The effects of dietary nickel exposure on growth and reproduction of Daphnia magna

Although there is growing evidence that dietborne metals can be toxic to various aquatic species, there is still insufficient knowledge to integrate this information in environmental risk assessment procedures. In this study, we investigated the effects of a 21-day exposure of Daphnia magna to a control diet (i.e. the green alga Pseudokirchneriella subcapitata containing <4.0 μg Ni/g dry wt) and five diets with elevated Ni concentrations (i.e. the same alga contaminated with Ni burdens between 33.7 and 837 μg Ni/g dry wt). A significant accumulation of dietborne Ni in D. magna, i.e. between 49.6 and 72.5 μg Ni/g dry wt, was observed when they were fed with diets containing between 85.6 and 837 μg Ni/g dry wt. This was paralleled by a significant reduction of reproduction (by 33.1%), measured as the total number of juvenile offspring per female and growth (by 9.1%), measured as the carapax length of 21-day-old females. Life-history analysis showed that the time to first brood of Ni exposed organisms was between 7.8 and 8.2 days, and occurred 0.7–1.1 days earlier than for the control organisms (time to first brood = 8.9 days). The number of offspring in the first brood was significantly reduced (by 21–33% compared to the control) in all dietary treatments. Longer exposure (≥8.9 days, i.e. from the second brood onwards) led to a reduction of brood size only when given diets containing 85.6 and 837 μg Ni/g dry wt. The results suggest that a variety of mechanisms may be involved in the effects of dietary Ni exposure, including altered resource allocation or targeted reproductive inhibition. While Ni exposure clearly altered the quality of the diet (measured as essential ω3 polyunsaturated fatty acid content and C:P ratio), we found no conclusive evidence that these diet quality shifts could have affected growth or total reproductive output. More research is required to fully understand the mechanisms of Ni toxicity associated with the dietary exposure route.     

In vitro antioxidant activity and total phenolic content of ethanolic leaf extract of Stevia rebaudiana Bert.

The aim of this study was to assess the in vitro potential of ethanolic leaf extract of Stevia rebaudiana as a natural antioxidant. The DPPH activity of the extract (20, 40, 50, 100 and 200 μg/ml) was increased in a dose dependent manner, which was found in the range of 36.93–68.76% as compared to ascorbic acid 64.26–82.58%. The IC₅₀ values of ethanolic extract and ascorbic acid in DPPH radical scavenging assay were obtained to be 93.46 and 26.75 μg/ml, respectively. The ethanolic extract was also found to scavenge the superoxide generated by EDTA/NBT system. Measurement of total phenolic content of the ethanolic extract of S. rebaudiana was achieved using Folin–Ciocalteau reagent containing 61.50 mg/g of phenolic content, which was found significantly higher when compared to reference standard gallic acid. The ethanolic extract also inhibited the hydroxyl radical, nitric oxide, superoxide anions with IC₅₀ values of 93.46, 132.05 and 81.08 μg/ml, respectively. However, the IC₅₀ values for the standard ascorbic acid were noted to be 26.75, 66.01 and 71.41 μg/ml respectively. The results obtained in this study clearly indicate that S. rebaudiana has a significant potential to use as a natural antioxidant agent.     

Biochemical indices of stress in the sandworm neanthes virens (Sars). I. responses to pentachlorophenol

Neanthes virens (Sars) were exposed to acute lethal and chronic sublethal concentrations of pentachlorophenol (PCP) in the laboratory. Coelomic fluid glucose and osmolality and tissue ascorbic acid and glycogen were monitored in order to assess the time-course response of this polychaete to pollutant stress. Glycemic responses and alterations in tissue ascorbic acid and glycogen levels were observed to be dose and time-dependent.During the acute lethal exposure (720 μg/I initially), a hypoglycemic response was evident during the first 8 h of exposure. At a less severe but still lethal exposure concentration (365 μg/I initially), a significant hyperglycemic response was observed after 21 h. The coelomic fluid glucose levels decreased gradually during the 4-day exposure as glycogen reserves were depleted. During the chronic (ca. 2-mth) exposure to 100 μg PCP/1. no unusual glycemic responses were observed. Glycogen reserves remained constant during the first month of exposure but were significantly depleted after 2 mth. Ascorbic acid levels became elevated in PCP-exposed worms after I wk in both parapodial tissue and posterior segments. This tendency continued throughout the exposure period and the difference between the exposed and control values was statistically significant at the termination of the experiment.An uptake and depuration experiment with¹⁴C-PCP was also conducted. Polychaetes exposed to 100 μg/l¹⁴C-PCP for 2 wk accumulated ca. 280 times the ambient exposure concentration on a wet weight basis. After 2 wk of depuration the mean PCP body burden of depurated animals had decreased by 29%.     

Absorption of andrographolides from Andrographis paniculata and its effect on CCl4-induced oxidative stress in rats

A simple and validated high-performance liquid chromatography (HPLC) method with UV detection has been used to determine the content of andrographolide (AP) and 14-deoxy-11,12-didehydroandrographolide (DIAP) in rat plasma after oral dose of methanol extract (1 g/kg body weight) of Andrographis paniculata leaf. An increase in plasma concentration of AP and DIAP was observed from 30 min to 3 h after oral administration of the extract. The maximum plasma concentrations of AP and DIAP were 1.42 ± 0.09 μg/ml and 1.31 ± 0.04 μg/ml, respectively. Fourteen days oral treatment of rats with the methanol extract (1 g/kg body weight) followed by CCl₄ administration preserved catalase (CAT), and superoxide dismutase (SOD) activities in erythrocytes, whereas plasma lipid peroxidation, alanine transaminase (ALT) and aspartate transaminase (AST) activities were restored to values comparable with control values. Treatment of rats with CCl₄ did not showed significant alteration (p > 0.05) in plasma total antioxidant status (TAS) as compare to values of control group.     

Comparative evaluation of the hypolipidemic effects of coconut water and lovastatin in rats fed fat–cholesterol enriched diet

The coconut water presents a series of nutritional and therapeutic properties, being a natural, acid and sterile solution, which contains several biologically active components, l-arginine, ascorbic acid, minerals such as calcium, magnesium and potassium, which have beneficial effects on lipid levels. Recent studies in our laboratory showed that both tender and mature coconut water feeding significantly (P < 0.05) reduced hyperlipidemia in cholesterol fed rats [Sandhya, V.G., Rajamohan, T., 2006. Beneficial effects of coconut water feeding on lipid metabolism in cholesterol fed rats. J. Med. Food 9, 400–407]. The current study evaluated the hypolipidemic effect of coconut water (4 ml/100 g body weight) with a lipid lowering drug, lovastatin (0.1/100 g diet) in rats fed fat–cholesterol enriched diet ad libitum for 45 days. Coconut water or lovastatin supplementation lowered the levels of serum total cholesterol, VLDL + LDL cholesterol, triglycerides and increased HDL cholesterol in experimental rats (P < 0.05). Coconut water feeding decreased activities of hepatic lipogenic enzymes and increased HMG CoA reductase and lipoprotein lipase activity (P < 0.05). Incorporation of radioactive acetate into free and ester cholesterol in the liver were higher in coconut water treated rats. Coconut water supplementation increased hepatic bile acid and fecal bile acids and neutral sterols (P < 0.05). Coconut water has lipid lowering effect similar to the drug lovastatin in rats fed fat–cholesterol enriched diet.     

In vitro analyses of the combination of high-dose doxycycline and antifungal agents against Candida albicans biofilms

The potential of antifungal agents used as antimicrobial lock therapy (ALT) for the conservative management of catheter-related candidemia has not been fully defined. We sought to determine the antifungal effect of high-dose doxycycline (DOX), alone or in combination with standard concentrations of amphotericin B (AMB), caspofungin (CAS) or fluconazole (FLC), against biofilms formed by Candida albicans in vitro. DOX alone (at 2048 μg/mL and 1024 μg/mL) demonstrated up to an 85% reduction of the metabolic activity of the C. albicans biofilm. Regardless of the concentration tested, FLC alone showed minimal activity (mean 22.9% reduction) against the C. albicans biofilm. When DOX 2048 μg/mL was used in combination with FLC, antifungal activity also increased up to 85%, suggesting an additive effect. DOX 128 μg/mL in combination with FLC demonstrated synergy (mean 58.3% reduction). The combination of DOX 2048 μg/mL or 512 μg/mL and AMB was superior to AMB alone at low concentrations (0.25–0.03125 μg/mL). However, DOX 128 μg/mL was antagonistic in combination with low concentrations of AMB. Maximal efficacy against the biofilm was observed with CAS at 8–0.25 μg/mL compared with FLC and AMB alone. A paradoxical effect (PE) occurred with CAS at 16 μg/mL, which showed a marked reduction in antifungal activity compared with lower concentrations of CAS. CAS at 16 μg/mL in combination with either DOX 2048 μg/mL or 512 μg/mL resulted in attenuation of the PE. These findings suggest that a high-dose DOX-based ALT strategy in combination with traditional antifungal agents may be useful for the treatment of C. albicans biofilms.     

Purification and characterization of a new l-amino acid oxidase from Daboia russellii siamensis venom

A new l-amino acid oxidase (designated as DRS-LAAO) was purified from Daboia russellii siamensis venom by ion-exchange, gel filtration and affinity chromatographies. DRS-LAAO is a homodimeric enzyme with a molecular weight of 120.0 kDa as measured by size exclusion chromatography and the monomeric molecular weight of 58.0 kDa as measured by SDS-PAGE under both non-reducing and reducing conditions. The N-terminal amino acid sequence (ADDKNPLEECFREDD) of DRS-LAAO shares high identity with other snake venom l-amino acid oxidases, especially with those isolated from viperid venoms. The enzyme displayed high specificity towards hydrophobic l-amino acids. The best substrate of DRS-LAAO was L-Leu followed by L-Phe and L-Ile, while five substrates — L-Pro, L-Asn, L-Gly, L-Ser and L-Cys were not oxidized. Optimal pH of DRS-LAAO was 8.8. The enzyme showed no hemorrhagic activity even at a dosage of 55.0 μg. DRS-LAAO dose-dependently inhibited platelet aggregation induced by ADP (83.33 μM) and TMVA (55.0 nM) with an IC₅₀ value of 32.8 μg/ml and 32.3 μg/ml, respectively. The minimum inhibitory concentrations (MICs) of DRS-LAAO against Staphylococci aureus (ATCC 25923), Pseudomonas aeruginosa (ATCC 27853) and Escherichia coli (ATCC 25922) were 9.0, 144.0 and 288.0 μg/ml, respectively. The minimum bactericidal concentrations (MBCs) of the enzyme for these strains were twice of the MIC values. These results showed that DRS-LAAO had the strongest antimicrobial activity against S. aureus among these three international standard stains. Antibacterial-activities of DRS-LAAO against eight clinical methicillin-resistant Staphylococcus aureus (MRSA) isolates were also tested. The MICs of DRS-LAAO against these isolates ranged from 4.5 to 36.0 μg/ml. And the MBCs of the enzyme against these isolates ranged from 9.0 to 72.0 μg/ml.     

Systematic evaluation of the antioxidant potential of different parts of Foeniculum vulgare Mill. from Portugal

Fennel (Foeniculum vulgare Mill.) is a widespread perennial umbeliferous (Apiaceae) herb, traditionally used for medicinal purposes and human consumption. It is highly recommended for diabetes, bronchitis and chronic coughs, and for the treatment of kidney stones; some of those chronic diseases are related to the production of radical species involved in the oxidative stress. Therefore, the antioxidant potential of this herb might explain some of their empirical uses in folk medicine. This is the first time that a systematic study on different parts of fennel is performed, in order to understand differences in the antioxidant potential of shoots, leaves, steams, and inflorescences, particularly related to their composition in antioxidant compounds such as vitamins (ascorbic acid and tocopherols) and phenolics. The shoots seems to have the highest radical-scavenging activity and lipid peroxidation inhibition capacity (EC₅₀ values < 1.4 mg/ml), which is in agreement with the highest content in phenolics (65.85 ± 0.74 mg/g) and ascorbic acid (570.89 ± 0.01 μg/g) found in this part. The shoots also revealed high concentration of tocopherols (34.54 ± 1.28 μg/g) and were the only part with flavonoids.     

Group 1 metabotropic glutamate receptors contribute to slow-onset potentiation in the rat CA1 region in vivo

It has been demonstrated in the CA1 region of the hippocampus in vitro, and in the dentate gyms and CA1 region in vivo, that application of the metabotropic glutamate receptor (mGluR) agonist, 1S, 3R-amino cyclopentane 2,3-dicarboxylic acid triggers a slow-onset potentiation of synaptic transmission in the hippocampus. This study examined the involvement of group 1 and 2 mGluRs in this phenomenon in the CA1 region of freely moving rats. Drugs were applied via the lateral cerebral ventricle, and measurements were obtained from the CA1 region via permanently implanted electrodes. The group 1 mGluR agonists, 3,5-dihydroxyphenylglycine (DHPG, 20–100 nmol/5 μl) and trans-azetidine-2,4-dicarboxylic acid (ADA, 100 nmol-1 μmol/5 μl) induced a dose-dependent potentiation of basal synaptic transmission. The mGluR antagonist R,S-α-methyl-carboxyphenylglycine (MCPG, 1 μmol), and the group1 mGluR antagonist, S-4- carboxyphenylglycine (4CPG, 100 nmol) competely inhibited the effects of both DHPG and ADA. The group 2 mGluR agonist, (S)-4-carboxy-3-hydroxy phenylglycine (4C3H-PG, 50–200 nmol/5 μl) induced a dosedependent decrease of basal synaptic transmission. These results suggest that in the CA1 region in vivo, slowonset potentiation may be mediated by group 1 mGluRs.     

Separation and determination of coumarins in Fructus cnidii extracts by pressurized capillary electrochromatography using a packed column with a monolithic outlet frit

The pressurized capillary electrochromatography (pCEC) was utilized for the separation and determination of coumarins in Fructus cnidii extracts from 12 different regions. After a thorough study of analytical parameters such as acetonitrile content of the mobile phase, the concentration and pH of the buffer, and the applied voltage, a methodology was proposed to separate and determine six coumarins of F. cnidii extracts in less than 15 min. The experiments were performed in an in-house packed column with a monolithic outlet frit under the optimal conditions: pH 4.0 ammonium acetate buffer at 10 mM containing 50% acetonitrile at −6 kV applied voltage. The calibration curves were linear in the range of 10.0–100.0 μg/mL for bergapten, 20.0–200.0 μg/mL for imperatorin, 5.0–400.0 μg/mL for osthole, 10.0–100.0 μg/mL for 2′-acetylangelicin, 10.0–200.0 μg/mL for oroselone, and 10.0–200.0 μg/mL for O-acetylcolumbianetin. The correlation coefficients were between 0.9967 and 0.9995. With this pCEC system, fingerprints of F. cnidii extracts were preliminarily established to distinguish three types of coumarins by characteristic peaks, and the quality of various sources of raw materials was evaluated by determining the contents of six coumarins.     

Antioxidant and antidermatophytic activities of essential oil and extracts of Magnolia liliflora Desr.

This study was carried out to assess the antioxidant and antidermatophytic activities of the essential oil and extracts of Magnolia liliflora Desr. Antioxidant activity was evaluated by using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. The free radical scavenging activities of the oil and ethyl acetate extract were found to be superior (IC₅₀ values = 10.11 and 16.17 μg/ml, respectively) as compared to butylatedhydreoxyanisole (BHA), (IC₅₀ value = 18.27 μg/ml). Also the ethyl acetate extract revealed the highest phenolic contents (96.13 mg/g of dry wt) as compared to the other extracts. Further, the oil (1000 μg/disc) and extracts (1500 μg/disc) revealed 42.36–63.12% and 19.07–54.14% antidermatophytic effect, respectively along with their respective MIC values ranging from 62.5 to 500 and 250 to 2000 μg/ml against the members of Trichophyton and Microsporum spp. Also the oil had strong detrimental effect on spore germination of tested fungal pathogens as well as concentration and time dependent kinetic inhibition of Microsporum canis KCTC 6348. The results of this study justify a potential role of M. liliflora to serve as a natural antioxidant and antidermatophytic agent.     

Effects of Tityus serrulatus scorpion venom on lung mechanics and inflammation in mice

The present study evaluated the effects of an intramuscular injection of Tityus serrulatus venom (TsV) (0.67 μg/g) on lung mechanics and lung inflammation at 15, 30, 60 and 180 min after inoculation. TsV inoculation resulted in increased lung elastance when compared with the control group (p < 0.001); these values were significantly higher at 60 min than at 15 and 180 min (p < 0.05). Resistive pressure (ΔP₁) values decreased significantly at 30, 60 and 180 min after TsV injection (p < 0.001). TsV inoculation resulted in increased lung inflammation, characterised by an increased density of mononuclear cells at 15, 30, 60 and 180 min after TsV injection when compared with the control group (p < 0.001). TsV inoculation also resulted in an increased pulmonary density of polymorphonuclear cells at 15, 30 and 60 min following injection when compared to the control group (p < 0.001). In conclusion, T. serrulatus venom leads to acute lung injury, characterised by altered lung mechanics and increased pulmonary inflammation.     

The occurrence of domoic acid linked to a toxic diatom bloom in a new potential vector: The tunicate Pyura chilensis (piure)

The tunicate Pyura chilensis (Molina, 1782); Phylum Chordata; Subphylum Urochordata; Class Ascidiacea, common local name “piure” or sea squirt; a filter-feeder (plankton and suspended particles) sessile species; may play an important role in monitoring domoic acid (DA) the principal toxic component of Amnesic Shellfish Poisoning (ASP). Significant DA concentrations have been determined in tunicate samples, collected during a recent ASP outbreak in Bahía Inglesa, an important scallop (Argopecten purpuratus) farming area. Several infaunal species were tested for the presence of DA, in addition to the usual scallop monitoring programme. DA was found at sub-toxic levels in filtering bivalves such as mussels (Mytilus chilensis), large mussels (Aulacomya ater) and clams (Protothaca thaca) (6.4, 5.4 and 4.7 μg DA/g tissue respectively). Of particular interest was the observation of significant accumulations of toxic Pseudo-nitzschia sp. diatoms in the internal siphon and atrium spaces of the tunicate.Toxin distribution within major tunicate organs was heterogeneous with 8.7–15.5 μg DA/g in edible tissues, 14.9–17.9 μg DA/g in the fecal material and 13.6–32.7 μg DA/g in the gut content. DA was determined by HPLC–UV and confirmed by diode-array detection and LC–MS/MS analysis. This is the first report of the presence of DA in a tunicate that is regularly consumed by coastal populations. These results confirm the need to include these organisms in sanitation programs for marine toxins.     

Antioxidant activities, metal contents, total phenolics and flavonoids of seven Morchella species

Seven Morchella species were analyzed for their antioxidant activities in different test systems namely β-carotene/linoleic acid, DPPH, reducing power, chelating effect and scavenging effect (%) on the stable ABTS⁺, in addition to their heavy metals, total phenolic and flavonoid contents. In β-carotene/linoleic acid system, the most active mushrooms were M. esculenta var. umbrina and M. angusticeps. In the case of DPPH, methanol extract of M. conica showed high antioxidant activity. The reducing power of the methanol extracts of mushrooms increased with concentration. Chelating capacity of the extracts was also increased with the concentration. On the other hand, in 40 μg ml⁻¹ concentration, methanol extract of M. conica, exhibited the highest radical scavenging activity (78.66 ± 2.07%) when reacted with the ABTS⁺ radical. Amounts of seven elements (Cu, Mn, Co, Zn, Fe, Ca, and Mg) and five heavy metals (Ni, Pb, Cd, Cr, and Al) were also determined in all species. M. conica was found to have the highest phenolic content among the samples. Flavonoid content of M. rotunda was also found superior (0.59 ± 0.01 μg QEs/mg extract).     

Development and validation of a capillary electrophoresis method for the simultaneous determination of impurities of escitalopram including the R-enantiomer

A stereospecific capillary electrophoresis assay for the simultaneous determination of related substances and the enantiomeric purity of escitalopram was developed by a central composite face-centered factorial design and subsequently validated. Separations were carried out in a 50 μm, 47/40 cm fused-silica capillary. The optimized conditions included 20 mM phosphate buffer, pH 2.5, containing 0.5 mg/ml β-cyclodextrin and 22 mg/ml sulfated β-cyclodextrin as background electrolyte, an applied voltage of −20 kV and a temperature of 28 °C. Salicylic acid was used as internal standard. The assay was validated for the (R)-enantiomer of citalopram and the enantiomers of the impurity citadiol in the range of 2.5–150 μg/ml and 2.5–50 μg/ml, respectively. The limit of detection was 0.02% for all compounds, the limit of quantitation 0.05%, relative to a concentration of escitalopram of 5 mg/ml. Intraday precision of migration time and peak area ratio were in the range of 0.17–0.44% and 1.64% and 6.25%, respectively. Relative standard deviations of interday precision ranged between 0.84% and 1.85% in the case of migration times and between 5.20% and 9.28% for peak area ratio. The assay was applied to the determination of the purity of escitalopram in bulk drug and tablets. (R)-Citalopram and (S)-citadiol were detected as impurities.     

Developmental anomalies induced by a non-selective COX inhibitor (ibuprofen) in zebrafish (Danio rerio)

Effects of ibuprofen (a non-selective COX inhibitor) on the embryonic development, hatching success, larval growth, behavioral pattern and survival competence were studied in Danio rerio. Embryos at 2/4 celled stage were exposed to graded doses (0, 1, 5, 10, 50 and 100 μg/L distilled water) of ibuprofen in triplicate sets (n = 30). The experiment was repeated thrice. The results indicate that developing embryos tolerated lower (1 and 5 μg/L) doses of the drug readily but, exposure to higher doses (>10 μg/L) caused retarded development, decreased hatching rate and growth, cardiac anomalies, spinal curvature, pectoral fin malformation and behavioral alterations resulting in greater mortality of experimental embryos. This study suggests that, ibuprofen which is marketed as over-the-counter (OTC) drug is embryotoxic at least at higher (>10 μg/L) dose level to zebrafish embryos.     

Dose tolerability of chronically inhaled voriconazole solution in rodents

Invasive pulmonary aspergillosis (IPA) is a fungal disease of the lung associated with high mortality rates in immunosuppressed patients despite treatment. Targeted drug delivery of aqueous voriconazole solutions has been shown in previous studies to produce high tissue and plasma drug concentrations as well as improved survival in a murine model of IPA. In the present study, rats were exposed to 20 min nebulizations of normal saline (control group) or aerosolized aqueous solutions of voriconazole at 15.625 mg (low dose group) or 31.25 mg (high dose group). Peak voriconazole concentrations in rat lung tissue and plasma after 3 days of twice daily dosing in the high dose group were 0.85 ± 0.63 μg/g wet lung weight and 0.58 ± 0.30 μg/mL, with low dose group lung and plasma concentrations of 0.38 ± 0.01 μg/g wet lung weight and 0.09 ± 0.06 μg/mL, respectively. Trough plasma concentrations were low but demonstrated some drug accumulation over 21 days of inhaled voriconazole administered twice daily. Following multiple inhaled doses, statistically significant but clinically irrelevant abnormalities in laboratory values were observed. Histopathology also revealed an increase in the number of alveolar macrophages but without inflammation or ulceration of the airway, interstitial changes, or edema. Inhaled voriconazole was well tolerated in a rat model of drug inhalation.     

Optimization and validation of a SPE-HPLC-PDA-fluorescence method for the simultaneous determination of drugs used in combined cardiovascular therapy in human plasma

This paper reports the chemometrical optimization and the validation of a quantitative high performance liquid chromatography-photodiode array-fluorescence (HPLC-PDA-Fluo) method for the simultaneous analysis, in human plasma, of drugs usually combined in cardiovascular therapy. Separation of chlorthalidone (CLTD), valsartan (VAL), valsartan-M1 (VAL-M1), fluvastatin (FLUV) and the internal standard (IS) candesartan cilexetil was performed on a dC18 Atlantis column (100 mm × 3.9 mm, 3 μm) using a gradient with a run time of 15 min. The mobile phase consisted of a mixture of acetonitrile and water containing 0.01% of formic acid and 10 mM of ammonium formate at pH 4.1. UV and fluorimetric (valsartan, its metabolite and fluvastatin) detectors were used. The sample preparation consisted of protein precipitation using acetonitrile suited to a solid-phase extraction (SPE) on a Strata-X cartridge for sample clean-up. Method validation was developed following the recommendations for bioanalytical method validation of International Conference on Harmonisation (ICH) and Food and Drug Administration (FDA) organizations. The method showed good linearity (31–3000 μg/l for chlorthalidone, 20–1000 μg/l for valsartan-M1, 10–5000 μg/l for valsartan and 14–1000 μg/l for fluvastatin), precision and accuracy. Recoveries were in the range of 78–91%. This method allowed the determination of these drugs in human plasma samples obtained from patients under cardiovascular treatment.     

Chemical composition and cytotoxic, mutagenic and genotoxic activities of the essential oil from Piper gaudichaudianum Kunth leaves

We have investigated the chemical composition of Piper gaudichaudianum essential oil, as well as its cytotoxic, mutagenic and genotoxic effects in V79 cells. The chemical analyses showed that the major compounds are (E)-nerolidol (22.4%), α-humulene (16.5%), (E)-caryophyllene (8.9%) and bicyclogermacrene (7.4%). Dose-dependent cytotoxic effects were observed in V79 cells treated with essential oil by using clonal survival, 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide reduction assay (MTT) and trypan blue exclusion assay (TB), and a significant decrease in survival was observed at concentrations of 0.5 μg/mL and higher. The P. gaudichaudianum essential oil treatment caused DNA strand breaks in V79 cells at concentrations up to 2 μg/mL, as detected by the alkaline comet assay, but did not induce double-strand breaks, as verified by neutral comet assay. It induced a significant increase in the frequency of micronucleated cells at 4, 6 and 10 μg/mL. Moreover, P. gaudichaudianum essential oil significantly increased lipid peroxidation at doses of 0.5 μg/mL and higher, suggesting that the observed oxidant potential can be responsible, at least in part, for its cytotoxic and genotoxic effects.     

Minimum tolerable exposure period and maximum threshold dietary intake of ochratoxin A for causing renal cancer in male Dark Agouti rats

In rats fed dietary ochratoxin A (5 ppm for 3, 6 or 9 months) no renal tumours occurred throughout natural life of the group treated for 3 months, during which the ochratoxin dose was 3 times that in the high dose group of the NTP study. Bilateral renal carcinoma occurred in one rat in the 6 month group. Four rats treated for 9 months developed unilateral renal carcinoma. Overall latency between ceasing toxin exposure and discovering tumours was 35–97 weeks. Experimental verification of a ‘no observable effect level’ was made for feed containing 400 ppb, equivalent to 7 μg ochratoxin A/day for Dark Agouti rats for up to 2 years, during which mean daily dose commenced at 50 μg/kg, but later for adults was in the range 30–20 μg/kg. This data doubles the daily in vivo threshold dose from the NTP study (15 μg/kg), and could influence human risk assessment. An at least 3 month threshold period for exposure to exceptionally high daily OTA intake (90 μg; 640–450 μg/kg) raises doubts over interpretation of experimental molecular data for OTA exposure at lower dose for up to 3 months in studies aimed at understanding carcinogenic mechanism.