Functional characterization of a basic D49 phospholipase A2 (LmTX-I) from the venom of the snake Lachesis muta muta (bushmaster).

The whole venom of Lachesis muta muta is preponderantly neurotoxic but moderately myotoxic on the chick biventer cervicis preparation (BCp). We have now examined these toxic activities of a basic phospholipase A(2), LmTX-I, isolated from the whole venom. LmTX-I caused a significant concentration-dependent neuromuscular blockade in the BCp. The time to produce 50% neuromuscular blockade was 14.7+/-0.75 min (30 microg/ml), 23.6+/-0.9 min (10 microg/ml), 34+/-1.7 min (2.5 microg/ml) and 39.2+/-3.6 min (1 microg/ml), (n=5/concentration; p<0.05). Complete blockade with all tested concentrations was not accompanied by inhibition of the response to ACh. At the highest concentration, LmTX-I (30 microg/ml) significantly reduced contractures elicited by exogenous KCl (20mM), increased the release of creatine kinase (1542.5+/-183.9 IU/L vs 442.7+/-39.8 IU/L for controls after 120 min, p<0.05), and induced the appearance of degenerating muscle fibers ( approximately 15%). Quantification of myonecrosis indicated 14.8+/-0.8 and 2.0+/-0.4%, with 30 and 10 microg/mlvenom concentration, respectively, against 1.07+/-0.4% for control preparations. The findings indicate that the basic PLA(2) present on venom from L. m. muta (LmTX-I) possesses a dominant neurotoxic action on isolated chick nerve-muscle preparations, whereas myotoxicity was mainly observed at the highest concentration used (30 microg/ml). These effects of LmTX-I closely reproduce the effects of the whole venom of L. m. muta in chick neuromuscular preparations.     

Potential human health risks from metals (Hg, Cd, and Pb) and polychlorinated biphenyls (PCBs) via seafood consumption: Estimation of target hazard quotients (THQs) and toxic equivalents (TEQs)

Edible marine species (fish, cephalopod molluscs, crustaceans) from the Adriatic Sea were analyzed for content in heavy metals (Hg, Cd and Pb) and polychlorinated biphenyls (PCBs). Health risks to human via dietary intake of seafood were assessed by the target hazard quotients (THQs) and the toxic equivalent factors (TEFs). Mercury maximum concentrations corresponded to fish (0.07-1.56 microg g(-1)w.w.), followed by cephalopod molluscs (0.10-0.55 microg g(-1)w.w.), and crustaceans (0.27-0.33 microg g(-1)w.w.). Cadmium levels in cephalopods (0.18-0.59 microg g(-1)w.w.) were higher than those in fish (0.01-0.05 microg g(-1)w.w.) and crustaceans (0.02-0.04 microg g(-1)w.w.), while for Pb the concentrations were generally low (fish: ND-1.18 microg g(-1)w.w., cephalopods: ND-0.17 microg g(-1)w.w., crustaceans: ND-0.03 microg g(-1)w.w.). For PCBs, concentrations in fish, cephalopods and crustaceans ranged between 141 and 3,406 ng g(-1)l.w., 190 and 542 ng g(-1)l.w., and 202 and 429 ng g(-1)l.w., respectively. Cd and Pb THQ values as well as estimates of PCB TEQ exposure indicated the absence of health risks through consumption of the various seafood. In contrast, mercury TEQs values due to consumption of certain fish species (albacore, rosefish and thornback ray) indicated that human health risk might be of concern.     

Anatoxin-a elicits an increase in peroxidase and glutathione S-transferase activity in aquatic plants

Although the toxic effects of cyanotoxins on animals have been examined extensively, little research has focused on their effects on macrophytes and macroalgae. To date only microcystins have been found to be detrimental to aquatic plants. Peroxidase activity of the free floating aquatic plant Lemna minor and the filamentous macroalga Chladophora fracta was measured after exposure to several concentrations of the cyanotoxin, anatoxin-a. Peroxidase activity (POD) was significantly (P < 0.05) increased after 4 days of exposure to an anatoxin-a concentration of 25 microg mL(-1) for both L. minor and C. fracta. Peroxidase activity was not significantly increased at test concentrations of 15 microg mL(-1) or lower. In another experiment, the effects of various concentrations of anatoxin-a on the detoxication enzyme, glutathione S-transferase (GST) in L. minor were investigated. GST activity was significantly elevated at anatoxin-a concentrations of 5 and 20 microg mL(-1). Photosynthetic oxygen production by L. minor was also found to be reduced at these concentrations. This is the first report to our knowledge of the cyanotoxin anatoxin-a being harmful to aquatic plants.     

Microcystin accumulation in liver and muscle of tilapia in two large Brazilian hydroelectric reservoirs.

The objective of this study was to measure levels of the toxin microcystin in different tissues of fish known to feed on cyanobacteria during toxic bloom events. Wild Nile and redbreast tilapia (Oreochromis niloticus and Tilapia rendalli) were sampled from the catch of artisanal fishermen at eutrophic stations of Funil and Furnas reservoirs in southeastern Brazil. Phytoplankton communities in the two reservoirs were quite different taxonomically, but not dissimilar in microcystin content (200 microg g dry weight (DW) seston(-1) at Funil, 800 microg gDW seston(-1) at Furnas). All of the 27 fish sampled contained microcystin, ranging from 0.8 to 32.1 microg g liver(-1) and from 0.9 to 12.0 ng g muscle(-1). Most microcystin variants found in seston were also found in fish liver. T. rendalli had the lowest concentration in both tissues when compared to O. niloticus. In both reservoirs, one of every four fish sampled, always O. niloticus, had a level of microcystins beyond the World Health Organization tolerable daily intake (8 ng g tissue(-1)) and represented a risk for consumers. It is possible that closer study of inter-species variability in toxin burden in cyanobacteria-impacted water bodies will permit the development of guidelines for fish consumption that will better protect public health.     

Exposure of beef cattle to sub-clinical doses of Microcystis aeruginosa: toxin bioaccumulation, physiological effects and human health risk assessment.

Yearling beef cattle were fed 1 x 10(5) cells ml(-1) of toxic Microcystis aeruginosa in their drinking water for a period of 28 d. Feed and water intakes of four control and four treated animals remained unchanged following introduction of M. aeruginosa into the drinking water of the treatment animals, and there were no significant differences in feed and water intakes between control and treated animals. We tested the blood plasma of both control and treated animals twice each week for elevated concentrations of the liver enzymes gamma-glutamyl transferase (GGT), glyceraldehyde dehydrogenase (GADH), amino aspartate transferase (AST) and bilirubin. All tests were negative indicating no measurable liver dysfunction resulting from microcystin intoxication. We also tested for the presence of free microcystin in the liver and blood plasma by HPLC and ELISA and for total microcystin (free+bound) in the liver by GC-MS. If all the ingested microcystin was bioaccumulated within the liver, the concentration would have exceeded 3 microg MCYST-LR g(-1) fresh weight. HPLC and GC-MS analysis of the liver tissue and blood plasma from treated animals failed to detect any microcystins. ELISA analysis of liver tissue extracts from the treated animals indicated microcystin concentrations as high as 0.92 microg MCYST-LR equivalents g(-1) fresh weight although none was indicated in the blood plasma. The microcystin concentrations measured by ELISA in livers of treated animals were more than 1000 times higher than the limit of quantification by HPLC and GC-MS indicating the ELISA results were almost certainly due to cross reactivity with something other than intact MCYST-LR. Based on the detection limits of the HPLC and the per capita daily consumption of beef in Australia, it appears that consumption by beef cattle of water containing M. aeruginosa cell concentrations up to 1 x 10(5)cells ml(-1) for 4 weeks would not produce concentrations of microcystin within the liver or blood plasma that would present an unacceptable risk to human health based on World Health Organization protocols for determining such risks.     

In vitro cytotoxicity of polycyclic aromatic hydrocarbon residues arising through repeated fish fried oil in human hepatoma Hep G2 cell line.

Repeated frying of vegetarian and non-vegetarian foods in edible oil is a common practice round the globe. Our studies suggest that repeated fish fried oil (RFFO) generates polycyclic aromatic hydrocarbons (PAHs), which may lead to hazardous effect on human health. In order to understand the mechanism of toxicity of RFFO extracts containing a mixture of PAHs, the in vitro cytotoxicity assays in human hepatoma cell line, Hep G2 was undertaken. In addition to RFFO extract, benzo(a)pyrene (BP) and chrysene were used as prototype compounds for heavy and light PAHs, respectively. Doses of BP and chrysene were made in such a way, that it could represent the appropriate content of heavy and light PAHs found in the RFFO extract. Out of total content of PAHs (1240.4 microg/kg) in RFFO, major composition is of light PAHs (854.8 microg/kg) while heavy PAHs showed the concentration of 385.7 microg/kg. Treatment of cells with 1 microg/ml RFFO extract for 48 h showed significant induction in ethoxyresorufin-O-deethylase (EROD) activity. Exposure of cells to higher doses of RFFO extract (10-100 microg/ml) for 24, 48 and 72 h caused 3.5-5.2, 4.3-8.5 and 1.8-2.3-fold enhancement in EROD activity, respectively. Further, RFFO extract caused a dose dependent increase (2.1-3.5-fold) in aryl hydrocarbon hydroxylase (AHH) activity at 48 h. Induction of EROD and AHH activity in Hep G2 cells was found to be relatively more following BP or chrysene treatment as compared to RFFO extract. RFFO extract did not cause any significant effect on cell viability at 1 microg/ml and 10 microg/ml. However, at 100 microg/ml concentration RFFO extract significantly decreased the cell viability at 24, 48 and 72 h. Exposure of 10 microg/ml RFFO extract reduced the colony forming ability (CFA) of Hep G2 cells with maximum decrease of 33.5% at 72 h. However, exposure of cells to RFFO extract at highest concentration of assay (100 microg/ml) reduced CFA (35-52%) at 24, 48 and 72 h. RFFO extract (1-100 microg/ml) had no significant effect on growth inhibition of cell up to 48 h of exposure. However, exposure of RFFO extract at all doses showed significant growth inhibition (20-25%) at 72 h. In conclusion, the results suggest that RFFO extract has substantial cytotoxic potential through the metabolic activation process of PAHs generated per se.     

Mediation of glutamatergic receptors and nitric oxide on striatal dopamine release evoked by anatoxin-a. An in vivo microdialysis study

In this work, the involvement of ionotropic glutamatergic receptors and nitric oxide on striatal dopamine release induced by anatoxin-a was investigated in conscious and freely-moving rats. To study the participation of glutamatergic receptors, the effects of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptors antagonist, 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), and N-methyl-D-aspartate (NMDA) receptor antagonists, dizocilpine (MK-801) and d(-)-2-amino-5-phosphonopentanoic acid (APV), were examined. The perfusion of 3.5 mM anatoxin-a increased the extracellular dopamine levels to 701% relative to the basal. When CNQX was administered with 3.5 mM anatoxin-a, the increase of dopamine levels was 29% smaller than that observed with anatoxin-a alone. When MK-801 and APV were administered, the effect of anatoxin-a was attenuated 26% and 25% respectively in terms of that observed with anatoxin-a alone. And with CNQX plus MK-801, the effect of anatoxin-a was 53% inhibited in terms of the effect of anatoxin-a alone. These results suggest that the striatal dopamine release induced by anatoxin-a is partly mediated by activation of both ionotropic glutamatergic receptors. Since the neuronal form of nitric oxide synthase (nNOS) produces nitric oxide (NO) primarily in response to activation of NMDA receptors, it was tested if NO could play any role in the effect of anatoxin-a. Treatment with NOS inhibitors, L-nitro-arginine methyl ester (L-NAME) and d(-)-2-amino-5-phosphonopentanoic acid (7-NI), induced decreased anatoxin-a effects of 22% and 26% respectively. In conclusion, the present in vivo results demonstrate that anatoxin-a induced an indirect activation of ionotropic glutamatergic receptors (NMDA and AMPA/kainite receptors), which stimulate striatal dopamine release. On the other hand, activation of NMDA receptors may elicit NO increased levels enhancing dopamine release.     

Effect of sodium diclofenac on serum and tissue concentration of amoxicillin and on staphylococcal infection

The effect of sodium diclofenac on serum and tissue amoxicillin concentration as well as their effect against staphylococcal infection was observed. Four polyurethane sponges were placed in the back of thirty rats. After 14 d, two granulomatous tissues received 0.5 ml of 10(8) cfu/ml (Staphylococcus aureus). Two days later, the rats were divided into five groups: group 1 received amoxicillin 50 mg/kg/p.o., group 2 received amoxicillin 25 mg/kg/p.o., group 3 received sodium diclofenac 2.5 mg/kg/i.m. and amoxicillin 50 mg/kg/p.o., group 4 received sodium diclofenac 2.5 mg/kg/i.m., and group 5 (control group) received NaCl 1 ml/p.o. After six hours of drug administration, blood serum (10 microl) and noninfected granulomatous tissues were placed on Mueller-Hinton agar inoculated with 10(8) cfu/ml (S. aureus). Infected tissues were dispersed in a sonic system and were spread (10 microl) on salt mannitol agar. Microorganisms were counted and the inhibition zones were measured after 18 h of incubation at 37 degrees C. Amoxicillin tissue concentration was 6.27 microg/g for group 1, 2.18 microg/g for group 2, and 0.72 microg/g for group 3. The serum concentrations were 11.56 microg/ml for group 1, 5.36 microg/ml for group 2, and 1.34 microg/ml for group 3. No differences were observed among group 1, 2, and 3 regarding staphylococci counts (Kruskall-Wallis test p>0.05). Group 4 reduced (p<0.05) staphylococci counts comparing to group 5. It was concluded that sodium diclofenac reduced serum and tissue amoxicillin concentration and, even in large doses, amoxicillin was not effective in eradicating the staphylococcal infection after 6 h of administration.     

First-derivative spectrophotometric and LC determination of cefuroxime and cefadroxil in urine

Two methods are presented for the determination of cefuroxime and cefadroxil in human urine using first (1D) derivative spectrophotometry and high-performance liquid chromatography. Cefuroxime and cefadroxil were determined by measurement of their first-derivative amplitude in 0.1 N sodium hydroxide at 292.5 and 267.3 nm, respectively in the concentration range of 2-10 microg ml(-1) for each drug. The HPLC method depends upon using a LiChrospher 100 RP-18 (5 microm) column at ambient temperature for cefuroxime and 35 degrees C for cefadroxil with mobile phases consisting of water-acetonitrile-acetic acid (85:15:0.1 v/v) at a flow rate of 1.5 ml min(-1) for cefuroxime; and 0.02 M potassium dihydrogen phosphate-acetonitrile (95:5 v/v) containing 0.003% (w/v) hexanesulphonic acid sodium salt and adjusted to apparent pH 3 with phosphoric acid at a flow rate of 2 ml min(-1) for cefadroxil. Quantitation was achieved with UV detection at 275 and 260 nm for cefuroxime and cefadroxil, respectively, based on peak area with linear calibration curves at the concentration ranges of 2-10 microg ml(-1) for cefuroxime and 5-20 microg ml(-1) for cefadroxil. The proposed methods were applied to the determination of dissolution rate for tablets and capsules containing each drug. The urinary excretion patterns as the cumulative amounts excreted have been calculated for each drug using the proposed methods.     

Estimating the accumulation and transfer of Nodularia spumigena toxins by the blue mussel Mytilus edulis: an appraisal from culture and mesocosm experiments.

Accumulation of Nodularia spumigena toxins by Mytilus edulis was studied during laboratory and mesocosm experiments in order to investigate the possible pathways of nodularin in mussels and calculate toxin budgets. Mussels were exposed to 0.2-15.6 microg nodularin l(-1), fed for up to 5 days with Nodularia cells from culture, or blooming in different nutrient-treated seawater. Toxin concentration was monitored with LC-ESI-MS. During different exposures, the amount of nodularin detected in mussels increased linearly with increasing toxin concentration in food and attained 0.28-13.8 microg of nodularin g dw(-1) of the mussel whole body tissue after 12 h. The digestive gland was found to be the tissue with the highest toxin concentration. Nodularin concentration in faeces was not proportional to faeces production or to toxin concentration in food; however, it seemed to be mostly related to food quality as well as to food availability. The percentage of nodularin taken up by the mussels, relative to the amount contained in the offered food, varied from 10% to 20%, depending on food quality. During a 5-day toxin accumulation experiment, the acute reduction of the toxin in mussel tissues the second day and the following stabilization, showed that probably mussels maintain low toxin levels via efficient elimination and/or toxin metabolism. After a 72 h depuration period, mussels showed 75% reduction in their toxin content.     

Renal,Biliary and Intestinal Clearance of a Quaternary Ammonium Compound,Emepronium in the Dog

Abstract Different routes of elimination of emepronium have been quantitated in conscious and anaesthetized dogs. Smoothed plasma concentration time curves and urinary excretion data obtained following intravenous bolus injection of 5 mg/kg indicate at least four different phases with the following half-lives: 5 min., I hour, 10 hours and 9 days. The initial dilution space, 0.2-0.6 l/kg, is rather similar to the extracellular volume (0.20-0.35 l/kg). From infusion experiments plasma clearance of emepronium have been found to be 33±2 ml. min^-1.kg^-1 (meani ± S.E.M., n = 14). The drug is secreted by the renal tubules at a maximum rate of 20 μg/min. at a plasma concentration of about 200 μg/l. Compared with other quaternary ammonium compounds, this is an easily saturated mechanism with a low capacity. The renal clearance constitutes about 30 % of the plasma clearance at a plasma concentration of 200 μg/l. The biliary excretion rate increases proportionally to a plasma concentration up to 100 μg/l and then reaches a maximal rate which is dependent on the bile flow. The biliary clearance decreases from 7 to 2 ml.min.^-1.kg^-1 when the bile flow changes from 7 to 4 g/ hour and the plasma concentration is 200 μg/l. Experiments with bile diversion and saline perfusion of isolated intestinal segments reveal an intestinal route of elimination of both emepronium and metabolites. The bile diversion experiments indicate that about 5-15% of an intravenous dose is excreted through the gastrointestinal epithelium. Perfusion of isolated intestinal loops indicates an apparent clearance of 4-5 ml. min^-1.m^-1 over a wide range of steady state plasma concentrations (20-1200 μ/l). An apparent total intestinal clearance could be calculated to about 7 ml. min.^-1.kg^-1. The remaining part of the plasma clearance (about 8 ml. min.^-1. kg^-1 in an anaesthetized dog) is accounted for by metabolism. The degree of protein binding has been shown to be low, about 20 per cent, and the red blood cell penetration in vitro is only 1-10% of the total amount present in whole blood.     

Potential developmental toxicity of anatoxin-a, a cyanobacterial toxin

Some 2000 species of cyanobacteria (blue-green algae) occur globally in aquatic habitats. They are able to survive under a wide range of environmental conditions and some produce potent toxins. Toxin production is correlated with periods of rapid growth (blooms) and 25%-70% of blooms may be toxic. Anatoxin-a is an alkaloid neurotoxin that acts as a potent neuro-muscular blocking agent at the nicotinic receptor. Acute toxicity, following consumption of contaminated water, is characterized by rapid onset of paralysis, tremors, convulsions and death. Human exposures may occur from recreational water activities and dietary supplements, but are primarily through drinking water. The current studies were conducted to examine the effect of in utero exposure on postnatal viability, growth and neurodevelopment, to evaluate the potential of in vitro embryotoxicity, and to explore the synergistic relationship between anatoxin-a and the algal toxin microcystin-LR by the oral route. The results of preliminary studies on amphibian toxicity are also reported. Time-pregnant mice received 125 or 200 microg kg(-1) anatoxin-a by intraperitoneal injection on gestation days (GD) 8-12 or 13-17. Pup viability and weight were monitored over a 6-day period. Maternal toxicity (decreased motor activity) was observed at 200 microg kg(-1) in both treatment periods. There were no significant treatment-related effects on pup viability or weight on postnatal day (PND) 1 or 6. The GD 13-17 pups were evaluated on PND 6, 12 and 20 for standard markers of neurodevelopmental maturation (righting reflex, negative geotaxis and hanging grip time). No significant postnatal neurotoxicity was observed. In vitro developmental toxicity was evaluated in GD 8 mouse embryos exposed to 0.1-25 microm anatoxin-a for 26-28 h. Perturbations in mouse yolk sac vasculature were noted from the 1.0 microm concentration in the absence of significant embryonic dysmorphology. Potential algal toxin synergism was tested in mice receiving either 0, 500 or 1,000 microg kg(-1) microcystin-LR by gavage and approximately 50 min later receiving either 0, 500, 1,000 or 2,500 microg kg(-1) anatoxin-a by the same route. No deaths occurred at any dose and no definitive signs of intoxication were observed. Stages 17 and 25 toad embryos (Bufo arenarum) were exposed to 0.03-30.0 mg l(-1) of anatoxin-a for 10 days. Adverse effects included a dose-dependent transient narcosis, edema and loss of equilibrium. Most notable was the occurrence of 100% mortality at the high dose in both groups 6-13 days post-exposure. The observed delay between initial exposure and death is highly unusual for anatoxin-a.     

The toxicity of cyanobacterial toxins in the mouse: II anatoxin-a

Blooms of cyanobacteria are known to have caused poisoning in fish, waterfowl, animals and man. One of the low molecular weight toxins responsible for this is the neurotoxin anatoxin-a which has been detected in reservoirs used for domestic water supplies. While the acute behaviour of this alkaloid is clear, there is uncertainty regarding the effects on man of ingestion of anatoxin-a at low levels over longer periods. In order to assess this risk, a series of in vitro and in vivo experiments were undertaken to investigate the pharmacology, subacute toxicity, and the teratogenicity of anatoxin-a in the mouse. The results of this work were as follows: (1) Pharmacological screening studies confirmed that anatoxin-a is a potent nicotinic agonist which can produce neuromuscular blockade and death by respiratory arrest. Recovery from a single sub-lethal dose is rapid and complete; (2) Repeated sub-lethal oral administration over 28 days in the mouse did not produce any reliable evidence of treatment-related toxicity; (3) From a preliminary screening study anatoxin-a does not appear to be a developmental toxicant in the mouse. These results indicate that a guideline value for anatoxin-a in drinking water of 1 microg l(-1) would provide an adequate margin of safety.     

Synthesis, X-ray crystal structure study, and cytostatic and antiviral evaluation of the novel cycloalkyl-N-aryl-hydroxamic acids

In vitro evaluation of the novel cycloalkyl-N-(4-chlorophenyl)-hydroxamic acids (2a-g) demonstrated that 2b,d,e exhibited rather marked inhibitory activity (IC50 = 7-10 microM) against pancreatic carcinoma, 2b-d against colon carcinoma, 2d against laryngeal carcinoma, and 2b,d against breast carcinoma. 2e showed the most pronounced anti-cytomegalovirus activity (EC50 = 1.5 and 0.8 microg mL(-1)) only at > or = 5-fold lower than the cytotoxic concentration. 2d and 2f showed modest, albeit selective, activity against cytomegalovirus (2d, EC50 = 7.3-8.9 microg mL(-1), selectivity index 7-10; 2f, EC50 = 7-13 microg mL(-1), selectivity index 10).     

Spectrophotometric determination of labetalol in pharmaceutical preparations and spiked human urine

Two simple and sensitive spectrophotometric methods were developed for the spectrophotometric determination of labetaolol (LBT). Both methods are based on the phenolic nature of the drug. The first method (Method I) is based on coupling LBT with diazotized benzocaine in presence of trimethylamine. A yellow colour peaking at 410 nm was produced and its absorbance is linear with the concentration over the range 1-10 microg ml(-1) with correlation coefficient (n=5) of 0.9993. The molar absorptivity was 2.633 x 10(4) l mol(-1) cm(-1). The second method (Method II) involves coupling LBT with diazotized p-nitroaniline in presence of sodium carbonate. An orange colour peaking at 456 nm was obtained and its absorbance is linear with concentration over the range 1-10 microg ml(-1) with correlation coefficient (n=5) of 0.99935. The stoichiometry of the reaction in both cases was accomplished adopting the limiting logarithmic method and was found to be 1:1. The developed method could be successfully applied to commercial tablets. The results obtained were in good agreement with those obtained using the official methods. No interference was encountered from co-formulated drugs, such as hydrochlorothiazide. The method was further extended to the in-vitro determination of LBT in spiked human urine. The % recovery (n=4) were 97.7+/-5.75 and 103.27+/-5.42 using the Methods I and II, respectively.     

Neurotoxic and myotoxic actions from Lachesis muta muta (surucucu) whole venom on the mouse and chick nerve-muscle preparations.

Lachesis genus is one of the less studied among others from Viperidae's genera, mainly due to difficulties in obtaining the venom. Accidents by Lachesis snakes cause severe envenoming syndrome, eventually leading victims to shock. This work is part of a comprehensive study aimed at studying the venom and its effects. Herein the neurotoxicity and myotoxicity of L. muta muta venom were investigated on mouse phrenic nerve-diaphragm (PNDp) and chick biventer cervicis (BCp) preparations. For both preparations the time required to venom produces 50% neuromuscular blockade was indirectly concentration-dependent, being for PNDp: 117.6+/-6.5 min (20 microg/ml), 70.1+/-8.6 min (50 microg/ml) and 43.6+/-3.8 min (100 microg/ml), and for BCp: 28+/-1.8 min (50 microg/ml), 30.4+/-2.3 min (10 microg/ml), 50.4+/-4.3 min (5 microg/ml) and 75.2+/-0.7 min (2 microg/ml), (n=5/dose). In BCp, a venom dose of 50 microg/ml significantly reduced contractures elicited by exogenous acetylcholine (55 microM) and KCl (20 mM), as well as increased the release of creatine kinase (442.7+/-39.8 IU/l in controls vs 4322.6+/-395.2 IU/l, after 120 min of venom incubation (P<0.05). Quantification of myonecrosis in BCp indicated the doses 50 and 10 microg/ml as significantly myotoxic affecting 59.7+/-6.2%, and 20.8+/-1.2% of fibers, respectively, whereas 5 and 2 microg/ml that affected 13.5+/-0.8% and 5.4+/-0.6% of fibers, were considered weakly- and non-myotoxic, respectively. We concluded that there are neurotoxins present in the venom, the concentration of which governs its pre- (if low) or postsynaptic (if high) activity. Since myotoxicity in the avian preparation is negligible at lower venom doses, but not neurotoxicity, we suggest that this effect may contribute minimum to the venom neurotoxic effect. The BCp is more sensible than PNDp to Lachesis m. muta venom.     

In vitro studies of cellular and molecular developmental toxicity of adjuvants, herbicides, and fungicides commonly used in Red River Valley, Minnesota

Recent epidemiologic studies showed increased frequency of birth defects in pesticide applicators and general population of the Red River Valley, Minnesota. These studies further indicated that this crop growing area used more chlorophenoxy herbicides and fungicides than elsewhere in Minnesota. Based on frequency of use and known biology, certain herbicides, pesticide additives, fungicides, and mycotoxins are suspect agents. To define whether these agents affect developmental endpoints in vitro, 16 selected agrochemicals were examined using the MCF-7 breast cancer cell line. In the flow cytometric assay, cell proliferation in this estrogen-responsive cell line indicates xenobiotic-mediated estrogenic effects. Cell viability, morphology, ploidy, and apoptosis were incorporated in this assay. Data showed that the adjuvants X-77 and Activate Plus induced significant cell proliferation at 0.1 and 1 microg/ml. The commercial-grade herbicides 2,4-D LV4 and 2,4-D amine induced cell proliferation at 1 and 10 microg/ml. The reagent-grade 2,4-D products failed to induce proliferation over the same concentration range, suggesting that other ingredients in the commercial products, presumably adjuvants, could be a factor in these results. The fungicides triphenyltin and mancozeb induced apoptosis at concentrations of 4.1 microg/ml (10(-5) M) and 50 microg/ml, respectively. Triphenyltin also induced aneuploidy (C2/M arrest) at 0.41 microg/ml (10(-6) M). Data provide a mechanistic step to understanding human reproductive and developmental effects in populations exposed to these agrochemicals, and initiative to focusing limited resources for future in vivo animal developmental toxicity studies.     

Spectrophotometric method for the determination of nifedipine with 4-(methylamino)phenol and potassium dichromate

A new simple, sensitive and reproducible spectrophotometric method for the determination of nifedipine in pure and dosage forms has been proposed. It is based on the reduction of nifedipine with Zn/NNH4Cl, followed by coupling with N-methyl-1,4-benzoquinoneimine--the oxidation product of 4-(methylamino)phenol, to give a chromophore which absorbed maximally at 525 nm. The experimental conditions were optimised and Beer's law was obeyed over the concentration range of 5-175 microg ml(-1). The molar absorptivity, detection limit, recovery and RSD were found to be 1.9 x 10(3) l mol(-1) cm(-1), 1.1 microg ml(-1), 99.7-100.5% and 0.3-0.8%, respectively. The proposed method was compared favourably with the official B.P. method.     

Anti-tumor activity of an antibiotic peptide derived from apoprotein E

BACKGROUND: Recently, we found that a 30-mer peptide derived from apoprotein E (apoE) 133-162 has antibiotic activity that is comparable with the classic antibiotics and neutrophil-derived antibiotic peptide. In this study, we tested if apoE 133-162 also has anti-tumor activity against several cancer cell lines. MATERIALS AND METHODS: Two gastric cancer cell lines (MKN-7, MNN-1), two pancreatic cancer cell lines (PANC-1, Paca-2) and one colon cancer cell line (COLO201) were used for MTT cytotoxic assay. Calcein leakage from artificial liposomes was also tested, varying the composition of liposome. RESULTS: The apoE 133-162 peptide had cytotoxic activity against all tested human cancer cell lines in a dose-dependent manner. In the Paca-2 cell, an equivalent cytotoxic activity to 5-FU (10 microg/ml) was observed at about 40 microg/ml of apoE 133-162 peptide, but no synergistic effect of apoE 133-162 (40 microg/ml ) with 5-FU (10 microg/ml), nor inhibitory effect by heparin(100 microg/ml), was observed. In the calcein leakage test, in the presence of 150 mM NaCl, the presence of cholesterol attenuated the membrane perturbation activity of apoE 133-162, and the more acidic membrane was susceptible to lysis. CONCLUSION: ApoE 133-162 has anti-tumor activity, probably through perturbation and formation of ion-permeable "pores" in membranes.