O,O,S-Trimethyl phosphorothioate effects on immunocompetence

O,O,S-Trimethyl phosphorothioate (OOS), a contaminant of technical formulations of some organophosphorus pesticides, was found to be immunotoxic at subtoxic doses in female C57Bl/6 mice. Mice treated orally with acute doses of 10 mg/kg OOS show no overt toxic signs such as weight loss or malaise. In addition, the levels of serum cholinesterase was not decreased. Histopathologic investigation demonstrated no alterations in liver, lung, kidney, heart, skin, brain, spleen, or gut. The LD₅₀ for delayed toxicity was approximately 35 mg/kg. Despite the lack of general toxic changes at doses of 5–10 mg/kg OOS, specific immunotoxic changes were found. The humoral or cell-mediated immune response of splenocytes from mice treated with 10 mg/kg OOS to in vivo immunization was diminished with respect to control animals. Responses were measured in ex vivo assays. Cytotoxic T-lymphocyte (CTL) responses were assessed by alloimmunization with the tumor P815 followed by a ⁵¹Cr release assay done ex vivo with splenic lymphocytes. Humoral responses were assessed by immunization with sheep red blood cells followed by a Jerne plaque assay to determine anti-sheep red blood cell antibody. Both cellular and humoral responses could be stimulated in vitro using cells from OOS-pretreated, primed animals, thus indicating that no permanent cellular elterations had occurred.     

In vitro effects of malathion and O,O,S-trimethyl phosphorothioate on cytotoxic T-lymphocyte responses

Oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in technical formulations of malathion, has been shown to be associated with a high incidence of pneumonia in rats and to be highly immunosuppressive in mice. Based on these findings, an in vitro model was established to study the effect of this and other organophosphorus compounds on murine cytotoxic T-lymphocyte (CTL) responses. The organophosphorus compounds were tested for their ability to block in vitro generation of CTL responses to alloantigen and/or the expression of these cytotoxic responses. Responses were generated in C57Bl/6 (H-2^b) spleen cells to mitomycin C-blocked P815 (H-2^d) tumor cells. The cytotoxicity of the cultured splenocytes to P815 target was measured using a 4-hr chromium release assay. These data demonstrated that malathion was able to block the ability of splenocytes to sensitize to P815 at concentrations as low as 25 μg/ml, but was not able to block the expression of cytotoxicity by mature killer T cells. The same was true for OOS which had been activated by preincubation with rat liver postmitochondrial supernatant (PMS). Activated OOS blocked the generation of CTL responses at concentrations as low as 75 μg/ml while having no effect on mature cytotoxic cells. In fact, both malathion and activated OOS were no longer able to suppress CTL responses if treatment was performed as early as 24 hr after exposure to antigen. Additionally, it was demonstrated that when malathion was preincubated with PMS it was no longer suppressive and that OOS without activation failed to suppress CTL responses.     

Lack of immunosuppressive effects of acute and subacute administration of malathion on murine cellular and humoral immune responses

Malathion has been previously shown to cause allergic responses and suppress the generation of a humoral immune response in vivo. In this study, the effect of in vivo administration of malathion on cellular, humoral and mitogenic responses was examined. Acute (50% LD₅₀) or subacute (10% LD₅₀ per day for 14 days) treatment with malathion in vivo did not affect the in vivo generation of specific antibody secreting cells to sheep red blood cells (SRBC) or cytotoxic T lymphocytes (CTL) to allogeneic tumor. However, 5 days following acute administration of malathion, there was a slight increase in humoral immune responsiveness. Acute treatment with 50% LD₅₀ purified malathion did not affect body weight, splenic cell number, or thymus size. However, mitogenic responses to Concanavalin A (Con A) and lipopolysaccharide (LPS) was significantly enhanced on all days tested following acute administration of malathion. In contrast, subacute treatment with malathion did not affect mitogenic response to Con A or LPS, but led to a significant decrease in thymic cell number.     

Comparative effects of organophosphorus insecticides on the activities of acetylcholinesterase,diacylglycerol kinase,and phosphatidylinositol phosphodiesterase in rat brain microsomes

The activities of acetylcholinesterase, diacylglycerol kinase, and phosphatidylinositol phosphodiesterase in rat brain microsomes were measured in the presence and absence of the organophosphorus insecticides, parathion and diazinon, and their respective oxon analogs, paraoxon and diazoxon. Marked inhibition of acetylcholinesterase (by 45–99%) was observed in the presence of paraoxon (10^?2–10^?6M) and diazoxon (10^?2–10^?4M). Reduction of acetylcholinesterase activity (by 22–33%) was achieved with the parent insecticides at high concentrations only (10^?2M). In most cases, diacylglycerol kinase was insensitive to the pesticides. Marked stimulation of phosphatidylinositol phosphodiesterase (by 10–57%) was observed in the presence of all pesticides (10^?2–10^?3M). The phosphodiesterase exhibited slightly greater sensitivity to the parent compounds compared to the oxon derivatives. Stimulation of the phosphodiesterase by the insecticides was not correlated with acetylcholinesterase inhibition. Accordingly, the increase in phosphodiesterase activity was judged not to be acetylcholine mediated, but rather represented a direct effect of the pesticides on the enzyme or its microenvironment. Based on the present in vitro observations, it is proposed that certain organophosphorus pesticides may interfere with the normal process of synaptic transmission through both the inhibition of acetylcholinesterase and the stimulation of phosphatidylinositol phosphodiesterase. In view of the high concentrations of pesticides required to elicit the latter effect, interpretation of its physiological significance must await results from further studies performed in vivo.     

Evaluation of pesticide effects on humoral response to sheep erythrocytes and mouse hepatitis virus 3 by immunosorbent analysis

Effect of selected organochlorine, organophosphorus, and carbamate pesticides on the humoral immune IgM response was examined upon immunization of inbred C57B1/6 mice with neutral, polyvalent, T-dependent sheep erythrocytes (SRBC) and T-independent lipopolysaccharide (LPS). In addition, a pathogenic antigen, mouse hepatitis virus 3 (MHV3) was used for determination for the interaction of selected pesticides on the primary IgG immune response in a model of viral infection, of the genetically resistant A/J mouse strain. Single, sublethal doses of dieldrin, carbofuran, and matacil induced a marked immunosuppression of the humoral responses to both neutral and pathogenic antigens. The data showed that single, sublethal doses (0.4 ≤ LD₅₀ ≤ 0.6) of dieldrin, carbofuran, and matacil inhibited the number of SRBC-primed cells without any direct cytotoxic effect on the activated plasmocyte, as the titer of specific antibody measured by an enzyme-linked immunosorbent assay (ELISA) per activated cell, was constant. In contrast, exposure to malathion at 10–14 days prior to the assay increased the number of plaque-forming cells (PFC) and the anti-SRBC IgM and anti-MHV3 IgG antibody titer, suggesting therefore an increase in the humoral response to neutral and pathogenic antigens in C57B1/6 and A/J mice. Immunomodulation of the humoral IgM response by selected pesticides was shown to take place at a stage prior to antibody secretion from the activated cell, as the ELISA/PFC index was similar to the control value. The data obtained for dieldrin-induced inhibition of the humoral response to SRBC and LPS antigens suggest a mechanism of immunosuppression common for both T-dependent and T-independent antigens. Good correlations were obtained for the immunomodulatory effects of selected pesticides, as measured by PFC and ELISA, which encourages support for the latter technique in the immunotoxicological screening of pesticides.     

In vitro and in vivo effects of some pesticides on carbonic anhydrase enzyme from rainbow trout (Oncorhynchus mykiss) gills

Here we investigated the in vitro and in vivo effects of the pesticides, deltamethrin, diazinon, propoxur and cypermethrin, on the activity of rainbow trout (rt) gill carbonic anhydrase (CA). The enzyme was purified from rainbow trout gills using Sepharose 4B-aniline-sulfanilamide affinity chromatography method. The overall purification was approx. 214-fold. SDS-polyacrylamide gel electrophoresis showed a single band corresponding to a molecular weight of approx. 29 kDa. The four pesticides dose-dependently inhibited in vitro CA activity. IC₅₀ values for deltamethrin, diazinon, propoxur and cypermethrin were 0.137, 0.267, 0.420 and 0.460 μM, respectively. In vitro results showed that pesticides inhibit rtCA activity with rank order of deltamethrin > diazinon > propoxur > cypermethrin. Besides, in vivo studies of deltamethrin were performed on CA activity of rainbow trout gill. rtCA was significantly inhibited at three concentrations (0.25, 1.0 and 2.5 μg/L) at 24 and 48 h.     

Risk assessment of pesticides and fungicides for acid-base regulation and salt transport in rainbow trout tissues

Carbonic anhydrase enzymes have been shown to play an important role in ion transport and pH regulation in several organisms including fish. Despite this information and the wealth of knowledge regarding the significance of CA enzymes in fish, few studies have reported the hazardous effects of chemicals on fish CA. Using rainbow trout (Oncorhynchus mykiss) as a model, this study aimed to determine the risk of pesticides and fungicides on fish carbonic anhydrase enzymes. For this purpose, screening of rainbow trout carbonic anhydrase activities was carried out in the presence of some pesticides and fungicides. Carbonic anhydrase enzymes were initially purified from rainbow trout liver, muscle, kidney and brain, and exposed to pesticides and fungicides, including mancozeb, cypermethrin, deltamethrin and dinocap. Pesticides and fungicides dose-dependently decreased in vitro CA activity at micromolar concentrations. Since deltamethrin was the most powerful inhibitor for all tissues in in vitro experiments, it was chosen for in vivo analysis as well. rtCAs were significantly inhibited by the three concentrations of deltamethrin (0.25, 1.0 and 2.5 μg/L) at 24th and 48th hours. Our findings indicate that deltamethrin, dinocap, mancozeb and cypermethrin are potent inhibitors for fish CA enzymes, and might cause undesirable results by disrupting acid-base regulation as well as salt transport in freshwater or seawater adapted fish.     

Cholinesterase (ChE) inhibition in pumpkinseed (Lepomis gibbosus) as environmental biomarker: ChE characterization and potential neurotoxic effects of xenobiotics

Inhibition of cholinesterases (ChEs) has been widely used as an environmental biomarker of exposure to organophosphates (OP) and carbamate (CB) pesticides. More recently, this biomarker has been suggested as a putative biomarker for exposure to detergents. The use of cholinesterase inhibition as effect criterion in Ecotoxicology requires the previous characterization of the specific enzymatic forms that may be present in different tissues or organs. Different ChEs isoforms may be present in the same tissue and may exhibit distinct sensitivities towards environmental contaminants. This work intended to characterize the soluble ChEs present in pumpkinseed sunfish (Lepomis gibbosus) total head and dorsal muscle homogenates, through the use of different substrates and selective inhibitors of cholinesterasic activity. Also, the in vitro effects of sodium dodecylsulphate (SDS - anionic detergent) and chlorfenvinphos (organophosphate pesticide) on the enzymatic activity of the mentioned species were investigated. In general terms, the predominant cholinesterasic form present in both tissues was acetylcholinesterase. Chlorfenvinphos was responsible for inhibitory effects on AChE activity, while SDS did not cause any significant effect. These results suggest that in environmental monitoring programs, L. gibbosus head and dorsal muscle AChE can be an adequate diagnostic tool for exposure to OP pesticides; this conclusion however is not applicable to detergent residues. We also discuss the usefulness of L. gibbosus as an alternative model system and valuable option for freshwater ecotoxicological monitoring programs.     

Acetylcholinesterase of Aphis citricola: Properties and significance in determining toxicity of systemic organophosphorus and carbamate compounds

In apterous adults of the spirea aphid, Aphis citricola van der Goot, the optimum conditions for determining acetylcholinesterase (AChE) activity consist of reaction mixture of 0.1 M phosphate buffer (pH 7.5), 10^?3M acetylthiocholine (ASCh), and enzyme extract equivalent to 80 ± 3 μg protein incubated for 15 min at 30°C. The K_m value for ASCh (6.7 × 10^?5M) was much lower than that of butyrylthiocholine (BuSCh) (1.25 × 10^?2M). The enzyme activity was almost completely inhibited by 10^?6M paraoxon or 10^?5M eserine and was 84% inhibited by 10^?5M BW284C51 (a specific AChE inhibitor). DTNB was found to inhibit the enzyme activity and was therefore added at the end of the reaction. AChE activity of A. citricola was inhibited in vitro and in vivo by dimethoxon > dimethoate, and aldicarb sulfoxide > aldicarb > aldicarb sulfone. The in vivo effect correlates well with the toxicity level of the various toxicants. A neurotoxicity index which combines both mortality and in vivo inhibition of the aphid AChE activity is suggested as a measure for determining the toxicity of organophosphorus and carbamate compounds toward aphids.     

Insecticidal properties,antiesterase activities,and metabolism of methamidophos

Methamidophos is highly toxic to insects but at best is a moderate cholinesterase inhibitor. Evaluation of the kinetics of its housefly cholinesterase inhibition showed that its affinity for the enzyme and its phosphorylation and bimolecular inhibition rates are all relatively low. In vivo metabolism studies in houseflies provided evidence that it is not activated to a more effective cholinesterase inhibitor and indirect evidence also was obtained for its slow degradation. In vitro metabolism studies in housefly and mouse tissues provided additional evidence for its lack of activation and slow metabolic degradation. Compared to other effective organophosphorus insecticides, methamidophos was slow in producing acute symptoms of poisoning and cholinesterase inhibition and required the accumulation of comparatively high internal levels for toxic effects. However, in vivo cholinesterase inhibition studies provided evidence for the interrelationships of cholinesterase inhibition and toxic effects. Thus, its relative stability and low in vivo degradation appeared to be of critical importance in accumulating and maintaining a sufficient internal concentration for a sufficiently long period of time to permit the development of its slowly expressed toxicity.     

Synergism of organophosphorus insecticides by diethyl maleate and related compounds in house flies

The toxicity of several organophosphorus and one carbamate insecticide for house flies is enhanced by simultaneous administration of diethyl maleate. Synergism factors vary from 2 to 116 and are strongly dependent on the combination of strain and insecticide studied. In general, it seems that thiono compounds are less synergized than their oxon analogs. Comparison of some analogs of maleic acid esters showed that the diethyl ester was not the most active compound. trans-Phenylbutenone was found to be a good alternative for diethyl maleate. Both diethyl maleate and trans-phenylbutenone deplete glutathione in the flies, but this depletion per se is certainly not the most important mode of action. In vitro experiments made clear that sufficient glutathione remained to permit a substantial insecticide degradation rate. Moreover, treatment of the flies with diethyl maleate 2–4 hr before the insecticide application resulted in a partial or complete disappearance of synergism in a period in which the glutathione concentration in the flies was still very low. Apart from this effect on the glutathione levels in the insect tissues it appeared from further experimental work in vitro that both compounds had a direct inhibitory effect on glutathione-dependent transferase(s) as well as on oxidative enzymes involved in insecticide degradation. The contribution of these reactions to the eventual synergism factor is discussed.     

Herbicidal interference with the photochemical activities of chloroplasts (in vivo and in vitro) of certain crop and weed species

The independent modes of action of diuron and atrazine on the photochemical activities of chloroplasts (In vivo and in vitro) from the leaves of crop plants Pisum sativum and Pennisetum typhoides and the weeds Amaranthus viridis and Cyperus rotundus were investigated. Hill reaction activity (DCPIP photoreduction) of in vivo chloroplasts (chloroplasts isolated from herbicide-sprayed plants) was unaffected by treatment at sublethal or intermediate levels of diuron or atrazine while that of in vitro chloroplasts (chloroplasts incubated in the required herbicidal concentration) was severely inhibited. The ferricyanide catalyzed noncyclic photophosphorylation was markedly reduced in both the in vivo and in vitro chloroplast systems. N-Methyl phenozonium sulfate (PMS)-mediated cyclic photophosphorylation was inhibited in the in vivo system while a pronounced enhancement of activity was noticed in the in vitro chloroplasts. The rate of NADP⁺ photoreduction was severely inhibited in the in vitro chloroplasts. The unaffected in the in vivo system. The herbicidal effects on the photoreactions of isolated chloroplasts were compared with chloroplasts isolated from herbicide-sprayed plants.     

白腐真菌降解农药研究进展

白腐真菌是一类腐生的丝状真菌，其分泌的非特异性胞外氧化酶可以降解多种结构不同、毒性较高且难以降解的有机农药。本研究总结了白腐真菌对有机氯、有机磷、氨基甲酸酯和拟除虫菊酯类农药的降解效果，并对部分农药的降解途径研究进展进行了综述，分析了白腐真菌降解农药的影响因素，同时对其应用前景进行了展望。     

Methylparathion- and carbofuran-induced mitochondrial dysfunction and oxidative stress in Helicoverpa armigera (Noctuidae: Lepidoptera)

The cotton bollworm, Helicoverpa armigera is a polyphagous pest of several crops in Asia, Africa, and the Mediterranean Europe. Organophosphate and carbamate insecticides are used on a large-scale to control Helicoverpa. Therefore, we studied the effect of methylparathion and carbofuran, an organophosphate and carbamate insecticide, respectively, on oxidative phosphorylation and oxidative stress in H. armigera larvae to gain an understanding of the different target sites of these insecticides. It was observed that state III and state IV respiration, respiratory control index (RCI), and P/O ratios were inhibited in a dose-dependent manner by methylparathion and carbofuran under in vitro and in vivo conditions. Methylparathion and carbofuran inhibited complex II by ∼45% and 30%, respectively. Lipid peroxidation, H₂O₂ content, and lactate dehydrogenase (LDH) activity increased and glutathione reductase (GR) activity decreased in a time- and dose-dependent manner in insecticide-fed larvae. However, catalase activity was not affected in insecticide-fed larvae. Larval growth decreased by ∼64% and 67% in larvae fed on diets with 100 μM of methylparathion and carbofuran. The results suggested that both the insecticides impede the mitochondrial respiratory functions and induced lipid peroxidation, H₂O₂, and LDH leak, leading to oxidative stress in cells, which contribute to deleterious effects of these insecticides on the growth of H. armigera larvae, along with their neurotoxic effects.     

Is insecticide-induced release of insect neurohormones a secondary effect of hyperactivity of the central nervous system?

Organochlorine and organophosphorus insecticides, in the concentration range of 1–10 μM, induce the release of hyperlipemic (adipokinetic) hormone from the isolated corpora cardiaca of the locust Locusta migratoria. Treatment of locusts with these insecticides in vivo also provokes the release of hyperlipemic hormone. The insecticide-induced release of hormone in vivo was found to precede the onset of poisoning symptoms. The insecticides tested in this study modulate the electrical activity of the isolated corpora cardiaca at doses lower than those required to have similar effects on the central nervous system. The results indicate that the insecticide-induced release of hormone may be mediated by the action of insecticides directly on neurosecretory cells.     

Implication of human drug transporters to toxicokinetics and toxicity of pesticides

Human membrane drug transporters are recognized as major actors of pharmacokinetics. Pesticides also interact with human drug transporters, which may have consequences for pesticide toxicokinetics and toxicity. The present review summarizes key findings about this topic. In vitro assays have demonstrated that some pesticides, belonging to various chemical classes, modulate drug transporter activity, regulate transporter expression and/or are substrates, thus bringing the proof of concept for pesticide‐transporter relationships. The expected low human concentration of pesticides in response to environmental exposure constitutes a key‐parameter to be kept in mind for judging the in vivo relevance of such pesticide‐transporter interactions and their consequences for human health. Existing data about interactions of pesticides with drug transporters remain, however, rather sparse; more extensive and systematic characterization of pesticide‐transporter relationships, through the use of high throughput in vitro assays and/or in silico methods, is, therefore, required. In addition, consideration of transporter polymorphisms, pesticide mixture effects and physiological and pathological factors governing drug transporter expression may help to better define the in vivo relevance of pesticide‐transporter interactions in terms of toxicokinetics and toxicity for humans. © 2019 Society of Chemical Industry     

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

The metabolism in vivo and in vitro of [¹⁴C]parathion and [¹⁴C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina. Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.     

The effect of insecticides containing the thionophosphoryl group on peptide neurotransmitter action in the hindgut of Periplaneta americana

Application of phosphorothionate and phosphorothiolothionate insecticides at micromolar concentrations inhibited the hindgut contraction of Periplaneta americana induced by proctolin or proctodeal nerve stimulation. At identical concentrations, oxon analogues of these insecticides had no effect on proctolin or neural responses in the hindgut while a phosphonate insecticide, EPN, had a long-lasting potentiating action on contraction. The present investigation identifies a new site of organophosphorus insecticide action that may be a useful model system for the design and testing of pesticides that interfere with peptidergic transmission.     

Researches on the evaluation of pesticide safety in humans using a pharmacokinetic approach

Similar to the pharmaceutical compounds, pesticides require human safety assessment for their registration and distribution; however, it is absolutely impossible to assess human safety by dosing humans with pesticides. Thus, how to appropriately evaluate the safety of pesticides in humans remains a great subject of debate. In this article, we present some examples of pesticide toxicity studies that identify species differences in toxicity and evaluate human safety by applying combinations of novel in vivo, in vitro, and in silico techniques to separately assess the key toxicodynamic (i.e., sensitivity) and/or toxicokinetic (i.e., exposure) factors. Because it is scientifically sound, the safety assessment strategy illustrated for three compounds in this article is expected to play an important role in the human safety assessment of agricultural compounds.     

From in vitro hepatic metabolic studies towards human health risk assessment: Two case studies of diuron and carbosulfan

Risk assessment of environmental pollutants is an absolutely essential tool in the overall process of protecting public health. Risk assessment needs reliable scientific information and one source of information is the characterization of metabolic fate and toxicokinetics of environmental pollutants. The aim of in vitro characterization is to produce relevant information on metabolism and interactions to anticipate and ultimately predict what happens in vivo in humans. Because human data is most appropriate to improve human risk assessment, the best option is to rely upon human-derived in vitro models and obtain quantitative toxicokinetics data from in vitro systems for the comparison between species or individuals. In short, based on our studies of pesticide metabolism and interactions, we have used in vitro metabolism data in human and animal hepatic in vitro models and clearance testing data to calculate chemical-specific adjustment factors, instead of fixed uncertainty factors, to be employed as an alternative and more realistic model for human health risk assessment of pesticides and other environmental pollutants.