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Organophosphate pesticides (OPs) are known to inhibit acetylcholine esterase (AChE), a critical effect used to establish health-based guidance values. This study developed a combined in vitro-in silico approach to predict AChE inhibition by the OP profenofos in rats and humans. A physiologically based kinetic (PBK) model was developed for both species. Parameter values for profenofos conversion to 4-bromo-2-chlorophenol (BCP) were derived from in vitro incubations with liver microsomes, liver cytosol, and plasma from rats (catalytic efficiencies of 1.1, 2.8, and 0.19 ml/min/mg protein, respectively) and humans (catalytic efficiencies of 0.17, 0.79, and 0.063 ml/min/mg protein, respectively), whereas other chemical-related parameter values were derived using in silico calculations. The rat PBK model was evaluated against literature data on urinary excretion of conjugated BCP. Concentration-dependent inhibition of rat and human AChE was determined in vitro and these data were translated with the PBK models to predicted dose-dependent AChE inhibition in rats and humans in vivo. Comparing predicted dose-dependent AChE inhibition in rats to literature data on profenofos-induced AChE inhibition revealed an accurate prediction of in vivo effect levels. Comparison of rat predictions (BMDL10 of predicted dose-response data of 0.45 mg/kg bw) and human predictions (BMDL10 of predicted dose-response data of 0.01 mg/kg bw) suggests that humans are more sensitive than rats, being mainly due to differences in kinetics. Altogether, the results demonstrate that in vivo AChE inhibition upon acute exposure to profenofos was closely predicted in rats, indicating the potential of this novel approach method in chemical hazard assessment.

The dietary specialist fruit fly Drosophila sechellia has evolved resistance to the secondary defence compounds produced by the fruit of its host plant, Morinda citrifolia. The primary chemicals that contribute to lethality of M. citrifolia are the medium-chain fatty acids octanoic acid (OA) and hexanoic acid. At least five genomic regions contribute to this adaptation in D. sechellia and whereas the fine-mapped major effect locus for OA resistance on chromosome 3R has been thoroughly analysed, the remaining four genomic regions that contribute to toxin resistance remain uncharacterized. To begin to identify the genetic basis of toxin resistance in this species, we removed the function of well-known detoxification gene families to determine whether they contribute to toxin resistance. Previous work found that evolution of cytochrome P450 enzymatic activity or expression is not responsible for the OA resistance in D. sechellia. Here, we tested the role of the two other major detoxification gene families in resistance to Morinda fruit toxins - glutathione-S-transferases and esterases - through the use of the pesticide synergists diethyl maleate and tribufos that inhibit the function of these gene families. This work suggests that one or more esterase(s) contribute to evolved OA resistance in D. sechellia., (© 2019 The Royal Entomological Society.)

The effect of triazophos (O, O-diethyl O-1-phenyl-1 H-1, 2, 4-triazol-3-yl phosphorothioate), a widely used insecticide was studied on the induction of oxidative stress and histological alterations at sub-chronic doses in male albino rats. Oral administration of triazophos at concentrations of 1.64, 3.2 and 8.2 mg/kg body wt for 30 days produced dose as well as time-dependent increase in the lipid peroxidation (determined by malondialdehyde levels) and glutathione-S-transferase (GST) activity in serum with aconcomitant decrease in ferric reducing ability of plasma (FRAP) and blood glutathione (GSH) content. Histopathological examination of liver of triazophos-treated rats showed significant and progressive degenerative changes as compared to control, which could be due to induction of oxidative stress. However, no significant histopathological changes were observed in spleen, kidney and brain at either dose of triazophos with respect to control. These results indicated that oral administration of triazophos was associated with enhanced lipid peroxidation and compromised antioxidant defence in rats in dose and time-dependent manner. Thus the present study demonstrated for the first time the role of oxidative stress as the important mechanism involved in the stimulation of hepatic histoarchitectural alterations at sub-chronic doses of triazophos in rats.

O,O,S-Trimethylphosphorothioate (OOS-TMP) has been shown to induce hypophagia and hypopraxia. Recent studies suggest that OOS-TMP-induced anorexia is partly mediated by its effect on the central nervous system. In this study, we examined the profiles of N-acylethanolamines (NEAs), including five amide-linked compounds, in the gastrointestinal system in C57BL/6J (B6) mice. The present results shown an orexigenic profile of the levels of NEAs with downregulation of the anorectic lipid, N-stearoylethanolamine (SEA), upregulation of the orexigenic lipid, 2-arachidonoyl glycerol (2-AG), at 2 h and upregulation of 2-AG at 24 h albeit with significant anorexia. However, the data indicated that the high level of 2-AG may be responsible for the hypopraxia. We next explored whether OOS-TMP may affect two models of hyperphagia and hyperglycemia, ins2(+/Akita) B6 (Akita) and B6-lepr(db)/lepr(db) mice (db/db). We identified potential anorexigenic effects in B6, Akita and db/db mice. Moreover, OOS-TMP was found to reduce blood glucose in Akita mice but not in db/db mice. Collectively, these findings suggest that N-acylethanolamines are not involved in the hypophagia but rather hypopraxia, and may play multiple physiological roles in this process. OOS-TMP might be a promising candidate for anti-obesity and anti-diabetic drug development.

Acute toxicity, morphological alterations and histological effects of an organophosphorus insecticide, profenofos (PFF) to earthworm, Eisenia foetida were evaluated by direct contact through a filter paper. The 24 and 48 h LC(50) was 4.56 and 3.55 microg cm(-2), respectively. Morphological and histological observations showed body ruptures, bloody lesions, and internal excessive formation of glandular cell mass and disintegration of circular and longitudinal muscles, which failed to regulate the internal coelomic pressure, leading to fragmentation in earthworms. Neurotoxic potentiality of PFF was assessed by measuring acetylcholinesterase (AChE). AChE basal K(m) and V(max) values were 0.26 mM and 0.15 micromol min(-1) mg(-1) protein, respectively. PFF alters the in vitro K(m) value, resulting in a competitive type of inhibition. The inhibitory constant K(i) was 2.52x10(-6) M. Morphological alterations and histological effects should be monitored in addition to AChE inhibition to assess OP pesticide contamination in soil ecosystems.

Six-week-old male and female Japanese quails (Coturnix japonica) received two organophosphate pesticides, isazofos and pyraclofos, for a 21-day dietary toxicity test, based on the OECD workshop report. During the treatment period, body weight and food consumption of the quail decreased with exposure to either isazofos or pyraclofos. Using the up-and-down procedure to determine the 50% mortality value, we found that the 21-day LC(50) of isazofos and pyraclofos were 40 and 87 mg/kg body weight, respectively. Ataxia, salivation, diarrhea, ruffled feathers, and convulsions at a dead point were observed with both pesticides. The tips of the villi were necrotic in the high dosage groups of isazofos- and pyraclofos-treated quail. Based on these results, body weight, food consumption, clinical signs, and histopathological findings may be useful parameters for detecting the dietary toxicity associated with isazofos and pyraclofos exposure. In addition, Japanese quail could be an excellent bird model for monitoring the toxicological risks of pesticides in Korea.

To investigate the effect of the phosphorothoate insecticide profenofos on male specific gene expression on rat testis, 16-week-old Wistar rats were orally administered at dose of 17.8 mg/kg twice weekly for 65 days. Gene expression in the testes was monitored by DNA microarray analysis and real-time RT-PCR, which revealed that genes related to steroidogenesis including cytochrome P450 17A1 (CYP17A1), steroidogenic acute regulatory protein (StAR) and CYP11A1 were significantly increased. Besides the testes were histopathologicaly examined, which revealed testicular destruction and degeneration represented by a layer of columnar epithelium, oedematous changes surrounding the seminiferous tubules besides vacuolated spermatogonial cells and more elongated Leydig cells. These data suggest that profenofos considered as one of the male reproductive toxicants. Furthermore, we propose that the above three steroidogenic-related genes and the gene of acrosomal reaction as potential biomarkers of testicular toxicity.

The safety of azamethiphos (AZA), an organophosphorous insecticide and the active ingredient of Salmosan, was evaluated in the European eel, seabass and rainbow trout. Fish were bathed in 0.1 ppm AZA for a period of 60, 120 or 240 min. After termination of each treatment fish were transferred to clean aquaria and randomly sampled over 21 days. Compared to controls, brain acetylcholinesterase (AChE) was inhibited up to 44, 56 and 62% in eels, seabass and trout, respectively, with the inhibition being significant for up to 4 days in eels and seabass and 7 days in trout. As result of the AChE depression, fish displayed motor hyperactivity and erratic jumping at the onset of treatment. Mortality was observed only in trout following exposure for 240 min. A variable correlation observed among species between the level of exposure, the reduced activity of brain AChE and the signs of toxicity suggest that brain AChE should be considered as an indicator of exposure rather than as an index of toxicity of AZA. The present data indicate that at the therapeutic dosage of 0.1 ppm AZA for 1h can be safely used in eels, seabass and trout. The extended treatment times up to 240 min were equally safe for eels and seabass but not for trout.

The use of combination pesticide (profenofos 40% + cypermethrin 4% EC) is on the rise in India and elsewhere. Cypermethrin is reported to be a tumor promoter, but its tumor-promoting potential along with profenofos is yet to be studied. A short-term, in vivo test for detection of a tumor promoter is cost-effective, saves time, and requires fewer animals than conventional methods. We report on a short-term test based on mechanisms of significant importance in tumor promotion. In this study, we have tested the tumor-promoting potential of a combination pesticide containing profenofos 40% and cypermethrin 4% EC in Wistar rats. Although cypermethrin has been reported as a tumor promoter, our study shows that the combination pesticide containing profenofos 40% and cypermethrin 4% EC is not a tumor promoter.

Several observations, both in humans and laboratory animals, have suggested that proanthocyanidins exhibit a broad spectrum of pharmacological, therapeutic and chemoprotective properties. Specifically, some of our earlier studies have shown that IH636 grape seed proanthocyanidin extract (GSPE, commercially known as ActiVin) provides excellent concentration- and dose-dependent protection against toxicities induced by diverse agents, such as acetaminophen, hydrogen peroxide, 12-O-tetradecanoylphorbol-13-acetate (TPA), smokeless-tobacco extract, idarubicin and 4-hydroxyperoxycyclophosphamide in both in vitro and in vivo models. In some models, GSPE proved to be a better cytoprotectant than vitamins C, E and beta-carotene. The purpose of this investigation was three fold: (i) to indirectly assess the bioavailability of GSPE in multiple target organs, (ii) quantify GSPE's capacity to avert cadmium chloride (CdCl2)-induced nephrotoxicity, dimethylnitrosamine (DMN)-induced splenotoxicity and O-ethyl-S,S-dipropyl phosphorodithioate (MOCAP)-induced neurotoxicity, and lastly (iii) to evaluate possible mechanisms of protection in mice. In order to determine all these, three separate experiments were designed and each experiment consisted of four groups, such as vehicle control, GSPE alone, toxicant alone and GSPE + toxicant. GSPE was administered orally (100 mg/Kg) for 7-8 days prior to the toxicant exposure. Parameters of the analyses included evaluation of serum chemistry changes (ALT, BUN and CK), histopathology and integrity of genomic DNA, both quantitatively and qualitatively. Results indicate that GSPE preexposure prior to cadmium chloride and DMN provided near complete protection in terms of serum chemistry changes (ALT, BUN and CK) and inhibition of both forms of cell death. e.g., apoptosis and necrosis. DNA damage, a common denominator usually associated with both apoptosis and necrosis was significantly reduced by GSPE treatment. Histopathological examination of organs correlated strongly with the changes in serum chemistry and the DNA modification data. Surprisingly, MOCAP exposure showed symptoms of neurotoxicity coupled with serum chemistry changes in the absence of any significant genomic DNA damage or brain pathology. Although, GSPE appeared to partially protect the neural tissue, it powerfully antagonized MOCAP-induced mortality. Taken together, this study suggests that in vivo GSPE-preexposure may protect multiple target organs from a variety of toxic assaults induced by diverse chemical entities.

Certain esterase inhibitors such as O-(2-chloro-2,3,3-trifluorocyclobutyl) O-ethyl S-propyl phosphorothioate (KBR-2822) and phenylmethanesulfonyl fluoride (PMSF) cause exacerbation (promotion) of toxic and traumatic axonopathies. Although these chemicals are capable of inhibiting neuropathy target esterase (NTE), which is the target for organophosphate induced delayed neuropathy, the target for promotion is unlikely to be NTE. Experiments were aimed to ascertain if neuropathy is caused by repeated dosing with a promoter not causing NTE inhibition and in the absence of deliberate injury to axons. Hens were treated with KBR-2822 (0.2 or 0.4 mg/kg per day) by gavage for 90 days and observed for clinical signs up to 21-23 days after treatment when histopathological examination was carried out. NTE and acetylcholinesterase (AChE) were measured at intervals and mean percentages of inhibition at steady state of inhibition/resynthesis (on day 20) were as follows: mean inhibition NTE was < or = 8% in the 0.2 mg/kg group and between 15 and 18% in the 0.4 mg/kg group in brain, spinal cord and peripheral nerve; mean AChE inhibition in brain was 31 and 57% in the two experimental groups, respectively. Controls treated with paraoxon (not neuropathic or a promoter and given at 0.05 mg/kg per day by gavage) showed 45% mean AChE inhibition and no NTE inhibition. Neither clinical nor morphological signs of neuropathy were observed in any group. To ascertain whether subclinical lesions were produced by the repeated treatment with KBR-2822, hens were given KBR-2822 (0.2 mg/kg per day) for 21 days by gavage followed by PMSF (120 mg/kg s.c. 24 h after the last dose of KBR-2822). A control group of hens was treated with the neuropathic DFP (0.03 mg/kg s.c. daily for 21 days causing 40-50% NTE inhibition) followed by PMSF (120 mg/kg s.c.). After PMSF, the KBR-2822 treated hens did not develop neuropathy whereas DFP treated hens did. Lack of neuropathy after repeated treatment with KBR-2822 indicates that a continuous promoting 'pressure' on hen axons is harmless in the absence of a concurrent biochemical or neurotoxic injury.

The serum cholinesterase (ChE) isoenzyme in rats shows 6 bands after polyacrylamide gradient gel electrophoresis. The effects of organophosphates (fenthion, chlorpyrifos, diazinon, bromophos, propaphos, haloxon, and DFP) on serum ChE isoenzyme bands were studied in 32 male and 32 female 6-w-old Sprague-Dawley rats. Each organophosphate was randomly administered to 4 male and 4 female rats. Blood samples were collected from the abdominal aorta under halothane anesthesia 6 h after dosing. The isoenzyme patterns were determined simultaneously with erythrocyte and serum ChE activities. Changes were observed in all 6 bands of the serum ChE isoenzymes after administration of fenthion, chlorpyrifos and propaphos. Diazinon had no influence on band 6, and DFP and bromophos had no influence on band 5. Haloxon did not effect any of the serum ChE isoenzyme bands. Serum ChE was most suppressed by fenthion, followed by DFP, bromophos, chlorpyrifos, propaphos and diazinon in that order of effect. Serum ChE activity was not suppressed by haloxon. Erythrocyte ChE activity was suppressed by every organophosphate. This experiment demonstrated a correlation between the organophosphate suppression of serum ChE activity and the concentration of serum ChE isoenzyme band 6.

O,O,S-Trimethyl phosphorothioate (OOS-TMP) is known to induce unique symptoms, which are characterized by hypophagia, progressive weight loss, and hypothermia. To determine whether there is the possibility of a causal relationship between these toxic symptoms and a direct action of OOS-TMP on the central nervous system, we investigated the development of these symptoms in Fischer 344 female rats after oral or intracerebral treatment with OOS-TMP. Oral administration of OOS-TMP at 20 mg/kg induced marked hypophagia, progressive weight loss and hypothermia. Moreover, inhibition of respiratory rate was observed immediately after treatment. It lasts during the entire experimental period. Profound hypothermia below 34 degrees C was observed more frequently in the rats, which became hypercapnic (PaCO2 > or = 50 mmHg). In contrast, administration of OOS-TMP at 20 mg/kg (as much as the oral dose) into the cerebral lateral ventricle succeeded in inducing hypophagia, progressive weight loss and lowered respiratory rates. On the other hand, by this route of administration, OOS-TMP at 20 mg/kg failed to induce hypothermia, hypercapnia and lung injury. The present results suggest that hypophagia and inhibitions of respiratory rate are attributable to the direct action of OOS-TMP on the central nervous system, while other symptoms are associated with lung injury.

Organophosphate-induced delayed polyneuropathy (OPIDP) is thought to be initiated by a variety of neuropathy target esterase (NTE) inhibitors. However, certain inhibitors such as phenylmethanesulfonyl fluoride, phenyl N-methyl N-benzyl carbamate, and phenyl di-n-pentyl phosphinate protect from OPIDP when given to hens before organophosphorus esters. They protect from neuropathy by preventing the binding of neuropathic inhibitors to NTE catalytic site. In contrast, when such NTE inhibitors are given afterward, the resulting clinical effect is more severe. This phenomenon was called promotion of OPIDP. Promotion has been tentatively explained by the interaction of promoters with a target other than the catalytic center of NTE. However, the doses of promoters which cause the effect have, so far, been found to always be inhibitory of NTE. We report that the phosphorothioic acid O-(2-chloro-2,3,3-trifluorocyclobutyl) O-ethyl S propyl ester (KBR-2822) given to hens at doses which did not inhibit NTE (2.5 mg/kg p.o.) promoted the neuropathies initiated by either dibutyl-2,2-dichlorovinyl phosphate (DBDCVP, 0.4 mg/kg s.c., 24 hr earlier) or diisopropyl phosphorofluoridate (DFP, 0.3 mg/kg sc or 0.5 mg/kg s.c., 24 hr earlier). When given alone, DBDCVP and DFP (0.5 mg/kg) caused mild OPIDP, whereas the lower dose of DFP did not cause clinical effects. Dose-response relationships with KBR-2822 indicated that clinical effects of the combined treatments are unlikely to be additive because the compound did not cause OPIDP up to the maximum tolerated dose (10 mg/kg p.o.). Promotion also occurred when KBR-2822 (2.5 mg/kg p.o.) was given before either DBDCVP (0.4 mg/kg s.c.) or DFP (0.3 mg/kg s.c.). NTE inhibitions in the nervous tissues caused by DBDCVP or DFP were not affected by pretreatment with KBR-2822, suggesting that the delivery of neuropathic. NTE inhibitors was not modified. We conclude that KBR-2822 promotes OPIDP initiated by either DBDCVP or DFP by affecting a target other than NTE catalytic site.

Administration of a single oral dose of the malathion impurity, O,O,S-trimethyl phosphorothioate (OOS-Me) or O,S,S-trimethyl phosphorodithioate (OSS-Me), to the rat resulted in hemostatic disorders, e.g. prolongation of blood clotting, prothrombin and thrombin time. Deficiency of coagulation Factors II, V and VII was also observed. OOS-Me and OSS-Me also caused dose-dependent increases of beta-glucuronidase in the blood with a maximum of 15- and 31-fold observed following treatment with 60 mg/kg OOS-Me and 40 mg/kg OSS-Me, respectively. Analysis of serum beta-glucuronidase by isoelectrofocusing electrophoresis showed that the liver endoplasmic reticulum was the source of this enzyme released into the blood. Co-treatment of OOS-Me with 5% O,O,O-trimethyl phosphorothioate (OOO-Me), a potent antagonist of OOS-Me-induced delayed toxicity, prevented hemostatic disorders but had no effect in reducing beta-glucuronidase levels. However, pretreatment of rats with piperonyl butoxide reduced the amount of beta-glucuronidase released into the blood. Of other O,O,S-trialkyl phosphorothioates examined, the O,O-diethyl S-alkyl phosphorothioates showed the highest activity in increasing beta-glucuronidase levels.

Protection afforded by trialkyl phosphorothionates against the lung injury caused by trialkyl phosphorothiolates probably results from the inhibition by the P = S moiety of the thionates, of one or more pulmonary cytochrome P-450 isozymes. The aromatic hydrocarbons p-xylene and pseudocumene also protect against this injury and inhibit some P-450 isozymes, but by a different mechanism. OOS-Trimethylphosphorothionate and p-xylene were compared as protective agents against the effect of OOS-trimethylphosphorothiolate and two other lung toxins ipomeanol and 1-nitronaphthalene that are known to be activated by cytochrome P-450. The effects of these protective compounds, in vivo, on pulmonary cytochrome P-450 activity were also determined. Both compounds inhibited pentoxyresorufin O-deethylase activity, but not ethoxyresorufin O-deethylase. The phosphorothionate was most effective against lung injury caused by the phosphorothiolates and 1-nitronaphthalene, whereas p-xylene was much more effective against ipomeanol. beta-Naphthoflavone, which induces pulmonary ethoxyresorufin O-deethylase activity, did not protect against phosphorothiolate or 1-nitronaphthalene injury, and it was only marginally effective in decreasing the toxicity of ipomeanol.

Circadian and ultradian rhythms of blood acetylcholinesterase (ACE) and rectal temperature were investigated during hypokinesia and administration of a toxic organic phosphate compound in various doses. Hypokinesia was found to produce a strong desynchronizing effect on ACE and thermal regulation. It is maintained that the use of chemicals with a well documented mechanism of action may yield reliable information about biorhythm restructuring in response to exogenous effects. It is emphasized that biorhythm changes should be taken into consideration when arranging work schedules in hypokinetic conditions.

Male Baladi rabbits were acutely and sub-chronically intoxicated with cyanofenphos and profenophos. The levels of cholesterol, triglycerides, B-lipoproteins and total proteins were determined in the serum, brain, spinal cord and sciatic nerve of rabbits. Moreover, the activities of alkaline phosphatase, acid phosphatase and glutamic-pyruvic transaminase were determined in the liver of the animals. The whole studies revealed that the biochemical constituents were highly affected by the tested insecticides. Also, the liver function suffered from adverse effects of the tested insecticides.

Oral administration of O,O,S-trimethyl phosphorothioate (OOS), an impurity present in widely used organophosphorus insecticides, causes delayed toxicity in rats, i.e., death occurring as late as 28 days after the treatment. The signs of toxicity include body weight loss (maximum on day 3), red staining around the nose, mouth and eyes, and an increased level of lactate dehydrogenase (LDH) in bronchopulmonary lavage fluid accompanied by morphological alteration of non-ciliated bronchiolar epithelial Clara cells. Pretreatment with phenobarbital, piperonyl butoxide (2 h), SKF 525-A, or small multiple doses of OOS protected against the OOS-induced elevated level of bronchopulmonary lavage LDH, and the other signs of delayed toxicity including morphological alteration of Clara cells. These studies support the view that OOS-induced delayed toxicity is mediated by the cytochrome P-450 dependent metabolism of OOS, and the lung may be the major target organ of delayed toxicity produced by OOS.