific toxicologic conditions.

Effect of acrolein and glutathione depleting agents on thioredoxin.

Acrolein is a widespread environmental pollutant that reacts rapidly with nucleophiles, especially cellular thiols. In addition to glutathione (GSH), thioredoxin (Trx) and thioredoxin reductase (TR) contain thiol groups and may react with electrophiles. In the present study, A549 cells treated with 5-25 microM acrolein for 30 min lost cellular Trx activity in a dose-dependent fashion. Over 90% of Trx activity was lost at concentrations of 25 microM or greater. In contrast, Trx protein content, as assessed by western blotting, was not altered immediately after the 30 min acrolein treatment. Both Trx activity and protein levels increased 4h after the acrolein treatment. However, Trx activity remained below control levels at 24h. A similar dose-response relationship was seen with TR in A549 cells exposed to acrolein. There was, however, a rapid recovery of TR activity such that it attained normal levels by 4h after doses < or = 75 microM acrolein. Diethyl maleate (DEM), a common but not highly specific, agent used to deplete GSH, also inactivated Trx. A 2 h exposure of A549 cells to 1 mM DEM depleted cellular GSH by ~50% and diminished Trx activity by over 67%. Lower DEM doses (0.125 mM and 0.25 mM) for 1h had no significant effect on GSH but significantly decreased Trx activity 12 and 23%, respectively. Similar to immediately after acrolein exposure, DEM did not affect Trx protein levels. A Trx-1-GFP fusion protein was transfected into A549 cells. While the fusion protein was expressed, the Trx component was inactive by the insulin reducing assay. In summary, Trx and TR are inactivated by acrolein. In addition, the GSH depleting agent DEM inactivates Trx somewhat more effectively than it depletes GSH. The Trx-1-GFP fusion protein, while readily expressed, appears to have little or no activity, perhaps because the small size of Trx-1 (12 kDa) is affected by the larger GFP.

Acrolein-induced cell death: a caspase-influenced decision between apoptosis and oncosis/necrosis.

Due to the dominating roles that caspases play in the apoptotic cascade, their activities appear to be a primary factor in the death pathway (apoptosis versus oncosis/necrosis) decision. In murine FL5.12 proB lymphocytes, the cellular consequences of acrolein treatment included a lack of typical apoptotic features in preference to oncosis/necrosis. Oncosis/necrosis was apparent by detection of a reduction in intracellular ATP concentration, increased plasma membrane leakage (measured by LDH release and flow cytometric detection of propidium iodide uptake) and morphological criteria. Analysis of acrolein-treated cell lysates or recombinant caspase enzymes showed overall dose-dependent decreases in caspase-3, -8 and -9 activities. In addition to acrolein's effect on intracellular caspases, it was also able to alter caspase-dependent apoptosis induced by secondary treatment with etoposide or following cytokine withdrawal. Acrolein at doses > or =20 microM circumvented etoposide or interleukin-3 withdrawal induced apoptosis. When acrolein was combined with mechlorethamine, another alkylating agent not dependent on caspases for its cell death signaling, necrosis was increased in a dose-dependent manner. Overall, these data suggest that caspase inhibition plays an important role in the cell death pathway decision, particularly with treatments dependent on the caspase cascade to induce apoptosis.

Fatty acid release and oxidation are factors in lipoxygenase inhibitor-induced apoptosis.

MK886, an inhibitor of 5-lipoxygenase activating protein (FLAP), and the lipoxygenase (LOX) inhibitors baicalein and nordihydroguaiaretic acid (NDGA), induce apoptosis by mechanisms independent of both LOX and FLAP. One possible mechanism for these agents is through an effect on the binding of fatty acids to LOX and fatty acid binding proteins resulting in increased intracellular levels of unbound fatty acids, particularly arachidonic acid (AA), that in turn, activate apoptosis signaling pathways either directly or following oxidation. In FL5.12 murine pro-B lymphocytic cells, exogenous fatty acids induced apoptosis proportional to their degree of unsaturation. MK886, baicalein, and NDGA significantly enhanced the release of [3H]-AA two to threefold within 2 h and induced apoptosis by 8 h. Neither MK886-induced AA release, nor apoptosis were affected by quinacrine, a phospholipase A2 inhibitor. The presence of peroxides 1 h after treatment of FL5.12 cells with these agents was evident by a two to threefold increase in the ferrous oxidation-xylenol orange (FOX) assay as well as dichlorofluorescein fluorescence measured with flow cytometry. Isoprostane formation, an additional index of lipid peroxidation, was increased threefold by 2 h, and fourfold at 4 h after MK886 or baicalein, but not after NDGA. Antioxidants were able to protect against NDGA-induced apoptosis but had no effect on baicalein and resulted in enhanced apoptosis with MK886. These data support the hypothesis that release of fatty acids and generation of oxidized species contribute to apoptosis induced by these LOX inhibitors, but that more complex mechanisms are likely involved.

Advancing pharmacist scholarship and research within academic pharmacy.

An appropriate balance between teaching, scholarship, and service is important for a faculty member to have a satisfying and successful career. The relative emphasis on each area normally changes during the course of a career. Although some level of scholarly output is an ongoing and fundamental expectation of all faculty members, this activity is too often given low priority, particularly among faculty members in practice areas who may have a minimal background in research and large demands on their time for teaching and clinical service. Addressing this issue requires establishing a shared commitment between administrators and faculty members, as well as identifying or developing education programs that will ensure research competence for practice faculty members. This paper provides insights into the role that scholarship and research should have for all pharmacy faculty members and provides suggestions for how to better advance this critical component within academic pharmacy.

University-based continuing education for pharmacists.

University-based continuing education (CE) fulfills an important role to support the professional development of pharmacists, advance the practice of pharmacy, and contribute to societal needs for research and healthcare services. Opportunities for pharmacists to engage in new models of patient care are numerous worldwide, particularly as pharmacists' scope of practice has expanded. Approaches to CE have changed to address the changing needs of pharmacists and now include a variety of approaches to support development of knowledge and skills. There is emphasis on the learning process as well as the knowledge, with the introduction of the concept of continuing professional development (CPD).As institutions of research and education, universities are uniquely positioned to bridge the gap between academic and practice environments, providing opportunities for translation of knowledge to practice. The Faculty of Pharmacy and Pharmaceutical Sciences at the University of Alberta is a provider of CE in Alberta, Canada, where an expanded scope of pharmacy practice includes prescribing, administering injections, accessing electronic patient records, and ordering laboratory tests. In this paper, the Faculty offers views about future directions for CE, including the integration of CE with core faculty activities, expanding the audience for CE, areas of focus for learning, and partnerships. Finally, we hope to ignite dialogue with others in the profession about the role and function of university-based CE.

Cooperation in pharmacy education in Canada and the United States.

Although the education of student pharmacists and the practice of pharmacy in Canada have many similarities with that in the United States, there also are differences. The planning of curricula in pharmacy education is of particular importance to the advancement of pharmacy in Canada because of significant changes in the scope of practice in several provinces, and in how community pharmacy is reimbursed for the services it can, or should, provide. Greater dialog between Canadian and American pharmacists has the potential not only to impact practice on both sides of the border but also to improve collaborations among Canadian and American pharmacy educators. This article provides background information and some suggestions on how to build partnerships in pharmacy education between Canada and the United States. Consortia-like arrangements have some particular promise, as does engaging border-states and provinces in regional meetings and other activities. By working together, Canadian and US pharmacy educators have the opportunity to implement the best of what each has to offer and to devise new and better ways to educate future and existing pharmacists.

Factors influencing the pharmacy faculty workforce.

In 2005, the Council of Faculties and the Council of Deans within the American Association of Colleges of Pharmacy (AACP) formed a task force to review the status of the pharmacy faculty workforce and to identify factors that may influence the supply of and demand for pharmacy faculty members. This manuscript summarizes the Task Force on Faculty Workforce's findings and describes specific strategies needed to address the various issues facing the academy. Based on Task Force predictions, the academy will need approximately 1200 new faculty members over the next 10 years due to the creation of new pharmacy programs, the expansion of existing programs, faculty retirements, and recurring vacant faculty positions.

Thioredoxin-related mechanisms in hyperoxic lung injury in mice.

Reduction of glutathione disulfide (GSSG) to glutathione (GSH) by glutathione reductase (GR) enhances the efficiency of GSH-dependent antioxidant activities. However, GR-deficient (a1Neu) mice are less susceptible to acute lung injury from continuous exposure to > 95% O(2) (96 h: 6.9 +/- 0.1 g right lung/kg body versus room air 3.6 +/- 0.3) than are C3H/HeN control mice (10.6 +/- 1.3 versus 4.2 +/- 0.3, P < 0.001). a1Neu mice have greater hepatic thioredoxin (Trx)1 and Trx2 levels than do C3H/HeN mice, suggesting compensation for the absence of GR. a1Neu mice exposed to hyperoxia for 96 hours showed lower levels of inflammatory infiltrates in lungs than did similarly exposed C3H/HeN mice. Pretreatment with aurothioglucose (ATG), a thioredoxin reductase (TrxR) inhibitor, exacerbated the effects of hyperoxia on lung injury in a1Neu mice (11.6 +/- 0.8, P < 0.001), but attenuated hyperoxic lung edema and inflammation in C3H/HeN mice (6.3 +/- 0.4, P < 0.001). No consistent alterations were observed in lung GSH contents or liver GSH or GSSG levels after ATG pretreatment. The data suggest that modulation of Trx/TrxR systems might provide therapeutically useful alterations of cellular resistance to oxidant stresses. The protective effects of ATG against hyperoxic lung injury could prove to be particularly useful therapeutically.

Mechanisms of N-acetylcysteine-driven enhancement of MK886-induced apoptosis.

N-Acetylcysteine (NAC), besides being a precursor of glutathione, has an array of other effects including an ability to scavenge free radicals, modulate gene expression and signal transduction pathways, and regulate cell survival and apoptosis. At concentrations lower than 20 mmol/L, NAC is nontoxic to cultured cells and can protect against apoptosis induced by a number of agents. A few recent reports, however, have indicated that NAC can also increase apoptosis. MK886, a 5-lipoxygenase activating protein (FLAP) inhibitor, induces apoptosis in many cell lines by an unknown mechanism that is independent of FLAP and lipoxygenase activity but is possibly related to effects on kinases such as Akt. In Jurkat T lymphocytes, NAC pretreatment (10 mmol/L) enhanced MK886-induced apoptosis by 2.4-fold. Following NAC-MK886 treatment, there was a significant increase in caspase-3 activity, and a decrease in mitochondrial transmembrane potential compared to MK886 alone. However, the extent of cytochrome c release was comparable between MK886 alone and MK886-NAC treatments. The enhancement of MK886-induced apoptosis by 10 mmol/L NAC appears to be partly related to a decrease in pH caused by this concentration of NAC, because an acidic environment favors activation of effector caspases and triggering of mitochondrial apoptosis. However, because neutralized NAC also enhanced apoptosis (1.6-fold), a direct role for NAC in augmenting the apoptotic pathways initiated by MK886 is suggested.