Anti-oxidative hormetic effects of cellular autophagy induced by nutrient deprivation in a molluscan animal model.

This investigation using a molluscan animal model tested the hypothesis that experimentally induced lysosomal autophagy protects against oxidative cell injury. Induction of augmented lysosomal autophagy has previously been implicated in this protective process. Four treatment groups of blue mussels (Mytilus galloprovincialis) were used: Group 1 (fed - control), Group 2 (fasted), Group 3 (copper + fed) and Group 4 (copper + fasted). Groups 2 and 4 were fasted in order to trigger autophagy; and samples of hepatopancreas (liver analogue or digestive gland) from all 4 groups were taken at 3, 6 and 15 days. Treatment with copper provided a positive reference for oxidative stress: Groups 3 and 4 were treated with copper (10 µg Cu2+/animal/day) for three days only. Oxidative damage and cellular injury in hepatopancreatic digestive cells was found to decrease in Group 2 (fasted) compared to Group 1 (fed - control). Group 3 (fed + copper) showed clear evidence of oxidative stress and cell injury, as well as induction of antioxidant activities. Group 4 (copper + fasted) had a reduced uptake of copper and toxicity of copper was also reduced, compared with Group 3. It was concluded that augmented autophagy had a hormetic cytoprotective anti-oxidant effect.

Oxidative stress, lysosomal damage and dysfunctional autophagy in molluscan hepatopancreas (digestive gland) induced by chemical contaminants.

Autophagy is a highly conserved evolutionary survival or defence process that enables cells and organisms to survive periods of environmental stress by breaking down cellular organelles and macromolecules in autolysosomes to provide a supply of nutrients for cell maintenance. However, autophagy is also a part of normal cellular physiology that facilitates the turnover of cellular constituents under normal conditions: it can be readily augmented by mild environmental stress; but becomes dysfunctional with severe oxidative stress leading to cellular pathology. The molluscan hepatopancreas or digestive gland provides a versatile and environmentally relevant model to investigate lysosomal autophagy and stress-induced dysfunctional autophagy. This latter process has been implicated in many animal and human disease conditions, including degenerative and neurodegenerative diseases, as well as obesity related conditions. Many environmental pollutants have also been found to induce dysfunctional autophagy in molluscan hepatopancreatic digestive cells, and in this study, the marine blue mussel Mytilus galloprovincialis was exposed for 7 days to: 0.1 µM, 1 µM and 10 µM concentrations of fluoranthene and phenanthrene (PAHs); chlorpyrifos and malathion (organophosphorus compounds); atrazine (triazine herbicide); copper (transition metal) and dodecylbenzene sulphonic acid (LAS, surfactant). The marine snail or periwinkle, Littorina littorea, was also exposed to phenanthrene, chlorpyrifos and copper. Indices of oxidative stress, cell injury and dysfunctional autophagy were measured (i.e., lysosomal membrane stability, protein carbonyls, lipofuscin, and lysosomal accumulation of lipid or lipidosis). Evidence of oxidative stress, based on the elevation of lipofuscin and protein carbonyls, was found for all compounds tested; with chlorpyrifos being the most toxic to both species. Dysfunctional autophagy was induced by all of the compounds tested in both species, except for atrazine in mussels. This failure of normal autophagy was consistently associated with oxidative stress. Autophagic dysfunction is an important emerging feature in the aetiology of many disease conditions in animals and humans; and an explanatory conceptual mechanistic model has been developed for dysregulation of autophagy in response to oxidative stress.

Integration of biochemical, histochemical and toxicogenomic indices for the assessment of health status of mussels from the Tamar Estuary, U.K.

The aim of this study was to examine whether a combination of biochemical, histopathological and toxicogenomic data could be used as a valuable tool for the assessment of biological risk associated with pollutants within the Tamar River and Estuary, S.W. England, U.K. Accordingly, biochemical and histopathological biomarkers (protein carbonyls, lipofuscin, neutral lipids, lysosomal stability [N-acetyl-ß-hexosaminidase and neutral red], lysosomal volume, ferric reducing antioxidant power [FRAP] and malonaldehyde [MDA]) and gene expression profiles were assessed in 5 sites from the Tamar River and Estuary (Neal Point, Town Quay, Wilcove, Cremyll Ferry and Whitsand; and a reference site, Trebarwith Strand, N. Cornwall). PAHs were measured in mussel tissue and sediment and metals were measured in mussel tissue only. Data from the biomarkers was integrated into a Mussel Expert System (MES) model to produce a simple assessment of mussel stress. Clear gradients of mussel toxicity were identified by the biomarkers (with the exception of neutral lipids) with the highest impacted animals found furthest up the Tamar, whilst the MES was unable to identify a gradient of effect. Gene expression profiles also indicated a gradient of stress with the greatest number of significantly up- or down- regulated genes found at the uppermost 2 sites. The MES did, however, determine that mussels from all sites, except the reference site, were highly stressed; a conclusion that could not be inferred from the biomarker data alone. It is concluded that the MES is a valuable tool that permits integration and interpretation of complex sets of biomarker data by identifying the biological meaning of biomarker changes.

A multivalent approach to the design and discovery of orally efficacious 5-HT4 receptor agonists.

5-HT(4) receptor agonists such as tegaserod have demonstrated efficacy in the treatment of constipation predominant irritable bowel syndrome (IBS-C), a highly prevalent disorder characterized by chronic constipation and impairment of intestinal propulsion, abdominal bloating, and pain. The 5-HT(4) receptor binding site can accommodate functionally and sterically diverse groups attached to the amine nitrogen atom of common ligands, occupying what may be termed a "secondary" binding site. Using a multivalent approach to lead discovery, we have investigated how varying the position and nature of the secondary binding group can be used as a strategy to achieve the desired 5-HT(4) agonist pharmacological profile. During this study, we discovered the ability of amine-based secondary binding groups to impart exceptional gains in the binding affinity, selectivity, and functional potency of 5-HT(4) agonists. Optimization of the leads generated by this approach afforded compound 26, a selective, orally efficacious 5-HT(4) agonist for the potential treatment of gastrointestinal motility-related disorders.

The in vitro pharmacological profile of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity.

The in vitro pharmacological profile of TD-5108, a novel, selective 5-HT(4) receptor agonist, was compared to that of clinically efficacious gastroprokinetic 5-HT(4) receptor agonists. TD-5108 produced an elevation of cyclic adenosine monophosphate in human embryonic kidney 293 cells expressing the human recombinant 5-HT(4(c)) (h5-HT(4(c))) receptor (pEC(50) = 8.3) and 5-HT(4) receptor-mediated relaxation of the rat esophagus (pEC(50) = 7.9) and contraction of the guinea pig colon (pEC(50) = 7.9). In all in vitro assays, TD-5108 was a high intrinsic activity agonist, unlike tegaserod, mosapride, and cisapride which, in the majority of test systems, had lower intrinsic activity. TD-5108 had high affinity (pK (i) = 7.7) and selectivity (> or =25-fold) for h5-HT(4(c)) receptors over other biogenic amine receptors. TD-5108 was >500-fold selective over other 5-HT receptors (including h5-HT(2B) and h5-HT(3A)) and, at 3 microM, had no effect on human ether-à-go-go-related gene K+ channels. In conclusion, TD-5108 is a selective 5-HT(4) receptor agonist in vitro. The high intrinsic activity and preferential binding of TD-5108 to 5-HT4 over other 5-HT receptors may result in an improved clinical profile for the treatment of gastrointestinal disorders of reduced motility.

The in vivo gastrointestinal activity of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity.

The in vivo preclinical pharmacodynamic profile of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity, was compared to that of the clinically studied gastrointestinal pro-kinetic agents, tegaserod, cisapride and mosapride. The activity of TD-5108 was evaluated in guinea pig colonic transit, rat oesophageal relaxation and dog gastrointestinal smooth muscle contractility models. Subcutaneous administration of TD-5108, tegaserod, cisapride and mosapride increased guinea pig colonic transit (rank order of potencies: TD-5108 > tegaserod > cisapride > mosapride). Following intravenous and intraduodenal dosing, TD-5108, tegaserod, cisapride and mosapride produced dose-dependent relaxation of the rat oesophagus. On a molar basis, TD-5108 was approximately twofold less potent than tegaserod following intravenous dosing but 6- or 86-fold more potent than cisapride or mosapride, respectively, and 9- or 18-fold more potent than tegaserod or cisapride, respectively, after intraduodenal administration. Orally dosed TD-5108 increased the contractility of the canine antrum, duodenum and jejunum with higher potency than tegaserod. The selective 5-HT(4) receptor agonist, TD-5108, demonstrates robust in vivo activity in the guinea pig, rat and dog gastrointestinal tracts.

Pharmacokinetics, serum inhibitory and bactericidal activity, and safety of telavancin in healthy subjects.

The pharmacokinetics, tolerability, and serum inhibitory and bactericidal titers of telavancin, a new rapidly bactericidal lipoglycopeptide with multiple mechanisms of action against gram-positive pathogens, were assessed in a two-part, randomized, double-blind, placebo-controlled, ascending-dose study with 54 healthy men. In part 1, single ascending intravenous doses of 0.25 to 15 mg/kg of body weight were studied. In part 2, multiple ascending doses (30-min infusions of 7.5 to 15 mg/kg/day) were studied over 7 days. Following the administration of multiple doses, steady state was achieved by days 3 to 4. At day 7 after the administration of telavancin at 7.5, 12.5, and 15 mg/kg/day, peak concentrations in plasma were 96.7, 151.3, and 202.5 microg/ml, respectively, and steady-state area-under-the-curve values were 700, 1,033, and 1,165 microg x h/ml, respectively. The elimination half-life ranged from 6.9 to 9.1 h following the administration of doses > or =5 mg/kg. Most adverse events were mild in severity. At 24 h postinfusion, serum from subjects given telavancin demonstrated potent bactericidal activity against methicillin-resistant Staphylococcus aureus and penicillin-resistant Streptococcus pneumoniae strains. The results suggest that telavancin may be an effective once-daily therapy for serious bacterial infections caused by these pathogens.

Seasonal variation in cytochrome P450 immunopositive protein levels, lipid peroxidation and genetic toxicity in digestive gland of the mussel Mytilus edulis.

The relationship between cytochrome P450 1A- and 2E-immunopositive proteins, lipid peroxidation and DNA strand breaks (SBs) was studied in Mytilus edulis digestive gland at different seasons and at different sites around the UK coast. Cytochrome P4501A (CYP1A)-immunopositive protein and DNA strand breaks were generally lowest in December but there was no correlation between PAH exposure (indicated by chemical measurement and CYP1A-immunopositive protein expression) and DNA strand breaks which was highest at the relatively non-polluted site (Port Quin). As with CYP1A, CYP2E1-immunopositive protein was maximal at most sites in May. Lipid peroxidation, in contrast, did not alter markedly throughout the year. In conclusion, DNA strand breakage was not correlated with any of the above parameters although it did correlate with "scope for growth" as did the inverse of PAH levels. The study highlights the need to establish the relative contribution of DNA damage and DNA repair processes to the production of DNA strand breaks and emphasises the need to consider seasonal variation in interpretation of biomarkers.

Hematopoietic stem cells and endothelial cell precursors express Tie-2, CD31 and CD45.

Early theories of tumor angiogenesis suggested that preexisting vessels surrounding the tumor were the principal source of the tumor vasculature but recent evidence suggests that endothelial progenitor cells (EPC) migrate from the marrow play an important role in developing the tumor blood supply. In a mouse model, in which the vascularization of a transplantable tumor was studied after bone marrow (BM) transplantation, we show that cells that express Tie-2, Sca-1, CD31 and CD45 function as both BM EPC and primitive hematopoietic stem cells. BM cells from transgenic mice expressing green fluorescent protein (GFP) under the control of the endothelial lineage-specific Tie-2 promoter (Tie-2 /GFP) were used to reconstitute irradiated (12 Gy) wild-type mice. Five donor BM cell populations were studied: (1) whole BM; (2) Sca-1-enriched BMC; (3) GFP/Tie-2+, Sca-1+ BMC; (4) GFP/Tie-2-, Sca-1+ BMC and (5) Sca-1-depleted BMC. After 4 weeks, the mice were injected with Tg.AC tumor cells. Three weeks later, sections from the tumors were stained for CD31 and examined for Tie-2-driven GFP expression. BM-derived endothelial cells were found only in mice transplanted with bone marrow containing populations of Tie-2+, Sca-1+ cells. As few as 3500 of these cells were sufficient to radioprotect lethally irradiated mice. Thus, we conclude that a rare subset of BMC (approximately 4 x 10(-3)%) with the putative properties of hemangioblasts have an active Tie-2 promoter. Selection of Tie-2+Sca-1+ BMC enriches for marrow-derived EPCs that participate in tumor angiogenesis and cells that can provide hematopoietic reconstitution of marrow-ablated mice.

Different levels of mussel (Mytilus edulis) DNA strand breaks following chronic field and acute laboratory exposure to polycyclic aromatic hydrocarbons.

Levels of polycyclic aromatic hydrocarbons (PAHs) including benzo[a]pyrene (B[a]P) were at least seven-fold higher in mussels sampled from a polluted site (Loch Leven, in Scotland, UK) compared to a nearby clean reference site (Loch Etive) throughout the year 2000. Levels of DNA strand breaks (alkaline COMET assay) using both gill and digestive gland nuclei were similar at both sites despite the difference in contaminant load (total PAH). In contrast, mussels collected from a reference site (Port Quin, Cornwall, UK) had an increase in DNA strand breaks in digestive gland cells following laboratory exposure to B[a]P-dosed Isochrysis galbana. However, after 14 days high dose (20 ppb-exposed diet) animals had returned to levels similar to the controls. There was no evidence of increased necrosis or apoptosis after treatments. The results from these two studies suggest that an adaptive response may prevent ongoing DNA damage in mussels exposed to high levels of B[a]P and PAH contamination.

Elevation of cytochrome P450-immunopositive protein and DNA damage in mussels (Mytilus edulis) transplanted to a contaminated site.

Mytilus edulis were collected from a reference site (Port Quin) and an urban/industrial contaminated site (New Brighton) in the UK during June 1999. Levels of PCBs (sigma7 congeners) and CB-138 were determined to be, respectively, 21 fold and 16 fold higher in the mussel digestive glands from New Brighton. Levels of CYPIA-immunopositive protein were 1.5 fold higher (P < 0.05) at the polluted site but the levels of DNA strand breaks were 1.3 fold higher (P<0.05) at the reference site. Mussels from Port Quin were placed in cages at both sites and both transplanted and indigenous populations sampled in September (13 weeks). Mussels transplanted from the reference site to the industrial site, reported elevated levels of CYP1A-immunopositive protein (1.4 fold; P < 0.05) and higher levels of DNA damage (1.2 fold; P < 0.05) compared to caged populations at the reference site and a PCB loading similar to the populations from the polluted site. Moreover, transplanted mussels had DNA damage 1.8 fold greater (P < 0.05) than indigenous mussels at the transplant site. These changes were small but significant when compared to the observed temporal changes in the indigenous populations.

Laparoscopic appendectomy for Crohn's disease of the appendix presenting as acute appendicitis.

BACKGROUND: Crohn's disease confined to the appendix is rare but has been well described in the literature. It can mimic acute appendicitis clinically. After surgical treatment, recurrences of Crohn's disease are rare. We report the first case of treatment by laparoscopic appendectomy of Crohn's disease confined to the appendix. METHODS: A healthy 32-year old man presented with a week-long history of vague lower abdominal pain. Diagnostic work-up, which included CT, enteroclysis, and routine blood work, revealed a patent appendiceal lumen with an inflammatory mass in the right lower quadrant. RESULTS: Diagnostic laparoscopy revealed an inflamed appendix, and a laparoscopic appendectomy was performed, with frozen-section examination revealing Crohn's disease of the appendix. Two years after surgery, the patient has not had a recurrence of symptoms. CONCLUSIONS: Crohn's disease of the appendix can mimic acute appendicitis, although often with a more indolent course. The disease may be treated successfully by laparoscopic appendectomy, with good long-term results.

Three cases of small bowel intussusception in relation to a rare lesion: inflammatory fibrous polyps.

BACKGROUND/AIMS: Inflammatory fibrous polyps (IFPs), also known as inflammatory pseudotumors, occur rarely in the gastrointestinal tract. IFPs have variable presentations, often presenting as small bowel obstruction due to intussusception or, less commonly, as an incidental finding on radiological examinations or screening colonoscopies. The diagnosis and management of IFPs will be discussed through a review of the literature and a series of cases from our own institution. METHODS: A retrospective analysis of the diagnosis, management and complications of IFPs was performed by a literature review. This was accompanied by a series of 3 cases of IFPs, 2 of which causing intussusception, diagnosed and treated in our own institution. CONCLUSIONS: IFP is a rare disease and has a variable presentation, from asymptomatic to small bowel obstruction due to intussusception. IFPs cannot be differentiated from malignancy without histological examination. Therefore, whether diagnosed incidentally or in the setting of intussusception, the treatment of IFPs is surgical resection of the involved bowel.

The BBXB motif of RANTES is the principal site for heparin binding and controls receptor selectivity.

The chemokine RANTES (regulated on activation normal T cell expressed and secreted; CCL5) binds selectively to glycosaminoglycans (GAGs) such as heparin, chondroitin sulfate, and dermatan sulfate. The primary sequence of RANTES contains two clusters of basic residues, (44)RKNR(47) and (55)KKWVR(59). The first is a BBXB motif common in heparin-binding proteins, and the second is located in the loop directly preceding the C-terminal helix. We have mutated these residues to alanine, both as point mutations as well as triple mutations of the 40s and 50s clusters. Using a binding assay to heparin beads with radiolabeled proteins, the (44)AANA(47) mutant demonstrated an 80% reduction in its capacity to bind heparin, whereas the (55)AAWVA(59) mutant retained full binding capacity. Mutation of the (44)RKNR(47) site reduced the selectivity of RANTES binding to different GAGs. The mutants were tested for their integrity by receptor binding assays on CCR1 and CCR5 as well as their ability to induce chemotaxis in vitro. In all assays the single point mutations and the triple 50s cluster mutation caused no significant difference in activity compared with the wild type sequence. However, the triple 40s mutant showed a 80-fold reduction in affinity for CCR1, despite normal binding to CCR5. It was only able to induce monocyte chemotaxis at micromolar concentrations. The triple 40s mutant was also able to inhibit HIV-1 infectivity, but consistent with its abrogated GAG binding capacity, it no longer induced enhanced infectivity at high concentrations.

Substrate recognition domains within extracellular signal-regulated kinase mediate binding and catalytic activation of mitogen-activated protein kinase phosphatase-3.

Mitogen-activated protein (MAP) kinase phosphatase-3 (MKP-3) is a dual specificity phosphatase that inactivates extracellular signal-regulated kinase (ERK) MAP kinases. This reflects tight and specific binding between ERK and the MKP-3 amino terminus with consequent phosphatase activation and dephosphorylation of the bound MAP kinase. We have used a series of p38/ERK chimeric molecules to identify domains within ERK necessary for binding and catalytic activation of MKP-3. These studies demonstrate that ERK kinase subdomains V-XI are necessary and sufficient for binding and catalytic activation of MKP-3. These domains constitute the major COOH-terminal structural lobe of ERK. p38/ERK chimeras possessing these regions display increased sensitivity to inactivation by MKP-3. These data also reveal an overlap between ERK domains interacting with MKP-3 and those known to confer substrate specificity on the ERK MAP kinase. Consistent with this, we show that peptides representing docking sites within the target substrates Elk-1 and p90(rsk) inhibit ERK-dependent activation of MKP-3. In addition, abolition of ERK-dependent phosphatase activation following mutation of a putative kinase interaction motif (KIM) within the MKP-3 NH(2) terminus suggests that key sites of contact for the ERK COOH-terminal structural lobe include residues localized between the Cdc25 homology domains (CH2) found conserved between members of the DSP gene family.

Stereoselective glucuronidation of formoterol by human liver microsomes.

AIMS: Formoterol is a beta2-adrenoceptor agonist marketed as a racemic mixture of the active (R; R)- and inactive (S; S)-enantiomers (rac-formoterol). The drug produces prolonged bronchodilation by inhalation but there is significant interpatient variability in duration of effect. Previous work has shown that in humans formoterol is metabolized by conjugation with glucuronic acid but little is known about the stereoselectivity of this reaction. The aim of the present study was to investigate the glucuronidation of formoterol enantiomers in vitro by human liver microsomes. METHODS: The kinetics of formation of formoterol glucuronides during incubation of racemate and of single formoterol enantiomers with human liver microsomes (n=9) was characterized by chiral h.p.l.c. assay. RESULTS: The kinetics of glucuronidation of the two formoterol enantiomers obeyed the Michaelis-Menten equation. Glucuronidation of formoterol was stereoselective and occurred more than two times faster for (S; S)-formoterol than for (R; R)-formoterol. In incubations with single formoterol enantiomers, the median (n=9) Km values for (R; R)-glucuronide and (S; S)-glucuronide were 827.6 and 840.4 microm, respectively, and the median V max values were 2625 and 4304 pmol min-1 mg-1, respectively. Corresponding values determined in incubations with rac-formoterol were 357.2 and 312.1 microm and 1435 and 2086 pmol min-1 mg-1 for (R; R)- and (S; S)-glucuronide, respectively. Interindividual variation was large with the ratio of V max/Km (S; S/R; R) ranging from 0.57 to 6.90 for incubations with rac-formoterol. CONCLUSIONS: Our study demonstrates that glucuronidation of formoterol by human liver microsomes is stereoselective and subject to high interindividual variability. These findings suggest that clearance of formoterol in humans is subject to variable stereoselectivity which could explain the variation in duration of bronchodilation produced by inhaled formoterol in patients with asthma.

Jejunal diverticulosis: a rare entity with multiple presentations, a series of cases.

BACKGROUND/AIMS: Jejunal diverticulosis (JD) is a rare disease that has a variable presentation. The signs, symptoms, diagnosis, complications and treatment of JD will be discussed through a review of the literature and a series of cases from our own institution. METHODS: A retrospective analysis of the diagnosis, treatment and complications of JD was performed by a literature review. This was accompanied by a series of four cases of JD diagnosed and treated in our own institution. CONCLUSIONS: JD is a rare disease in which most patients are asymptomatic. However, JD's different complications are serious and can be fatal. The treatment is mainly surgical; however, there have been cases where nonsurgical management was successful.

Development of hepatic CYP1A and blood vitellogenin in eel (Anguilla anguilla) for use as biomarkers in the Thames Estuary, UK.

The potential of eel (Anguilla anguilla) as a monitoring species for the Thames Estuary, UK, was examined. Hepatic cytochrome P4501A [7-ethoxyresorufin O-deethylase (EROD) activity] and blood vitellogenin (Western analysis) were investigated as biomarkers of exposure to, respectively, organic contaminants and to contaminants showing estrogenic activity. Hepatic microsomal EROD activities in A. anguilla from seven sites in the Thames Estuary in May 1998 varied three-fold (111 +/- 24 to 355 +/- 42 pmol min-1 mg protein-1) (mean +/- S.E.M.) and showed correlation with salinity; however, the latter relationship was not maintained at other times of the year. The range of EROD activities was two- to eight-fold higher than the 37 +/- 8 pmol min-1 mg-1 for A. anguilla from the relatively clean Tamar Estuary. beta-Naphthoflavone treatment (5 mg kg-1 wet wt.; 2 days) of Thames A. anguilla produced a two-fold increase in hepatic microsomal EROD activity. Comparing the Thames EROD data with those for A. anguilla from well-characterised contaminated sites in the Netherlands (Van der Oost, R., Goksøyr, A., Celander, M., Heida, H., & Vermeulen, N. P. E. 1996. Aquatic Toxicology, 36, 189-222), the Thames is suggested to be moderately impacted by polycyclic aromatic hydrocarbons and related contaminants. 17-beta-Estradiol treatment produced the appearance of a plasma protein of 211 Kd app. mol. wt. (recognised by antibodies to vitellogenin of Morone saxatilis), but putative vitellogenin could not be detected in A. anguilla from selected sites in the Thames Estuary.

Seasonal variation in mussel Mytilus edulis digestive gland cytochrome P4501A- and 2E-immunoidentified protein levels and DNA strand breaks (Comet assay).

Mytilus edulis digestive gland microsomes were prepared from indigenous populations sampled from a clean reference site (Port Quin) and an urban-industrial contaminated site (Blackpool) in the UK. Samples were collected in March/April, May, August and December 1998. Western blot analysis was performed using polyclonal antibodies to fish CYP1A and rat CYP2E using partially purified M. edulis CYP as a positive control, to aid identification. CYP1A- and CYP2E-immunopositive protein levels showed different site-specific seasonal variation with higher levels of CYP2E determined in May (P < 0.05). At both sites, lower levels of CYP1A-immunopositive protein but not CYP2E-immunopositive protein were observed in the samples collected in December (P < 0.05). This correlated with lower levels of nuclear DNA damage (Comet assay expressed as per cent tail DNA) observed in December compared to August (P < 0.05).

Surveillance of a recently switched non-prescription medicine (Diclofenac) using a pharmacy-based approach.

PURPOSE: Postmarketing surveillance of prescription medicines is a routine practice, yet similar evaluation of non-prescription medicines, including those recently switched from prescription status, is uncommon. This study presents the methodologic issues and limitations of the use of pharmacies in the 'post-reclassification' surveillance of oral diclofenac potassium 25 mg which had been recently switched from physician prescription to non-prescription sale. METHODS: Consenting user-purchasers were recruited from 175 New Zealand pharmacies over 4 months. Purchasers were mailed a questionnaire for completion 7 days post-purchase. Those purchasers who met criteria for being potentially 'at risk' of adverse events were re-surveyed 30 days post-purchase. A descriptive analysis was carried out using t-test and chi-square as appropriate. These results were compared to those from other types of studies in this area. RESULTS: The 1240 recruited purchasers returned 990 valid questionnaires (80% response). Of these 557 (56%) met 'at risk' criteria and received the second questionnaire with 480 valid returns (86.2% response). CONCLUSIONS: Useful data was gathered on the 'real-life' usage of a medicine recently reclassified from prescription to non-prescription sale. The use of community pharmacies as recruiting centres was found to be effective. Copyright (c) 2000 John Wiley & Sons, Ltd.