Metabonomics analysis of urine and plasma from rats given long-term and low-dose dimethoate by ultra-performance liquid chromatography-mass spectrometry.

This study assessed the effects of long-term, low-dose dimethoate administration to rats by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Dimethoate (0.04, 0.12, and 0.36 mg/kg body weight/day) was administered daily to male Wistar rats through their drinking water for 24 weeks. Significant changes in serum clinical chemistry were observed in the middle- and high-dose groups. UPLC-MS revealed evident separate clustering among the different dose groups using global metabolic profiling by supervised partial least squares-discriminant analysis. Metabonomic analysis showed alterations in a number of metabolites (12 from urine and 13 from plasma), such as L-tyrosine, dimethylthiophosphate (DMTP), dimethyldithiophosphate (DMDTP), citric acid, uric acid, suberic acid, glycylproline, allantoin, isovalerylglutamic acid and kinds of lipids. The results suggest that long-term, low-dose exposure to dimethoate can cause disturbances in liver function, antioxidant and nervous systems, as well as the metabolisms of lipids, glucose, fatty acids, amino acids, and collagen in rats. DMTP and DMDTP, which had the most significant changes among all other studied biomarkers, were considered as early, sensitive biomarkers of exposure to dimethoate. The other aforementioned proposed toxicity biomarkers in metabonomic analysis may be useful in the risk assessment of the toxic effects of dimethoate. Metabonomics as a systems toxicology approach was able to provide comprehensive information on the dynamic process of dimethoate induced toxicity. In addition, the results indicate that metabonomic approach could detect systemic toxic effects at an earlier stage compared to clinical chemistry. The combination of metabonomics and clinical chemistry made the toxicity of dimethoate on rats more comprehensive.

Poisoning with the S-Alkyl organophosphorus insecticides profenofos and prothiofos.

BACKGROUND: Many organophosphorus (OP) insecticides have either two O-methyl or two O-ethyl groups attached to the phosphorus atom. This chemical structure affects their responsiveness to oxime-induced acetylcholinesterase (AChE) reactivation after poisoning. However, several OP insecticides are atypical and do not have these structures. AIM: We aimed to describe the clinical course and responsiveness to therapy of people poisoned with two S-alkyl OP insecticides-profenofos and prothiofos. DESIGN: We set up a prospective cohort of patients with acute profenofos or prothiofos self-poisoning admitted to acute medical wards in two Sri Lankan district hospitals. Clinical observation was carried out throughout their inpatient stay; blood samples were taken in a subgroup for assay of cholinesterases and insecticide. RESULTS: Ninety-five patients poisoned with profenofos and 12 with prothiofos were recruited over 5 years. Median time to admission was 4 (IQR 3-7) h. Eleven patients poisoned with profenofos died (11/95; 11.6%, 95% CI 5.9-20); one prothiofos patient died (1/12; 8.3%, 95% CI 0.2-38). Thirteen patients poisoned with profenofos required intubation for respiratory failure (13/95; 13.7%, 95% CI 7.5-22); two prothiofos-poisoned patients required intubation. Both intubations and death occurred late compared with other OP insecticides. Prolonged ventilation was needed in those who survived-a median of 310 (IQR 154-349) h. Unexpectedly, red cell AChE activity on admission did not correlate with clinical severity-all patients had severe AChE inhibition (about 1% of normal) but most had only mild cholinergic features, were conscious, and did not require ventilatory support. CONCLUSION: Compared with other commonly used OP insecticides, profenofos and prothiofos are of moderately severe toxicity, causing relatively delayed respiratory failure and death. There was no apparent response to oxime therapy. The lack of correlation between red cell AChE activity and clinical features suggests that this parameter may not always be a useful marker of synaptic AChE activity and severity after OP pesticide poisoning.

Albumin binding as a potential biomarker of exposure to moderately low levels of organophosphorus pesticides.

We have evaluated the potential of plasma albumin to provide a sensitive biomarker of exposure to commonly used organophosphorus pesticides in order to complement the widely used measure of acetylcholinesterase (AChE) inhibition. Rat or human plasma albumin binding by tritiated-diisopropylfluorophosphate ((3)H-DFP) was quantified by retention of albumin on glass microfibre filters. Preincubation with unlabelled pesticide in vitro or dosing of F344 rats with pesticide in vivo resulted in a reduction in subsequent albumin radiolabelling with (3)H-DFP, the decrease in which was used to quantify pesticide binding. At pesticide exposures producing approximately 30% inhibition of AChE, rat plasma albumin binding in vitro by azamethiphos (oxon), chlorfenvinphos (oxon), chlorpyrifos-oxon, diazinon-oxon and malaoxon was reduced from controls by 9+/-1%, 67+/-2%, 56+/-2%, 54+/-2% and 8+/-1%, respectively. After 1 h of incubation with 19 microM (3)H-DFP alone, the level of binding to rat or human plasma albumins reached 0.011 or 0.039 moles of DFP per mole of albumin, respectively. This level of binding could be further increased by raising the concentration of (3)H-DFP, increasing the (3)H-DFP incubation time, or by substitution of commercial albumins for native albumin. Pesticide binding to albumin was presumed covalent since it survived 24 h dialysis. After dosing rats with pirimiphos-methyl (dimethoxy) or chlorfenvinphos (oxon) (diethoxy) pesticides, the resultant albumin binding were still significant 7 days after dosing. As in vitro, dosing of rats with malathion did not result in significant albumin binding in vivo. Our results suggest albumin may be a useful additional biomonitor for moderately low-level exposures to several widely used pesticides, and that this binding differs markedly between pesticides.

[Studying chronic effects caused by alkaline products of from bituminous-salt masses obtained through destruction of sarin, soman and RVX].

The authors summarized study results on chronic effects caused by products of leaching from bituminous-salt masses obtained through destruction of sarin, soman and RVX. State of experimental rats was evaluated with integral informative tests (physiologic, biochemical, hematologic and morphologic) presenting changes in objective health parameters and revealing every disorder in organs and systems functioning.

[Pharmacokinetics and stability in the blood in vivo of phosphodiester and modified oligonucleotide derivatives]. / Issledovanie farmakokinetiki i stabil'nosti v krovi in vivo fosfodiéfirnykh i modifitsirovannykh proizvodnykh oligonukleotidov.

In vivo stability and distribution of deoxyribooligonucleotides and 3'-end modified oligodeoxyribooligonucleotides were studied in blood serum and cells. Substitution of two 3'-end internucleotide phosphodiester bonds increased the stability of modified oligonucleotides. Modified oligonucleotides were shown to be stable in blood serum for up to 24 hours. Leukocytes did not bind oligonucleotides significantly, whereas erythrocytes accumulate mono-, di-, three-, tetranucleotides appeared in the blood stream as degradation products of the parent oligonucleotide.

Dimethylphosphorus metabolites in serum and urine of persons poisoned by malathion or thiometon.

The urinary excretion rates of dimethyl-phosphate, -phosphorothioate and -phosphorodithioate were studied in six persons of whom four had ingested a concentrated solution of malathion and two of thiometon. The concentration decrease of single and total dimethylphosphorus metabolites was biphased, with a fast initial rate and a slow later rate. The excretion rate of total metabolites in the faster phase depended on the initial concentration in urine. At concentrations higher than 100 nmol/mg creatinine, the excretion half-times ranged from 7.5 to 15.4 h and at concentrations between 52 and 95 nmol/mg creatinine from 34.7 to 55.4 h. Non-metabolized malathion was detected only in one urine sample collected from one person immediately after hospitalization. Two persons poisoned with malathion were taken blood serum samples for the analysis of the parent pesticide and its metabolites on a daily basis after hospitalization. The parent pesticide was detectable in the serum only one day after the poisoning. The concentration of total malathion dimethylphosphorus metabolites in serum decreased very quickly within 1.5 days after hospitalization. The total metabolite elimination half-times were 4.1 and 4.7 h in the initial phase, and 53.3 and 69.3 days in the later slower elimination phase. There was no correlation between maximum concentrations of total metabolites measured in serum and/or urine on the day of admission to hospital and the initial depression of serum cholinesterase (BChE, EC 3.1.1.8) and erythrocyte acetylcholinesterase (AChE, EC 3.1.1.7).

Prothiofos metabolites in human poisoning.

CASE REPORT: A 63-year-old woman was admitted to a local hospital after the ingestion of 40% prothiofos preparation (Tokuthion) 370 mL. Gastric lavage was performed and cathartics, active charcoal, diuretics, atropine sulfate, and pralidoxime were administered. Serum cholinesterase activity was 1.3 IU/L (normal 200-460 IU/L). The patient's consciousness was gradually restored after 4 hours of charcoal hemoperfusion and she was discharged 5 days after admission with no sequelae. METHOD: Plasma and urine prothiofos and metabolites were detected by gas chromatography-flame photometry and gas chromatography-mass spectrometry. Two despropyl metabolites were synthesized for identification and estimation. RESULTS: The main metabolites were identified with authentic prothiofos and methyl esters of synthesized des-S-propyl prothiofos oxon (O-2,4-dichlorophenyl O-ethyl phosphate), despropyl prothiofos oxon (O-2,4-dichlorophenyl O-ethyl phospholothiolate), and des-S-propyl prothiofos (O-2,4-dichlorophenyl O-ethyl phosphorothioate). Despropyl prothiofos (O-2,4-dichlorophenyl O-ethyl phosphorodithioate) was also identified in plasma. Large amounts of the hydrolyzed product, 2,4-dichlorophenol and its conjugate were also found. The metabolic pattern of prothiofos in humans appears to be different from that in rats.

Study of phosphorothioate-modified oligonucleotide resistance to 3'-exonuclease using capillary electrophoresis.

The effect of phosphorothioate (PS) internucleotide linkages on the stability of phosphodiester oligodeoxyribonucleotides (ODNs) was investigated using 25-mer ODNs containing single or multiple PS backbone modifications. The in vitro stability of the oligomers was measured both in 3'-exonuclease solution and in plasma. For the separation of ODNs, capillary electrophoresis with a replaceable polymer separation matrix was used. As expected, DNA fragments with PS linkages at the 3'-end were found to be more resistant to 3'-exonuclease hydrolysis. Also increasing exonuclease resistance was the non-specific adsorption of phosphorothioate ODNs to enzyme.

Quantitation of postmortem profenofos levels.

CASE REPORT: An 88-year-old woman was found dead, and suicidal ingestion of profenofos, an organophosphate pesticide, was suspected. METHOD: Gas chromatography-nitrogen phosphorus detection was employed for quantitation of profenofos after its identification by gas chromatography/mass spectrometry. RESULTS: The levels of profenofos in whole blood, urine, and gastric contents were 1200 ng, 350 ng, and 3.35 mg/mL, respectively.

Experiences with acute organophosphate poisonings in Crete.

Nine human acute poisonings due to intentional ingestion of organophosphorous pesticides are presented. Six of the victims died. Six patients were treated in the Intensive Care Unit (ICU) from 34 h to 45 d, while 3 were found dead by relatives. Two of the patients treated in the ICU fully recovered after 15 and 24 d while the third survivor developed delayed neuropathy. Organophosphate blood levels were determined on admission and during therapy, and in 1 case atropine and pralidoxime levels were also detected. Significant fluctuations of the plasma cholinesterase activity were observed during therapy. Postmortem analysis revealed higher levels of pesticides in organs (eg 23.1 micrograms fenthion/g kidney) and in fat (135.2 micrograms fenthion/g) than in blood (eg 4.8 micrograms fenthion/ml) and vitreous humor. Considerable pesticide was measured in testis (eg 5.8 micrograms fenthion/g, 0.8 micrograms methidathion/g) and uterus (170.5 micrograms malathion/g). Extracorporeal decontamination to enhance pesticide elimination is a therapeutic challenge.

Chlorpyrifos metabolites in serum and urine of poisoned persons.

Concentrations of parent pesticide and corresponding diethylphosphorus metabolites in blood serum and urine were investigated in persons who had ingested a concentrated solution of organophosphorus pesticide chlorpyrifos. The organophosphate poisoning was indicated by a significant depression of blood cholinesterase (EC 3.1.1.7 and EC 3.1.1.8) activities. Blood and spot urine samples were collected daily after admission of the persons to hospital. Chlorpyrifos was detected only in serum samples in a period up to 15 days after poisoning. In the same samples chlorpyrifos oxygen analogue, chlorpyrifos oxon, was not detected. The presence of diethylphosphorothioate in all serum and urine samples confirmed that part of chlorpyrifos was hydrolysed before its oxidation. The maximum concentrations of chlorpyrifos in serum and of metabolites in serum and urine were measured on the day of admission. The decrease in concentrations followed the first-order kinetics with the initial rate constant faster and the later one slower. In the faster elimination phase chlorpyrifos was eliminated from serum twice as fast (t1/2 = 1.1-3.3 h) as the total diethylphosphorus metabolites (t1/2 = 2.2-5.5 h). The total urinary diethylphosphorus metabolites in six chlorpyrifos poisoned persons were excreted with an average elimination half-time of 6.10 +/- 2.25 h (mean +/- S.D.) in the faster and of 80.35 +/- 25.8 h in the slower elimination phase.

Diethylphosphorus metabolites in serum and urine of persons poisoned by phosalone.

The presence and elimination rate of phosalone and its diethylphosphorus metabolites in blood serum and urine were studied in persons who had ingested a concentrated phosalone solution. Phosalone was detected only in serum samples. As it was rapidly hydrolysed and eliminated from the body, its diethylphosphorus metabolites were a more sensitive indicator of exposure. The concentration decrease of phosalone in serum and of total diethylphosphorus metabolites in serum and urine followed the kinetics of a biphasic reaction. The faster elimination half-times in serum, calculated for two persons, were 2.3 and 3.4 h for phosalone and 3.4 and 38.6 h for total diethylphosphorus metabolites. In the faster phase the average elimination half-time of total urinary metabolites in five persons was 25 +/- 17 h. The kinetic data for total urinary metabolites in a person occupationally exposed to phosalone indicated an early and very fast elimination phase (elimination half-time 1.3 h), which was overlooked in poisoned persons. The proportions of single metabolites in total urinary metabolites in poisoned persons depended on whether the total amount of diethylphosphorus metabolites was above 1000 or below 1000 nmol/mg creatinine. Diethylphosphorodithioate predominated at high and diethylphosphate at low concentrations of total metabolites. The correlation between the maximum concentrations of total metabolites, measured in urine of poisoned persons on the day of admission to hospital or a day later, and the initial depression of serum cholinesterase (EC 3.1.1.8) and erythrocyte acetylcholinesterase (EC 3.1.1.7) activities was poor (r = 0.6).

Cholinesterase reactivation in organophosphorus poisoned patients depends on the plasma concentrations of the oxime pralidoxime methylsulphate and of the organophosphate.

We measured in nine patients, poisoned by organophosphorus agents (ethyl parathion, ethyl and methyl parathion, dimethoate, or bromophos), erythrocyte and serum cholinesterase activities, and plasma concentrations of the organophosphorus agent. These patients were treated with pralidoxime methylsulphate (Contrathion), administered as a bolus injection of 4.42 mg.kg-1 followed by a continuous infusion of 2.14 mg.kg-1/h, a dose regimen calculated to obtain the presumed "therapeutic" plasma level of 4 mg.l-1, or by a multiple of this infusion rate. Oxime plasma concentrations were also measured. The organophosphorus agent was still detectable in some patients after several days or weeks. In the patients with ethyl and methyl several days or weeks. In the patients with ethyl and methyl parathion poisoning, enzyme reactivation could be obtained in some at oxime concentrations as low as 2.88 mg.l-1; in others, however, oxime concentrations as high as 14.6 mg.l-1 remained without effect. The therapeutic effect of the oxime seemed to depend on the plasma concentrations of ethyl and methyl parathion, enzyme reactivation being absent as long as these concentrations remained above 30 micrograms.l-1. The bromophos poisoning was rather mild, cholinesterases were moderately inhibited and increased under oxime therapy. The omethoate inhibited enzyme could not be reactivated.

Acute poisoning with bromofosmethyl (bromophos).

One hour after suicidal ingestion of about 20 mL of a 38% solution of bromofosmethyl, CAS: 2104-96-3 (bromophos), a 52 year-old female was admitted to the hospital with extreme miosis, hypersalivation, hyperperistalsis and muscular fibrillation. Gastric lavage was performed and activated charcoal administered. Cholinergic symptoms were antagonized by repeated doses of 0.5 mg atropine. Because of the high dose of bromophos, hemoperfusion was performed with amberlite XAD4. The bromophos clearance during hemoperfusion was 95 mL/min (flow 200 mL/min). The patient received two doses of 500 mg obidoxime for recurrent muscular fibrillation. The further clinical course was uneventful. On day 4, the patient was transferred to a psychiatric ward because of persistent suicidality. In contrast to poisoning by most organophosphates, red blood cell acetyl cholinesterase was only minimally depressed but the plasma butyryl cholinesterase was initially decreased and normalized within a few days. The records of 25 patients reported to our Poison Control Center with ingestion of more than 1 g bromophos were also evaluated. The most frequent symptoms were miosis, hyperperistalsis, hypersalivation, agitation, nausea/vomiting and convulsions. Nine of the patients had no symptoms. Bromophos is relatively less toxic than its phosphate derivative, parathion.

The determination of O,S,S-trimethylphosphorodithioate in the plasma and various tissues of rats using high-resolution gas chromatography with nitrogen-phosphorus detection.

A method has been developed for the determination of O,S,S-trimethylphosphorodithioate in the plasma, lung, liver, brain and thymus of rats using high-resolution gas chromatography. The organophosphorus compound was extracted from the biological sample with ethyl acetate and analysed on a carbowax 20M fused-silica capillary column with a nitrogen-phosphorus specific detector. O,S,S-Triethylphosphorodithioate was used as an internal standard added to the sample before extraction. The sensitivity of the method allowed the compound to be measured in 0.1-ml aliquots of plasma or in 20-mg wet weight of tissue down to a level of 5 ng/sample. The method has been applied to a pharmacokinetic study in the rat after an oral or intravenous dosage with 25 mg/kg of O,S,S-trimethylphosphorodithioate.