Individual and combined effects of mycotoxins from typical indoor moulds

The mycotoxins patulin, gliotoxin and sterigmatocystin can be produced by common indoor moulds and enter the human body via inhalation of mycotoxin containing spores and particulates. There are various studies about the individual effects of these mycotoxins, but a lack of knowledge about their effects in mixtures. The aim of this study was to evaluate combined effects on the singe celled organism Tetrahymena pyriformis. Using the MIXTOX model (EU project NOMIRACLE) synergistic or antagonistic effects with dose level deviation or dose ratio dependent deviation were analyzed. The most toxic compound gliotoxin (EC₅₀ 0.37 μM) and patulin (EC₅₀ 9.3 μM) as shown by the MIXTOX model acted synergistic, caused by similar mode of actions. Within the combination with sterigmatocystin (maximum inhibition of 45% at 12.5 μM) antagonistic effects were observed with switch to synergism if the toxicity of the mixture is mainly caused by sterigmatocystin. In the end the MIXTOX model was applicable for the prediction of combined effects of toxic compounds.     

What are the differences between aerobic and anaerobic toxic effects of sulfonamides on Escherichia coli?

Bacteria in the environment face the threat of antibiotics. However, most studies investigating the toxicity and toxicity mechanisms of antibiotics have been conducted on microorganisms in aerobic conditions, while studies examining the anaerobic toxicity and toxicity mechanisms of antibiotics are still limited. In this study, we determined the aerobic and anaerobic toxicities of sulfonamides (SAs) on Escherichia coli. Next, a comparison of the aerobic and anaerobic toxicities indicated that the SAs could be divided into three groups: Group I: log(1/EC_50-anaerobic) > log(1/EC_50-aerobic) (EC_50-anaerobic/EC_50-aerobic, the median effective concentration under anaerobic/aerobic conditions), Group II: log(1/EC_50-anaerobic) ≈ log(1/EC_50-aerobic), and Group III: log(1/EC_50-anaerobic) < log(1/EC_50-aerobic). Furthermore, this division was not based on the reactive oxygen species (ROS) level or the interaction energy (E_binding) value, which represents the affinity between SAs and dihydropteroate synthase (dhps) but rather on the total binding energy. Furthermore, SAs with greatly similar structures were categorized into different groups. This deep insight into the difference between aerobic and anaerobic toxicities will benefit environmental science, and the results of this study will serve as a reference for the risk assessment of chemicals in the environment.     

Toxicological effects of phenol on four marine microalgae

The toxic effects of phenol on four marine microalgae (Dunaliella salina, Platymonas subcordiformis, Phaeodactylum tricornutum Bohlin, and Skeletonema costatum) were evaluated. The 96 h EC₅₀ values were 72.29, 92.97, 27.32, and 27.32 mg L⁻¹, respectively, which were lower than those values of freshwater microalgae reported in the literature. During a 96-h exposure to a sub-lethal concentration of phenol (1/2 96 h EC₅₀) with green alga (D. salina) and diatom (S. costatum), reactive oxygen species (ROS) accumulation, and chlorophyll a (Chl a) content decrease were simultaneously observed in diatom cells after 48 h treatment. On the contrary, other chlorophylls in both algae were unaffected. Under transmission electron microscopy (TEM), the phenol-induced ultrastructure alterations included disappearance, or shrinkage, of nucleolus and enlargement of vacuoles, which may result in programmed cell death (PCD). The increase in number of lipid droplets may be related to phenol detoxification. These results indicate that the sensitivity of marine microalgae to phenol was dependent on some biotic factors such as cell size, ROS production, and phenol degradation ability in algal cells.     

Potential method to determine irritant potency in vitro – Comparison of two reconstructed epidermal culture models with different barrier competency

Testing chemicals for their ability to cause skin irritation is required for all ingredients of products that come into contact with the skin. Here, we describe a potential method for determining the irritant potency of a chemical in vitro and apply the method to two different reconstructed epidermis models which exhibit different barrier properties. Two surfactants: sodium dodecyl sulphate, Triton X100 and two non-surfactants: 2-4-di-nitro-chloro-benzene, cinnamaldehyde were applied topically in a dose response for 24 h. Biomarkers IL-1alpha, IL-1RA, IL-8 and MTT were assessed and EC₅₀ values determined. Variation in barrier properties between the epidermal models led to variation in the extent of penetration of surfactants, but not of non-surfactants which in turn influenced the EC₅₀ value obtained from surfactants. Furthermore, EC₅₀ values showed that no single biomarker could be classed as the most sensitive biomarker since biomarker sensitivity differed between the different chemicals studied. However, the ranking of the chemicals in order of strong to weak irritant was the same irrespective of the model used and also independent of the biomarker used (Triton X100 > DNCB > SDS > CA). This study describes a method which not only distinguishes an irritant from a non-irritant but which may possibly also be used to determine irritant potency.     

Bispyridinium non-oximes: An evaluation of cardiac effects in isolated hearts and smooth muscle relaxing effects in jejunum

Bispyridinium non-oximes seem to be promising candidates for the generic treatment of nerve agent poisoning as they interact with nicotinic and muscarinic acetylcholine receptors. The lead compound MB327 showed therapeutic effectiveness in vitro and in vivo but was toxic at higher doses. In the present study, the effect of various bispyridinium non-oximes on isolated heart and small intestine function was investigated. Bispyridinium non-oximes and oximes were tested in at least seven different concentrations in rat jejunum preparations pre-treated with carbachol. All bispyridinium non-oximes showed classical dose response curves with MB327 being the most effective (EC₅₀ = 6.6 μM) and MB782 being slightly less effective (EC₅₀ = 10.4 μM). Neither the bispyridinium non-oximes nor the oximes showed cardiotoxic effects in the isolated Langendorff heart. The tested bispyridinum compounds showed no direct cardiac effect but had variable smooth muscle relaxing effects. Further in vivo studies are required to get more insight into potential toxic mechanisms of these promising nerve agent antidotes.     

Antiviral evaluation of octadecyloxyethyl esters of (S)-3-hydroxy-2-(phosphonomethoxy)propyl nucleosides against herpesviruses and orthopoxviruses

Our previous studies showed that esterification of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) or 1-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]cytosine (HPMPC) with alkoxyalkyl groups such as hexadecyloxypropyl (HDP) or octadecyloxyethyl (ODE) resulted in large increases in antiviral activity and oral bioavailability. The HDP and ODE esters of HPMPA were shown to be active in cells infected with human immunodeficiency virus, type 1 (HIV-1), while HPMPA itself was virtually inactive. To explore this approach in greater detail, we synthesized four new compounds in this series, the ODE esters of 9-(S)-[3-hydroxy-2-(phosphonomethoxy)-propyl]guanine (HPMPG), 1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]thymine (HPMPT), 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2,6-diaminopurine (HPMPDAP) and 9-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-2-amino-6-cyclopropylaminopurine (HPMP-cPrDAP) and evaluated their antiviral activity against herpes simplex virus, type 1 (HSV-1), human cytomegalovirus (HCMV), and vaccinia, cowpox and ectromelia. Against HSV-1, subnanomolar EC₅₀ values were observed with ODE–HPMPA and ODE–HPMPC while ODE–HPMPG had intermediate antiviral activity with an EC₅₀ of 40 nM. In HFF cells infected with HCMV, the lowest EC₅₀ values were observed with ODE–HPMPC, 0.9 nM. ODE–HPMPA was highly active with an EC₅₀ of 3 nM, while ODE–HPMPG and ODE–HPMPDAP were also highly active with EC₅₀s of 22 and 77 nM, respectively. Against vaccinia and cowpox viruses, ODE–HPMPG and ODE–HPMPDAP were the most active and selective compounds with EC₅₀ values of 20–60 nM and selectivity index values of 600–3500. ODE–HPMPG was also active against ectromelia virus with an EC₅₀ value of 410 nM and a selectivity index value of 166. ODE–HPMPG and ODE–HPMPDAP are proposed for further preclinical evaluation as possible candidates for treatment of HSV, HCMV or orthopoxvirus diseases.     

Comparative analysis of goitrogenic effects of phenylthiourea and methimazole in zebrafish embryos

Craniofacial malformations, reduced locomotion and induction of genes encoding for enzymes involved in thyroid hormone synthesis were assessed using methimazole and N-phenylthiourea in zebrafish embryos. Gene expression, the most sensitive endpoint (EC_{50_MMI} = 372–765 μM, EC_{50_PTU} = 7.6–8.6 μM), was analysed in wild-type and in a transgenic strain, tg(tg:mCherry), expressing mCherry fluorescence protein under the control of the thyroglobulin gene. Reduction of locomotion and craniofacial malformations were observed at one or two orders of magnitude above concentrations affecting gene expression, respectively. Both effects could be linked to the malformations caused by reduced thyroxin levels. Our results show that due to the presence of the autoregulatory loop of the hypothalamus–pituitary–thyroid axis, various molecular initiating events of thyroid disruption are amenable for the zebrafish embryo. We propose the tg(tg:mCherry) bioassay as a sensitive tool in medium scale screening of goitrogens, given the minimal effort for sample preparation and analysis of gene expression.     

Development and validation of an OECD reproductive toxicity test guideline with the pond snail Lymnaea stagnalis (Mollusca,Gastropoda)

The OECD test guideline development program has been extended in 2011 to establish a partial life-cycle protocol for assessing the reproductive toxicity of chemicals to several mollusk species, including the great pond snail Lymnaea stagnalis. In this paper, we summarize the standard draft protocol for a reproduction test with this species, and present inter-comparison results obtained in a 56-day prevalidation ring-test using this protocol.Seven European laboratories performed semi-static tests with cultured snails of the strain Renilys® exposed to nominal concentrations of cadmium chloride (from 53 to 608 μg Cd L⁻¹). Cd concentrations in test solutions were analytically determined to confirm accuracy in the metal exposure concentrations in all laboratories. Physico-chemical and biological validity criteria (namely dissolved oxygen content >60% ASV, water temperature 20 ± 1 °C, control snail survival >80% and control snail fecundity >8 egg-masses per snail over the test period) were met in all laboratories which consistently demonstrated the reproductive toxicity of Cd in snails using the proposed draft protocol. Effect concentrations for fecundity after 56 days were reproducible between laboratories (68 < EC_50–56d < 124 μg L⁻¹) and were consistent with literature data. EC_50–56d and EC_10–56d values were comprised within a factor of 1.8 and 3.6, respectively, which is in the range of acceptable variation defined for reference chemicals in OECD test guidelines for invertebrates. The inter-laboratory reproducibility coefficient of variation (CV) for the Cd LC_50–56d values was 8.19%. The inter-laboratory comparison of fecundity within the controls gave a CV of 29.12%, while exposure to Cd gave a CV of 25.49% based on the EC_50–56d values. The OECD has acknowledged the success of this prevalidation exercise and a validation ring-test involving 14 laboratories in Europe, North- and South-America is currently being implemented using four chemicals (Cd, prochloraz, trenbolone and tributyltin).     

Toxicity of the veterinary sulfonamide antibiotic sulfamonomethoxine to five aquatic organisms

The purpose of this study was to investigate the acute and chronic toxicity of sulfamonomethoxine (SMM) to aquatic organisms to evaluate its impact at different trophic levels in the ecosystem. Regarding the growth inhibition of microalgae, SMM exhibited 72-h median effective concentration (EC₅₀) values of 5.9 mg L⁻¹ for freshwater Chlorella vulgaris and 9.7 mg L⁻¹ for marine Isochrysis galbana. In a study on the cladocerans, SMM exhibited acute toxicity and 48-h median lethal concentrations of 48 mg L⁻¹ for Daphnia magna and 283 mg L⁻¹ for D. similis. An examination of chronic toxicity revealed that SMM inhibited the brook production of the cladocerans and exhibited 21-day EC₅₀ values of 14.9 mg L⁻¹ for D. magna and 41.9 mg L⁻¹ for D. similis. This study investigated the potentially adverse effects of SMM on aquatic organisms and revealed that microalgae exhibited higher sensitivity to SMM than cladocerans did. The residue of SMM in water is recommended to be carefully evaluated to reduce ecological impacts after applied to cultured animals.     

Comparison of PrestoBlue and MTT assays of cellular viability in the assessment of anti-proliferative effects of plant extracts on human endothelial cells

IntroductionPrestoBlue (PB) is a new, simple and extremely fast live assay to monitor cell viability and cytotoxicity.Herein, we compared two in vitro cytotoxicity assays, new (PB) and classic (MTT), in the assessment of viability of human umbilical vein endothelial cells (HUVECs) in the presence of selected plant extracts.MethodsThe anti-proliferative effects of two extracts from medicinal plants, i.e., walnut husk extract and spent hop extract, used at the concentration range of 1–200 μg/ml of gallic acid equivalent, were compared with the effects recorded for resveratrol — a natural polyphenolic compound. Reduction of dyes by endothelial cells was determined colorimetrically (MTT and PB) and fluorometrically (PB).ResultsAt higher concentrations, all tested compounds caused significant loss of cell viability. Regardless of plant compound, the PB assay, when measured colorimetrically, produced higher EC₅₀ values compared to other modes of measurement, however, the statistically significant differences in EC₅₀ values among the assays were revealed only for spent hop extract. Conversely, the EC₅₀ values for each plant compound obtained in MTT (colorimetric assay) and PB (fluorometric assay) were similar. According to EC₅₀ values, the cytotoxicity of plant compounds ranked as follows: spent hop extract > resveratrol > walnut husk extract. Furthermore, the MTT assay showed overall lower inter-assay variability and higher signal-to-noise ratio compared to PB assay.DiscussionIn conclusion, we recommend fluorometric PrestoBlue assay for cytotoxicity assessment in human endothelial cells. Due to substantial differences in EC₅₀ values and S/N ratios between spectrophotometric PB and MTT or fluorometric PB assays, colorimetric quantification of HUVECs' viability with the use of PB reagent should be avoided.     

Exposure of mice to benzo(a)pyrene impairs endometrial receptivity and reduces the number of implantation sites during early pregnancy

Benzo(a)pyrene (BaP) is a ubiquitous environmental pollutant. Studies have demonstrated it to be an endocrine-disrupting chemical that can cause adverse effects on the female reproductive system. However, the effect of BaP on early pregnancy has not been reported. We investigated the effect of BaP on endometrial receptivity and embryo implantation. Pregnant mice were dosed with BaP at 0.2, 2 and 20 mg/kg/day from day 1 (D1) to day 5 (D5) of gestation. Exposure to BaP impaired the morphology of the endometrium and decreased the number of implantation sites (p_0.2 = 0.006, p₂ = 0.167, p₂₀ = 0.003). Levels of estrodiol (p < 0.001, for three treatment group compare with control group) and progesterone-4 in plasma were elevated in BaP-treatment groups (p_0.2 < 0.001, p₂ < 0.001, p₂₀ = 0.032). Expression of estrogen receptor-α was up-regulated (p_0.2 = 0.002, p₂ = 0.131, p₂₀ = 0.024) whereas expression of the progesterone receptor was down-regulated (p_0.2 < 0.001, p₂ = 0.064, p₂₀ = 0.021). Levels of receptivity-related genes HoxA10 (p_0.2 < 0.001, p₂ = 0.135, p₂₀ < 0.001) and E-cadherin (p_0.2 = 0.002, p₂ = 0.624, p₂₀ = 0.137) were changed by BaP. These results revealed that BaP can disrupt the balance of estrogen and progesterone, influence expression of their receptors and downstream related genes, lead to changes in endometrium receptivity, and reduce of the number of implantation sites.     

Contractile effects of endothelins on isolated ampullar segment of human oviduct in luteal phase of menstrual cycle

Endothelin-1 induces contractions of human oviduct ampullar segment in follicular phase of menstrual cycle, acting on ET_A receptors. The aim of our study was to investigate effects of endothelin-1, endothelin-2 and endothelin-3 on isolated ampullar segment of human oviducts, taken from the patients in luteal phase of menstrual cycle. Fallopian tubes were taken from 20 female patients (one tube from each patient) during abdominal hysterectomy with adnexectomy, due to extensive uterine fibroids. The oviduct ampulla was mounted in an organ bath longitudinally, and the tension of the isolated preparation was recorded with the isometric transducer. Endothelin-1 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 6.80 ± 1.2 × 10^?10 M), and concentration-dependent inhibition of its rhythmic contractions (EC₅₀ = 7.86 ± 2.3 × 10^?10 M). Endothelin-2 produced concentration-dependent tonic contraction of the isolated ampullar segment (EC₅₀ = 4.56 ± 0.3 × 10^?10 M), without affecting its rhythmic contractions. Endothelin-3 did not affect either tone or rhythmic contractions of the isolated preparations. Selective antagonist for ET_A receptor subtype, BQ 123, produced inhibition of endothelin-1 effects on both tone (pA₂ = 9.50) and spontaneous rhythmic contractions (pA₂ = 10.73), while selective antagonist for ET_B receptor subtype, BQ 788, produced only inhibition of endothelin-1 effects on tone (pA₂ = 9.61), while the effect of endothelin-1 on spontaneous rhythmic contractions remained unaffected. The results of our study suggest that in the luteal phase both ET_A and ET_B receptors regulate tone, and only ET_A receptors regulate rhythmic activity of human oviduct's ampullar segment.     

Effects of un-ionized ammonia on histological,endocrine, and whole organism endpoints in slimy sculpin (Cottus cognatus)

Ammonia is known to be an important toxicant in aquatic environments. Although ammonia toxicity has been well studied in many fish species, effects of chronic exposure on slimy sculpin (Cottus cognatus), a critical biomonitoring species for northern aquatic habitats, are not well known. Further, with increasing mining development in Canada's north, this information is critical to better predict potential effects of mine effluent discharges on northern fish species. Slimy sculpin were exposed to six concentrations of un-ionized ammonia (NH₃) relevant to concentrations found in northern mining effluents: control (0 ppm), 0.278 ppm, 0.556 ppm, 0.834 ppm, 1.112 ppm, and 1.668 ppm. An LC₅₀ of 1.529 ppm was calculated from mortality data. Histopathological examination of gills indicated significant tissue damage, measured as lamellar fusion and epithelial lifting, at 0.834 ppm, 1.112 ppm, and 1.668 ppm. Using gill endpoints, NOEC and LOEC were calculated as 0.556 ppm and 0.834 ppm, respectively. An EC₅₀ of 0.775 ppm was determined for lamellar fusion and an EC₅₀ of 0.842 ppm for epithelial lifting. Hemorrhage of gills was present in mortalities, which occurred at 1.668 ppm of un-ionized ammonia. A significant decrease in liver somatic index (LSI) was seen in both male and female fish at 0.834 ppm and 1.112 ppm, respectively. Gonadosomatic index (GSI) in female fish significantly increased at 1.668 ppm un-ionized ammonia with an associated significant increase in total wholebody testosterone concentrations. GSI in male fish also significantly increased at 1.668 ppm but no differences were seen in testosterone concentrations. No significant differences were seen in gonad histopathological assessments or condition factor. Gill histopathology endpoints may be a more sensitive indicator for detecting effects in slimy sculpin exposed to ammonia than traditional chronic endpoints. Results from this study indicate that ammonia concentrations commonly reported in northern mine effluents hold potential to affect the health of slimy sculpin including acute, chronic, histological and endocrine endpoints.     

In vitro characterization and pharmacokinetics in mice following pulmonary delivery of itraconazole as cyclodextrin solubilized solution

This study aims to make a 2-hydroxypropyl-β-cyclodextrin (HPβCD) solubilized itraconazole (ITZ) solution (i.e., HPβCD-ITZ) suitable for pulmonary delivery by nebulization, and compare pharmacokinetics of inhaled nebulized aerosols of HPβCD-ITZ versus a colloidal dispersion of ITZ nanoparticulate formulation (i.e., URF-ITZ). Solid state characterizations of lyophilized HPβCD-ITZ by differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS) indicated the formation of dynamic inclusion complexes between ITZ and HPβCD. Nebulized aerosols of both HPβCD-ITZ and colloidal dispersion of URF-ITZ were confirmed suitable for deep lung delivery. Single doses of the nebulized aerosols (equivalent to 5.3 mg ITZ/mL in 5 mL) in mice produced similar ITZ lung depositions and pharmacokinetic profiles, with ITZ lung levels of approximately 4 μg/g wet lung weight upon completion of nebulization and remained above 0.5 μg/g at 24 h. HPβCD-ITZ demonstrated faster systemic absorption of ITZ across lung epithelium than URF-ITZ, with t_max values of 1.5 and 3.0 h, and AUC_0–∞ of 2513 and 3717 ng h/mL, respectively. The fast absorption of solubilized ITZ across lung mucosal surface may be due in part to the elimination of the phase-to-phase transition.     

Oxidation of siloxanes during biogas combustion and nanotoxicity of Si-based particles released to the atmosphere

Siloxanes have been detected in the biogas produced at municipal solid waste landfills and wastewater treatment plants. When oxidized, siloxanes are converted to silicon oxides. The objectives of this study were to evaluate the transformation of siloxanes and potential nanotoxicity of Si-based particles released to the atmosphere from the gas engines which utilize biogas. Data available from nanotoxicity studies were used to assess the potential health risks associated with the inhalation exposure to Si-based nanoparticles. Silicon dioxide formed from siloxanes can range from 5 nm to about 100 nm in diameter depending on the combustion temperature and particle clustering characteristics. In general, silicon dioxide particles formed during from combustion process are typically 40–70 nm in diameter and can be described as fibrous dusts and as carcinogenic, mutagenic, astmagenic or reproductive toxic (CMAR) nanoparticles. Nanoparticles deposit in the upper respiratory system, conducting airways, and the alveoli. Size ranges between 5 and 50 nm show effective deposition in the alveoli where toxic effects are higher. In this study the quantities for the SiO₂ formed and release during combustion of biogas were estimated based on biogas utilization characteristics (gas compositions, temperature). The exposure to Si-based particles and potential effects in humans were analyzed in relation to their particle size, release rates and availability in the atmosphere. The analyses showed that about 54.5 and 73 kg/yr of SiO₂ can be released during combustion of biogas containing D4 and D5 at 14.1 mg/m³ (1 ppm) and 15.1 mg/m³ (1 ppm), respectively, per MW energy yield.     

Determination of dynamic doxorubicin-EC50 value in an automated high-content workstation for cellular assays

To overcome the problems of endpoint tests routinely required for EC₅₀ determination, we utilized a novel automated high-content workstation and calculated a time-resolved EC₅₀ value of MCF-7 mamma carcinoma cells treated with a pharmacologic agent. Measuring parameters were the cellular oxygen consumption and the extracellular acidification. These parameters were detected in real-time and label free with optochemical sensor spots in a modified 24-well sensor plate. In particular, the objective was to compare the measuring data of the workstation with a classical standard resazurin cell assay and to transfer the benefit of continuously recorded metabolic data of the workstation to practical time-resolved information about the effect of the applied active reagent (doxorubicin). MCF-7 cells were treated with a broad range of doxorubicin concentrations (100 μM to 1 nM) over 24 h and cellular activities were investigated with both, the resazurin assay and the automated workstation. Twenty-four hours after treatment, the resazurin assay showed an EC₅₀ value (6.3 μM) which was about one decade above the value obtained from oxygen consumption rate (0.37 μM) and extracellular acidification rate (0.72 μM), measured with the workstation. Presumably, the differences are due to the different metabolic nature and regulation behind these measuring parameters. By polynomial fitting of continuously recorded metabolic data, we were able to point out a dynamic, time-resolved EC₅₀ characteristic for different time points. The workstation is a powerful tool to record in vitro kinetic data of pharmacologic effects in vital cells in an automated experimental run.     

Efficacy and dosing of antidotes applied to Daphnia intoxicated by nerve agent tabun

A new assay with Daphnia, which can be used as a time, cost, and human effort-saving tool in the development of effective antidotes against organophosphate intoxications, is presented. Five concentrations of atropine (antimuscarinic anticholinergics) as well as a reactivator (trimedoxime) were tested to define the optimal dosage. Various reactivators (trimedoxime, obidoxime) were used to examine difference in effectivity of treatments. The most effective dose of trimedoxime corresponded to the 75% of its EC₅₀(24) value, i.e. 77.85 mg l⁻¹. The most effective dose of atropine corresponded to the 25% of its EC₅₀(24) value, i.e. 104.70 mg l⁻¹. The most effective treatment was a combined atropine-obidoxime treatment, followed by the combined atropine-trimedoxime treatment, the atropine only and the obidoxime only treatments. The efficacy of the trimedoxime only treatment was doubtful. The surprisingly high efficacy of obidoxime in the obidoxime only treatment indicates that some oximes might act in daphnids not just as reactivators but also by some other mechanisms.     

Genetic modification of ALAD and VDR on lead-induced impairment of hearing in children

Polymorphisms in the δ-aminolevulinic acid dehydratase (ALAD) and the vitamin D receptor (VDR) genes may modify lead metabolism and neurotoxicity.Two cohorts of children were examined for hearing [pure-tone audiometry (PTA), brain stem auditory evoked potentials (BAEP)], acoustic otoemission (transient emission evoked by a click) and blood-lead concentrations (B–Pb). The children were genotyped for polymorphisms in ALAD and VDR.The median B–Pbs were 55 and 36 μg/L in the two cohorts (merged cohort 45 μg/L). B–Pb was significantly associated with impaired hearing when tested with PTA (correlation coefficient r_S = 0.12; P < 0.01), BAEP (r_S = 0.18; P < 0.001) and otoemission (r_S = −0.24; P < 0.001).VDR significantly modified the lead-induced effects on PTA. Carriers of the VDR alleles BsmI B, VDR TaqI t and VDR FokI F showed greater toxic effects on PTA, compared to BsmI bb, VDR TaqI TT and VDR FokI ff carriers. No significant interaction was found for ALAD.Lead impairs hearing functions in the route from the cochlea to the brain stem at low-level exposure, and polymorphisms in VDR significantly modify these effects.     

GABAA receptor modulation by piperine and a non-TRPV1 activating derivative

The action of piperine (the pungent component of pepper) and its derivative SCT-66 ((2E,4E)-5-(1,3-benzodioxol-5-yl))-N,N-diisobutyl-2,4-pentadienamide) on different gamma-aminobutyric acid (GABA) type A (GABA_A) receptors, transient-receptor-potential-vanilloid-1 (TRPV1) receptors and behavioural effects were investigated.GABA_A receptor subtypes and TRPV1 receptors were expressed in Xenopus laevis oocytes. Modulation of GABA-induced chloride currents (I_GABA) by piperine and SCT-66 and activation of TRPV1 was studied using the two-microelectrode-voltage-clamp technique and fast perfusion. Their effects on explorative behaviour, thermoregulation and seizure threshold were analysed in mice. Piperine acted with similar potency on all GABA_A receptor subtypes (EC₅₀ range: 42.8 ± 7.6 μM (α₂β₂)–59.6 ± 12.3 μM (α₃β₂)). I_GABA modulation by piperine did not require the presence of a γ_2S-subunit, suggesting a binding site involving only α and β subunits. I_GABA activation was slightly more efficacious on receptors formed from β_2/3 subunits (maximal I_GABA stimulation through α₁β₃ receptors: 332 ± 64% and α₁β₂: 271 ± 36% vs. α₁β₁: 171 ± 22%, p < 0.05) and α₃-subunits (α₃β₂: 375 ± 51% vs. α₅β₂:136 ± 22%, p < 0.05). Replacing the piperidine ring by a N,N-diisobutyl residue (SCT-66) prevents interactions with TRPV1 and simultaneously increases the potency and efficiency of GABA_A receptor modulation. SCT-66 displayed greater efficacy on GABA_A receptors than piperine, with different subunit-dependence. Both compounds induced anxiolytic, anticonvulsant effects and reduced locomotor activity; however, SCT-66 induced stronger anxiolysis without decreasing body temperature and without the proconvulsive effects of TRPV1 activation and thus may serve as a scaffold for the development of novel GABA_A receptor modulators.     

Inhibitory effect of Nymphaea pubescens Willd. flower extract on carrageenan-induced inflammation and CCl4-induced hepatotoxicity in rats

Nymphaea pubescens Willd. is used as ingredient of ethnic diet and folk medicine in South-East Asia. The water (NPW), methanol (NPM) and chloroform (NPC) extracts of N. pubescens flowers were investigated for NO, ${\text{O}}_2^{\text{<mglyph src="https://sdfestaticassets-us-east-1.sciencedirectassets.com/shared-assets/16/entities/rad"></mglyph>}-}$ and DPPH radical scavenging and iron chelating activities in vitro. NPW was found to be the most potent free radical scavenger (EC₅₀ < 100 μg/mL) whereas NPC did not show EC₅₀ at 500 μg/mL. Therefore, NPW was selected for further studies on anti-inflammatory and hepatoprotective activities, using standard in vitro and in vivo models. NPW exhibited inhibition of nitrogen radical generation in LPS-activated macrophages (IC₅₀ = 75.5 μg/mL) through suppression of iNOS protein, with no associated toxicity in the cells. Further, 500 mg/kg of NPW reduced rat paw edema by ∼50% after 6 h of carrageenan administration. Hepatoprotective activity of NPW was also evaluated in vivo on CCl4-induced hepatotoxicity model in rats. NPW treatment (500 mg/kg/day for ten days) attenuated CCl4-induced increase in serum enzymes, viz. alanine and aspartate aminotransferases (ALT and AST) and bilirubin. Also, glutathione and superoxide dismutase (SOD)-levels were restored towards normalcy in the liver of CCl4-treated rats, indicating the hepatoprotective role of NPW, which was found to contain a fair amount of flavonoids, phenolics, and saponin constituents.