A pharmacological investigation of the venom extract of the Australian box jellyfish, Chironex fleckeri, in cardiac and vascular tissues

The pharmacology of Australian box jellyfish, Chironex fleckeri, unpurified (crude) nematocyst venom extract (CVE) was investigated in rat isolated cardiac and vascular tissues and in anaesthetised rats.In small mesenteric arteries CVE (0.01-30 μg/ml) caused contractions (EC₅₀ 1.15 ± 0.19 μg/ml) that were unaffected by prazosin (0.1 μM), bosentan (10 μM), CGRP_8-37 (1 μM) or tetrodotoxin (1 μM). Box jellyfish antivenom (5-92.6 units/ml) caused rightward shifts of the CVE concentration-response curve with no change in the maximum. In the presence of l-NAME (100 μM) the sensitivity and maximum response to CVE were increased, whilst MgSO₄ (6 mM) decreased both parameters. CVE (1-10 μg/ml) caused inhibition of the contractile response to electrical sympathetic nerve stimulation.Left atrial responses to CVE (0.001-30 μg/ml) were bi-phasic, composed of an initial positive inotropy followed by a marked negative inotropy and atrial standstill. CVE (0.3 μg/ml) elicited a marked decrease in right atrial rate followed by atrial standstill at 3 μg/ml. These responses were unaffected by 1 μM of propranolol, atropine or CGRP_8-37. Antivenom (54 and 73 units/ml) caused rightward shifts of the CVE concentration-response curve and prevented atrial standstill in left and right atria.The effects of CVE do not appear to involve autonomic nerves, post-synaptic α₁- or β₁-adrenoceptors, or muscarinic, endothelin or CGRP receptors, but may occur through direct effects on the cardiac and vascular muscle. Box jellyfish antivenom was effective in attenuating CVE-induced responses in isolated cardiac and vascular tissues.     

A cell-based assay for screening of antidotes to,and antivenom against Chironex fleckeri (box jellyfish) venom

IntroductionChironex fleckeri is a large box jellyfish that has been labelled the ‘most venomous animal’ in the world. We have recently shown that the primary effect of C. fleckeri venom in vivo is cardiovascular collapse. This study utilised a cell-based assay to examine the effects of C. fleckeri venom on the proliferation of a rat aortic smooth muscle cell line. In addition, the ability of CSL box jellyfish antivenom and/or various potential treatment strategies to neutralise the effects of the venom was examined.MethodsA7r5 cells were cultured in media containing venom. The effect of CSL box jellyfish antivenom (5 U/mL), CSL polyvalent snake antivenom (5 U/mL), lanthanum (5 µM), MgSO₄ (50 mM), verapamil (5 µM) or felodipine (5 µM) was examined. Cell viability was determined using a Cell titer 96 AQueous One Solution cell proliferation assay.ResultsIncubation of A7r5 cells with serially diluted venom (2–0.004 µg/mL) caused a concentration-dependent inhibition of cell proliferation with an IC₅₀ value of 0.056 µg/mL. This response was not affected by the absence of calcium or the presence of lanthanum in the media. Box jellyfish antivenom (5 U/mL) prevented the inhibition of cell proliferation caused by the venom. Verapamil (5 µM) had no significant effect on the inhibition. In contrast, felodipine (5 µM) or MgSO₄ (50 mM) potentiated the effects of the venom and partially negated the protective effect of the antivenom.DiscussionThis study displayed the ability to utilise a cell-based assay to determine the effects of C. fleckeri venom on vascular cell viability. It showed that CSL box jellyfish can neutralise the effects of the venom but only if added prior to the venom. In addition, potential adjunct therapies verapamil, felodipine and MgSO₄ were found to be ineffective, with felodipine and MgSO₄ potentiating the detrimental effects of the venom.     

Cardiovascular actions of the venom from the Irukandji (Carukia barnesi) jellyfish: effects in human, rat and guinea-pig tissues in vitro and in pigs in vitro

1. We have investigated the cardiovascular pharmacology of the crude venom extract (CVE) from the potentially lethal, very small carybdeid jellyfish Carukia barnesi, in rat, guinea-pig and human isolated tissues and anaesthetized piglets. 2. In rat and guinea-pig isolated right atria, CVE (0.1-10 microg/mL) caused tachycardia in the presence of atropine (1 micromol/L), a response almost completely abolished by pretreatment with tetrodotoxin (TTX; 0.1 micromol/L). In paced left atria from guinea-pig or rat, CVE (0.1-3 microg/mL) caused a positive inotropic response in the presence of atropine (1 micromol/L). 3. In rat mesenteric small arteries, CVE (0.1-30 microg/mL) caused concentration-dependent contractions that were unaffected by 0.1 micromol/L TTX, 0.3 micromol/L prazosin or 0.1 micromol/L omega-conotoxin GVIA. 4. Neither the rat right atria tachycardic response nor the contraction of rat mesenteric arteries to CVE were affected by the presence of box jellyfish (Chironex fleckeri) antivenom (92.6 units/mL). 5. In human isolated driven right atrial trabeculae muscle strips, CVE (10 microg/mL) tended to cause an initial fall, followed by a more sustained increase, in contractile force. In the presence of atropine (1 micromol/L), CVE only caused a positive inotropic response. In separate experiments in the presence of propranolol (0.2 micromol/L), the negative inotropic effect of CVE was enhanced, whereas the positive inotropic response was markedly decreased. 6. In anaesthetized piglets, CVE (67 microg/kg, i.v.) caused sustained tachycardia and systemic and pulmonary hypertension. Venous blood samples demonstrated a marked elevation in circulating levels of noradrenaline and adrenaline. 7. We conclude that C. barnesi venom may contain a neural sodium channel activator (blocked by TTX) that, in isolated atrial tissue (and in vivo), causes the release of transmitter (and circulating) catecholamines. The venom may also contain a 'direct' vasoconstrictor component. These observations explain, at least in part, the clinical features of the potentially deadly Irukandji syndrome.     

Chemical profile,antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima Burm. and Citrus sinensis (L.) Osbeck essential oils and their cyclic monoterpene, dl-limonene

The study deals with antifungal, antiaflatoxigenic and antioxidant activity of Citrus maxima and Citrus sinensis essential oils (EOs) and their phytochemical composition. The EOs were obtained by hydrodistillation and their chemical profile was determined through GC and GC–MS analysis. Both the EOs and their 1:1 combination showed broad fungitoxic spectrum against different food contaminating moulds. The EOs and their combination completely inhibited aflatoxin B₁ (AFB₁) production at 500 ppm, whereas, dl-limonene, the major component of EOs showed better antiaflatoxigenic efficacy even at 250 ppm. Both the oils exhibited antioxidant activity as DPPH free radical scavenger in dose dependent manner. The IC₅₀ for radical scavenging efficacy of C. maxima and C. sinensis oils were to be 8.84 and 9.45 μl ml⁻¹, respectively. The EOs were found non-mammalian toxic showing high LD₅₀ for mice (oral, acute). The oils may be recommended as safe plant based antimicrobials as well as antioxidants for enhancement of shelf life of food commodities by checking their fungal infestation, aflatoxin production as well as lipid peroxidation.     

Pharmacokinetics of a F(ab′)2 scorpion antivenom administered intramuscularly in healthy human volunteers

This paper presents the first study of F(ab′)₂ scorpion antivenom pharmacokinetics in humans after intramuscular (im) administration. The specific anti-Centruroides scorpion antivenom was used in 6 human healthy volunteers. The fabotherapeutic was administered as a 47.5 mg im bolus. Blood samples were drawn at 0, 5, 15, 30, 45, 60 , 90, 120, and 180 min, 6 h and at 1, 2, 3, 4, 10 and 21 days after antivenom administration. We measured antivenom concentrations in serum using a specific high sensitivity ELISA method for F(ab′)₂. Antivenom concentration in serum was fit to a 3 compartment model (inoculation site, plasma and extra vascular extracellular space), it was assumed that the venom may also be irreversibly removed from plasma. Calculated time course of antivenom content shows that at any time no more that 16.6 (5.3, 31.9)% (median and 95% confidence interval) of the antivenom bolus is present in plasma. The time to peak plasma [F(ab′)₂] was 45 (33, 74) h. The most significant antivenom pharmacokinetic parameters determined were: AUC_im, ∞ = 803 (605, 1463) mg · h · L^− 1; V_c = 8.8 (2.8, 23.6) L; V_ss, im = 55 (47, 64) L; MRT_im = 776(326, 1335) h; CL_t = 3.7 (0.6, 1.9) mL · min^− 1; f_im, V_ss = 0.300 (0.153, 0.466). Comparing these parameters with the ones obtained intravenously by Vázquez et al. [2], the parameters were more disperse between subjects, determined with more uncertainty in each individual subject, and the peak F(ab′)₂ in plasma occurred with considerable delay; all indicating that the IM route should not be used to administer the antivenom, with the possible exceptionof cases occurring very far from hospitals, as an extreme means to provide some protection before the IV route becomes available.     

In vitro and in vivo antibacterial activities of garenoxacin against group G Streptococcus dysgalactiae subsp. equisimilis

In this study, garenoxacin showed potent in vitro activity against clinical isolates of group G Streptococcus dysgalactiae subsp. equisimilis [minimum inhibitory concentration for 90% of the organisms (MIC₉₀) = 0.125 μg/mL] and was superior to levofloxacin (MIC₉₀ = 1 μg/mL) and moxifloxacin (MIC₉₀ = 0.25 μg/mL). In experimental pneumonia caused by group G S. dysgalactiae subsp. equisimilis in mice, the effective dose for 50% survival (ED₅₀) of garenoxacin following single oral administration was 1.87 mg/kg, >10.7-fold and 4.6-fold less than the ED₅₀ values of levofloxacin (>20 mg/kg) and moxifloxacin (8.54 mg/kg), respectively. The area under the free serum concentration-time curve from 0-24 h (fAUC_0-24)/MIC ratio of garenoxacin in serum following oral administration of 20 mg/kg was 73.2, which was 8.7-11.4-fold and 1.4-fold greater than that of levofloxacin (6.44-8.46) and moxifloxacin (51.4), respectively. These results suggest that garenoxacin has potential for the treatment of infectious diseases caused by S. dysgalactiae subsp. equisimilis.     

Phenolic composition and biological activities of Juniperus drupacea Labill. berries from Turkey

The present study was designed to define the phenolic profile and the biological potential of berries methanol extract of Juniperus drupacea Labill. from Turkey.The total phenolic content (Folin-Ciocalteau assay) was 48.06 ± 0.99 mg GAE/g extract. The HPLC-DAD-ESI-MS analysis allowed the determination of the complete phenolic profile of J. drupacea berries. Phenolic acids represented more than 60% of the total phenolics, and tyrosol was the major one (1324 ± 0.64 μg/g extract); within the flavonoids amentoflavone was detected as the main constituent (927 ± 0.35 μg/g extract).The extract exhibited good antioxidant properties, as determined by different in vitro models: DPPH test (IC₅₀ 0.38 ± 0.02 mg/mL), reducing power (12.63 ± 0.14 ASE/mL), Fe²⁺ chelating ability (IC₅₀ 2.26 ± 0.06 mg/mL), and TBA test (IC₅₀ 2.47 ± 1.13 μg/mL).Cytotoxicity against Artemia salina was highlighted (LC₅₀ 489.47 ± 27.8 μg/mL), and a significant decrease (p ? 0.05; p ? 0.01) in HepG2 cells viability was observed at the higher concentrations (5-10 μg/mL).The extract displayed good antibacterial activity towards Gram-positive bacteria and in particular Staphylococcus aureus was the most susceptible strain (MIC 78.12 μg/mL).     

Genetic toxicology evaluation of essential oil of Alpinia zerumbet and its chemoprotective effects against H2O2-induced DNA damage in cultured human leukocytes

Essential oil (EO) of Alpinia zerumbet leaves, at non-toxic concentrations (50–300 μg/mL), did not induce genotoxicity in human leukocytes. However, at the highest concentration (500 μg/mL) tested caused a reduction in cell proliferation and viability, and an increase in DNA damage. Moreover, in vivo experiments showed that EO (400 mg/kg) did not exert mutagenicity on peripheral blood cells and bone marrow in mice. In DPPH test, EO showed scavenging effects against DPPH radicals, and other free radicals (determination of intracellular GSH and lipid peroxidation assays). Furthermore, EO was able to reduce the intracellular levels of ROS, and prevented leukocytes DNA against oxidative damage. The ability of EO to reduce H₂O₂ toxicity was observed only when cells were treated with EO during and after exposure to H₂O₂. With the co- and post-treatment procedures, EO decreased the frequency of apoptotic and micronucleated leukocytes as well DNA strand breaks. However, a synergistic effect was observed in cultures exposed to 500 μg/mL EO. In conclusion, EO at concentrations up to 300 μg/mL or doses up to 400 mg/kg are not mutagenic in leukocytes and in mice, but do have antioxidative and protective effects against the cytotoxicity and clastogenesis induced by H₂O₂.     

Tributyltin (TBT) and dibutyltin (DBT) differently inhibit the mitochondrial Mg-ATPase activity in mussel digestive gland

Tri-n-butyltin (TBT) has long been considered as the most toxic among organotins, especially to membrane systems. The partially dealkylated derivative di-n-butyltin (DBT) has up to now received poor attention and, whenever considered, shown to be less toxic than TBT except on the immune system. The present kinetic approach evidences that both TBT and DBT in vitro inhibit the Mg-ATPase in mussel digestive gland mitochondria by a different mechanism. DBT even displays a higher efficiency than TBT (IC₅₀ = 0.32 μM for TBT vs. 0.19 μM for DBT) in inhibiting the enzyme hydrolytic activity. Differently from TBT which at high concentrations (>1 μM) apparently decreases the oligomycin-sensitivity of the Mg-ATPase, DBT at any concentration tested does not affect the oligomycin sensitivity. TBT probably binds to F₀, either in the form of free enzyme or of enzyme-substrate complex (Ki = K′i), acting as non-competitive inhibitor with respect to the ATP substrate. Conversely DBT, which acts as uncompetitive inhibitor of ATP and as competitive inhibitor of Mg²⁺ cofactor, may bind strongly to F₁ subunit, thus preventing ATP hydrolysis. The Mg-ATPase inhibition by both organotins warns against a potential threat to crucial cell energy metabolism processes even after years from contamination and partial TBT debutylation.     

Volatile compounds in the stem bark of Sclerocarya birrea (Anacardiaceae) possess antimicrobial activity against drug-resistant strains of Helicobacter pylori

The aim of this study was to isolate and identify phytochemicals with anti-Helicobacter pylori activity from the stem bark of Sclerocarya birrea. The plant crude extract was fractionated by silica gel column and thin layer chromatography techniques, initially with ethyl acetate (EA) and subsequently with a combination of ethyl acetate/methanol/water (EMW). Further fractionation and identification of the phytoconstituents was achieved by gas chromatography and mass spectrometry (GC/MS) analysis. The antimicrobial activity of the fractions and compounds was evaluated against five metronidazole- and clarithromycin-resistant strains of H. pylori as well as a reference strain ATCC 43526 using the microbroth dilution technique. Amoxicillin was included in the experiments as a positive control antibiotic. Of the 18 fractions collected, 16 demonstrated anti-H. pylori activity with 50% minimum inhibitory concentration (MIC₅₀) values ranging from 310 μg/mL to 2500 μg/mL. Two of the fractions (EMW fraction 6 and EA fraction 1) revealed the presence of 5 and 24 compounds, respectively, representing 40.5% and 86.57% of the total composition. Most of the compounds were essential oils, with terpinen-4-ol being the most abundant agent (35.83%), followed by pyrrolidine (32.15%), aromadendrene (13.63%) and α-gurjunene (8.77%). MIC₅₀ ranges for amoxicillin, terpinen-4-ol and pyrrolidine were 0.0003-0.06 μg/mL, 0.004-0.06 μg/mL and 0.005-6.3 μg/mL, respectively. The inhibitory activities of terpinen-4-ol and pyrrolidine were similar to amoxicillin (P > 0.05). Most of these compounds are being reported in this plant for the first time and may represent new sources of therapeutically useful compounds against H. pylori.     

Isolation and characterization of lethal proteins in nematocyst venom of the jellyfish Cyanea nozakii Kishinouye

Cyanea nozakii Kishinouye, a jellyfish widely distributed in coastal areas of China, has garnered attention because of its stinging capacity and the resulting public health hazard. We used a recently developed technique to extract jellyfish venom from nematocysts; the present study investigates the lethality of C. nozakii venom. The nematocyst contents were extremely toxic to the grass carp, Ctenopharyngodon idellus, producing typical neurotoxin toxicity. The ID₅₀ was about 0.6 μg protein/g fish. Toxin samples were stable when kept at −80 ^°C, but after 48 h, an 80% decline in lethality occurred at −20 ^°C. Poor stability of the venom was observed within the range of 65-80 ^°C and at pH 3.5. The venom was hydrolyzed by a proteolytic enzyme, trypsin. Fractionation of the venom yielded two protein bands with molecular weights of 60 kDa and 50 kDa. Our results provide the first evidence that C. nozakii produces lethal toxins. These characteristics highlight the need for the isolation and molecular characterization of new active toxins in C. nozakii.     

Coordinated role of voltage-gated sodium channels and the Na/H exchanger in sustaining microglial activation during inflammation

Persistent neuroinflammation and microglial activation play an integral role in the pathogenesis of many neurological disorders. We investigated the role of voltage-gated sodium channels (VGSC) and Na⁺/H⁺ exchangers (NHE) in the activation of immortalized microglial cells (BV-2) after lipopolysaccharide (LPS) exposure. LPS (10 and 100 ng/ml) caused a dose- and time-dependent accumulation of intracellular sodium [(Na⁺)_i] in BV-2 cells. Pre-treatment of cells with the VGSC antagonist tetrodotoxin (TTX, 1 μM) abolished short-term Na⁺ influx, but was unable to prevent the accumulation of (Na⁺)_i observed at 6 and 24 h after LPS exposure. The NHE inhibitor cariporide (1 μM) significantly reduced accumulation of (Na⁺)_i 6 and 24 h after LPS exposure. Furthermore, LPS increased the mRNA expression and protein level of NHE-1 in a dose- and time-dependent manner, which was significantly reduced after co-treatment with TTX and/or cariporide. LPS increased production of TNF-α, ROS, and H₂O₂ and expression of gp91^phox, an active subunit of NADPH oxidase, in a dose- and time-dependent manner, which was significantly reduced by TTX or TTX + cariporide. Collectively, these data demonstrate a closely-linked temporal relationship between VGSC and NHE-1 in regulating function in activated microglia, which may provide avenues for therapeutic interventions aimed at reducing neuroinflammation.     

Antibacterial and antiparasitic effects of Bothrops marajoensis venom and its fractions: Phospholipase A2 and l-amino acid oxidase

Some proteins present in snake venom possess enzymatic activities, such as phospholipase A₂ and l-amino acid oxidase. In this study, we verify the action of the Bothrops marajoensis venom (BmarTV), PLA₂ (BmarPLA₂) and LAAO (BmarLAAO) on strains of bacteria, yeast, and Leishmania sp. The BmarTV was isolated by Protein Pack 5PW, and several fractions were obtained. Reverse phase HPLC showed that BmarPLA₂ was isolated from the venom, and N-terminal amino acid sequencing of sPLA₂ showed high amino acid identity with other lysine K49 sPLA₂s isolated from Bothrops snakes. The BmarLAAO was purified to high molecular homogeneity and its N-terminal amino acid sequence demonstrated a high degree of amino acid conservation with others LAAOs. BmarLAAO was able to inhibit the growth of P. aeruginosa, C. albicans and S. aureus in a dose-dependent manner. The inhibitory effect was more significant on S. aureus, with a MIC = 50 μg/mL and MLC = 200 μg/mL. However, the BmarTV and BmarPLA₂ did not demonstrate inhibitory capacity. BmarLAAO was able to inhibit the growth of promastigote forms of L. chagasi and L. amazonensis, with an IC₅₀ = 2.55 μg/mL and 2.86 μg/mL for L. amazonensis and L. chagasi, respectively. BmarTV also provided significant inhibition of parasitic growth, with an IC₅₀ of 86.56 μg/mL for L. amazonensis and 79.02 μg/mL for L. chagasi. BmarPLA₂ did not promote any inhibition of the growth of these parasites. The BmarLAAO and BmarTV presented low toxicity at the concentrations studied. In conclusion, whole venom as well as the l-amino acid oxidase from Bothrops marajoensis was able to inhibit the growth of several microorganisms, including S. aureus, Candida albicans, Pseudomonas aeruginosa, and Leishmania sp.     

Antihypertensive and vasorelaxant effects of tilianin isolated from Agastache mexicana are mediated by NO/cGMP pathway and potassium channel opening

Current investigation was undertaken to elucidate the mode of action of tilianin, isolated from Agastache mexicana, as a vasorelaxant agent on in vitro functional rat thoracic aorta test and to investigate the in vivo antihypertensive effect on spontaneously hypertensive rats (SHR). Tilianin (0.002-933 μM) induced significant relaxation in a concentration- and endothelium-dependent and -independent manners in aortic rings pre-contracted with noradrenaline (NA, 0.1 μM), and serotonin (5-HT, 100 μM). Effect was more significant (p < 0.05) in endothelium-intact (+E) aorta rings than when endothelium was removed (−E). Pre-treatment with N-nitro-l-arginine methyl ester (l-NAME; 10 μM) or 1-H-[1,2,4]-oxadiazolo-[4,3a]-quinoxalin-1-one (ODQ, 1 μM) produced a significant change of the relaxant response and activity was markedly inhibited, but not by indomethacin (10 μM) or atropine (1 μM). Furthermore, tilianin (130 μM) provoked a significant displacement to the left in the relaxation curve induced by sodium nitroprusside (SNP; 0.32 nM to 0.1 μM). Moreover, tilianin induced significant in vitro NO overproduction (1.49 ± 0.86 μM of nitrites/g of tissue) in rat aorta compared with vehicle (p < 0.05). In addition, pre-treatment with tetraethylammonium (TEA, 5 mM) and 2-aminopyridine (2-AP, 0.1 μM) shifted to the right the relaxant curve induced by tilianin (p < 0.05). Finally, a single oral administration of tilianin (50 mg/kg) exhibited a significant decrease in systolic and diastolic blood pressures (p < 0.05) in SHR model.Results indicate that tilianin mediates relaxation mainly by an endothelium-dependent manner, probably due to NO release, and also through an endothelium-independent pathway by opening K⁺ channels, both causing the antihypertensive effect.     

Chemical composition,antifungal and antiaflatoxigenic activities of Ocimum sanctum L. essential oil and its safety assessment as plant based antimicrobial

The study deals with the efficacy of Ocimum sanctum essential oil (EO) and its major component, eugenol against the fungi causing biodeterioration of food stuffs during storage. O. sanctum EO and eugenol were found efficacious in checking growth of Aspergillus flavus NKDHV8; and, their minimum inhibitory concentrations (MICs) were recorded as 0.3 and 0.2 μl ml⁻¹, respectively. The O. sanctum EO and eugenol also inhibited the aflatoxin B₁ production completely at 0.2 and 0.1 μl ml⁻¹, respectively. Both of these were found superior over some prevalent synthetic antifungals and exhibited broad fungitoxic spectrum against 12 commonly occurring fungi. The LD₅₀ value of O. sanctum EO on mice was found to be 4571.43 μl kg⁻¹ suggesting its non-mammalian toxic nature. The findings of present study reveals the possible exploitation of O. sanctum EO and eugenol as plant based safe preservatives against fungal spoilage of food stuffs during storage.     

Characterization of major phytocannabinoids, cannabidiol and cannabinol, as isoform-selective and potent inhibitors of human CYP1 enzymes

Inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, on catalytic activities of human cytochrome P450 (CYP) 1 enzymes were investigated. These cannabinoids inhibited 7-ethoxyresorufin O-deethylase activity of recombinant CYP1A1, CYP1A2, and CYP1B1 in a competitive manner. CBD most potently inhibited the CYP1A1 activity; the apparent K_i value (0.155 μM) was at least one-seventeenth of the values for other CYP1 isoforms. On the other hand, CBN more effectively decreased the activity of CYP1A2 and CYP1B1 (K_i = 0.0790 and 0.148 μM, respectively) compared with CYP1A1 (K_i = 0.541 μM). Δ⁹-THC less potently inhibited the CYP1 activity than CBD and CBN, and showed low selectivity against the CYP1 inhibition (K_i = 2.47-7.54 μM). The preincubation of CBD resulted in a time- and concentration-dependent decrease in catalytic activity of all the recombinant CYP1 enzymes and human liver microsomes. Similarly, the preincubation of Δ⁹-THC or CBN caused a time- and concentration-dependent inhibition of recombinant CYP1A1. The inactivation of CYP1A1 by CBD indicated the highest k_inact/K_I value (540 l/mmol/min) among the CYP1 enzyme sources tested. The inactivation of recombinant CYP1A1 and human liver microsomes by CBD required NADPH, was not influenced by dialysis and by glutathione, N-acetylcysteine, and superoxide dismutase as trapping agents. These results indicated that CBD and CBN showed CYP1 isoform-selective direct inhibition and that CBD was characterized as a potent mechanism-based inhibitor of human CYP1 enzymes, especially CYP1A1.     

Infection by mycotoxigenic fungal species and mycotoxin contamination of maize grain in Umbria, central Italy

Surveys were carried out in 2006 and 2007 in Umbria (central Italy) to evaluate the presence of mycotoxigenic fungi and mycotoxins in maize grain sampled at harvest. Fusarium spp., were the most abundant species detected in maize kernels, followed by Aspergillus species of sections Flavi and Nigri and by Penicillium spp. Among Fusarium species, F. verticillioides was the most prevalent species, as detected by PCR directly on the kernels and on the fungi isolated from the kernels, followed by F. proliferatum and F. subglutinans. Fumonisins were the predominant mycotoxins with values, on average, of 4.3 and 5.7 mg kg⁻¹, in 2006 and 2007, respectively, with a maximum of 76.3 mg kg⁻¹ in the second year. Deoxynivalenol ranged from 0.2 to 3.98 mg kg⁻¹ in 2006 (average 1.04 mg kg⁻¹) and from undetectable levels to 14 mg kg⁻¹ in 2007 (average 0.86 mg kg⁻¹). Aflatoxins, analyzed only in 2007, averaged 26.3 μg kg⁻¹, with a maximum of 820 μg kg⁻¹. Zearalenone content was always very low. Results indicate that EU legal limits for these mycotoxins were rarely exceeded with low levels across most of the examined area, suggesting that this region could be considered suitable for the production of healthy maize.     

Comparative analysis of the antioxidant and DNA protection capacities of Anadenanthera colubrina, Libidibia ferrea and Pityrocarpa moniliformis fruits

This study aimed to explore the antioxidant and DNA protection abilities of hydroalcoholic extracts from fruits of Anadenanthera colubrina (ACHE), Libidibia ferrea (LFHE) and Pityrocarpa moniliformis (PMHE). These extracts were tested by five antioxidant methods (phosphomolibdenium and reducing power assays; superoxide, hydrogen peroxide and nitric oxide scavenging) and DNA protection capacity. Total phenolic content was measured by Folin-Ciocalteu method. ACHE exhibited the highest phenolic content (578 mg/g GAE), followed by LFHE (460 mg/g GAE) and PMHE (448 mg/g GAE). In phosphomolibdenium assay, ACHE showed 24.81% of activity in relation to ascorbic acid, whereas LFHE and PMHE had 21.08% and 18.05%, respectively. These plants showed high ability to inhibit reactive species tested with IC50 values ranged from 10.66 to 14.37 μg/mL for superoxide radical; 26.05 to 45.43 μg/mL for hydrogen peroxide; 178.42 to 182.98 μg/mL for reducing power; and 199.2 to 283 μg/mL for nitric oxide. Furthermore, these extracts had capacity to break the DNA damage induced by hydroxyl radicals. The antioxidant activity of these plants is related with their higher phenolic content and show that they may be used as source of bioactive compounds, relevant to the maintenance of oxidative stability of the food matrix, cosmetics and/or pharmaceutical preparations.     

The critical importance of defined media conditions in Daphnia magna nanotoxicity studies

Due to the widespread use of silver nanoparticles (AgNPs), the likelihood of them entering the environment has increased and they are known to be potentially toxic. Currently, there is little information on the dynamic changes of AgNPs in ecotoxicity exposure media and how this may affect toxicity. Here, the colloidal stability of three different sizes of citrate-stabilized AgNPs was assessed in standard strength OECD ISO exposure media, and in 2-fold (media2) and 10-fold (media10) dilutions by transmission electron microscopy (TEM) and atomic force microscopy (AFM) and these characteristics were related to their toxicity towards Daphnia magna. Aggregation in undiluted media (media1) was rapid, and after diluting the medium by a factor of 2 or 10, aggregation was reduced, with minimal aggregation over 24 h occurring in media10. Acute toxicity measurements were performed using 7 nm diameter particles in media1 and media10. In media10 the EC₅₀ of the 7 nm particles for D. magna neonates was calculated to be 7.46 μg L⁻¹ with upper and lower 95% confidence intervals of 6.84 μg L⁻¹ and 8.13 μg L⁻¹ respectively. For media1, an EC₅₀ could not be calculated, the lowest observed adverse effect concentration (LOAEC) of 11.25 μg L⁻¹ indicating a significant reduction in toxicity compared to that in media10. The data suggest the increased dispersion of nanoparticles leads to enhanced toxicity, emphasising the importance of appropriate media composition to fully assess nanoparticle toxicity in aquatic ecotoxicity tests.