Elevated prolactin responses to <Emphasis Type="SmallCaps">l</Emphasis>-tryptophan infusion in medication-free depressed patients

Rationale Several previous neuroendocrine studies have demonstrated reduced 5-HT_1A receptor function in major depressive disorder (MDD). However, hypercortisolaemia or previous drug treatment may have been significant confounds. Objectives To replicate previous studies in subjects with MDD who had been drug free for at least 8 weeks and to relate the findings to measures of HPA axis function. Methods Hormonal responses to l-tryptophan infusion were measured in patients with MDD (n=20) and healthy controls (n=20). Basal salivary cortisol and DHEA were also profiled. Results No attenuation of 5-HT_1A receptor-dependent neuroendocrine responses (growth hormone, prolactin) was observed in patients with MDD. The prolactin response to l-tryptophan was significantly greater in MDD patients than in healthy controls (P=0.008). There was a significant negative correlation between prolactin response and basal salivary cortisol secretion over the 3 days prior to the test. Conclusions These data do not support previous findings of reduced 5-HT_1A function in MDD and suggest that hypercortisolaemia or psychotropic medication may have accounted for the attenuation. Basal cortisol, DHEA and the cortisol-DHEA ratio did not differ between patients and controls, and all patients were psychotropic medication-free. The greater prolactin response to l-tryptophan infusion in depressed subjects may be the result of an increase in dopamine receptor sensitivity, secondary to reduced dopamine levels.     

Investigations on the Humidity-Induced Transformations of Salbutamol Sulphate Particles Coated with <Emphasis Type="SmallCaps">l</Emphasis>-Leucine

PURPOSE: The crystallization and structural integrity of micron-sized inhalable salbutamol sulphate particles coated with L: -leucine by different methods are investigated at different humidities. The influence of the L: -leucine coating on the crystallization of salbutamol sulphate beneath the coating layer is explored. METHODS: The coated particles are prepared by an aerosol flow reactor method, the formation of the L: -leucine coating being controlled by the saturation conditions of the L: -leucine. The coating is formed by solute diffusion within a droplet and/or by vapour deposition of L: -leucine. The powders are humidified at 0%, 44%, 65% and 75% of relative humidity and the changes in physical properties of the powders are investigated with dynamic vapour sorption analysis (DVS), a differential scanning calorimeter (DSC), and a scanning electron microscope (SEM). RESULTS: Visual observation show that all the coated particles preserve their structural integrity whereas uncoated salbutamol sulphate particles are unstable at 65% of relative humidity. The coating layer formed by diffusion performs best in terms of its physical stability against moisture and moisture-induced crystallization. The degree of crystallization of salbutamol in the as-prepared powders is within the range 24-35%. The maximum degree of crystallization after drying ranges from 55 to 73% when the salbutamol crystallizes with the aid of moisture. In addition to providing protection against moisture, the L: -leucine coating also stabilizes the particle structure against heat at temperatures up to 250 degrees C. CONCLUSION: In order to preserve good flowability together with good physical stability, the best coating would contain two L: -leucine layers, the inner layer being formed by diffusion (physical stability) and the outer layer by vapour deposition (flowability).     

Role of a Novel Excipient Poly(Ethylene Glycol)-<Emphasis Type="BoldItalic">b</Emphasis>-Poly(<Emphasis Type="SmallCaps">L</Emphasis>-Histidine) in Retention of Physical Stability of Insulin in Aqueous Solutions

Purpose This study is to investigate whether poly(ethylene glycol) (PEG)-b-poly(L-histidine) [PEG-polyHis] can reduce aggregation of insulin in aqueous solutions on agitation by forming ionic complexes. Materials and Methods Insulin aggregation on agitation was monitored spectrophotometrically and by fibrillation studies with a dye Thioflavin T. Pluronic F-127 as a control and PEG-polyHis as a novel multifunctional excipient were added to prevent destabilization of insulin. Conformation of insulin was evaluated in a circular dichroism (CD) study. Results Ionic interactions between insulin and PEG-polyHis were induced in the pH range: 5.5–6.5. pH 5.5 was selected for further evaluation based on particle size/zeta potential studies. Ionic complexation with PEG-polyHis is more effective at pH 5.5 in stabilizing insulin (75% of insulin retained versus 0% with no excipient) than Pluronic F-127 (42% retained). PEG-polyHis guards against insulin aggregation in non-complexing pH conditions (pH 7.4), 64% insulin retained versus 58% with F-127 and 0% with no excipient) pointing to the potential role played by PEG in modulation of insulin surface adsorption. Rate of fibrillation was higher for plain insulin compared with addition of PEG-polyHis and Pluronic F-127 at both pH. Conclusions Understanding and manipulation of such polyelectrolyte-protein complexation will likely play a role in protein stabilization.     

Comparison of the Effect of Different pH Buffering Techniques on the Toxicity of Copper and Zinc to <Emphasis Type="Italic">Daphnia Magna</Emphasis> and <Emphasis Type="Italic">Pseudokirchneriella Subcapitata</Emphasis>

During the time-course of ecotoxicity tests with algae and chronic (reproductive) toxicity tests with daphnids, in which algae are present as a food source, pH can dramatically increase due to photosynthetic activity. As pH changes can significantly affect metal speciation and thus its bioavailability, it may be necessary to buffer the pH of the exposure medium. One class of buffers (Goods N-subtituted aminosulfonic acids) are increasingly being used in biological and chemical applications, including ecotoxicity testing. However, the potential effect of these buffers on metal toxicity has, so far, scarcely been examined. In this study we investigated if MOPS (3-N morpholino propane sulfonic acid) affected the toxicity of copper and zinc to two standard test organisms: the cladoceran Daphnia magna and the green alga Pseudokirchneriella subcapitata. First, we demonstrate that up to a concentration of 750 mg l^–1 (which proved to be sufficient for pH buffering) MOPS did not affect 21-day net reproduction of D. magna or the 72-h population growth of P. subcapitata. Second, we conducted bioassays in copper and zinc spiked standard media for the pH range 6 – 8. For D. magna the possible effect of 750 mg l^–1 MOPS on acute copper and zinc toxicity was investigated by performing parallel 48-h toxicity tests in NaHCO₃ and MOPS buffered test media. Seventy-two hour growth inhibition assays with P. subcapitata were performed in parallel in MOPS and NaHCO₃ buffered test media and in test media with daily manual pH adjustment with HCl. For daphnids no significant differences in copper and zinc toxicity were observed between MOPS or NaHCO₃ buffered test media. For algae no significant differences in metal toxicity were observed between MOPS and HCl buffered media, but in test media buffered with NaHCO₃ an increased copper and zinc toxicity was observed as a consequence of pH increases during the test. Clearly, the results of this study demonstrate the importance of pH buffering in metal toxicity testing and the suitability of the MOPS buffer for that purpose.     

Effect of tiron on remote organ injury in rats with severe acute pancreatitis induced by <Emphasis Type="SmallCaps">l</Emphasis>-arginine

Acute pancreatitis (AP) is an acute inflammatory disorder of the pancreas that can be complicated by involvement of other remote organs. Oxidative stress is known to have a crucial role in the development of pancreatic acinar damage and one of the main causes in multisystem organ failure in experimental AP. The aim of the study was to determine the effect of tiron on pancreas and remote organ damage in l-arginine (L-Arg) induced AP rat model. Thirty-two male rats were divided in random into four groups: control, tiron, L-Arg, and tiron with L-Arg. At the end of the experiment, blood samples were withdrawn for biochemical analysis. The pancreas, lung, kidney, and liver were collected for histopathological examination. Estimation of pancreatic water content was done. Analysis of pulmonary, hepatic, renal, and pancreatic lipid peroxide levels (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH) were carried out. Finally, nuclear factor kappa B (NF-κB) and transforming growth factor β1 (TGF-β1) expression in pancreatic tissue was determined. Results indicated that treatment with tiron significantly decreased lipid peroxide levels and markedly increased both SOD activity and GSH level. Moreover, histopathological analysis further confirmed that administration of tiron relatively ameliorates pancreatic acinar cells and remote organ damage. Increased immunoreactivity of NF-κB and TGF-β1 were reduced also by tiron treatment. These findings pointed out the protective role of the mitochondrial antioxidant, tiron against AP induced by L-Arg.     

Lipid-based Formulations for Danazol Containing a Digestible Surfactant,Labrafil M2125CS: <Emphasis Type="BoldItalic">In Vivo</Emphasis> Bioavailability and Dynamic <Emphasis Type="BoldItalic">In Vitro</Emphasis> Lipolysis

Purpose

To evaluate the use of Labrafil® M2125CS as a lipid vehicle for danazol. Further, the possibility of predicting the in vivo behavior with a dynamic in vitro lipolysis model was evaluated.

Methods

Danazol (28 mg/kg) was administered orally to rats in four formulations: an aqueous suspension, two suspensions in Labrafil® M2125CS (1 and 2 ml/kg) and a solution in Labrafil® M2125CS (4 ml/kg).

Results

The obtained absolute bioavailabilities of danazol were 1.5?±?0.8%; 7.1?±?0.6%; 13.6?±?1.4% and 13.3?±?3.4% for the aqueous suspension, 1, 2 and 4 ml Labrafil® M2125CS per kg respectively. Thus administration of danazol with Labrafil® M2125CS resulted in up to a ninefold increase in the bioavailability, and the bioavailability was dependent on the Labrafil® M2125CS dose. In vitro lipolysis of the formulations was able to predict the rank order of the bioavailability from the formulations, but not the absorption profile of the in vivo study.

Conclusions

The bioavailability of danazol increased when Labrafil® M2125CS was used as a vehicle, both when danazol was suspended and solubilized in the vehicle. The dynamic in vitro lipolysis model could be used to rank the bioavailabilities of the in vivo data.

     

Main phenolic compounds from the flower of <Emphasis Type="Italic">Trachelospermum asiaticum</Emphasis> var. <Emphasis Type="Italic">intermedium</Emphasis> (Apocynaceae)

Six phenolic compounds were isolated from the flowers of Trachelospermum asiaticum var. intermedium (Apocynaceae). These structures were determined on the basis of spectral data.     

<Emphasis Type="BoldItalic">In Vitro</Emphasis> and <Emphasis Type="BoldItalic">In Vivo</Emphasis> Release of Vascular Endothelial Growth Factor from Gelatin Microparticles and Biodegradable Composite Scaffolds

PURPOSE: This work evaluated gelatin microparticles and biodegradable composite scaffolds for the controlled release of vascular endothelial growth factor (VEGF) in vitro and in vivo. METHODS: Gelatin crosslinking, VEGF dose, and buffer type were investigated for their effects on VEGF release. Release was also evaluated from microparticles confined within porous polymer scaffolds (composites). In vitro and in vivo studies were conducted using radiolabeled VEGF. RESULTS: The effect of VEGF dose on its fractional release from gelatin microparticles in vitro was minimal, but the addition of collagenase to the buffer resulted in a higher cumulative release of VEGF. Gelatin crosslinking extent was a significant factor on release from both microparticles alone and composite scaffolds in vitro and in vivo. VEGF bioactivity from composite scaffolds in vitro was maintained above 90% of the expected bioactivity over 14 days. CONCLUSIONS: VEGF release kinetics were dependent on the extent of gelatin crosslinking and were characteristic of the specific growth factor due to the effects of growth factor size, charge, and conformation on its complexation with gelatin. These studies demonstrate the utility of gelatin microparticles and their composite scaffolds as delivery vehicles for the controlled release of VEGF for tissue engineering applications.     

Design of Biodegradable Nanoparticles for Oral Delivery of Doxorubicin: <Emphasis Type="BoldItalic">In vivo</Emphasis> Pharmacokinetics and Toxicity Studies in Rats

Purpose  Doxorubicin, a potent anticancer drug associated with cardiotoxicity and low oral bioavailability, was loaded into nanoparticles with a view to improve its performance. Methods  Doxorubicin loaded PLGA nanoparticles were prepared by a double emulsion method. The pH dependent stability of nanoparticles in simulated fluids was evaluated. DSC and XRD studies were carried out in order to ascertain the nature of doxorubicin in formulations in conjunction with accelerated stability studies. The in vitro release was investigated in phosphate buffer. The pharmacokinetic and toxicity studies were conducted in rats. Results  Nanoparticles had an average size of 185 nm, with 49% entrapment at 10% w/w of polymer. The particles displayed good pH dependent stability in the pH range 1.1–7.4. DSC and XRD studies revealed the amorphous nature of doxorubicin in nanoparticles and the accelerated stability studies revealed the integrity of formulations. Initial biphasic release (20%) followed by a sustained release (80%) for 24 days was observed under in vitro conditions. The doxorubicin loaded nanoparticles demonstrated superior performance in vivo as evident by enhanced bioavailability and lower toxicity. Conclusions  Together, the data indicates the potential of doxorubicin loaded nanoparticles for oral chemotherapy. Further, these formulations could be explored for new indications like leishmaniasis.     

<Emphasis Type="SmallCaps">d</Emphasis>-Cycloserine and performance under different states of anxiety in healthy volunteers

Rationale There is interest in the development of augmentation therapy in the treatment of anxiety disorders. Recent publications have shown that d-cycloserine can benefit exposure therapy in a group of acrophobic (height phobic) subjects and in patients with social anxiety disorder. These studies were based on the animal data suggesting that drugs acting to enhance glutamate function may be developed to accelerate the behavioural treatment of anxiety disorders. Perhaps by enhancing glutamate/N-methyl-d-aspartate receptor function, learning is thus enhanced. This study examines the effects of d-cycloserine 50 mg on a task that involves learning. We manipulated anxiety levels to model the effects of high anxiety. Objectives To evaluate performance and learning, we used the Manikin task. Two groups of 24 healthy volunteers participated in a double-blind, placebo-controlled study. One group received the inhalation of CO₂ 7.5% to model high anxiety, and the second group received air to represent lower anxiety. Subjects received d-cycloserine 50 mg or placebo, and the Manikin task was performed during the gas inhalation. Results There were significant differences in the group inhaling air, but not CO₂, with the d-cycloserine group showing an increase in correct responses. This difference was apparent at several time blocks during the 20-min task. These findings were supported by subjective measures in that participants who received d-cycloserine reported that the task was easier. Conclusions We have shown that at lower anxiety levels, d-cycloserine 50 mg improved the performance of this challenging visuospatial cognitive task. This increase in performance was not seen when anxiety was higher, and d-cycloserine did not appear to increase subjective anxiety. These data lend support to the use of d-cycloserine and related glutamate enhancers as cognitive modulators and suggest that the actions of d-cycloserine are not simply related to increased arousal or anxiety.     

Chromosomal Damage in Two Species of Aquatic Turtles (<Emphasis Type="Italic">Emys orbicularis</Emphasis> and <Emphasis Type="Italic">Mauremys caspica</Emphasis>) Inhabiting Contaminated Sites in Azerbaijan

The Caspian region and specifically the Apsheron peninsula of Azerbaijan are known to be polluted with a variety of environmental contaminants. These complex mixtures of contaminants make risk assessment difficult. We used the flow cytometry method (FCM) and the micronucleus assay (MN) to assess chromosomal damage in aquatic turtles (Emys orbicularis, the European pond turtle; and Mauremys caspica, the Caspian turtle) inhabiting contaminated wetlands in Azerbaijan. Evidence of genetic damage was found for two sites, Neftchala and Sumgayit, relative to a reference site, Ali Bairamly. Sediment samples from each site were analyzed for PAHs and mercury to evaluate potential contaminant associations with genetic damage. A significant positive correlation was documented between three-ring PAH sediment concentrations and FCM estimates of chromosomal damage in E. orbicularis. These data combine to show that the contaminated wetlands in Sumgayit and Neftchala are genotoxic and that three-ring PAHs are likely a significant influence on observed genotoxicity.     

Observed Differences in Life History Characteristics of Nematodes <Emphasis Type="Italic">Aphelenchus</Emphasis> and <Emphasis Type="BoldItalic">Acrobeloides</Emphasis> upon Exposure to Copper and Benzo(a)pyrene

Maturity index values reflect life history characteristics often inferred by morphology. We tested the hypothesis that Acrobeloides and Aphelenchus are sensitive to chemical pollutants, opposite of what their colonizer-persister (CP) value of 2 suggests. Acrobeloides and Aphelenchus were reared at 19°C and provided diets of Escherichia coli and Rhizoctonia solani, respectively. LC₅₀ values for Aphelenchus exposed to copper or benzo(a)pyrene (BaP) are greater than Acrobeloides. Copper impedes growth of Acrobeloides at 10 μg/g, and results in 100% mortality at 20 μg/g. In contrast, Aphelenchus is more resilient, with no visible impact at 20 μg/g. Acrobeloides and Aphelenchus were sensitive to much lower concentrations of BaP than copper, i.e., 0.5 μg/g inhibited development of Acrobeloides and 2 μg/g for Aphelenchus. Egg size and hatch were unaffected at 15 μg/g copper. In contrast, 0.5 μg/g BaP reduced both egg size and hatch for Aphelenchus but not Acrobeloides. Survival of Acrobeloides and reproduction of Aphelenchus responded differently to copper and BaP, implying the relationship between this classification and their sensitivity to short-term effects may be less straightforward than presumed. Refinement of index values based on empirical evidence can be used to improve sensitivity and interpretation of nematode community indices for environmental monitoring.     

Investigations of responses to metal pollution in land snail populations (<Emphasis Type="Italic">Cantareus aspersus</Emphasis> and <Emphasis Type="Italic">Cepaea nemoralis</Emphasis>) from a smelter-impacted area

A cross-transplantation field experiment was performed to investigate about possible adaptation/acclimatization to metal pollution in common garden snail Cantareus aspersus (ex-Helix aspersa) and brown-lipped grove snail Cepaea nemoralis populations. Adults were collected from an area surrounding a former smelter (ME), highly polluted by trace metals (TMs) for decades, and from an unpolluted site (BE). Subadults of first generation (F1) were exposed in microcosms in a 28-day kinetic study. Four exposure sites were chosen around the smelter along a soil pollution gradient (vegetation and soil otherwise comparable). Bioaccumulation in snail soft tissues globally increased with soil contamination, with Cd, Pb and Zn concentrations reaching 271, 187, 5527 μg g⁻¹, respectively. Accumulation kinetic patterns were similar between snail species but C. nemoralis showed greater TM levels than C. aspersus. Some inter-population differences were revealed in TM accumulation (bioaccumulation factors, accumulation kinetics) but did not suggest consistent adaptive responses. We did not detect negative effects of TM exposure on snail condition (body weight, shell size, shell weight). ME C. aspersus snails produced heavier shells than BE snails under exposure to TMs at the highest level, suggesting an adaptive response. The protocol used in this study, however, did not allow unambiguously distinguishing whether this response was due to genetic adaptation or to maternal effects. Abnormal but reversible shell development of adult ME C. nemoralis suggested physiological acclimatization. Differences in responses to TMs between populations are observed for conchological parameters, not for bioaccumulation, with different strategies according to the species (acclimatization or adaptation/maternal effects).     

<Emphasis Type="Italic">In Vivo</Emphasis> and <Emphasis Type="Italic">In Vitro</Emphasis> Evidence for Brain Uptake of 4-Phenylbutyrate by the Monocarboxylate Transporter 1 (MCT1)

Purpose

4-Phenylbutyrate (4-PBA) is expected to be a potential therapeutic for several neurodegenerative diseases. These activities require 4-PBA transport into the brain across the blood-brain barrier (BBB). The objective of the present study was to characterize the brain transport mechanism of 4-PBA through the BBB.

Methods

The brain transport of 4-PBA across the BBB was investigated following intravenous (IV) injection and internal carotid artery perfusion (ICAP) in vivo. The mechanism of transport was examined using TR-BBB cells, an in vitro model of the BBB.

Results

The volume of distribution (V_D) of 4-PBA by rat brain was about 7-fold greater than that of sucrose, a BBB impermeable vascular space marker, suggesting the blood-to-brain transport of 4-PBA through the BBB in the physiological state. [¹⁴C]4-PBA uptake by TR-BBB cells showed time-, pH- and concentration-dependence with a K _m of 13.4 mM at pH 7.4 and 3.22 mM at pH 6.0. The uptake was Na⁺ independent, and was significantly inhibited by alpha-cyano-4-hydroxycinnamate (a typical inhibitor for monocarboxylate transport), endogenous monocarboxylate compounds and monocarboxylic drugs. Lactate and valproate competitively inhibited [¹⁴C]4-PBA uptake with K _i value of 13.5 mM and 7.47 mM, respectively. These results indicate the role of monocarboxylate transporters (MCTs) in 4-PBA transport into the brain at the BBB. TR-BBB cells expressed mRNA of rMCT1, 2, and 4, especially, rMCT1 showed high mRNA expression level. In addition, [¹⁴C]4-PBA uptake was inhibited by rMCT1 specific small interfering RNA.

Conclusion

The transport mechanism of 4-PBA from blood to brain across the BBB likely involves MCT1.

     

Synthesis and antitumor evaluation of some 1,3,4-oxadiazole-2(3<Emphasis Type="Italic">H</Emphasis>)-thione and 1,2,4-triazole-5(1<Emphasis Type="Italic">H</Emphasis>)-thione derivatives

A series of 1,3,4-oxadiazole-2-thione and 1,2,4-triazole-5-thione derivatives have been successfully synthesized and studied for their antitumor activity. These compounds were prepared from carboxylic acids and chiral aminoacid esters. The prepared compounds were evaluated for their cytotoxicity against cancer in vitro using hydroxyurea (HU) as positive control. A fair number of compounds were found to have significant antitumor activity. To study the molecular basis of interaction and affinity of binding of the target molecules, all the compounds were docked into the ATPase domain of TP-II and tubulin using Schrödinger.     

Formulation and Characterization of Injectable Poly(<Emphasis Type="SmallCaps">dl</Emphasis>-lactide-<Emphasis Type="BoldItalic">co</Emphasis>-glycolide) Implants Loaded with N-Acetylcysteine,a MMP Inhibitor

Purpose The objective of this study was to develop poly(lactic-co-glycolic acid) (PLGA) injectable implants (i.e., millicylinders) with microencapsulated N-acetylcysteine (NAC) for site-specific controlled NAC release, for potential chemopreventive applications in persons with previously excised head and neck cancers. Methods PLGA 50:50 (i.v. = 0.57 dl/g) implants with 1–10 wt% NAC free acid or 10 wt% NAC salts (NAC–Na⁺, NAC–Mg²⁺ and NAC–Ca²⁺) were prepared by solvent extrusion and/or fluid energy micronization (FEM) methods. X-ray diffraction (XRD), scanning electron microscopy (SEM), and differential scanning calorimetry (DSC) studies were performed to evaluate the physical mixing of NAC with PLGA. PLGA implant degradation was studied by kinetics of polymer molecular weight decline (gel permeation chromatography) and mass loss. Release studies were conducted in N₂ purged PBS (pH 7.4) at 37°C in evacuated and sealed ampoules. NAC was quantified by HPLC at 210 nm. Results XRD, SEM and DSC studies indicated that NAC had dissolved in the polymer phase at 1–3.5% w/w loading, but became discretely suspended in the polymer at 6–10% w/w. Initial burst and long-term release rate increased with increased drug loading, and release was uncharacteristically rapid at higher loading (6–10% w/w). The cause of the rapid release was linked to extensive plasticization, matrix porosity and general acid catalysis of PLGA degradation caused by the NAC free acid. PLGA millicylinders loaded with 10% w/w NAC–Ca²⁺ and NAC–Mg²⁺salts exhibited reduced burst (34 vs 13–22% release within a day of incubation for NAC free acid vs NAC–Ca²⁺ and NAC–Mg²⁺salts, respectively) and slow and continuous complete release over 4 weeks without significant NAC-catalyzed degradation of PLGA. Release of NAC from NAC–Ca²⁺/PLGA implant was slower than that of NAC–Mg²⁺/PLGA consistent with the lower solubility of the former salt. NAC with its free thiol was rapidly converted to its cystine dimer in the presence of molecular oxygen. PLGA released samples in sealed and evacuated ampoules indicated >80% parent NAC remaining after the 1 month release analysis irrespective of initial NAC free acid and salt forms. Conclusion By encapsulating the NAC–Mg²⁺ and NAC–Ca²⁺ salts in PLGA implants, the high initial burst, short release duration, and the general acid catalysis caused by the NAC free acid were each prevented and 1-month slow and continuous release was attained with minimal instability of the free thiol group.     

Pollution-induced behavioural effects in the brown bullhead (<Emphasis Type="Italic">Ameiurus nebulosus</Emphasis>)

Aquatic ecosystems are major sinks for pollutants which can have adverse effects on biodiversity. Thus, it is important to understand the nature of pollution-induced change in aquatic ecosystems. We show that brown bullheads (Ameiurus nebulosus) may have evolved in response to chronic pollution exposure. We collected adults from the Detroit River (polluted site) and Belle River (control site). Both adults and common-garden raised juveniles were tested for aggression, locomotion, and escape response using consecutive unchallenged (clean) and challenged (polluted) trials. Detroit River fish were more aggressive than Belle River fish when challenged. Furthermore, Belle River fish showed increased locomotion when exposed to pollutants, whereas Detroit River fish were unaffected. The consistent difference in adult and juvenile behaviour across trials suggests a genetic response to pollution. Escape response on the other hand, showed inter-population differences, but no consistency between adults and juveniles, indicating that this behaviour is influenced by non-genetic factors. We discuss our data with respect to the potential adaptation of populations to pollution and the implications for prioritizing remediation efforts.     

Synthesis of 2-substituted-6-(4<Emphasis Type="Italic">H</Emphasis>-1,2,4-triazol-4-yl)benzo[<Emphasis Type="Italic">d</Emphasis>]oxazoles as potential anticonvulsant agents

A series of 2-substituted-6-(4H-1,2,4-triazol-4-yl)benzo[d]oxazoles were synthesized. The anticonvulsant effect and neurotoxicity of the compounds (intraperitoneally) were evaluated with the maximal electroshock (MES) test, subcutaneous pentylenetetrazole (sc-PTZ), and rotarod tests in mice. 2-Phenyl-6-(4H-1,2,4-triazol-4-yl)benzo[d]oxazole (3g) was the most active and also had the lowest toxicity. In the anti-MES potency test, it showed median effective dose (ED₅₀) of 29.5 mg/kg, a median toxicity dose (TD₅₀) of 285 mg/kg, and a protective index (PI) of 9.7, which is greater than the reference drug, carbamazepine, which has a PI of 6.4.     

<Emphasis Type="Italic">Morinda citrifolia</Emphasis> Linn. (Rubiaceae) leaf extracts mitigate UVB-induced erythema

Morinda citrifolia Linn. (Rubiaceae) leaves have been used in tropical folk medicine to treat topical inflammation and burns. A carbomer gel base, containing the ethanol extract and juice pressed from the leaves, was evaluated for potential allergenic properties in a repeat-insult patch test in 49 volunteers. To investigate the topical photo-protective properties, the combined ethanol extract and leaf juice were evaluated in a UVB-induced erythema model in 25 volunteers. The crude ethanol extract of M. citrifolia leaves was also evaluated in vitro for potential anti-inflammatory activity in a histamine H-1 receptor antagonism assay. There was no evidence of allergenic potential in the repeat-insult patch test. When the combination of ethanol extract and leaf juice was applied, the UVB dose required to induce erythema was almost 3.5 times greater than with untreated skin (P < 0.001). In the histamine H-1 receptor-binding assay, the crude ethanol extract of M. citrifolia leaves inhibited receptor binding by 57%. These results suggest that M. citrifolia leaves are safe for topical use and may be useful in mitigating UVB-induced injury to the skin.     

Genotoxicity monitoring of freshwater environments using caged carp (<Emphasis Type="Italic">Cyprinus carpio</Emphasis>)

The present study deals with genotoxicity assessment of freshwaters using caged carp (Cyprinus carpio). Carps were transplanted from a fish-farm to three differently polluted sites in eastern Croatia. Two polluted sites were situated in the river Drava, downstream from the cities of Belišće and Osijek, while the reference site was in the Nature Park Kopački rit, a preserved wetland area with limited anthropogenic influence. Exposure lasted for 3 weeks and was repeated for 3 years (2002–2004). DNA damage was assessed in erythrocytes of the exposed animals by the Comet assay and micronucleus test (MNT). In order to evaluate possible differences in stress responses to polluted water in situ and in aquaria a laboratory exposure was performed with water from the studied location in the second year of the study. Carp from the sites with high anthropogenic influence (Belišće and Osijek) had higher average DNA damage as expressed in both the MNT and Comet assay. Of the two, the Comet assay appeared to be more sensitive following both caging and aquaria exposures. The results from this study suggest that 3 weeks caging exposure of C. carpio may be a useful strategy to monitor for genotoxic agents in freshwater ecosystems.