Larvicidal action of ethanolic extracts from fruit endocarps of Melia azedarach and Azadirachta indica against the dengue mosquito Aedes aegypti

Ethanolic extracts from the kernels of ripe fruits from the Indian Lilac Melia azedarach and from the well-known Neem tree, Azadirachta indica were assayed against larvae of Aedes aegypti, the mosquito vector of dengue fever. The lethality bioassays were carried out according to the recommendations of the World Health Organization. Extracts were tested at doses ranging from 0.0033 to 0.05 g% in an aqueous medium for 24 and 48 h, at 25 or 30 °C, with or without feeding of the larvae. LC₅₀, LC₉₅ and LC₉₉ were determined. Both seed extracts proved lethal for third to fourth instar larvae. Non-fed A. aegypti larvae were more susceptible to Azadirachta extracts at both temperatures. Under a more realistic environmental situation, namely with fed larvae at 25 °C, the death rates caused by the Melia extract were higher, although at 30 °C the extract of Azadirachta had an even higher lethality. Inter allia, the LC₅₀ values for the crude extracts of these two members of the Meliaceae ranged from 0.017 to 0.034 g% while the LC₉₉ values ranged from 0.133 to 0.189 g%. Since no downstream processing was undertaken to purify the active agents in the extracts, our findings seem very promising, suggesting that it may be possible to increase the larvicidal activity further by improving the extraction and the fractionation of the crude limonoids, for instance removing the co-extracted natural fats.     

Acute toxicity of inorganic selenium to Daphnia magna (Straus) and the effect of sub-acute exposure upon growth and reproduction

The acute toxicities of sodium selenite (Na₂SeO₃) and sodium selenate (Na₂SeO₄) to Daphnia magna were determined in defined culture at 22°C. For adults, the 48-h LC₅₀ values were 0.68 ppm selenium as selenite and 0.75 ppm selenium as selenate. Juveniles were more sensitive, with a 48-h LC₅₀ of 0.55 ppm selenium as selenate. Eggs and embryos were found to be much less sensitive, with a 72-h LC₅₀ of 1.4 ppm selenium as selenate.

Sub-acute exposure of D. magna to sodium selenate caused suppression of growth over instars 1–5 and reduced egg production in instar 9 when adults were exposed to test solutions from instar 6 onwards. These sublethal effects were found at concentrations in the range proposed as suitable for the use of selenium in the amelioration of mercury contamination.     

Evaluation of acute toxicity of carbaryl and malathion to freshwater teleosts, Channa punctatus (Bloch) and Heteropneustes fossilis (Bloch)

Bioassay tests evaluated the acute toxicity of carbaryl (a carbamate) (50'% wettable powder, W.P.), and malathion (50% E.C.) (an organophosphate) (50% emulsifiable concentrate; E.C.) by determining their LC₅₀ and the acute toxic ranges for 24, 48 and 72, and 96-h exposure to Channa punctatus (Bl.) and Heteropneustes fossilis (Bl.). Regression equations and slope functions were determined for different time periods. The relative susceptibility of the fish, the relative toxicity, and the safe concentrations of these biocides were calculated on the basis of LC₅₀ for 96 h. C. punctatus was found to be relatively more susceptible to both the compounds, while malathion was the more toxic of the two biocides.     

Acute toxicity of three detergents and two insecticides in the lugworm, Arenicola marina (L.): a histological and a scanning electron microscopic study

The toxicity of three detergents (sodium dodecyl benzene sulfonate, sodium dodecyl sulfate and Triton X-100) and two insecticides (Carbaryl and Parathion-ethyl) on the lugworm, Arenicola marina, was investigated. The 48-h LC₅₀ values were established and the morphological alterations in epidermis, gills and intestine were analysed by light and scanning electron microscopy. The three detergents were equally toxic (LC₅₀ from 12 to 15 mg · l⁻¹) while the insecticides were more potent (LC₅₀ = 7.2 and 2.7 mg · l⁻¹, for the Carbaryl and the Parathion-ethyl, respectively). The gills and the epidermic receptors were the most sensitive sites of the lugworm, while the thoracic epidermis was the most resistant of the structures studied.     

Lethal and sublethal effects of malathion on three life stages of the grass shrimp, Palaemonetes pugio

The effects of malathion exposure on three life stages of the grass shrimp (Palaemonetes pugio) were evaluated. After 96-h exposures, malathion was most toxic to newly hatched larvae with an LC₅₀ of 9.06 μg l⁻¹ followed by an LC₅₀ of 13.24 μg l⁻¹ for 18-day-old larvae and an LC₅₀ of 38.19 μg l⁻¹ for adult shrimp. In a separate bioassay, to simulate field conditions, newly hatched larvae were exposed to malathion at 6 h day⁻¹ every 5 days at a salinity of 10‰ until metamorphosis to postlarvae. After four pulse dose exposures, mortality was highest in the two highest concentrations, of 15.0 and 30.0 μg l⁻¹. The number of instars to postlarvae was significantly lower in the highest concentration compared to control. The findings indicate that malathion may not directly affect growth in a measurable way but may alter natural metamorphic rhythms at the highest concentrations. Whole body acetylcholinesterase (AChE) activity was measured on Day 0 and Day 15 from the pulse exposure test. AChE activities were not significantly different from controls. Other factors than just AChE inhibition may have contributed to malathion toxicity.     

Ultrastructural effects of AAL-toxin TA from the fungus Alternaria alternata on black nightshade (Solanum nigrum L.) leaf discs and correlation with biochemical measures of toxicity

Ultrastructural effects of AAL-toxin T_A from Alternaria alternata on black nightshade (Solanum nigrum L.) leaf discs and correlation with biochemical measures of toxicity. In black nightshade (Solanum nigrum L.) leaf discs floating in solutions of AAL-toxin T_A (0.01–200 μM) under continuous light at 25°C, electrolyte leakage, chlorophyll loss, autolysis, and photobleaching were observed within 24 h. Electrolyte leakage, measured by the conductivity increase in the culture medium, began after 12 h with 200 μM AAL-toxin T_A, but was observed after 24 h with 0.01 to 50 μM AAL-toxin T_A, when it ranged from 25% to 63% of total releasable electrolytes, respectively. After 48 h incubation, leakage ranged from 39% to 79% of total for 0.01 to 200 μM AAL-toxin T_A, respectively, while chlorophyll loss ranged from 5% to 32% of total, respectively. Ultrastructural examination of black nightshade leaf discs floating in 10 μM AAL-toxin T_A under continuous light at 25°C revealed cytological damage beginning at 30 h, consistent with the time electrolyte leakage and chlorophyll reduction were observed. After 30 h incubation chloroplast starch grains were enlarged in control leaf discs, but not in AAL-toxin T_A-treated discs, and the thylakoids of treated tissue contained structural abnormalities. After 36–48 h incubation with 10 μM AAL-toxin T_A, all tissues were destroyed with only cell walls, starch grains, and thylakoid fragments remaining. Toxicity was light-dependent, because leaf discs incubated with AAL-toxin T_A in darkness for up to 72 h showed little phytotoxic damage. Within 6 h of exposure to ≥0.5 μM toxin, phytosphingosine and sphinganine in black nightshade leaf discs increased markedly, and continued to increase up to 24 h exposure. Thus, physiological and ultrastructural changes occurred in parallel with disruption of sphingolipid synthesis, consistent with the hypothesis that AAL-toxin T_A causes phytotoxicity by interrupting sphingolipid biosynthesis, thereby damaging cellular membranes.     

Toxicity assessment of fumonisins using the brine shrimp (Artemia salina) bioassay.

The Fusarium mycotoxins fumonisin B₁ (FB₁) (1) and B₂ (FB₂) (2), their hydrolysed analogues HFB₁ (3) and HFB₂ (4) and the recently discovered fumonisin derivatives N-palmitoyl-HFB₁ (5) and N-carboxymethyl-FB₁ (6) were compared for their toxicity in a short term bioassay using brine shrimp (Artemia salina). The brine shrimp were hatched in artificial sea water and exposed to the fumonisins in microwell plates with a mortality endpoint after 48 hours. LC₅₀ values were calculated after Probit transformation of the resulting data. Of the substances tested, fumonisin B₁ emerged to be the most toxic whereas its N-carboxymethyl analogue was 100-fold less effective. The hydrolysed fumonisins showed a four- to sixfold reduced toxicity compared to FB₁. N-Palmitoyl-HFB₁ had a higher LC₅₀ value than its precursor HFB₁. The brine shrimp assay proved to be a convenient and rapid system for toxicity assessment of this group of mycotoxins.     

Microcystin uptake inhibits growth and protein phosphatase activity in mustard (Sinapis alba L.) seedlings

Mustard (Sinapis alba L.) seeds were cultivated for seven days on a solid nutrient medium supplemented with 0–40 μg microcystin-RR per ml. Microcystin-RR affected seedling growth ( ₅₀ 0.8 μg/ml) and microcystin concentrations ≥5.0 μg/ml produced malformed plants. The inhibition of protein phosphatase 1 and 2A activity correlated with the growth inhibition. The seedlings were also shown to take up ³H-dihydromicrocystin-LR derived radioactivity up to a level corresponding to ca. 80 ng toxin per mg plant protein.     

Purification and biological effects of C-type lectin isolated from Bothrops insularis venom

Bothrops insularis is a snake from Queimada Grande Island, which is an island located about 20 miles away from the southeastern coast of Brazil. Compared to other Brazilian species of Bothrops, the toxinology of B. insularis is still poorly understood. Its C-type lectin is involved in several biological processes including anticoagulant and platelet-modulating activities. We purified the C-type lectin (BiLec) from Bothrops insularis venom and investigated its effect in the isolated kidney. BiLec was purified after two chromatographic steps; firstly, the whole venom was submitted to an HPLC molecular exclusion chromatography followed by a second purification through affinity chromatography. B. insularis lectin (BiLec) was studied as to its effect on the renal function of isolated perfused rat kidneys with the use of six Wistar rats. The concentration of 10 μg/mL increased perfusion pressure (PP; control₆₀=108.27±4.9; BiLec₆₀=112.9±5.4 mmHg; *p<0.05) and renal vascular resistance (RVR; control₆₀=5.38±0.51; BiLec₆₀=6.01±0.57 mmHg; *p<0.05). The urinary flow reduced significantly at 90 and 120 min of perfusion (UF; control₁₂₀=0.160±0.020; BiLec₁₂₀=0.082±0.008 mL g⁻¹ min⁻¹; *p<0.05). Glomerular filtration rate (GFR; control₁₂₀=0.697±0.084; BiLec₁₂₀=0.394±0.063 mL g⁻¹ min⁻¹; *p<0.05) diminished only at 120 min. BiLec did not change the percentage of sodium (TNa⁺), potassium (TK⁺) and chloride tubular transport (TCl⁻). The histological alterations probably reflected direct injury on glomerular and tubular renal cells, as demonstrated by the rise in permeability of glomerular endothelial cells, revealed by the presence of a proteinaceous material in the Bowman space. We postulate that the C-type lectin B. insularis promoted its effects probably through interactions with endothelial cells or through the release of other mediators by tubular, mesangial and endothelial cells.     

Bothroalternin, a thrombin inhibitor from the venom of Bothrops alternatus

Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin–Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by -thrombin ( ₅₀=28 μg/ml). A single band of 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation ( ₅₀=0.19 μg/ml) compared to bothrojaracin ( ₅₀=0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.     

In vitro cytotoxicity tests for the prediction of acute toxicity in vivo

Investigations of the use of in vitro cytotoxicity tests for the prediction of acute toxicity in vivo have been reviewed with particular emphasis on those studies that have been published during the past 5 years. Numerous cell types, endpoints and exposure periods have been used in cytotoxicity tests, although these appear generally to have little effect on the resulting correlation between in vitro IC₅₀ values and in vivo LD₅₀ values. The in vitro data correlate better with rodent parenteral (ip or iv) LD₅₀ values than with oral LD₅₀ values due to kinetic considerations. For certain groups of related chemicals (e.g. antitumour compounds, metal salts), and for some sets of unrelated chemicals, the in vitro data correlate very well with LD₅₀ values. However, while cytotoxicity tests are useful for screening chemicals for their intrinsic and relative toxicities, it is impossible to tell whether predictions based on cytotoxicity data alone would be sufficiently accurate for labelling and classifying a new chemical according to its likely acute toxicity in vivo. The in vitro endpoints need to be of greater relevance to the possible mechanisms of chemically-induced acute toxicity in vivo than most of those that are used at present.     

Crystal structure of a novel phospholipase A2 from Naja naja sagittifera with a strong anticoagulant activity

This is the first PLA₂ crystal structure from group I that shows a strong anticoagulant property. The monomeric PLA₂ was purified from the venom of Naja naja sagittifera (Indian cobra). Its amino acid sequence has been determined using cDNA technique. The amino acid sequence of sPLA₂ contains three positively charged and two negatively charged residues in the segment 54–71 (numbering scheme of sPLA₂) thus giving this region an overall cationic amphiphilic surface. This suggested the presence of an anticoagulant activity in sPLA₂. The enzyme was crystallized using hanging drop vapour diffusion method in the presence of calcium chloride. The crystals belong to space group P4₁ with cell dimensions of a=b=42.0 Å, c=65.9 Å. The X-ray crystal structure was determined at 1.8 Å resolution using molecular replacement method and refined to an R value of 0.179 for 10,023 reflections. The overall scaffolding of sPLA₂ is essentially similar to those observed for other group I PLA₂s. However, the conformations of various surface loops were found to be significantly different. The most significant observation pertains to the anticoagulant loop in which both the acidic residues are engaged in intramolecular interactions whereas all the three basic residues are free to interact with other molecules. This makes the sPLA₂ a potentially strong anticoagulating molecule.     

Bioactive properties and chemical composition of six walnut (Juglans regia L.) cultivars

The chemical composition, antioxidant potential and antimicrobial activity were studied in six walnuts (Juglans regia L.) cultivars (cv. Franquette, Lara, Marbot, Mayette, Mellanaise and Parisienne) produced in Portugal. Concerning their chemical composition the main constituent of fruits was fat ranging from 78.83% to 82.14%, being the nutritional value around 720 kcal per 100 g of fruits. Linoleic acid was the major fatty acid reaching the maximum value of 60.30% (cv. Lara) followed by oleic, linolenic and palmitic acids. The aqueous extracts of walnut cultivars were investigated by the reducing power assay, the scavenging effect on DPPH (2,2-diphenyl-1-picrylhydrazyl) radicals and β-carotene linoleate model system. All the walnut extracts exhibited antioxidant capacity in a concentration-dependent manner being the lowest EC₅₀ values obtained with extracts of cv. Parisienne. Their antimicrobial capacity was also checked against gram positive (Bacillus cereus, Bacillus subtilis, Staphylococcus aureus) and gram negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae) and fungi (Candida albicans, Cryptococcus neoformans), revealing activity against the different tested microorganisms.     

Modeling inflammation–drug interactions in vitro: A rat Kupffer cell-hepatocyte coculture system

Xenobiotic–inflammation interactions lead to hepatotoxicity in vivo. Selected xenobiotic agents (acetaminophen, APAP; chlorpromazine, CPZ; allyl alcohol, AlOH; monocrotaline, MCT) for which this occurs were evaluated for ability to elicit the release of Kupffer cell (KC)-derived inflammatory mediators and to modulate lipopolysaccharide (LPS)-stimulated release of these mediators. Using KCs and hepatocytes (HPCs) isolated from rat, KC/HPC cocultures were treated with either LPS, xenobiotic, vehicle or a combination. Six hours later, the release of inflammatory mediators was assessed. LPS alone caused a concentration-dependent increase in TNF- release but had no significant effect on the release of PGE₂. APAP by itself did not alter release of TNF-, PGE₂, IL-10, Gro/KC or IFN-γ; however, in the presence of LPS, APAP enhanced LPS-induced TNF- and Gro/KC release. APAP also attenuated LPS-induced increases in IL-10 and MCP-1. CPZ alone caused a concentration-dependent increase in TNF- release, which was approximately additive in the presence of LPS. AlOH alone did not affect TNF- release, but decreased TNF- production in the presence of LPS. AlOH increased PGE₂ production, and this effect was potentiated in the presence of LPS. MCT by itself did not affect release of TNF- but increased the response to LPS. Neither MCT, LPS, nor the combination affected production of PGE₂. These results demonstrate that KC/HPC cocultures can be used to evaluate interactions of xenobiotics with LPS. Furthermore, data from these studies qualitatively mirror reported data from whole animal studies, suggesting that this model could be useful for predicting aspects of xenobiotic–inflammation interactions in vivo.     

Behavioural and gill histopathological effects of acute exposure to sodium chloride in moneda (Metynnis orinocensis)

To evaluate the toxicity of sodium chloride (NaCl), juveniles and adult Metynnis orinocensis were exposed for 96 h to 0, 5, 10, 15, 20 or 40 g L⁻¹ of salt. Food intake, behaviour, opercular frequency (OF), mortality, body weight and gill microscopic alterations were evaluated. Behavioural changes were observed in fish exposed to concentrations higher than 10 g L⁻¹. Juveniles and adults showed a progressive decrease in the OF and body weight. Food intake decreased in concentrations below 15 g L⁻¹. Juveniles and adults exposed to 15, 20 or 40 g L⁻¹ had 100% mortality. Lamellar congestion, hyperplasia and fusion were the common microscopic alterations at higher concentrations. The gill congestion severity increased with salt concentration. The LC₅₀ for juveniles and adults were 10.5 g L⁻¹ and 10.8 g L⁻¹, respectively. These results suggest that salt concentrations lower than 5 g L⁻¹ are safe for preventive and therapeutic practices in Metynnis orinocensis; whereas prolonged exposure higher than 10 g L⁻¹ is deleterious in this species.     

GSTM1 and GSTT1 polymorphism influences protection against induced oxidative DNA damage by quercetin and ascorbic acid in human lymphocytes in vitro

Antioxidants are of major importance in the protection against cellular oxidative damage caused by endogenous as well as exogenous free radicals. This study aims to establish the impact of genetic polymorphisms in GSTM1 and GSTT1, which encode for enzymatic antioxidative defence, on H₂O₂-induced oxidative DNA damage and on the effectiveness of quercetin and ascorbic acid in preventing this induced damage in human lymphocytes. Lymphocytes from 12 healthy volunteers were pre-incubated either with 10 μM of quercetin or with 10 μM of ascorbic acid, and exposed to 25 μM H₂O₂ for 1 h. The induction of oxidative DNA damage was quantified using the Comet assay. Genotyping of these 12 subjects showed that six individuals were GSTM1+ and six were GSTM1−; eight were GSTT1+ and four GSTT1−.

Results

Baseline levels of oxidative DNA damage did not differ between GSTM1 or GSTT1 variants and their respective wild types. Also with respect to ex vivo induced levels of oxidative DNA damage, no significant difference was seen between variants and wild types of both genotypes. The protection against H₂O₂-induced oxidative DNA damage by quercetin was significantly higher in GSTT1 wild types than in GSTT1 variants (57% and 9% decrease, respectively; p = 0.01); furthermore, GSTT1 wild types were protected against induced oxidative DNA damage by ascorbic acid pre-incubation while GSTT1 variants showed an increase of damage (16% decrease vs. 91% increase; p = 0.01). For GSTM1 variants and wild types, observed differences in protective effects of quercetin or ascorbic acid were not statistically significant. Overall, quercetin proves to be better in protecting human lymphocytes in vitro against oxidative DNA damage upon H₂O₂ challenge than ascorbic acid.     

Ameliorative action of cyanobacterial phycoerythrin on CCl(4)-induced toxicity in rats

Carbon tetrachloride (CCl₄) is largely used as solvent in chemical industries. Carbon tetrachloride is also well known for hepatic and renal toxic actions. The in vivo metabolism of carbon tetrachloride to trichloromethyl (CCl₃) and peroxy trichloromethyl (OOCCl₃) radicals has been extensively reported to cause acute liver damage like cirrhosis, steatosis and necrosis. We have evaluated protective action of purified cyanobacterial phycoerythrin (C-PE) on carbon tetrachloride-induced hepatic and renal toxicity in male rats. Rats were orally treated with 25 and 50 mg/kg BW of C-PE along with CCl₄ (50% CCl₄, 0.5 ml/kg BW, intraperitoneally) for 28 consecutive days. Results demonstrated that C-PE dose-responsively ameliorates CCl₄-toxicity by significantly decreasing (P < 0.05) organs weight, aminotransferases, alkaline phosphatase, glucose, lipid profile, creatinine, uric acid and malondialdehyde (MDA) concentrations with rise in body weight, food intake, hemoglobin, protein, bilirubin and FRAP values. Neither C-PE nor CCl₄ influenced on serum minerals. Hepatic and renal tissues showed significant decline (P < 0.05) in malondialdehyde, lipid hydroperoxides and conjugated dienes with rise in SOD, catalase, GPx, GSH, vitamin-E and vitamin-C levels. Presently observed pharmacological effect on CCl₄ toxicity were from tetrapyrrole molecule and to some extent bilirubin biotransformations, as well as metabolic (dietary protein) actions of C-PE.     

Comparative toxicity of sulfur mustard and nitrogen mustard on tracheal epithelial cells in primary culture

Sulfur mustard (SM) and mechlorethamine (HN2) are two alkylating agents. SM represents a potential chemical warfare agent and HN2 is used in cancer chemotherapy. Based on the similarities of their action, although few comparative studies of their effects have been performed on the same model, many compounds effective against HN2 side-effects have been proposed, unsuccessfully, against SM-induced lesions. We performed this study to compare the toxic effects of these two alkylating agents on rabbit tracheal epithelium in primary culture. Using neutral red uptake, we evidenced that for a time of contact of 24 hr, HN2 LC₅₀ was significantly lower than SM LC₅₀ (0.034±0.009 and 0.132±0.023 m , respectively; P<0.001). On the other hand, for exposure at 10⁻³ , the time necessary to decrease the cell viability rate to 50% was shorter with SM than with HN2 (11±1 min and 54±2 min, respectively; P<0.0001). These two alkylating agents induced apoptosis which was evidenced by DNA ladder and by 4′,6-diamidino-2-phenylindole (DAPI) DAPI staining. The apoptosis rates were time and dose dependent for the two toxics: mild doses induced apoptosis, while higher doses induced necrosis.