Serum Organophosphorus compound levels as a surrogate marker for severity assessment and management in Acute Organophosphorus Poisoning - A novel approach.

Organophosphorus (OP) compounds are the most extensively used pesticides' class worldwide; cause most self­poisoning deaths especially in India. Thus, it is utmost important for early identification and aggressive management of OP poisoning from the clinical perspective to prevent serious complications by using sophisticated LC-MS/MS approach. This was a prospective study involving 103 patients of OP cases admitted to Karnataka Institute of Medical Sciences from June 2022 to May 2023, based on the inclusion and exclusion criteria patients were subjected to study. On admission, venous blood was collected from patient with Malathion and Profenofos OP poisoning history and subjected to serum biomarker and to LC-MS/MS analysis. Out of the 103 patients, 68 patients consumed Profenofos (66%) and 35 patients consumed Malathion (34%). Pseudocholinesterase levels among the of OP cases revealed that the 33 patients had mild toxicity, 40 patients had moderate toxicity and 30 patients had severe toxicity of OP poisoning. Subsequently LC-MS/MS analysis showed that the results obtained are not in correlation with indirect serum marker pseudocholinesterase levels. On the other side, LC-MS/MS results are in correlation with the clinical outcome of the patients with respect to morbidity and mortality. Thus, LC-MS/MS approach to assess the OP levels in patients could be used as potential diagnostic and prognostic marker for the absolute quantification of OP compounds compared to indirect OP levels estimation.

Exposure to environmentally relevant concentrations of malathion induces significant cellular, biochemical and histological alterations in Labeo rohita.

The extensive use of malathion, an organophosphate pesticide, raises the possibility of its undesirable toxicity to non-target organisms. Agricultural run-off and vector control sprays are the major sources of exposure to this pesticide for aquatic organisms. Some earlier studies have reported the presence of malathion at concentrations ranging from 18.12µg/L to 105.2µg/L in various water samples. In this study, we have tested the hypothesis that these sub-lethal yet environmentally significant concentrations of malathion has serious toxicological implications on the fingerlings of Labeo rohita. Exposure to increasing concentration of malathion (10, 50 and 100µg/L) was reflected in the serum concentration of the pesticide and also in the inhibition of acetylcholinesterase activity in fish brains. Increased abnormalities in liver function test coupled with a rise in the oxidative stress response were observed in gills, liver and kidney. However, the increase in antioxidant enzyme activities like superoxide dismutase and glutathione-S-transferase by malathion exposure suggested a hormetic response. Tissue injury due to malathion was evident from the morphological and nuclear anomalies in the H-E stained sections of gill, liver and kidney. Cell cycle analysis of these organs further fortified the histopathological findings. This study elucidates the sub-lethal toxicity of environmentally relevant malathion concentrations on Labeo rohita which indicates the potential health hazard posed to human beings consuming this fish. This calls for careful application of malathion in areas adjoining to inland fisheries.

Combined subchronic toxicity of dichlorvos with malathion or pirimicarb in mice liver and serum: a metabonomic study.

Organophosphorus (OP) and carbamate (CM) pesticides are widely used in agriculture. These pesticides are highly toxic to humans and their residues in food pose potential threat to human health. In this study, we investigated the effect of subchronic low-level exposure of OPs (dichlorvos, DDVP; malathion, MAL), CM pirimicarb (PI), or their mixtures (DDVP+MAL, DDVP+PI) on mice liver. Metabonomic analysis based on (1)H nuclear magnetic resonance spectroscopy was carried out in combination with biochemical assays. Serum metabonomic analysis showed that levels of trimethylamine-N-oxide, lactate, acetone, very low- and low-density lipoprotein and 3-hydroxybutyrate changed after exposure to the pesticides. In the liver extracts, lactate, glucose, choline, glutathione, alanine, glutamine and isoleucine levels changed after the treatment by pesticides. Our results indicated that exposure to low dose DDVP, MAL and PI, either alone or in combination lead to alteration of liver glucose, fat and protein metabolism, energy metabolism and oxidative balance. This study also showed that metabonomics is of potential use in food toxicity study.

Experimental measurements for the effect of dilution procedure in blood esterases as animals biomarker for exposure to OP compounds.

Organophosphate compounds can bind to carboxylesterase, which may lower the concentration of organophosphate pesticides at the target site enzyme, cholinesterase. It is unclear from the literature whether it is the carboxylesterase affinity for the organophosphate and/or the number of carboxylesterase molecules that is the dominant factor in determining the protective potential of carboxylesterase. The fundamental dilutions and kinetic effects of esterase enzyme are still poorly understood. This study aims to confirm and extend our current knowledge about the effects of dilutions on esterases activities in the blood for birds with respect to protecting the enzyme from organophosphate inhibition. There was significantly higher esterases activities in dilution 1 : 10 in the all blood samples from quail, duck, and chick compared to other dilutions (1 : 5, 1 : 15, 1 : 20, and 1 : 25) in all cases. Furthermore, our results also pointed to the importance of estimating different dilutions effects prior to using in birds as biomarker tools of environmental exposure. Concentration-inhibition curves were determined for the inhibitor in the presence of dilutions 1 : 5, 1 : 10, plus 1 : 15 (to stimulate carboxylesterase). Point estimates (concentrations calculated to produce 20, 50, and 80% inhibition) were compared across conditions and served as a measure of esterase-mediated detoxification. Results with well-known inhibitors (malathion) were in agreement with the literature, serving to support the use of this assay. Among the thiol-esters dilution 1 : 5 was observed to have the highest specificity constant (k(cat)/K(m)), and the K m and k cat values were 176 µM and 16,765 s(-1), respectively, for S-phenyl thioacetate ester, while detected in dilution 1: 15 was the lowest specificity constant (k(cat)/K(m)), and the Km and k cat values were 943 µM and 1154 s(-1), respectively, for acetylthiocholine iodide ester.

Malathion exposure and insulin resistance among a group of farmers in Al-Sharkia governorate.

OBJECTIVES: Exposure to certain environmental toxins may be associated with increased risk of developing diabetes mellitus. The aim of the present study was to investigate the relation between chronic exposure to malathion and insulin resistance among farmers. DESIGN AND METHODS: The study included 98 non diabetic farmers who handle agricultural insecticides during their field work. The range of the exposure period for agricultural pesticides was 15-20 years. All farmers were males with mean age 39±12 years. Another 90 administrative employees at Zagazig University Hospitals, non diabetic males age matched were selected as controls. History taking including family history for diabetes, assessment of blood pressure, height, weight, waist circumference and body mass index was done for all participants. Blood samples were withdrawn for measurement of malathion concentration, fasting blood glucose level and fasting insulin level for calculation of homeostasis model assessment of insulin resistance (HOMA-IR). RESULTS: 24.5% had positive family history for diabetes. It was observed that there was a significant increase in the mean values of malathion blood concentration among studied farmers compared to corresponding controls. There was a positive correlation between malathion blood concentration, waist circumference and insulin resistance. It was also observed that the increase in the mean values of waist circumference and body mass index was accompanied by a significant increase in the mean values of malathion blood concentration. CONCLUSION: The current results suggested that chronic exposure of non diabetic farmers to organophosphorus malathion pesticides may induce insulin resistance. This effect tended to strengthen as waist circumference increases.

Malathion-induced oxidative stress in human erythrocytes and the protective effect of vitamins C and E in vitro.

Malathion is an organophosphate (OP) pesticide that has been shown to induce oxidative stress in erythrocytes through the generation of free radicals and alteration of the cellular antioxidant defense system. We examined the effect of several different doses of malathion (25, 75, 200 microM), or malathion in combination with vitamin C (VC; 10 microM) or vitamin E (VE; 30 microM), on the levels of malondialdehyde (MDA), and superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities in human erythrocytes in vitro. Erythrocytes were incubated under various treatment conditions (malathion alone, vitamins alone, or malathion plus vitamin) at 37 degrees C for 60 min, and the levels of MDA, and SOD, CAT and GPx activities, were determined. Treatment with malathion alone increased the levels of MDA and decreased SOD, CAT, and GPx activities in erythrocytes (P < 0.05). There were no statistical differences among VC-treated, VE-treated, or VC + VE-treated erythrocyes, as compared with nontreated control cells. Treatment of cells with malathion + VC, malathion + VE, or a combination of all three agents prevented malathion-induced changes in antioxidant enzyme activity and lipid peroxidation. However, this effect was seen only at low concentrations of malathion (25 and 75 microM), and the combination of VC + VE had a more protective effect than VC or VE alone. These results indicated that the presence of vitamins at concentrations that are similar to the levels found in plasma have no effect on malathion-induced toxicity in erythrocytes at a concentration of malathion (200 microM) that is typically used in pesticides.

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.

Biochemical evidence for free radical-induced lipid peroxidation as a mechanism for subchronic toxicity of malathion in blood and liver of rats.

Organophosphorus compounds may induce oxidative stress leading to generation of free radicals and alterations in antioxidant and scavengers of oxygen free radicals (OFRs). The effect of subchronic exposure to malathion in the production of oxidative stress was evaluated in male Wistar rats. Administration of malathion (100, 316, 1000, 1500 ppm) for 4 weeks increased catalase (CAT), superoxide dismutase (SOD) activities as well as malondialdehyde (MDA) concentration in red blood cells (RBC) and liver. However, acetylcholinesterase (AChE) and cholinesterase (ChE) activities were decreased in these samples. The increase in RBC and liver lipid peroxidation correlated well with the inhibition in RBC AChE and liver ChE activities. Elevation of MDA concentrations and increased activities of CAT and SOD showed significant correlations in both RBC and liver samples when different doses of malathion were used. The results of the present study suggest the usefulness of RBC AChE measurement as a good biomarker in the estimation of malathion-induced oxidative stress affecting blood and liver.

Simultaneous determination of malathion, permethrin, DEET (N,N-diethyl-m-toluamide), and their metabolites in rat plasma and urine using high performance liquid chromatography.

A method was developed for the separation and quantification of the insecticide malathion (O,O-dimethyl-S-(1,2-carbethoxyethyl) phosphorodithioate), its metabolite malaoxon (O,O-dimethyl-S-(1,2-carbethoxyethyl) phosphorothioate), the insecticide permethrin (3-(2,2-dichloro-ethenyl)-2,2-dimethylcyclopropanecarboxylic acid(3-phenoxyphenyl)methylester), two of its metabolites m-phenoxybenzyl alcohol and m-phenoxybenzoic acid, the insect repellent N,N-diethyl-m-toluamide (DEET), and its metabolites m-toluamide and m-toluic acid in rat plasma and urine. The method used high performance liquid chromatography (HPLC) with reversed phase C(18) column, and UV detection at 210 nm. The compounds were separated using gradient of 45--99% acetonitrile in water (pH 3.5) at a flow rate ranging between 0.5 and 2 ml/min in a period of 15 min. The retention times ranged from 7.4 to 12.3 min. The limits of detection ranged between 20 and 100 ng/ml, while limits of quantitation were 50-150 ng/ml. Average percentage recovery of five spiked plasma samples were 80.1+/-4.2, 75.2+/-4.6, 84.5+/-4.0, 84.3+/-3.4, 82.8+/-3.9, 83.9+/-5.5, 82.2+/-6.0, 83.1+/-4.3, and from urine 78.8+/-3.9, 76.4+/-4.9, 82.3+/-4.5, 82.5+/-3.9, 81.4+/-4.0, 83.9+/-4.3, 81.5+/-5.0, and 84.5+/-3.8 for, malathion, malaoxon, DEET, m-toluamide, m-toluic acid, permethrin, m-phenoxybenzyl alcohol, and m-phenoxybenzoic acid, respectively. The method was reproducible and linear over range between 100 and 1000 ng/ml. This method was applied to analyze the above chemicals and metabolites following combined dermal administration in rats.

Detoxification of organophosphates by A-esterases in human serum.

1. In vitro detoxification of the organophosphate (OP) insecticides paraoxon, chlorpyrifos-oxon and malaoxon has been investigated in human serum. 2. Specific A-esterase activity to each OP substrate was measured in the serum of 100 individuals using established spectrophotometric methods for paraoxonase and chlorpyrifos-oxonase and a novel assay for malaoxonase activity. 3. Dose-effect inhibition of serum cholinesterase by the three OPs was measured in pooled human serum. Inhibition of calcium dependent A-esterases by addition of EDTA resulted in increased inhibition of cholinesterase at a given OP concentration. 4. Data from both the direct spectrophotometric measurement of A-esterase activity and inhibition of serum cholinesterase in the presence and absence of A-esterase activity indicated that human serum A-esterase catalysed detoxification of chlorpyrifos-oxon> paraoxon> malaoxon. Our data also confirms the wide variation in potency to inhibit cholinesterase between the three OPs. 5. Malaoxonase activity in human serum does not appear to be polymorphic, however, there is large inter-individual variation as has been previously found for other A-esterases. 6. This study has demonstrated two approaches to investigate the inter-individual variation towards specific OPs and the relative ability of human serum A-esterase to detoxify specific OP compounds.

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).

Rapid analysis of malathion in blood using head space-solid phase microextraction and selected ion monitoring.

A simple and rapid method for analysis of malathion in blood was developed using head space-solid phase microextraction (HS-SPME) and gas chromatography mass spectrometry/ electron impact ionization-selected ion monitoring (GC-MS/EI-SIM). A vial containing a blood sample, ammonium sulphate, sulphuric acid and fenitrothion as an internal standard, was heated at 90 degrees C for 15 min. The extraction fiber of the SPME was exposed for 5 min in the head space of the vial. The compounds absorbed on the fiber were detached by exposing the fibre in the injection port of GC-MS. A straight calibration curve was obtained between malathion concentrations of 2.5 to 50.0 micrograms g-1 in blood. No interfering substances were found, and the time for analysis was 40 min for one sample.

Relapse and elevation of blood urea nitrogen in acute fenitrothion and malathion poisoning.

We observed 6 patients with severe fenitrothion and/or malathion poisoning necessitating artificial ventilation and intensive care monitoring. Three developed relapse following acute cholinergic crisis. In these patients the blood urea nitrogen (BUN) abnormally elevated before the development of relapse and the initial high concentration of plasma organophosphate (OP) decreased only gradually. However, the patients who did not develop relapse showed no elevation of BUN and a relatively low concentration of plasma OP. This observation was confirmed in a retrospective search of 14 patients. In addition, erythrocyte cholinesterase (EChE) activities were more helpful to diagnose the development of relapse than plasma cholinesterase activities. Therefore, careful monitoring of BUN in addition to plasma OP concentration may be useful to predict the development of relapse.

Comparison of myoglobin, creatine kinase-MB, and cardiac troponin I for diagnosis of acute myocardial infarction.

Serial plasma concentrations of myoglobin, creatine kinase MB (CK-MB) isoenzyme, and cardiac troponin I (cTnI) were measured in 25 patients with a confirmed diagnosis of acute myocardial infarction (AMI), and 74 patients who were suspected of AMI but were subsequently ruled out for this diagnosis. The cutoff concentration for the cTnI assay was optimally determined to be 2.5 ng/mL. Of the three markers, myoglobin had the highest clinical sensitivity (50 percent) when blood was collected between 0 to 6 h after the onset of chest pain. Assays for all serum markers used had high clinical sensitivity (> 93 percent) 6 to 24 h after onset. The CK-MB remained highly sensitive for 48 h, while cTnI was sensitive for up to 72 h. Between 72 and 150 h, cTnI had a clinical sensitivity of 70 percent as compared to 21 percent and 18 percent for myoglobin and CK-MB, respectively. The clinical specificity of cTnI for non-AMI patients was equivalent to CK-MB and significantly higher than for myoglobin. The clinical efficiency of cTnI for all samples was better than either CK-MB or myoglobin, owing mainly to the wider diagnostic window. The specificity of cTnI for 59 patients with chronic renal failure, skeletal muscle trauma and disease was better than all of these markers including cardiac troponin T (cTnT). Results of this study show that cTnI is an effective marker for the retrospective diagnosis of AMI, and consideration should be given to its use in place of CK-MB.

Gas chromatographic determination of parathion and paraoxon in fish plasma and tissues.

A sensitive capillary gas chromatographic (GC) method for the simultaneous determination of the organophosphate insecticide, parathion, and its active metabolite, paraoxon, in biological samples was developed. This method involved a simple liquid-liquid extraction of parathion and paraoxon from water, plasma, or tissues and capillary GC determination using electron-capture detection and splitless injection; malathion was used as the internal standard. A gradient oven temperature program was used; the injection port and detector temperatures were 200 and 300 degrees C, respectively. These techniques allowed quantitative determination of parathion and paraoxon at 9-210-ng/ml. concentrations;recoveries ranged from 79.4 to 110.3% for tissues and from 91.9 to 100.0% for plasma and water. The within-day and between-day coefficients of variation were less than 8.0%. The method was used to characterize the pharmacokinetics of parathion and paraoxon and the tissue distribution of paraoxon in rainbow trout.

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.

Malathion disposition in dermally and orally treated rats and its impact on the blood serum acetylcholine esterase and protein profile.

14C-methoxy-malathion with either pure or 50% E.C. formulated malathion were applied orally or dermally at one tenth of their LD50 to two batches of male albino rats. More than 90% of 14C was released with urine after 24 hours. The rest of 14C was detected in the feces, blood, intestines, liver and kidney in a descending order. No significant 14C was detected in other organs. Comparing the oral pure and formulated malathion treatments, there was no significant variation in the rate of disposition or excretion of 14C-malathion. However, the dermal treatment revealed that the 14C-formulated malathion was released faster than the pure one in urine in the first 24 hours; while the 14C-pure malathion showed relatively higher levels in the feces and blood in the first 24 hours. In a third batch of male albino rats, the effect of the same level of dermal treatment by either pure or 50% E.C. formulated malathion on serum acetylcholine-esterase (A. Ch. E.) activity and serum protein profile was studied. The serum A. Ch. E. activity was found to be inhibited to 40% activity after 6 to 24 hours for both treatments. However, after 96 hours the serum of the pure malathion treated rats showed full recovery of A.Ch.E. activity, while the formulated malathion treated showed only 60% activity. The SDS-PAGE analysis showed a differentiation in the serum protein bands of the 48 hours exposed rats to formulated malathion which was confirmed by the scanned gel profile. The FPLC integrated chromatograms proved an initiation of a new protein band accompanied with rearrangement of the albumin and pre-albumin bands. Thus it can be concluded that, the impact on the blood serum protein profile and A. Ch. E. activity can be used as reliable criteria to detect acute toxicity of malathion and other choline-esterase inhibitors in exposed field workers. Further research is needed to elucidate the specificity and sensitivity of such criteria as biomarkers for human exposure.