Development and identification of monoclonal antibodies against parathion.

An amino-derivative of parathion was prepared and conjugated to human serum albumin (HSA) and bovine thyroglobulin (BTG) via diazonium condensation. Spleen cells producing high titer antibody were removed and fused with myeloma cells of SP2/0 origin. Using a conventional immunization protocol, we generated nine stable murine monoclonal antibodies (MAbs) producing cell lines to parathion. After four successive limiting dilutions, antibodies produced by nine clones had high affinities, ranging from 10(9) to 10(12) M(-1). These clones were found to be of IgG class and IgM class with k light chain. Subclass determination showed that the clones produced IgG(1), IgG(2a), IgG(2b), and IgM types of antibody. One clone (2H(9)) was used to establish the calibration curve with a sensitivity of 26 ng/mL, a practical working range of 46.8-6000 ng/mL parathion.

Aminoparathion: a highly reactive metabolite of parathion. 1. Reactions with polyphenols and polyphenol oxidase.

Spiking of tomato and apple fruits with parathion at different levels of about 1-4 mg/kg irradiation and under simulated sunlight conditions resulted in nearly complete photodegradation within 13 h, but extractable parathion degradation products could not be found in any case. However, after irradiation of an unrealistically spiked apple (134 mg/kg) different photoproducts including aminoparathion (AP) were detectable by HPLC, proving that the hitherto postulated photochemistry of parathion indeed takes place in the fruit cuticle environment. Besides the photoreduction pathway it was shown for the first time that AP is also easily formed by reduction of the primary photoproduct nitrosoparathion with thiols (cysteine, glutathione), while ascorbic acid only leaves hydroxylaminoparathion. In the presence of polyphenols, AP was effectively bound to quinone intermediates formed by both silver oxide and polyphenol oxidases. For pyrocatechol, a disubstituted o-quinone derivative could be isolated as a dark red addition product and structurally be elucidated. However, in the presence of caffeic acid, catechol, naringin, and quercetin, respectively, insoluble dark colored polymers precipitated within 48 h, while in the supernatants AP was not detectable any more. Polymer-bound and nonextractable AP was proven by transesterification with sodium ethoxide releasing O,O,O-triethyl thiophosphate which was determined by GC. Additionally, AP itself was a substrate for polyphenol oxidases, resulting in a quinone imine intermediate which in turn reacted with excessive AP yielding deep red colored di- and trimerization products.

Effect of organophosphorus insecticides and their metabolites on astroglial cell proliferation.

Though little attention has been given to the possibility that glial cells may represent a target for the developmental neurotoxicity of organophosphorus (OP) insecticides, recent evidence, obtained in particular with chlorpyrifos (CP), suggests that developmental exposure to this compound may indeed target astrocytes. To substantiate and expand these observations, we carried out a series of in vitro studies utilizing fetal rat astrocytes and a human astrocytoma cell line, 1321N1 cells, to investigate the effect of the OPs CP, diazinon (DZ) and parathion (P), their oxygen analogs chlorpyrifos oxon (CPO), diazoxon (DZO) and paraoxon (PO), and their metabolites 3,5,6-trichloro-2-pyridinol (TCP), 2-isopropyl-6-methyl-4-pyrimidol (IMP) and para-nitrophenol (PNP), on cell proliferation. In fetal rat astrocytes and astrocytoma cells maintained in serum, CP, DZ, P, CPO, DZO, and PO induced a concentration-dependent inhibition in [(3)H]thymidine incorporation with a very similar potency (IC(50) between 45 and 57 microM). Among the other metabolites, PNP was the most potent (IC(50)=70-80 microM), while TCP and IMP were much less effective (IC(50)>100 microM). Cytotoxicity appears to account only for a small part of the effect on DNA synthesis. OP insecticides and their oxons were three- to six-fold more potent in inhibiting [(3)H]thymidine incorporation when cells were synchronized in the G(0)/G(1) phase of the cell cycle and re-stimulated by carbachol or epidermal growth factor. These results suggest that OP insecticides and their oxons affect astroglial cell proliferation and that the transition from the G(0)/G(1) to the S/G(2) phase of the cell cycle may be particularly sensitive to the action of these compounds.

Maturation-dependent effects of chlorpyrifos and parathion and their oxygen analogs on acetylcholinesterase and neuronal and glial markers in aggregating brain cell cultures.

An in vitro model, the aggregating brain cell culture of fetal rat telencephalon, has been used to study the maturation-dependent sensitivity of brain cells to two organophosphorus pesticides (OPs), chlorpyrifos and parathion, and to their oxon derivatives. Immature (DIV 5-15) or differentiated (DIV 25-35) brain cells were treated continuously for 10 days. Acetylcholinesterase (AChE) inhibitory potency for the OPs was compared to that of eserine (physostigmine), a reversible AChE inhibitor. Oxon derivatives were more potent AChE inhibitors than the parent compounds, and parathion was more potent than chlorpyrifos. No maturation-dependent differences for AChE inhibition were found for chlorpyrifos and eserine, whereas for parathion and paraoxon there was a tendency to be more effective in immature cultures, while the opposite was true for chlorpyrifos-oxon. Toxic effects, assessed by measuring protein content as an index of general cytotoxicity, and various enzyme activities as cell-type-specific neuronal and glial markers (ChAT and GAD, for cholinergic and GABAergic neurons, respectively, and GS and CNP, for astrocytes and oligodendrocytes, respectively) were only found at more than 70% of AChE inhibition. Immature compared to differentiated cholinergic neurons appeared to be more sensitive to OP treatments. The oxon derivates were found to be more toxic on neurons than the parent compounds, and chlorpyrifos was more toxic than parathion. Eserine was not neurotoxic. These results indicate that inhibition of AChE remains the most sensitive macromolecular target of OP exposure, since toxic effects were found at concentrations in which AChE was inhibited. Furthermore, the compound-specific reactions, the differential pattern of toxicity of OPs compared to eserine, and the higher sensitivity of immature brain cells suggest that the toxic effects and inhibition of AChE are unrelated.

Pesticide decontamination from fabric by laundering and simulated weathering.

This laboratory study investigated the effectiveness of selected detergents and the phenomenon of simulated environmental conditions (weathering) on the removal of a commercial-grade mixture of parathion and methyl parathion from a three-layer laminated fabric. The weathering treatment consisted of exposure and non-exposure to simulated environmental conditions of heat, light, and humidity. Contaminated fabric samples were laundered in one of three detergents containing an anionic, a nonionic, and a combined anionic and nonionic surfactant. The test fabric, a three-layer fabric containing an impermeable microporous film laminated between two layers of nylon, was pipette-contaminated with 400 microliters of field strength pesticide solution and allowed to dry. Half of the contaminated samples were weathered in an Atlas Fade-Ometer. All of the contaminated samples were subsequently laundered in a Launder-Ometer. Percent of pesticide residue was determined by gas chromatography. Weathering did significantly reduce both parathion and methyl parathion residues remaining in the test fabric. No statistically significant difference was found among the three detergents. High amounts of both parathion and methyl parathion remained in the test fabric after weathering and laundry treatments. Before the test fabric can be recommended for use in protective garments further research is needed to develop more effective decontamination procedures.

In vivo genotoxic effects in mice of Metacid 50, an organophosphorus insecticide.

The genotoxic effect of methyl parathion, an organophosphorus insecticide commercially available as Metacid 50, was studied in Swiss albino mice using bone marrow and peripheral blood micronucleus tests. Single acute oral doses of the insecticide at concentrations of 75.0, 37.5, 18.75 and 9.375 mg/kg body weight, equivalent to 1/2 LD50, 1/4 LD50, 1/8 LD50 and 1/16 LD50, administered to female mice, elicited positive responses in bone marrow and peripheral blood micronuclei tests. Statistically significant increases in the frequency of micronuclei were observed at higher doses in both the tests performed. The data obtained for our experiments suggest that methyl parathion is a potent mutagen and so it is also likely to have a genotoxic effect in humans.

Behavioral effects of subchronic intoxication with parathion-methyl in male Wistar rats.

The behavioral effects of subchronic exposure of male Wistar rats to the organophosphorus compound parathion-methyl (for 6 weeks 1/50 or 1/100 of LD50, PO) were studied. Open-field (OF) and elevated plus-maze (EPM) tasks were used to decide whether or not the compound can affect behavior. Significant effects were measured in the OF activity during the first minute, on the activity of crossing outer squares, increasing latencies to leave center, start of rearing, grooming, and defecation. EPM parameters showed an increased amount of time spent in the open arms and a clear tendency to enter more frequently open arms. The defecation rate in the EPM was significantly decreased. Results are discussed in terms of effects on emotional behavioral components (arousal) and on physiological functioning of the peripheral nervous system (lengthening of latencies).

Impairment of the regulation of gonadal function in Channa punctatus by metacid-50 and carbaryl under laboratory and field conditions.

Regulation of gonadal function by gonadotropic hormone (GtH) and gonadotropin-releasing hormone (GnRH) in Channa punctatus was significantly affected by nonlethal levels of Metacid-50 and Carbaryl. Under laboratory conditions, the time-dependent decrease in serum GtH level was higher in Carbaryl-treated fish than in Metacid-50-treated fish. The situation was reversed in the field, with a higher inhibitory effect of Metacid-50 being recorded. On the other hand, pituitary GtH content and GnRH activity were inhibited to a greater extent by Metacid-50 than by Carbaryl under both field and laboratory conditions. The present findings highlight that even low doses of Metacid-50 and Carbaryl are effective enough to cause reproductive damage, as evidenced by homeostatic unbalance of the reproductive regulatory system.

Evaluation of estimations in vitro of tissue/blood distribution coefficients for organothiophosphate insecticides.

Physiologically based pharmacokinetic (PBPK) modeling of foreign chemicals is dependent on the accurate determination of their tissue/blood distribution coefficients, Kp (partition coefficients). The present study was undertaken to evaluate the validity of the in vitro estimation of the Kp values of the organothiophosphate insecticides parathion and methyl parathion by equilibrium dialysis. Data derived from previously published studies that utilized single-pass perfusions of mouse livers in situ with parathion or methyl parathion were analyzed to determine liver/perfusate Kp values from the equation Kp = (t 1/2ss) (Q)/(0.693) (VH), where Kp is the liver/perfusate distribution ratio, t 1/2ss is the half-life for approach to steady state of the chemical, VH is the liver volume, and Q is the perfusate flow rate. Kp values for methyl parathion were calculated to be 16.4 +/- 7.5 and 9.5 +/- 2.7 (mean +/- SD) for perfused livers and equilibrium dialysis, respectively, while estimates of Kp for parathion were 15.6 +/- 6.3 and 19.5 +/- 5.5 for perfused livers and equilibrium dialysis, respectively. These results indicate that equilibrium dialysis can be utilized to give an accurate estimate of tissue partitioning of parathion and methyl parathion from perfusate into perfused mouse livers.

Experimental study of the effects of a new protective cream against skin irritation provoked by the organic solvents n-hexane, trichlorethylene and toluene.

In a first experiment, the potential effectiveness of three cream formulations (A, B, C) against skin irritation due to n-hexane was evaluated using a guinea pig model. Semi-quantitative histopathological assessment of skin lesions were carried out. Due to interesting protective effects, cream formulation A (Travabon) was selected and further investigated. In a second series of experiments, this cream showed some protective effects against irritation due to toluene, but was not found efficacious against trichlorethylene irritation.

Studies on some kinetic parameters of aldolases in selected tissues of the freshwater fish (Tilapia mossambica) under the toxic impact of methyl parathion.

Kinetic parameters of aldolases in muscle, gill, liver and brain tissues of the teleost Tilapia mossambica were studied at sublethal concentrations with methyl parathion (MP). The pH activity profiles were optimal at pH 7.0 and 9.0 in gill, liver and brain tissues, whereas only a single peak at pH 7.0 was observed in muscle tissue of both control and MP-exposed fish. The pH 7.0-specific peak was confirmed as aldolase A and the pH 9.0-specific peak represents the tissue-specific aldolases: aldolase B in liver, aldolase C in brain and aldolase B-C in gill. It is further confirmed with the inhibitor sensitivity of aldolases at two peaks with 6 x 10(-3) M ATP or AMP. The pH-based substrate-dependent kinetics of aldolases showed a variable trend. The tissue-specific activity of pH 7.0-specific aldolases showed low Km values for gill followed by muscle, liver and brain tissues, suggestive of its high enzyme-substrate affinity. During MP exposure, the Vmax values of pH 7.0-specific aldolases in muscle, gill and brain were unchanged compared to controls, but Km values were decreased. The pH 9.0-specific aldolases of gill and liver from MP-exposed fish showed decreased Km values with a slight increase in Vmax values. Effectors such as lysine, arginine and Ca2+ inhibited, while histidine, cysteine, aspartic acid and alpha-ketoglutaric acid elevated the activity levels of aldolases.(ABSTRACT TRUNCATED AT 250 WORDS)

Laundry parameters as factors in lowering methyl parathion residue in cotton/polyester fabrics.

Cotton/polyester fabrics were contaminated with a 1.25% solution of methyl parathion (MeP) emulsifiable concentrate formulation, then laboratory laundered. The laundering variables were detergent type, concentration, water volume, and mechanical agitation. Specimens were unfinished (UN) and soil repellent finished (SR) fabrics. Heavy duty liquid and phosphate-built powdered detergents were equally effective when used in combination with a prewash product. A negative linear relationship between detergent concentration and MeP residue remaining after laundering was established. An interaction between detergent concentration and fabric finish was observed. Soil repellent finished fabrics required detergent concentrations above the recommended amount for more efficient soil removal. A negative linear relationship between water volume and after-laundering residue was observed. Water volume played a more significant role in pesticide removal than agitation during laundering.

Impact of nonlethal levels of Metacid-50 and carbaryl on thyroid function and cholinergic system of Channa punctatus.

With regard to brain acetylcholinesterase and acetylcholine and serum triiodothyronine (T3) and thyroxine (T4) profiles, a biphasic response pattern was elicited in Channa punctatus chronically exposed to nonlethal doses of locally used pesticides, namely, Metacid-50 and Carbaryl. Data revealed that these xenobiotics caused significant inhibition of brain acetylcholinesterase activity and a decrease in thyroxine level accompanied by a concurrent increase in acetylcholine accumulation and T3 level. It is surmised that Metacid-50 and Carbaryl influence both neural and hormonal functions.