Effect of malathion on Bufo arenarum Hensel development--I. Esterase inhibition and recovery.

Newly fertilized Bufo arenarum Hensel embryos were exposed continuously or for a brief period (72-120 hr) to malathion (44 ppm) and then resuspended in amphibian Ringer's solution. Continuous exposure depressed acetylcholinesterase (EC 3.1.1.7), butyrylcholinesterase (EC 3.1.1.8) and carboxylesterase (EC 3.1.1.1) activities. The activities of the three enzymes in embryos treated for 72 hr recovered after a delay of 24 hr, but these enzymes showed different rates of recovery in embryos treated for 120 hr. Acrylamide disc electrophoresis showed several bands of esterase activity in control embryos. Continuous exposure to malathion abolished all esterase activity within 48 hr, but if the exposure continued new bands of esterase activity appeared at 120 hr of exposure. The zymograms of embryos exposed for 72 or 120 hr to malathion and then transferred to uncontaminated medium for 120 hr were similar to that of control embryos.

Fibronectin and laminin enhance gingival cell attachment to dental implant surfaces in vitro.

The objective of this study was to determine if the biologically active molecules laminin and fibronectin could influence gingival cell attachment to implant surfaces in vitro. Flat squares of the following implant materials were tested: smooth, plasma-sprayed, and hydroxyapatite-coated titanium. The surfaces were coated with equimolar quantities of either laminin, fibronectin, or bovine serum albumin. Fibronectin coating of implant surfaces resulted in two to three times the enhancement of gingival fibroblast binding on all implant surfaces tested (P < .01), with a lesser effect on epithelial cells. Laminin coating resulted in three to four times the enhancement of gingival epithelial cell binding on all implant surfaces tested (P < .01), with a lesser effect on fibroblasts. The two cell types exhibited distinct preferences for the different molecules tested with the surface roughness having some influence.

Groundwater contamination by azinphos methyl in the northern Patagonic Region (Argentina).

Approximately 30 groundwater monitoring wells, under a fruit production field, in the Valley of the Neuquen River (Northern Patagonic, Argentina), to which different pesticides have been applied, were sampled eleven times between October 1995 and March 1997. Azinphos methyl was the main pesticide applied and it was detected with the highest frequency in groundwater wells during the period of intensive pesticide application in the Southern Hemisphere. Dimethoate, methidathion, fosmet, cipermethrin, carbaryl, propoxur, carbofuran, benomyl and carbendazim were also detected with lower frequency. The characteristic of the area under study was alkaline soil, with an organic matter content below 2.5% and texture sandy clay loam. The half life of azinphos methyl in soils was 166.2 days in the sun light for horizon A and 194.15 in the dark for horizon B. Leaching of azinphos methyl through the different soil horizons was minimum. On the basis of our lysimeter laboratory data, in which most of the pesticide was adsorbed into the soil column and only small quantities leachate, we inferred that the impact of azinphos methyl on groundwater would be minimal. However, field data indicates that there is a persistence of azinphos methyl in groundwater during the application season.

Removal of azinphos methyl by alfalfa plants (Medicago sativa L.) in a soil-free system.

The objective of this study was to investigate the removal of azinphos methyl assisted by alfalfa plants, with special emphasis on the effects of this compound on some plant's physiological parameters. Hydroponic cultures of alfalfa (Medicago sativa L., var Romagnola) were employed as a model system. These cultures were exposed to a nutrient medium containing 10 mg/l of azinphos methyl. A first-order kinetic approach was used to describe the removal of azinphos methyl from the solution. After 20 days of culture, the initial amount of azinphos methyl was reduced to non-detectable levels in the presence of plants. In the absence of plants, 20% of azinphos methyl remained in the solution after 30 days of treatment. The half-life of the pesticide was reduced from 10.8 to 3.4 days in the presence of plants. The growth index of alfalfa plants exposed to azinphos methyl was negatively affected. Chlorophyll contents were reduced after 24 h of treatment and thereafter the levels were comparable to that of control plants. The peroxidase activity of alfalfa roots was not affected by the presence of azinphos methyl. In conclusion, alfalfa plants were able to survive when exposed to an effective concentration of 10 mg/l of azinphos methyl in the root zone, with some alterations on their physiological parameters.

Dieldrin modifies the hydrolysis of PIP2 and decreases the fertilization rate in Bufo arenarum oocytes.

Carbachol treatment in Bufo arenarum oocytes decreases the radioactivity in [32P]PIP2 in the following 20 min after stimulation and increases the [3H]glycerol labeling of 1,2-DAG at 1 min of stimulation. On the contrary, in Dieldrin treated oocytes carbachol stimulation produces an increase in [32P]PIP2 labeling without changes in [3H]1,2-DAG radioactivity. The sustained hydrolysis of PIP2 observed in Control oocytes is necessary to generate the intracellular second messengers which initiate the fertilization pathway. The lack of response to muscarinic stimulation in Dieldrin treated oocytes, may be associated with an early activation of PIP2-PLC by the insecticide, producing a depletion of the PIP2 pool previous to the stimulation with carbachol. These changes take place simultaneously with a decrease in the ability of Bufo arenarum oocytes to be fertilized in vitro, suggesting a correlation between impairment in the PIP2 cascade and a decrease in the fertilization rate.

Azinphos methyl residues in shallow groundwater from the fruit production region of northern Patagonia, Argentina.

Groundwater samples were collected from monitoring wells along an area of intensive fruit production. Different types of correlations were investigated between soil physicochemical characteristics, depths and agricultural practices with pesticide detection frequency. In the three analyzed periods azinphos methyl, S-(3,4-diydro-4-oxobenzo[d]-[1,2,3]-triazin-3-ylmethyl)-O,O-dimethyl phosphorodithioate, showed a definite seasonal behavior related to the application pattern, increasing its concentration in the aquifer from October to November-December and then decreasing towards March. Samples obtained during the non-spraying season showed that azinphos methyl residues were lower than detection limit (LOD). An inverse correlation was observed between azinphos methyl concentration and the time elapsed since the last pesticide application. Seasonal events such as rain and irrigation influence the chemical concentration in the well, while no correlation was obtained between soil characteristics and azinphos methyl concentration. The soil attenuation capacity was not enough to prevent the presence of azinphos methyl in the aquifer during the application season.

Prenatal exposure to pesticides: analysis of human placental acetylcholinesterase, glutathione S-transferase and catalase as biomarkers of effect.

Pre- and perinatal exposure to pesticides is deleterious on foetal and neonatal development, but information regarding possible effects on environmental low-dose exposure to pesticides is scarce. Most epidemiological studies of the health effect of pesticides have been based on self-reported information. However, detailed information on past pesticide use is difficult to reconstruct. This is a current study conducted among pregnant mothers attending a delivery care and perinatal programme at a public hospital. The study investigates biomarkers of early effects in placentas from women living in an area with an intensive use of pesticides in the northern part of Patagonia, province of Río Negro, Argentina, and it assesses the consistency of the information provided by self-reports. The study confirms that placental acetylcholinesterase and catalase activities are significantly associated with periods of organophosphorus pesticides application, while glutathione S-transferase is not affected. We found a positive correlation between environmental exposure to organophosphorus pesticides and carbamate insecticides and newborn head circumference. The findings provide a further indication of a link between placenta acetylcholinesterase and catalase activity and prenatal exposure to pesticides in population studies. Both placenta enzymes may be used as biomarkers in health surveillance programmes for early diagnosis of exposure related alterations produced by organophosphorus pesticides and carbamate pesticides.

Phosphoinositide phosphorylation and shape changes produced by phosmet-oxon in human erythrocytes.

1. "In vitro" incubation of red blood cells with phosmetoxon induced crenated and invaginated forms. 2. [32P] phosphate incorporation was greater in membranes from erythrocytes exposed to 300 nM phosmetoxon for 10 min than in control cells. 3. The highest incorporation was for phosphatidylinositol (PI), followed by phosphatidylinositol phosphate (PIP) and phosphatidylinositolbiphosphate (PIP2). 4. An activation of phosphatidylinositol (PI) kinase was detected with 150 and 300 nM of the pesticide, while there was no change in poliphosphoinositides (PPI) phosphodiesterase activity. 5. Results suggest an association between changes in PI kinase activity, the phosphorylation cycle of phosphatidylinositols and alterations in erythrocyte morphology induced by phosmetoxon.

The turnover of phospholipid fatty acyl chains is activated by the insecticide Dieldrin in Bufo arenarum oocytes.

Dieldrin is a widespread environmental contaminant hazardous to many wildlife species. Some evidence obtained with Bufo arenarum oocytes indicates that Dieldrin decreases the fertilization rate in amphibian oocytes, but little is known about mechanisms by which the pesticide affects fertilization. Therefore, we investigated the effect of Dieldrin on oocyte phospholipid metabolism. Freshly obtained oocytes, prelabeled with 2 3H-glycerol or 9-10 3H palmitate, were exposed to 4 mg/L Dieldrin for 2 hours. Dieldrin reduced the amount of 2 3H-glycerol incorporation in all phosphoglycerides classes: PI, PA, PS, and SPH were affected in 80% of the cases and PC and PE were only reduced in 39% of the cases. The incorporation in neutral lipids was not affected. On the contrary, 9,10 3H-palmitate incorporation increased in PC, PI, and PA, but TAG and FFA decreased. The more efficient incorporation of 3H-palmitate compared with 3H-glycerol in Dieldrin-treated oocytes suggests the operation of an alternative route other than de novo synthesis for phospholipids. The retailoring of phosphoglycerides via a deacylation-acylation pathway was demonstrated. These changes in phospholipid metabolism could be associated with the activation of certain enzymes produced by the pesticide.

The role of glutathion conjugation in the regulation of early toad embryos' tolerance to pesticides.

Reduced glutathion (GSH) content and glutathione S-transferase (GSH S-transferase) activity were investigated in developing toad embryos exposed to parathion, malathion, lindane and dieldrin. The embryonic GSH content was reduced after 96 h of incubation with 20.00 ppm malathion and 2.00 ppm lindane. Parathion and dieldrin did not produce any change. A similar effect was obtained in advanced stages of development (6-days larvae), but only with malathion. No correlation between the decrease in GSH level and mortality or morphologic abnormalities was observed. The four pesticides increased the activity of GSH S-transferase indicating that the enzyme is susceptible to induction during early development. The higher effect depicted by malathion may be related with an enhanced conjugation of the pesticide. Both GSH decrease and GSHS-transferase induction modifies the cell redox status and may indirectly influence transcription and translation. The early expression of GST genes provides the embryo with a useful mechanism for the regulation of tolerance against chemical stress.

Effect of exogenously applied polyamines on malathion toxicity in the toad Bufo arenarum Hensel.

The polyamines putrescine, spermidine, and spermine, are able to potentiate the toxicity of malathion in Bufo arenarum Hensel toad larvae. This action is synergistic and maximal with spermidine, which elevated up to 13-fold the mortality produced by this organophosphorus compound. Spermidine increased the degree of inhibition of acetylcholinesterase activity elicited by malathion, and impaired the recovery of this activity at the end of the treatment. Spermidine had no effect on the enzyme when applied alone. Toxic effects were also observed with the polyamines themselves when applied at concentrations similar to the intracellular levels described for rapid-growing organisms.

Is aging related to cell signalling alterations in amphibian oocytes?

The storage of Bufo arenarum oocytes decreased their ability to become fertilized "in vitro". The stimulation with carbachol of "young oocytes" showed a persistent hydrolysis with phosphatidylinositol 4,5 diphosphate (PIP2) while in "aged oocytes" both phosphatidylinositol 4 phosphate (PIP) and PIP2 were hydrolyzed at a non-significant level. These results and the lower 32P-phosphoinositide levels found in "aged oocytes" at time of stimulation agree with diminished phosphoinositide kinase and phospholipase C activities, as the consequence of a non-specific phosphoinositide hydrolysis probably occurring during storage.

Effect of pesticide use in fruit production orchards on shallow ground water.

As a part of landscape-scale study, ground water samples were collected from 30 wells located in fruit production farms belonging to the valley of Neuquen river during the period 1995-1998 and analyzed for organophosphate pesticides. As a consequence of the leaching process, ground water from the Valley of Neuquen River frequently contained concentrations of organophosphorus pesticides that exceeded acute toxicity risk ratios established to protect aquatic life. It was found that some pesticides, as azinphos methyl, had a high detection frequency, 66% of the samples, with concentrations varying from no detection to 48.9 ppb. Dimethoate, metidathion and phosmet were also detected with frequencies of 14.1, 13.6 and 10.8% and with concentration ranks from no detection to a maximum value of 10.9, 2.0 and 15.5 ppb, respectively. Seasonal variations and temporal trends were found for these compounds in ground water.

Is buffering capacity the principle role of the jelly coat in Bufo arenarum fertilization?

1. Dejellied Bufo arenarum oocytes can be fertilized in Ringer-Phosphate buffer with the same efficiency as jellied (control) oocytes. 2. Ringer-Phosphate buffer at pH = 7.4 not only provides buffering capacity but also the delta pH necessary for the acrosomal reaction. 3. The use of Ringer-TRIS buffer at pH = 7.4 does not render as good as Ringer-Phosphate buffer results, in terms of fertilization percentages. 4. Insemination of oocytes in Ringer-TRIS buffer interferes with early development.

Lipid metabolic pathways operating in amphibian full-grown oocytes.

The utilization of (2-3H)-glycerol in lipid biosynthesis was analyzed in Bufo arenarum and Xenopus laevis full-grown oocytes. The precursor was more actively incorporated in Xenopus laevis oocytes. Neutral glycerides were the lipids displaying the highest levels of radioactivity followed by phosphatidylcholine and phosphatidylethanolamine. After reincubation of prelabeled oocytes in a saline buffer solution, a net fall in labeled phosphatidic acid concomitant with an increase in phosphatidylcholine were detected. The present findings establish that glycerolipid biosynthesis is operative in full-grown oocytes. In addition, Xenopus laevis oocytes seem to be metabolically more active.

Thiols and polyamines in the potentiation of malathion toxicity in larval stages of the toad Bufo arenarum.

Treatment with exogenous spermidine enhanced acute malathion toxicity during larval development of the toad Bufo arenarum Hensel. The polyamine was rapidly incorporated in the larvae with a subsequent metabolization to putrescine and spermine, which were excreted to the media. Endogenous polyamine levels were not changed by either spermidine or malathion treatments. However, 0.5-mM spermidine modified malathion uptake and bioavailability increasing the concentration of the xenobiotic in the larvae. The amount of reduced thiols was decreased by both compounds, but the depletion was insufficient to induce cytotoxicity. The oxidative degradation of polyamines competes for the pool of reduced glutathione used in the conjugation of malathion in the larvae, thus leading to the reported potentiation of toxicity. Our results suggest that exposure to thiols-depleting agents may induce alteration of organophosphate degradation in amphibian larvae.

Comparative study of dieldrin effects on amphibian liver and early embryo phospholipids.

1. The effect of Dieldrin on phospholipid content and composition was compared in adult frog liver and in early embryos. 2. Male frogs are more resistant to acute Dieldrin treatment than females. Total phospholipids content increases in the liver of male animals while in females decreases. 3. Early embryo development was not affected morphologically by the addition of 0.02-10.0 mg of Dieldrin/1; however phospholipid content and composition was altered. 4. Adaptive changes in the embryo due to chronically administered Dieldrin is suggested.